PEPTIDOMIMETIC MACROCYCLES AND USES THEREOF

Abstract

The present disclosure describes the synthesis of peptidomimetic macrocycles and methods of using peptidomimetic macrocycles to treat a condition. The present disclosure also describes methods of using peptidomimetic macrocycles in combination with at least one additional pharmaceutically-active agent for the treatment of a condition, for example, cancer.

Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 31, 2018, is named 35224-823_201_SL.txt and is 1,195,677 bytes in size.

BACKGROUND

The human transcription factor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase MDM2, also known as HDM2, negatively regulates p53 function through a direct binding interaction, which neutralizes the p53 transactivation activity. Loss of p53 activity, either by deletion, mutation, or MDM2 overexpression, is the most common defect in human cancers.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a peptidomimetic macrocycle and at least one pharmaceutically-active agent, wherein the peptidomimetic macrocycle and the at least one pharmaceutically-active agent are administered with a time separation of more than 61 minutes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that treatment with SP262 and SP154 resulted in decreased PD-L1 expression in HCT-116 p53^+/+ cells, but not HCT-116 p53^−/− cells.

FIG. 2 illustrates the dosing regiments (DRs) used in the “3+3” dose escalation trial.

FIG. 3 shows drug concentration levels in patient plasma at all dose levels tested in Arm A (LEFT PANEL) and Arm B (RIGHT PANEL).

FIG. 4 shows fold-increase levels from baseline levels of plasma MIC-1 on cycle one, day one, two, or three (C1D1, C1D2, C1D3) at dose levels at or above 0.83 mg/kg.

FIG. 5 shows a waterfall plot that illustrates the anti-tumor activity of AP1 in patients of the Phase 1 dose-escalation trial.

FIG. 6 shows results of the anti-tumor activity study for 33 patients.

FIG. 7 shows the time-on-drug for evaluable p53-WT patients who had CRs, PRs, and SDs when dosed with AP1 at ≥3.2 mg/kg/cycle.

FIG. 8 PANEL A shows a 50-year-old patient with peripheral T-Cell Lymphoma (PTCL). FIG. 8 PANEL B shows that the lymph node returned to its normal size and was no longer detected by the PET tracer as being cancerous after six cycles of AP1 treatment. FIG. 8 PANEL C shows images of a 73-year-old patient with Merkel Cell Carcinoma (MCC). FIG. 8 PANEL D shows that skin lesions diminished in size and left only mild scar tissue after one cycle of AP1 treatment.

FIG. 9 LEFT PANEL shows PET scans from the first patient enrolled in the Phase 2 study prior to treatment with AP1. FIG. 9 RIGHT PANEL shows PET scans from the first patient enrolled in the Phase 2 study after 2 cycles of treatment with AP1.

FIG. 10 TOP PANEL shows percentage of human CD45 engraftment in bone marrow for the vehicle, and treatment with 20 mg/kg AP1. FIG. 10 BOTTOM PANEL shows the percentage survival of mice upon treatment with the vehicle or administration of AP1.

FIG. 11 shows a graph of MCF-7 cell proliferation determined using a WST-1 assay measured at the indicated time points after different numbers of MCF-7 cells were grown at 37° C. for a 24 hour growth period.

FIG. 12 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of ribociclib.

FIG. 13 shows MCF-7 cell proliferation when the cells were treated with AP1 or AP1 with varying concentrations of ribociclib.

FIG. 14 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of AP1. MCF-7 cells were treated with ribociclib or a combination of ribociclib and AP1 at concentrations of 0.1 μM, 0.3 μM, and 1 μM.

FIG. 15 shows MCF-7 cell proliferation when the cells were treated with ribociclib or ribociclib with varying concentrations of AP1.

FIG. 16 shows a combination index plot of ribociclib in MCF-7 cells.

FIG. 17 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of abemaciclib.

FIG. 18 shows MCF-7 cell proliferation when the cells were treated with AP1 or AP1 with varying concentrations of abemaciclib.

FIG. 19 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of AP1.

FIG. 20 shows MCF-7 cell proliferation when the cells were treated with abemaciclib or abemaciclib with varying concentrations of AP1.

FIG. 21 shows cell proliferation of MCF-7 cells when the cells were treated with palbociclib alone.

FIG. 22 shows cell proliferation of MCF-7 cells when the cells were treated with AP1 alone.

FIG. 23 shows MCF-7 cell proliferation when the cells were treated simultaneously with a fixed amount of AP1 and varying amounts of palbociclib.

FIG. 24 shows MCF-7 cell proliferation when the cells were treated simultaneously with a fixed amount of palbociclib and varying amounts of AP1.

FIG. 25 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of AP1 and palbociclib in different orders over a period of 72 h.

FIG. 26 shows MCF-7 cell proliferation when the cells were pre-treated with AP1 for 24 h and subsequently treated with varying concentrations of palbociclib; and when the cells were pre-treated with varying concentrations of palbociclib for 24 h and subsequently treated with a fixed amount of AP1.

FIG. 27 shows MCF-7 cell proliferation when the cells were pre-treated with varying concentrations of AP1 for 24 h and subsequently treated with fixed amounts of palbociclib; and when the cells were pre-treated with fixed amounts of palbociclib and subsequently treated with varying concentrations of AP1.

FIG. 28 shows MOLT-3 cell proliferation when the cells were treated with palbociclib alone.

FIG. 29 shows MOLT-3 cell proliferation when the cells were treated with AP1 alone.

FIG. 30 shows the combination index plot of the treatment of MCF-7 cells with AP1 and palbociclib using a WST-1 assay.

FIG. 31 shows the combination index plot of the treatment of MCF-7 cells with AP1 and palbociclib using CyQUANT.

FIG. 32 shows the effects of AP1, palbociclib, or combination treatment with AP1+palbociclib on the median tumor volumes in the SJSA-1 osteosarcoma xenograft model.

FIG. 33 shows the effects of AP1, palbociclib, or combination treatment with AP1+palbociclib on the median tumor volumes in the MCF-7.1 human breast carcinoma xenograft model.

FIG. 34 shows individual tumor volumes of mice treated with MCF-7.1 human breast carcinoma xenografts treated with the vehicle.

FIG. 35 PANEL A shows the individual tumor volumes of mice treated with AP1 20 mg/kg qwk×4. FIG. 35 PANEL B shows the individual tumor volumes of mice treated with palbociclib 75 mg/kg qd×22. FIG. 35 PANEL C shows the individual tumor volumes of mice treated with AP1, and treated with palbociclib 6 h after administration of AP1. FIG. 35 PANEL D shows the individual tumor volumes of mice treated with palbociclib, and treated with AP1 6 h after administration of AP1.

FIG. 36 shows the effects of AP1, palbociclib, or combination treatment with AP1+palbociclib on the median tumor volumes in the A549 xenograft model.

FIG. 37 PANEL A shows the effect of vehicle treatment on median tumor volumes in the A549 xenograft model. FIG. 37 PANEL B shows the effect of vehicle treatment on median tumor volumes in the A549 xenograft model.

FIG. 38 shows C32 cell proliferation when the cells were treated with trametinib alone or trametinib in combination with varying concentrations of AP1.

FIG. 39 shows the combination index plot of the treatment of C32 cells with AP1 and trametinib.

FIG. 40 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 with varying concentrations of trametinib.

FIG. 41 shows C32 cell proliferation when the cells were treated with varying concentrations of AP1 and varying concentrations of trametinib.

FIG. 42 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 and varying concentrations of trametinib.

FIG. 43 shows MEL-JUSO cell proliferation when the cells were treated with no agent, AP1 alone, trametinib alone, or 0.03 μM AP1 and 1.0 nM trametinib.

FIG. 44 shows MEL-JUSO cell proliferation when the cells were treated with trametinib alone or trametinib with varying concentrations of AP1

FIG. 45 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and trametinib.

FIG. 46 shows A375 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of trametinib.

FIG. 47 shows A375 cell proliferation when the cells were treated with trametinib alone or trametinib in combination with varying concentrations of AP1.

FIG. 48 shows the combination index plot of the treatment of A375 melanoma cells with AP1 and trametinib.

FIG. 49 shows C32 cell proliferation when the cells were treated with varying concentrations of binimetinib and AP1.

FIG. 50 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of binimetinib.

FIG. 51 shows C32 cell proliferation when the cells were treated with binimetinib alone or binimetinib in combination with varying concentrations of AP1.

FIG. 52 shows the combination index plot of the treatment of C32 cells with AP1 and binimetinib.

FIG. 53 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of binimetinib.

FIG. 54 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of binimetinib.

FIG. 55 shows MEL-JUSO cell proliferation when the cells were treated with binimetinib alone or binimetinib in combination with varying concentrations of AP1.

FIG. 56 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and binimetinib.

FIG. 57 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying combinations of pimasertib.

FIG. 58 shows C32 cell proliferation when the cells were treated with varying concentrations of AP1 and pimasertib.

FIG. 59 shows C32 cell proliferation when the cells were treated with pimasertib alone or pimasertib in combination with varying concentrations of AP1.

FIG. 60 shows the combination index plot of the treatment of C32 cells with AP1 and pimasertib.

FIG. 61 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of pimasertib.

FIG. 62 shows MEL-JUSO cell proliferation when the cells were treated with AP1 and pimasertib.

FIG. 63 shows MEL-JUSO cell proliferation when the cells were treated with pimasertib alone or pimasertib in combination with varying concentrations of AP1.

FIG. 64 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and pimasertib.

FIG. 65 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying combinations of selumetinib.

FIG. 66 shows C32 cell proliferation when the cells were treated with varying concentrations of AP1 and selumetinib.

FIG. 67 shows C32 cell proliferation when the cells were treated with selumetinib alone or selumetinib in combination with varying concentrations of AP1.

FIG. 68 shows the combination index plot of the treatment of C32 cells with AP1 and selumetinib.

FIG. 69 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of pimasertib.

FIG. 70 shows MEL-JUSO cell proliferation when the cells were treated with AP1 and pimasertib.

FIG. 71 shows MEL-JUSO cell proliferation when the cells were treated with pimasertib alone or pimasertib in combination with varying concentrations of AP1.

FIG. 72 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and pimasertib.

FIG. 73 shows combination treatment and dosing regimens used to study the effects of AP1 to treat AML.

FIG. 74 shows the results of treatment with AP1 or Paclitaxel on individual mouse tumor volume by day.

FIG. 75 shows the results of combination treatment with AP1+paclitaxel on individual mouse tumor volume by day.

FIG. 76 shows the results of treatment with AP1 or Paclitaxel on individual mouse tumor volume by day on a Log₁₀ axis to show growth.

FIG. 77 shows the results of combination treatment with AP1+paclitaxel on individual mouse tumor volume by day on a Log₁₀ axis to show growth.

FIG. 78 shows the results of treatment with AP1 or Paclitaxel on individual mouse tumor volume % change from baseline by day.

FIG. 79 shows the results of combination treatment with AP1+paclitaxel on individual mouse tumor volume % change from baseline by day.

FIG. 80 shows the results of treatment with AP1 or Paclitaxel on median tumor volume % change from baseline by day.

FIG. 81 shows the results of combination treatment with AP1+paclitaxel on median tumor volume % change from baseline by day.

FIG. 82 shows the results of treatment with AP1 or Paclitaxel on average (±1 StDev) tumor volume % change from baseline by day.

FIG. 83 shows the results of combination treatment with AP1+paclitaxel on average (±1 StDev) tumor volume % change from baseline by day.

FIG. 84 compares the results of treatment with AP1, paclitaxel, or combination treatment with AP1+paclitaxel on the average % change in tumor volume from baseline per day.

FIG. 85 compares the results of treatment with AP1, paclitaxel, or combination treatment with AP1+paclitaxel on the average % change in tumor volume from baseline per day.

FIG. 86 shows the effect of treatment with AP1, paclitaxel, or combination treatment with AP1+paclitaxel on individual tumor volume % change from baseline on Day 28 per study group.

FIG. 87 shows the effect of treatment with AP1, eribulin, or combination treatment with AP1+eribulin on the average % change of tumor volume.

FIG. 88 shows the effect of treatment with AP1, eribulin, or combination treatment with AP1+eribulin on individual tumor volume % change from baseline on Day 28

FIG. 89 shows changes in the normalized body weights of mice treated under various dosing regimens of AP1, Abraxane®, or combination treatment with AP1+Abraxane® over a period of 12 days in the MCF-7.1 human breast carcinoma xenograft model.

FIG. 90 shows changes in tumor volumes (mm³) of mice treated under various dosing regimens over a period of 12 days in the MCF-7.1 human breast carcinoma xenograft model.

FIG. 91 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 91 PANEL B shows the results of treatment with anti-PD-1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 91 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 91 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model.

FIG. 92 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 92 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 92 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 92 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model.

FIG. 93 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 93 PANEL B shows the results of treatment with anti-PD-1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 93 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 93 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model.

FIG. 94 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 94 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 94 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 94 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model.

FIG. 95 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 95 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 95 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 95 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model.

FIG. 96 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 96 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 96 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 96 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model.

FIG. 97 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. FIG. 97 PANEL B shows the results of treatment with anti-CTLA-4 9H10 on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. FIG. 97 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. FIG. 97 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-CTLA-4 on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line.

DETAILED DESCRIPTION

The human transcription factor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase MDM2, also known as HDM2, negatively regulates p53 function through a direct binding interaction that neutralizes the p53 transactivation activity. Neutralization of p53 transactivation activity leads to export from the nucleus of p53 protein, which targets p53 for degradation via the ubiquitylation-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or MDM2 overexpression, is the most common defect in human cancers. Tumors that express wild type p53 are vulnerable to pharmacologic agents that stabilize or increase the concentration of active p53.

MDMX (MDM4) is a negative regulator of p53, and there is significant structural homology between the p53 binding interfaces of MDM2 and MDMX. The p53-MDM2 and p53-MDMX protein-protein interactions are mediated by the same 15-residue alpha-helical transactivation domain of p53, which inserts into hydrophobic clefts on the surface of MDM2 and MDMX. Three residues within this domain of p53 (F19, W23, and L26) are essential for binding to MDM2 and MDMX.

Provided herein are p53-based peptidomimetic macrocycles that modulate an activity of p53 and p53-based peptidomimetic macrocycles that inhibit the interactions between p53 and MDM2 and/or p53 and MDMX proteins. Also provided herein are the use of p53-based peptidomimetic macrocycles and an additional therapeutic agent for the treatment of a condition. Further, provided herein are p53-based peptidomimetic macrocycles and additional therapeutic agents that can be used for treating diseases, for example, cancer and other hyperproliferative diseases.

Definitions

As used herein, the term “macrocycle” refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinked polypeptide” refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analogue) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analogue) within the same molecule. Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the α-carbon of the first amino acid residue (or analogue) to the α-carbon of the second amino acid residue (or analogue). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analogue residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analogue residues in addition to any which form the macrocycle. A “corresponding uncrosslinked polypeptide” when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.

AP1 is an alpha helical hydrocarbon crosslinked polypeptide macrocycle with an amino acid sequence less than 20 amino acids long that is derived from the transactivation domain of wild type human p53 protein. AP1 contains a phenylalanine, a tryptophan and a leucine amino acid in the same positions relative to each other as in the transactivation domain of wild type human p53 protein. AP1 has a single cross link spanning amino acids in the i to the i+7 position of the amino acid sequence and has more than three amino acids between the i+7 position and the carboxyl terminus. AP1 binds to human MDM2 and MDM4 and has an observed mass of 950-975 m/e as measured by electrospray ionization-mass spectrometry.

As used herein, the term “stability” refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo. Non-limiting examples of secondary structures contemplated herein are α-helices, 3₁₀ helices, β-turns, and β-pleated sheets.

As used herein, the term “helical stability” refers to the maintenance of an α-helical structure by a peptidomimetic macrocycle as measured by circular dichroism or NMR. In some embodiments, a peptidomimetic macrocycle can exhibit at least a 1.25, 1.5, 1.75, or 2-fold increase in α-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.

The term “amino acid” refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally-occurring amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes, without limitation, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogues.

The term “α-amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the α-carbon.

The term “β-amino acid” refers to a molecule containing both an amino group and a carboxyl group in a β configuration.

The term “naturally-occurring amino acid” refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

The following table shows a summary of the properties of natural amino acids:

	3-	1-	Side-	Side-chain
	Letter	Letter	chain	charge	Hydropathy
Amino Acid	Code	Code	Polarity	(pH 7.4)	Index
Alanine	Ala	A	nonpolar	neutral	1.8
Arginine	Arg	R	polar	positive	−4.5
Asparagine	Asn	N	polar	neutral	−3.5
Aspartic acid	Asp	D	polar	negative	−3.5
Cysteine	Cys	C	polar	neutral	2.5
Glutamic acid	Glu	E	polar	negative	−3.5
Glutamine	Gln	Q	polar	neutral	−3.5
Glycine	Gly	G	nonpolar	neutral	−0.4
Histidine	His	H	polar	Positive (10%)	−3.2
				Neutral (90%)
Isoleucine	Ile	I	nonpolar	neutral	4.5
Leucine	Leu	L	nonpolar	neutral	3.8
Lysine	Lys	K	polar	positive	−3.9
Methionine	Met	M	nonpolar	neutral	1.9
Phenylalanine	Phe	F	nonpolar	neutral	2.8
Proline	Pro	P	nonpolar	neutral	−1.6
Serine	Ser	S	polar	neutral	−0.8
Threonine	Thr	T	polar	neutral	−0.7
Tryptophan	Trp	W	nonpolar	neutral	−0.9
Tyrosine	Tyr	Y	polar	neutral	−1.3
Valine	Val	V	nonpolar	neutral	4.2

“Hydrophobic amino acids” include small hydrophobic amino acids and large hydrophobic amino acids. “Small hydrophobic amino acids” are glycine, alanine, proline, and analogues thereof. “Large hydrophobic amino acids” are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogues thereof. “Polar amino acids” are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogues thereof. “Charged amino acids” are lysine, arginine, histidine, aspartate, glutamate, and analogues thereof.

The term “amino acid analogue” refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogues include, without limitation, β-amino acids and amino acids wherein the amino or carboxy group is substituted by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).

The term “non-natural amino acid” refers to an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acids or amino acid analogues include, without limitation, structures according to the following:

Amino acid analogues include β-amino acid analogues. Examples of β-amino acid analogues include, but are not limited to, the following: cyclic β-amino acid analogues; β-alanine; (R)-β-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid; (R)-3-amino-4-(2-methylphenyl)-butyric acid; (R)-3-amino-4-(2-naphthyl)-butyric acid; (R)-3-amino-4-(2-thienyl)-butyric acid; (R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(3,4-difluorophenyl)butyric acid; (R)-3-amino-4-(3-benzothienyl)-butyric acid; (R)-3-amino-4-(3-chlorophenyl)-butyric acid; (R)-3-amino-4-(3-cyanophenyl)-butyric acid; (R)-3-amino-4-(3-fluorophenyl)-butyric acid; (R)-3-amino-4-(3-methylphenyl)-butyric acid; (R)-3-amino-4-(3-pyridyl)-butyric acid; (R)-3-amino-4-(3-thienyl)-butyric acid; (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(4-bromophenyl)-butyric acid; (R)-3-amino-4-(4-chlorophenyl)-butyric acid; (R)-3-amino-4-(4-cyanophenyl)-butyric acid; (R)-3-amino-4-(4-fluorophenyl)-butyric acid; (R)-3-amino-4-(4-iodophenyl)-butyric acid; (R)-3-amino-4-(4-methylphenyl)-butyric acid; (R)-3-amino-4-(4-nitrophenyl)-butyric acid; (R)-3-amino-4-(4-pyridyl)-butyric acid; (R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (S)-3-amino-4-(1-naphthyl)-butyric acid; (S)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(2-chlorophenyl)-butyric acid; (S)-3-amino-4-(2-cyanophenyl)-butyric acid; (S)-3-amino-4-(2-fluorophenyl)-butyric acid; (S)-3-amino-4-(2-furyl)-butyric acid; (S)-3-amino-4-(2-methylphenyl)-butyric acid; (S)-3-amino-4-(2-naphthyl)-butyric acid; (S)-3-amino-4-(2-thienyl)-butyric acid; (S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(3,4-difluorophenyl)butyric acid; (S)-3-amino-4-(3-benzothienyl)-butyric acid; (S)-3-amino-4-(3-chlorophenyl)-butyric acid; (S)-3-amino-4-(3-cyanophenyl)-butyric acid; (S)-3-amino-4-(3-fluorophenyl)-butyric acid; (S)-3-amino-4-(3-methylphenyl)-butyric acid; (S)-3-amino-4-(3-pyridyl)-butyric acid; (S)-3-amino-4-(3-thienyl)-butyric acid; (S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(4-bromophenyl)-butyric acid; (S)-3-amino-4-(4-chlorophenyl)-butyric acid; (S)-3-amino-4-(4-cyanophenyl)-butyric acid; (S)-3-amino-4-(4-fluorophenyl)-butyric acid; (S)-3-amino-4-(4-iodophenyl)-butyric acid; (S)-3-amino-4-(4-methylphenyl)-butyric acid; (S)-3-amino-4-(4-nitrophenyl)-butyric acid; (S)-3-amino-4-(4-pyridyl)-butyric acid; (S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydropyridine-4-carboxylic acid; 3-amino-3-(2-chlorophenyl)-propionic acid; 3-amino-3-(2-thienyl)-propionic acid; 3-amino-3-(3-bromophenyl)-propionic acid; 3-amino-3-(4-chlorophenyl)-propionic acid; 3-amino-3-(4-methoxyphenyl)-propionic acid; 3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid; D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartic acid γ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester; L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine; L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan; L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolysine; Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine; (R)-β-phenylalanine; L-β-homoaspartic acid γ-t-butyl ester; L-β-homoglutamic acid δ-t-butyl ester; L-Nω-β-homolysine; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine; O-t-butyl-L-β-homohydroxy-proline; O-t-butyl-L-β-homoserine; O-t-butyl-L-β-homothreonine; O-t-butyl-L-β-homotyrosine; 2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylic acid.

Amino acid analogues include analogues of alanine, valine, glycine or leucine. Examples of amino acid analogues of alanine, valine, glycine, and leucine include, but are not limited to, the following: α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid; α-methyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine; β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine; β-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine; β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine; β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; β-chloro-L-alanine; β-cyano-L-alanine; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine; β-cyclopenten-1-yl-alanine; β-cyclopentyl-alanine; β-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; β-t-butyl-D-alanine; β-t-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid; 2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine; 2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine; 3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine; 4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt; cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-α,β-diaminopropionic acid; D-α-aminobutyric acid; D-α-t-butylglycine; D-(2-thienyl)glycine; D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine; D-allylglycine.dicyclohexylammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyric acid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine; (2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine; 2-amino-3-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid; L-α-aminobutyric acid; L-α-t-butylglycine; L-(3-thienyl)glycine; L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid dicyclohexyl-ammonium salt; L-2-indanylglycine; L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine; L-phenylglycine; L-propargylglycine; L-norvaline; N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid; L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine; (N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionic acid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid; (N-3-allyloxycarbonyl)-L-α,β-diaminopropionic acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyric acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid; (N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

Amino acid analogues include analogues of arginine or lysine. Examples of amino acid analogues of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)₂-OH; Lys(N₃)—OH; Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine; Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptanedioic acid; L-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithine; (Nδ-4-methyltrityl)-D-omithine; (Nδ-4-methyltrityl)-L-ornithine; D-omithine; L-omithine; Arg(Me)(Pbf)-OH; Arg(Me)₂-OH (asymmetrical); Arg(Me)₂-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)₂-OH.HCl; Lys(Me₃)-OH chloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

Amino acid analogues include analogues of aspartic or glutamic acids. Examples of amino acid analogues of aspartic and glutamic acids include, but are not limited to, the following: α-methyl-D-aspartic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid; D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid; L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamic acid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

Amino acid analogues include analogues of cysteine and methionine. Examples of amino acid analogues of cysteine and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.

Amino acid analogues include analogues of phenylalanine and tyrosine. Examples of amino acid analogues of phenylalanine and tyrosine include β-methyl-phenylalanine, β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3′-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)-D-phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, and methyl-tyrosine.

Amino acid analogues include analogues of proline. Examples of amino acid analogues of proline include, but are not limited to, 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.

Amino acid analogues include analogues of serine and threonine. Examples of amino acid analogues of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and α-methylserine.

Amino acid analogues include analogues of tryptophan. Examples of amino acid analogues of tryptophan include, but are not limited to, the following: α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

In some embodiments, amino acid analogues are racemic. In some embodiments, the D isomer of the amino acid analogue is used. In some embodiments, the L isomer of the amino acid analogue is used. In other embodiments, the amino acid analogue comprises chiral centers that are in the R or S configuration. In still other embodiments, the amino group(s) of a β-amino acid analogue is substituted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. In yet other embodiments, the carboxylic acid functional group of a β-amino acid analogue is protected, e.g., as its ester derivative. In some embodiments the salt of the amino acid analogue is used.

A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially abolishing its essential biological or biochemical activity (e.g., receptor binding or activation). An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a polypeptide, e.g., is replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g., norleucine for methionine) or other properties (e.g., 2-thienylalanine for phenylalanine, or 6-Cl-tryptophan for tryptophan).

The term “capping group” refers to the chemical moiety occurring at either the carboxy or amino terminus of the polypeptide chain of the subject peptidomimetic macrocycle. The capping group of a carboxy terminus includes an unmodified carboxylic acid (i.e. —COOH) or a carboxylic acid with a substituent. For example, the carboxy terminus can be substituted with an amino group to yield a carboxamide at the C-terminus. Various substituents include but are not limited to primary, secondary, and tertiary amines, including pegylated secondary amines. Representative secondary amine capping groups for the C-terminus include:

The capping group of an amino terminus includes an unmodified amine (i.e. —NH₂) or an amine with a substituent. For example, the amino terminus can be substituted with an acyl group to yield a carboxamide at the N-terminus. Various substituents include but are not limited to substituted acyl groups, including C₁-C₆ carbonyls, C₇-C₃₀ carbonyls, and pegylated carbamates. Representative capping groups for the N-terminus include, but are not limited to, 4-FBzl (4-fluoro-benzyl) and the following:

The term “member” as used herein in conjunction with macrocycles or macrocycle-forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.

The symbol “” when used as part of a molecular structure refers to a single bond or a trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to the α-carbon (or another backbone atom) in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally-occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an α,α di-substituted amino acid).

The term “α,α di-substituted amino” acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the β-carbon) that is attached to two natural or non-natural amino acid side chains.

The term “polypeptide” encompasses two or more naturally- or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).

The term “first C-terminal amino acid” refers to the amino acid which is closest to the C-terminus. The term “second C-terminal amino acid” refers to the amino acid attached at the N-terminus of the first C-terminal amino acid.

The term “macrocyclization reagent” or “macrocycle-forming reagent” as used herein refers to any reagent which can be used to prepare a peptidomimetic macrocycle by mediating the reaction between two reactive groups. Reactive groups can be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II) salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents can additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which can provide a reactive Ru(II) species. In other cases, the reactive groups are terminal olefins. In such embodiments, the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated. In other examples, catalysts have W or Mo centers. In some embodiments, the reactive groups are thiol groups. In some embodiments, the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups such as halogen groups.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine or iodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, “alkyl” is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. For example, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive) carbon atoms. The term “lower alkenyl” refers to a C₂-C₆ alkenyl chain. In the absence of any numerical designation, “alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms.

The term “alkynyl” refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl moiety contains the indicated number of carbon atoms. For example, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive) carbon atoms. The term “lower alkynyl” refers to a C₂-C₆ alkynyl chain. In the absence of any numerical designation, “alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkyl group, as defined above. Representative examples of an arylalkyl group include, but are not limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more —C(O)NH₂ groups. Representative examples of an arylamido group include 2-C(O)NH₂-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl, 3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl.

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a heterocycle. Representative examples of an alkylheterocycle group include, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine, —CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a —C(O)NH₂ group. Representative examples of an alkylamido group include, but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂, —CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃, —C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH3, and —CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a hydroxyl group. Representative examples of an alkanol group include, but are not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂ CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and —C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a —COOH group. Representative examples of an alkylcarboxy group include, but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH, —CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted. Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring are substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom or group such as a hydrogen atom on any molecule, compound or moiety. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds disclosed herein contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included unless expressly provided otherwise. In some embodiments, the compounds disclosed herein are also represented in multiple tautomeric forms, in such instances, the compounds include all tautomeric forms of the compounds described herein (e.g., if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included unless expressly provided otherwise. All crystal forms of the compounds described herein are included unless expressly provided otherwise.

As used herein, the terms “increase” and “decrease” mean, respectively, to cause a statistically significantly (i.e., p<0.1) increase or decrease of at least 5%.

As used herein, the recitation of a numerical range for a variable is intended to convey that the variable is equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable is equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable is equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 takes the values 0, 1 or 2 if the variable is inherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and ≤2 if the variable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or”.

The term “on average” represents the mean value derived from performing at least three independent replicates for each data point.

The term “biological activity” encompasses structural and functional properties of a macrocycle. Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.

The term “binding affinity” refers to the strength of a binding interaction, for example between a peptidomimetic macrocycle and a target. Binding affinity can be expressed, for example, as equilibrium dissociation constant (“K_D”), which is expressed in units which are a measure of concentration (e.g. M, mM, μM, nM etc). Numerically, binding affinity and K_D values vary inversely, such that a lower binding affinity corresponds to a higher K_D value, while a higher binding affinity corresponds to a lower K_D value. Where high binding affinity is desirable, “improved” binding affinity refers to higher binding affinity and therefore lower K_D values.

As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

The terms “combination therapy” or “combined treatment” or in “combination” as used herein denotes any form of concurrent or parallel treatment with at least two distinct therapeutic agents.

The term “in vitro efficacy” refers to the extent to which a test compound, such as a peptidomimetic macrocycle, produces a beneficial result in an in vitro test system or assay. In vitro efficacy can be measured, for example, as an “IC₅₀” or “EC₅₀” value, which represents the concentration of the test compound which produces 50% of the maximal effect in the test system.

The term “ratio of in vitro efficacies” or “in vitro efficacy ratio” refers to the ratio of IC₅₀ or EC₅₀ values from a first assay (the numerator) versus a second assay (the denominator). Consequently, an improved in vitro efficacy ratio for Assay 1 versus Assay 2 refers to a lower value for the ratio expressed as IC₅₀(Assay 1)/IC₅₀(Assay 2) or alternatively as EC₅₀(Assay 1)/EC₅₀(Assay 2). This concept can also be characterized as “improved selectivity” in Assay 1 versus Assay 2, which can be due either to a decrease in the IC₅₀ or EC₅₀ value for Target 1 or an increase in the value for the IC₅₀ or EC₅₀ value for Target 2.

As used in the present application, “biological sample” means any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus, and the like. Also included within the meaning of the term “biological sample” is an organ or tissue extract and culture fluid in which any cells or tissue preparation from a subject has been incubated. The biological samples can be any samples from which genetic material can be obtained. Biological samples can also include solid or liquid cancer cell samples or specimens. The cancer cell sample can be a cancer cell tissue sample. In some embodiments, the cancer cell tissue sample can obtained from surgically excised tissue. Exemplary sources of biological samples include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In some cases, the biological samples comprise fine needle aspiration samples. In some embodiments, the biological samples comprise tissue samples, including, for example, excisional biopsy, incisional biopsy, or other biopsy. The biological samples can comprise a mixture of two or more sources; for example, fine needle aspirates and tissue samples. Tissue samples and cellular samples can also be obtained without invasive surgery, for example by punctuating the chest wall or the abdominal wall or from masses of breast, thyroid or other sites with a fine needle and withdrawing cellular material (fine needle aspiration biopsy). In some embodiments, a biological sample is a bone marrow aspirate sample. A biological sample can be obtained by methods known in the art such as the biopsy methods provided herein, swabbing, scraping, phlebotomy, or any other suitable method.

The term “solid tumor” or “solid cancer” as used herein refers to tumors that usually do not contain cysts or liquid areas. Solid tumors as used herein include sarcomas, carcinomas and lymphomas. In various embodiments, leukemia (cancer of blood) is not solid tumor.

Solid tumor cancers that can be treated by the methods provided herein include, but are not limited to, sarcomas, carcinomas, and lymphomas. In specific embodiments, solid tumors that can be treated in accordance with the methods described include, but are not limited to, cancer of the breast, liver, neuroblastoma, head, neck, eye, mouth, throat, esophagus, esophagus, chest, bone, lung, kidney, colon, rectum or other gastrointestinal tract organs, stomach, spleen, skeletal muscle, subcutaneous tissue, prostate, breast, ovaries, testicles or other reproductive organs, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, and brain or central nervous system. Solid tumors that can be treated by the instant methods include tumors and/or metastasis (wherever located) other than lymphatic cancer, for example brain and other central nervous system tumors (including but not limited to tumors of the meninges, brain, spinal cord, cranial nerves and other parts of central nervous system, e.g. glioblastomas or medulla blastemas); head and/or neck cancer; breast tumors; circulatory system tumors (including but not limited to heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue); excretory system tumors (including but not limited to tumors of kidney, renal pelvis, ureter, bladder, other and unspecified urinary organs); gastrointestinal tract tumors (including but not limited to tumors of the esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus and anal canal, tumors involving the liver and intrahepatic bile ducts, gall bladder, other and unspecified parts of biliary tract, pancreas, other and digestive organs); oral cavity tumors (including but not limited to tumors of lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx); reproductive system tumors (including but not limited to tumors of vulva, vagina, Cervix uteri, Corpus uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); respiratory tract tumors (including but not limited to tumors of nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung, e.g. small cell lung cancer or non-small cell lung cancer); skeletal system tumors (including but not limited to tumors of bone and articular cartilage of limbs, bone articular cartilage and other sites); skin tumors (including but not limited to malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.

In some examples, the solid tumor treated by the methods of the instant disclosure is pancreatic cancer, bladder cancer, colon cancer, liver cancer, colorectal cancer (colon cancer or rectal cancer), breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancers, CNS cancers, brain tumors, bone cancer, skin cancer, ocular tumor, choriocarcinoma (tumor of the placenta), sarcoma or soft tissue cancer.

In some examples, the solid tumor to be treated by the methods of the instant disclosure is selected bladder cancer, bone cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, ocular tumor, renal cancer, liver cancer, lung cancer, pancreatic cancer, choriocarcinoma (tumor of the placenta), prostate cancer, sarcoma, skin cancer, soft tissue cancer or gastric cancer.

In some examples, the solid tumor treated by the methods of the instant disclosure is breast cancer. Non limiting examples of breast cancer that can be treated by the instant methods include ductal carcinoma in situ (DCIS or intraductal carcinoma), lobular carcinoma in situ (LCIS), invasive (or infiltrating) ductal carcinoma, invasive (or infiltrating) lobular carcinoma, inflammatory breast cancer, triple-negative breast cancer, paget disease of the nipple, phyllodes tumor (phylloides tumor or cystosarcoma phyllodes), angiosarcoma, adenoid cystic (or adenocystic) carcinoma, low-grade adenosquamous carcinoma, medullary carcinoma, papillary carcinoma, tubular carcinoma, metaplastic carcinoma, micropapillary carcinoma, and mixed carcinoma.

In some examples, the solid tumor treated by the methods of the instant disclosure is bone cancer. Non limiting examples of bone cancer that can be treated by the instant methods include osteosarcoma, chondrosarcoma, the Ewing Sarcoma Family of Tumors (ESFTs).

In some examples, the solid tumor treated by the methods of the instant disclosure is skin cancer. Non limiting examples of skin cancer that can be treated by the instant methods include melanoma, basal cell skin cancer, and squamous cell skin cancer.

In some examples, the solid tumor treated by the methods of the instant disclosure is ocular tumor. Non limiting examples of ocular tumor that can be treated by the methods of the instant disclosure include ocular tumor is choroidal nevus, choroidal melanoma, choroidal metastasis, choroidal hemangioma, choroidal osteoma, iris melanoma, uveal melanoma, intraocular lymphoma, melanocytoma, metastasis retinal capillary hemangiomas, congenital hypertrophy of the RPE, RPE adenoma or retinoblastoma.

In some embodiments solid tumors treated by the methods disclosed herein exclude cancers that are known to be associated with HPV (Human papillomavirus). The excluded group includes HPV positive cervical cancer, HPV positive anal cancer, and HPV head and neck cancers, such as oropharyngeal cancers.

The term “liquid cancer” as used herein refers to cancer cells that are present in body fluids, such as blood, lymph and bone marrow. Liquid cancers include leukemia, myeloma and liquid lymphomas. Liquid lymphomas include lymphomas that contain cysts or liquid areas. Liquid cancers as used herein do not include solid tumors, such as sarcomas and carcinomas or solid lymphomas that do not contain cysts or liquid areas.

Liquid cancer cancers that can be treated by the methods provided herein include, but are not limited to, leukemias, myelomas, and liquid lymphomas. In specific embodiments, liquid cancers that can be treated in accordance with the methods described include, but are not limited to, liquid lymphomas, lekemias, and myelomas. Exemplary liquid lymphomas and leukemias that can be treated in accordance with the methods described include, but are not limited to, chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted), and nodular lymphocyte-predominant Hodgkin lymphoma.

Examples of liquid cancers include cancers involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Exemplary disorders include: acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), multiple mylenoma, hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant liquid lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), periphieral T-cell lymphoma (PTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. For example, liquid cancers include, but are not limited to, acute lymphocytic leukemia (ALL); T-cell acute lymphocytic leukemia (T-ALL); anaplastic large cell lymphoma (ALCL); chronic myelogenous leukemia (CML); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); B-cell chronic lymphocytic leukemia (B-CLL); diffuse large B-cell lymphomas (DLBCL); hyper eosinophilia/chronic eosinophilia; and Burkitt's lymphoma.

In some embodiments, the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; AIDS-related cancers; AIDS-related lymphoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL); Hodgkin lymphoma; multiple myeloma; multiple myeloma/plasma cell neoplasm; Non-Hodgkin lymphoma; or primary central nervous system (CNS) lymphoma. In various embodiments, the liquid cancer can be B-cell chronic lymphocytic leukemia, B-cell lymphoma-DLBCL, B-cell lymphoma-DLBCL-germinal center-like, B-cell lymphoma-DLBCL-activated B-cell-like, or Burkitt's lymphoma.

In some embodiments, a subject treated in accordance with the methods provided herein is a human who has or is diagnosed with cancer lacking p53 deactivating mutation and/or expressing wild type p53. In some embodiments, a subject treated for cancer in accordance with the methods provided herein is a human predisposed or susceptible to cancer lacking p53 deactivating mutation and/or expressing wild type p53. In some embodiments, a subject treated for cancer in accordance with the methods provided herein is a human at risk of developing cancer lacking p53 deactivating mutation and/or expressing wild type p53. A p53 deactivating mutation in some example can be a mutation in DNA-binding domain of the p53 protein. In some examples the p53 deactivating mutation can be a missense mutation. In various examples, the cancer can be determined to lack one or more p53 deactivating mutations selected from mutations at one or more of residues R175, G245, R248, R249, R273, and R282. The lack of p53 deactivating mutation and/or the presence of wild type p53 in the cancer can be determined by any suitable method known in art, for example by sequencing, array based testing, RNA analysis and amplifications methods like PCR.

In certain embodiments, the human subject is refractory and/or intolerant to one or more other standard treatment of the cancer known in art. In some embodiments, the human subject has had at least one unsuccessful prior treatment and/or therapy of the cancer.

In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor.

In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor, determined to lack a p53 deactivating mutation and/or expressing wild type p53. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor, determined to lack a p53 deactivating mutation and/or expressing wild type p53. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor, determined to lack a p53 deactivating mutation and/or expressing wild type p53. A p53 deactivating mutation, as used herein is any mutation that leads to loss of (or a decrease in) the in vitro apoptotic activity of p53.

In some embodiments, the subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor, determined to have a p53 gain of function mutation. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor, determined to have a p53 gain of function mutation. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor, determined to have a p53 gain of function mutation. A p53 gain of function mutation, as used herein is any mutation such that the mutant p53 exerts oncogenic functions beyond their negative domination over the wild-type p53 tumor suppressor functions. The p53 gain of function mutant protein mat exhibit new activities that can contribute actively to various stages of tumor progression and to increased resistance to anticancer treatments. Accordingly, in some embodiments, a subject with a tumor in accordance with the composition as provided herein is a human who has or is diagnosed with a tumor that is determined to have a p53 gain of function mutation.

In some embodiments, the subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor that is not p53 negative. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor that is not p53 negative. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor that is not p53 negative.

In some embodiments, the subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor that expresses p53 with partial loss of function mutation. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor that expresses p53 with partial loss of function mutation. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor that expresses p53 with partial loss of function mutation. As used herein “a partial loss of p53 function” mutation means that the mutant p53 exhibits some level of function of normal p53, but to a lesser or slower extent. For example, a partial loss of p53 function can mean that the cells become arrested in cell division to a lesser or slower extent.

In some embodiments, the subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor that expresses p53 with a copy loss mutation and a deactivating mutation. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor that expresses p53 with a copy loss mutation and a deactivating mutation. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor that expresses p53 with a copy loss mutation and a deactivating mutation.

In some embodiments, the subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor that expresses p53 with a copy loss mutation. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor that expresses p53 with a copy loss mutation. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor that expresses p53 with a copy loss mutation.

In some embodiments, the subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor that expresses p53 with one or more silent mutations. In other embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, predisposed or susceptible to a tumor that expresses p53 with one or more silent mutations. In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, at risk of developing a tumor that expresses p53 with one or more silent mutations. Silent mutations as used herein are mutations which cause no change in the encoded p53 amino acid sequence.

In some embodiments, a subject treated for tumor in accordance with the methods provided herein is a human, who has or is diagnosed with a tumor, determined to lack a dominant p53 deactivating mutation. Dominant p53 deactivating mutation or dominant negative mutation, as used herein, is a mutation wherein the mutated p53 inhibits or disrupt the activity of the wild-type p53 gene.

Peptidomimetic Macrocycles

In some embodiments, a peptidomimetic macrocycle has the Formula (I):

wherein:

• • each A, C, D, and E is independently a natural or non-natural amino acid or an amino acid analog, and each terminal D and E independently optionally includes a capping group;
  • each B is independently a natural or non-natural amino acid, an amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v and w is independently an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10;
  • u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
  • each x, y, and z is independently an integer from 0-10, for example the sum of x+y+z is 2, 3, or 6; and
  • n is an integer from 1-5.

In some embodiments, v and w are integers from 1-30. In some embodiments, w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.

In some embodiments, w is an integer from 3-10, for example 3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other embodiments, w is 6. In some embodiments, v is an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10. In some embodiments, v is 2.

In an embodiment of any of the Formulas described herein, L₁ and L₂, either alone or in combination, do not form a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl that is unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl that is unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 3. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_x, when x is 3, encompasses embodiments wherein the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments wherein the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound can encompass peptidomimetic macrocycles which are the same or different. For example, a compound can comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is an α-helix and R₈ is —H, allowing for intra-helical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

In some embodiments, peptidomimetic macrocycles are also provided of the formula:

wherein:

• • each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8), wherein each X is an amino acid;
  • each D and E is independently a natural or non-natural amino acid or an amino acid analog;
  • R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • v is an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50, 1-30, 1-20 or 1-10;
  • w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10; and
  • n is an integer from 1-5.

In some embodiments, v and w are integers from 1-30. In some embodiments, w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.

In some embodiments of any of the Formulas described herein, at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least four of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least five of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least six of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8). In other embodiments, at least seven of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8).

In some embodiments, a peptidomimetic macrocycle has the Formula:

wherein:

• • each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9), wherein each X is an amino acid;
  • each D is independently a natural or non-natural amino acid or an amino acid analog;
  • each E is independently a natural or non-natural amino acid or an amino acid analog, for example an amino acid selected from Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine);
  • R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • v is an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10;
  • w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10; and
  • n is an integer from 1-5.

In some embodiments of the above Formula, at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least four of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least five of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least six of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9). In other embodiments of the above Formula, at least seven of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9).

In some embodiments, w is an integer from 3-10, for example 3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other embodiments, w is 6. In some embodiments, v is an integer from 1-10. In some embodiments, v is 2.

In an embodiment of any of the Formulas described herein, L₁ and L₂, either alone or in combination, do not form a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 3. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_x, when x is 3, encompasses embodiments wherein the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments wherein the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound can encompass peptidomimetic macrocycles which are the same or different. For example, a compound can comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is an α-helix and R₈ is —H, allowing intra-helical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

In some embodiments, a peptidomimetic macrocycle of Formula (I) has Formula (Ia):

wherein:

• • each A, C, D, and E is independently a natural or non-natural amino acid or an amino acid analog;
  • each B is independently a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each L is independently a macrocycle-forming linker;
  • each L′ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each being optionally substituted with R₅, or a bond, or together with R₁ and the atom to which both R₁ and L′ are bound forms a ring;
  • each L″ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each being optionally substituted with R₅, or a bond, or together with R₂ and the atom to which both R₂ and L″ are bound forms a ring;
  • each R₁ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or together with L′ and the atom to which both R₁ and L′ are bound forms a ring;
  • each R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or together with L″ and the atom to which both R₂ and L″ are bound forms a ring;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • n is an integer from 1-5;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v and w is independently an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1-15, or 1-10;
  • each x, y and z is independently an integer from 0-10, for example x+y+z is 2, 3, or 6; and
  • u is an integer from 1-10, for example 1-5, 1-3, or 1-2.

In some embodiments, L is a macrocycle-forming linker of the formula -L₁-L₂-. In some embodiments, each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅; each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; and n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 2. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments wherein the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound can encompass peptidomimetic macrocycles which are the same or different. For example, a compound can comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is a helix and R₈ is —H, allowing intra-helical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as a helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁′ and R₂′ is independently an amino acid.

In other embodiments, the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chain and “” is [D]_v, [E]_w as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shown below.

In other embodiments, D and/or E in the compound of Formula I are further modified to facilitate cellular uptake. In some embodiments, lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound of Formula I represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers. In a specific embodiment, a peptidomimetic macrocycle comprises two macrocycle-forming linkers. In an embodiment, u is 2.

In some embodiments, the peptidomimetic macrocycles have the Formula (I):

wherein:

• • each A, C, D, and E is independently a natural or non-natural amino acid or an amino acid analog;
  • each B is independently a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently macrocycle-forming linker of the formula

• • • wherein each L₁, L₂ and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v and w is independently an integer from 1-1000;
  • each x, y and z is independently an integer from 0-10;
  • us is an integer from 1-10; and
  • n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl that is unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl that are unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 2. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments wherein the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.

In some embodiments, each of the first two amino acid represented by E comprises an uncharged side chain or a negatively charged side chain. In some embodiments, each of the first three amino acid represented by E comprises an uncharged side chain or a negatively charged side chain. In some embodiments, each of the first four amino acid represented by E comprises an uncharged side chain or a negatively charged side chain. In some embodiments, one or more or each of the amino acid that is i+1, i+2, i+3, i+4, i+5, and/or i+6 with respect to Xaa₁₃ represented by E comprises an uncharged side chain or a negatively charged side chain.

In some embodiments, the first C-terminal amino acid and/or the second C-terminal amino acid represented by E comprise a hydrophobic side chain. For example, the first C-terminal amino acid and/or the second C-terminal amino acid represented by E comprises a hydrophobic side chain, for example a small hydrophobic side chain. In some embodiments, the first C-terminal amino acid, the second C-terminal amino acid, and/or the third C-terminal amino acid represented by E comprise a hydrophobic side chain. For example, the first C-terminal amino acid, the second C-terminal amino acid, and/or the third C-terminal amino acid represented by E comprises a hydrophobic side chain, for example a small hydrophobic side chain. In some embodiments, one or more or each of the amino acid that is i+1, i+2, i+3, i+4, i+5, and/or i+6 with respect to Xaa₁₃ represented by E comprises an uncharged side chain or a negatively charged side chain.

In some embodiments, w is between 1 and 1000. For example, the first amino acid represented by E comprises a small hydrophobic side chain. In some embodiments, w is between 2 and 1000. For example, the second amino acid represented by E comprises a small hydrophobic side chain. In some embodiments, w is between 3 and 1000. For example, the third amino acid represented by E comprises a small hydrophobic side chain. For example, the third amino acid represented by E comprises a small hydrophobic side chain. In some embodiments, w is between 4 and 1000. In some embodiments, w is between 5 and 1000. In some embodiments, w is between 6 and 1000. In some embodiments, w is between 7 and 1000. In some embodiments, w is between 8 and 1000.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is a helix and R₈ is —H, allowing intra-helical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as a helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

In some embodiments, L is a macrocycle-forming linker of the formula

In some embodiments, L is a macrocycle-forming linker of the formula

or a tautomer thereof.

Exemplary embodiments of the macrocycle-forming linker L are shown below:

Amino acids which are used in the formation of triazole crosslinkers are represented according to the legend indicated below. Stereochemistry at the alpha position of each amino acid is S unless otherwise indicated. For azide amino acids, the number of carbon atoms indicated refers to the number of methylene units between the alpha carbon and the terminal azide. For alkyne amino acids, the number of carbon atoms indicated is the number of methylene units between the alpha position and the triazole moiety plus the two carbon atoms within the triazole group derived from the alkyne.

• • $5a5 Alpha-Me alkyne 1,5 triazole (5 carbon)
  • $5n3 Alpha-Me azide 1,5 triazole (3 carbon)
  • $4rn6 Alpha-Me R-azide 1,4 triazole (6 carbon)
  • $4a5 Alpha-Me alkyne 1,4 triazole (5 carbon)

In some embodiments, any of the macrocycle-forming linkers described herein can be used in any combination with any of the sequences shown in TABLE 1, TABLE 1a, TABLE 1b, or TABLE 1c and also with any of the R-substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at least one α-helix motif. For example, A, B and/or C in the compound of Formula I include one or more α-helices. As a general matter, α-helices include between 3 and 4 amino acid residues per turn. In some embodiments, the α-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues. In specific embodiments, the α-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In some embodiments, the macrocycle-forming linker stabilizes an α-helix motif included within the peptidomimetic macrocycle. Thus, in some embodiments, the length of the macrocycle-forming linker L from a first Cα to a second Cα is selected to increase the stability of an α-helix.

In some embodiments, the macrocycle-forming linker spans from 1 turn to 5 turns of the α-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the α-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 Å to 9 Å per turn of the α-helix, or approximately 6 Å to 8 Å per turn of the α-helix.

Where the macrocycle-forming linker spans approximately 1 turn of an α-helix, the length is equal to approximately 5 carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 2 turns of an α-helix, the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 3 turns of an α-helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 4 turns of an α-helix, the length is equal to approximately 20 carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 5 turns of an α-helix, the length is equal to approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 1 turn of an α-helix, the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming linker spans approximately 2 turns of the α-helix, the linkage contains approximately 7 atoms to 15 atoms, approximately 9 atoms to 13 atoms, or approximately 11 atoms. Where the macrocycle-forming linker spans approximately 3 turns of the α-helix, the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms. Where the macrocycle-forming linker spans approximately 4 turns of the α-helix, the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms. Where the macrocycle-forming linker spans approximately 5 turns of the α-helix, the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms.

Where the macrocycle-forming linker spans approximately 1 turn of the α-helix, the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members. Where the macrocycle-forming linker spans approximately 2 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members. Where the macrocycle-forming linker spans approximately 3 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members. Where the macrocycle-forming linker spans approximately 4 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members. Where the macrocycle-forming linker spans approximately 5 turns of the α-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.

In other embodiments, provided are peptidomimetic macrocycles of Formula (II) or (IIa):

wherein:

• • each A, C, D, and E is independently a natural or non-natural amino acid or an amino acid analog, and the terminal D and E independently optionally include a capping group;
  • each B is independently a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each v and w is independently an integer from 1-1000;
  • u is an integer from 1-10;
  • each x, y, and z is independently integers from 0-10; and
  • n is an integer from 1-5.

In one example, L₁ and L₂, either alone or in combination, do not form a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted or substituted with halo-. In another example, both R₁ and R₂ are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R₁ and R₂ is methyl. In other embodiments, R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 1. In other embodiments, x+y+z is at least 2. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor is independently selected. For example, a sequence represented by the formula [A]_x, when x is 3, encompasses embodiments wherein the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments wherein the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle comprises a secondary structure which is an α-helix and R₈ is —H, allowing intra-helical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For example, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L as measured from a first Cα to a second Cα is selected to stabilize a desired secondary peptide structure, such as an α-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker -L₁-L₂- are shown below.

In some embodiments, the peptidomimetic macrocycle has the Formula (III) or Formula (IIIa):

wherein:

• • each A_a, C_a, D_a, E_a, A_b, C_b, and D_b is independently a natural or non-natural amino acid or an amino acid analog;
  • each B_a and B_b is independently a natural or non-natural amino acid, amino acid analog,

[—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-];

• • each R_a1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or R_a1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the D_a or E_a amino acids; or together with L_a forms a ring that is unsubstituted or substituted;
  • each R_a2 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or R_a2 forms a macrocycle-forming linker L′ connected to the alpha position of one of the D_a or E_a amino acids; or together with L_a forms a ring that is unsubstituted or substituted;
  • each R_b1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or R_b1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the D_b amino acids; or together with L_b forms a ring that is unsubstituted or substituted;
  • each R₃ is independently alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted, or H;
  • each L_a is independently a macrocycle-forming linker, and optionally forms a ring with R_a1 or R_a2 that is unsubstituted or substituted;
  • each L_b is independently a macrocycle-forming linker, and optionally forms a ring with R_b1 that is unsubstituted or substituted;
  • each L′ is independently a macrocycle-forming linker;
  • each L₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄-]_n, any of which is unsubstituted or substituted;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any of which is unsubstituted or substituted;
  • each K is independently O, S, SO, SO₂, CO, CO₂, OCO₂, NR₃, CONR₃, OCONR₃, OSO₂NR₃, NR_3q, CONR_3q, OCONR_3q, or OSO₂NR_3q, wherein each R_3q is independently a point of attachment to R_a1, R_a2, or R_b1;
  • R_a7 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted; or H; or part of a cyclic structure with a D_a amino acid;
  • R_b7 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted; or H; or part of a cyclic structure with a D_b amino acid;
  • R_a8 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted; or H; or part of a cyclic structure with an E_a amino acid;
  • R_b8 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted; or H; or an amino acid sequence of 1-1000 amino acid residues;
  • each va and vb is independently an integer from 0-1000;
  • each wa and wb is independently an integer from 0-1000;
  • each ua and ub is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein ua+ub is at least 1;
  • each xa and xb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each ya and yb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each za and zb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and
  • each n is independently 1, 2, 3, 4, or 5,or a pharmaceutically-acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has the Formula (III) or Formula (IIIa):

wherein:

• • each A_a, C_a, D_a, E_a, A_b, C_b, and D_b is independently a natural or non-natural amino acid or an amino acid analogue;
  • each B_a and B_b is independently a natural or non-natural amino acid, amino acid analog,

[—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-];

• • each R_a1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or R_a1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the D_a or E_a amino acids; or together with L_a forms a ring that is unsubstituted or substituted;
  • each R_a2 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or R_a2 forms a macrocycle-forming linker L′ connected to the alpha position of one of the D_a or E_a amino acids; or together with L_a forms a ring that is unsubstituted or substituted;
  • each R_b1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or R_b1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the D_b amino acids; or together with L_b forms a ring that is unsubstituted or substituted;
  • each R₃ is independently alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted with R₅, or H;
  • each L_a is independently a macrocycle-forming linker, and optionally forms a ring with R_a1 or R_a2 that is unsubstituted or substituted;
  • each L_b is independently a macrocycle-forming linker, and optionally forms a ring with R_b1 that is unsubstituted or substituted;
  • each L′ is independently a macrocycle-forming linker;
  • each L₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄-]_n, any of which is unsubstituted or substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any of which is unsubstituted or substituted with R₅;
  • each K is independently O, S, SO, SO₂, CO, CO₂, OCO₂, NR₃, CONR₃, OCONR₃, OSO₂NR₃, NR_3q, CONR_3q, OCONR_3q, or OSO₂NR_3q, wherein each R_3q is independently a point of attachment to R_a1, R_a2, or R_b1;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope, or a therapeutic agent;
  • each R₆ is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R_a is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted with R₅; or H; or part of a cyclic structure with a D_a amino acid;
  • R_b7 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted with R₅; or H; or part of a cyclic structure with a D_b amino acid;
  • each R_a8 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted with R₅; or H; or part of a cyclic structure with an E_a amino acid;
  • R_b8 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is unsubstituted or substituted with R₅; or H; or an amino acid sequence of 1-1000 amino acid residues;
  • each va and vb is independently an integer from 0-1000;
  • each wa and wb is independently an integer from 0-1000;
  • each ua and ub is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein ua+ub is at least 1;
  • each xa and xb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each ya and yb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each za and zb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and
  • each n is independently 1, 2, 3, 4, or 5,or a pharmaceutically-acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle of the invention has the formula defined above, wherein:

• • each L_a is independently a macrocycle-forming linker of the formula -L₁-L₂-, and optionally forms a ring with R_a1 or R_a2 that is unsubstituted or substituted;
  • each L_b is independently a macrocycle-forming linker of the formula -L₁-L₂-, and optionally forms a ring with R_b1 that is unsubstituted or substituted;
  • each L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R₄—K—R₄-]_n, any of which is unsubstituted or substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any of which is unsubstituted or substituted with R₅;
  • each K is independently O, S, SO, SO₂, CO, CO₂, OCO₂, NR₃, CONR₃, OCONR₃, OSO₂NR₃, NR_3q, CONR_3q, OCONR_3q, or OSO₂NR_3q, wherein each R_3q is independently a point of attachment to R_a1, R_a2, or R_b1;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope, or a therapeutic agent; and
  • each R₆ is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent,or a pharmaceutically-acceptable salt thereof.

In some embodiments, the peptidomimetic macrocycle has the formula defined above wherein one of L_a and L_b is a bis-thioether-containing macrocycle-forming linker. In some embodiments, one of L_a and L_b is a macrocycle-forming linker of the formula -L₁-S-L₂-S-L₃-.

In some embodiments, the peptidomimetic macrocycle has the formula defined above wherein one of L_a and L_b is a bis-sulfone-containing macrocycle-forming linker. In some embodiments, one of L_a and L_b is a macrocycle-forming linker of the formula -L₁-SO₂-L₂-SO₂-L₃-.

In some embodiments, the peptidomimetic macrocycle has the formula defined above wherein one of L_a and L_b is a bis-sulfoxide-containing macrocycle-forming linker. In some embodiments, one of L_a and L_b is a macrocycle-forming linker of the formula -L₁-S(O)-L₂-S(O)-L₃-.

In some embodiments, a peptidomimetic macrocycle of the invention comprises one or more secondary structures. In some embodiments, the peptidomimetic macrocycle comprises a secondary structure that is an α-helix. In some embodiments, the peptidomimetic macrocycle comprises a secondary structure that is a β-hairpin turn.

In some embodiments, u_a is 0. In some embodiments, u_a is 0, and L_b is a macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, u_a is 0, and L_b is a macrocycle-forming linker that crosslinks a β-hairpin secondary structure. In some embodiments, u_a is 0, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, u_a is 0, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure.

In some embodiments, u_b is 0. In some embodiments, u_b is 0, and L_a is a macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, u_b is 0, and L_a is a macrocycle-forming linker that crosslinks a 3-hairpin secondary structure. In some embodiments, u_b is 0, and L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, u_b is 0, and L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks a 3-hairpin secondary structure.

In some embodiments, the peptidomimetic macrocycle comprises only α-helical secondary structures. In other embodiments, the peptidomimetic macrocycle comprises only β-hairpin secondary structures.

In other embodiments, the peptidomimetic macrocycle comprises a combination of secondary structures, wherein the secondary structures are α-helical and β-hairpin structures. In some embodiments, L_a and L_b are a combination of hydrocarbon-, triazole, or sulfur-containing macrocycle-forming linkers. In some embodiments, the peptidomimetic macrocycle comprises L_a and L_b, wherein L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and L_b is a triazole-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, the peptidomimetic macrocycle comprises L_a and L_b, wherein L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure, and L_b is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, the peptidomimetic macrocycle comprises L_a and L_b, wherein L_a is a triazole-containing macrocycle-forming linker that crosslinks an α-helical structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, the peptidomimetic macrocycle comprises L_a and L_b, wherein L_a is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure.

In some embodiments, u_a+u_b is at least 1. In some embodiments, u_a+u_b=2.

In some embodiments, u_a is 1, u_b is 1, L_a is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, u_a is 1, u_b is 1, L_a is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, u_a is 1, u_b is 1, L_a is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, u_a is 1, u_b is 1, L_a is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure.

In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and L_b is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and L_b is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and L_b is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and L_b is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure.

In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker with an α-helical secondary structure, and L_b is a sulfur-containing macrocycle-forming linker. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker with a β-hairpin secondary structure, and L_b is a sulfur-containing macrocycle-forming linker.

In some embodiments, u_a is 1, u_b is 1, L_a is a sulfur-containing macrocycle-forming linker, and L_b is a hydrocarbon-containing macrocycle-forming linker with an α-helical secondary structure. In some embodiments, u_a is 1, u_b is 1, L_a is a sulfur-containing macrocycle-forming linker, and L_b is a hydrocarbon-containing macrocycle-forming linker with a β-hairpin secondary structure.

In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, u_a is 1, u_b is 1, L_a is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and L_b is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure.

In some embodiments, R_b1 is H.

Unless otherwise stated, any compounds (including peptidomimetic macrocycles, macrocycle precursors, and other compositions) are also meant to encompass compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the described structures except for the replacement of a hydrogen atom by deuterium or tritium, or the replacement of a carbon atom by ¹³C or ¹⁴C are contemplated.

In some embodiments, the compounds disclosed herein can contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds can be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). In other embodiments, one or more carbon atoms is replaced with a silicon atom. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are contemplated herein.

In some embodiments, the peptidomimetic macrocycle comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle comprises an amino acid sequence that is at least 60% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle comprises an amino acid sequence that is at least 65% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle comprises an amino acid sequence that is at least 70% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle comprises an amino acid sequence that is at least 75% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

In some embodiments, the peptidomimetic macrocycle is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle is at least 60% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle is at least 65% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle is at least 70% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b. In some embodiments, the peptidomimetic macrocycle is at least 75% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles can be prepared by any of a variety of methods known in the art. For example, any of the residues indicated by “$” or “$r8” in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b can be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.

α,α-Disubstituted amino acids and amino acid precursors can be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides. For example, the “S5-olefin amino acid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is (R)-α-(2′-octenyl) alanine. Following incorporation of such amino acids into precursor polypeptides, the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle. In various embodiments, the following amino acids can be employed in the synthesis of the peptidomimetic macrocycle:

In other embodiments, the peptidomimetic macrocycles are of Formula IV or IVa. In such embodiments, amino acid precursors are used containing an additional substituent R—at the alpha position. Such amino acids are incorporated into the macrocycle precursor at the desired positions, which can be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then effected according to the indicated method.

Pharmaceutically-Acceptable Salts

The invention provides the use of pharmaceutically-acceptable salts of any therapeutic compound described herein. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically-acceptable salt is an ammonium salt.

Metal salts can arise from the addition of an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the invention. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine.

In some embodiments, an ammonium salt is a triethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or a pipyrazine salt.

Acid addition salts can arise from the addition of an acid to a compound of the invention. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccarate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate salt, a succinate salt, a methanesulfonate (mesylate) salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt.

Purity of Compounds of the Invention

Any compound herein can be purified. A compound herein can be least 1% pure, at least 2% pure, at least 3% pure, at least 4% pure, at least 5% pure, at least 6% pure, at least 7% pure, at least 8% pure, at least 9% pure, at least 10% pure, at least 11% pure, at least 12% pure, at least 13% pure, at least 14% pure, at least 15% pure, at least 16% pure, at least 17% pure, at least 18% pure, at least 19% pure, at least 20% pure, at least 21% pure, at least 22% pure, at least 23% pure, at least 24% pure, at least 25% pure, at least 26% pure, at least 27% pure, at least 28% pure, at least 29% pure, at least 30% pure, at least 31% pure, at least 32% pure, at least 33% pure, at least 34% pure, at least 35% pure, at least 36% pure, at least 37% pure, at least 38% pure, at least 39% pure, at least 40% pure, at least 41% pure, at least 42% pure, at least 43% pure, at least 44% pure, at least 45% pure, at least 46% pure, at least 47% pure, at least 48% pure, at least 49% pure, at least 50% pure, at least 51% pure, at least 52% pure, at least 53% pure, at least 54% pure, at least 55% pure, at least 56% pure, at least 57% pure, at least 58% pure, at least 59% pure, at least 60% pure, at least 61% pure, at least 62% pure, at least 63% pure, at least 64% pure, at least 65% pure, at least 66% pure, at least 67% pure, at least 68% pure, at least 69% pure, at least 70% pure, at least 71% pure, at least 72% pure, at least 73% pure, at least 74% pure, at least 75% pure, at least 76% pure, at least 77% pure, at least 78% pure, at least 79% pure, at least 80% pure, at least 81% pure, at least 82% pure, at least 83% pure, at least 84% pure, at least 85% pure, at least 86% pure, at least 87% pure, at least 88% pure, at least 89% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.2% pure, at least 99.3% pure, at least 99.4% pure, at least 99.5% pure, at least 99.6% pure, at least 99.7% pure, at least 99.8% pure, or at least 99.9% pure.

Formulation and Administration

Pharmaceutical Compositions

Pharmaceutical compositions disclosed herein include peptidomimetic macrocycles and pharmaceutically-acceptable derivatives or prodrugs thereof. A “pharmaceutically-acceptable derivative” means any pharmaceutically-acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound disclosed herein which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound disclosed herein. Particularly favored pharmaceutically-acceptable derivatives are those that increase the bioavailability of the compounds when administered to a mammal (e.g., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Some pharmaceutically-acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.

In some embodiments, peptidomimetic macrocycles are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties. Such modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.

For preparing pharmaceutical compositions from the compounds disclosed herein, pharmaceutically-acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the crosslinked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

The pharmaceutical preparation can be in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

When one or more compositions disclosed herein comprise a combination of a peptidomimetic macrocycle and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. In some embodiments, the additional agents are administered separately, as part of a multiple dose regimen, from one or more compounds disclosed herein. Alternatively, those agents are part of a single dosage form, mixed together with the compounds disclosed herein in a single composition.

Mode of Administration

An effective amount of a peptidomimetic macrocycles of the disclosure can be administered in either single or multiple doses by any of the accepted modes of administration. In some embodiments, the peptidomimetic macrocycles of the disclosure are administered parenterally, for example, by subcutaneous, intramuscular, intrathecal, intravenous or epidural injection. For example, the peptidomimetic macrocycle is administered intravenously, intra-arterially, subcutaneously or by infusion. In some examples, the peptidomimetic macrocycle is administered intravenously. In some examples, the peptidomimetic macrocycle is administered intra-arterially.

Regardless of the route of administration selected, the peptidomimetic macrocycles of the present disclosure, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms. The peptidomimetic macrocycles according to the disclosure can be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

In one aspect, the disclosure provides pharmaceutical formulation comprising a therapeutically-effective amount of one or more of the peptidomimetic macrocycles described above, formulated together with one or more pharmaceutically-acceptable carriers (additives) and/or diluents. In one embodiment, one or more of the peptidomimetic macrocycles described herein are formulated for parenteral administration for parenteral administration, one or more peptidomimetic macrocycles disclosed herein can be formulated as aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use. Such formulations can comprise sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. If desired the formulation can be diluted prior to use with, for example, an isotonic saline solution or a dextrose solution. In some examples, the peptidomimetic macrocycle is formulated as an aqueous solution and is administered intravenously.

Amount and Frequency of Administration

Dosing can be determined using various techniques. The selected dosage level can depend upon a variety of factors including the activity of the particular peptidomimetic macrocycle employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular peptidomimetic macrocycle being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular peptidomimetic macrocycle employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. The dosage values can also vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

A physician or veterinarian can prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In some embodiments, a suitable daily dose of a peptidomimetic macrocycle of the disclosure can be that amount of the peptidomimetic macrocycle which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. The precise time of administration and amount of any particular peptidomimetic macrocycle that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular peptidomimetic macrocycle, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like.

Dosage can be based on the amount of the peptidomimetic macrocycle per kg body weight of the patient. Alternatively, the dosage of the subject disclosure can be determined by reference to the plasma concentrations of the peptidomimetic macrocycle. For example, the maximum plasma concentration (C_max) and the area under the plasma concentration-time curve from time 0 to infinity (AUC) can be used.

The amount of the peptidomimetic macrocycle that is administered to a subject can be from about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 i g/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg per body weight of the subject.

The amount of the peptidomimetic macrocycle that is administered to a subject can be from about 0.01 mg/kg to about 100 mg/kg body weight of the subject. In some embodiments, the amount of the peptidomimetic macrocycle administered is about 0.01-10 mg/kg, about 0.01-20 mg/kg, about 0.01-50 mg/kg, about 0.1-10 mg/kg, about 0.1-20 mg/kg, about 0.1-50 mg/kg, about 0.1-100 mg/kg, about 0.5-10 mg/kg, about 0.5-20 mg/kg, about 0.5-50 mg/kg, about 0.5-100 mg/kg, about 1-10 mg/kg, about 1-20 mg/kg, about 1-50 mg/kg, or about 1-100 mg/kg body weight of the human subject. In some embodiments, the amount of the peptidomimetic macrocycle administered is about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg body weight of the subject. In some embodiments, the amount of the peptidomimetic macrocycle administered is about 5 mg/kg. In some embodiments, the amount of the peptidomimetic macrocycle administered is about 10 mg/kg. In some embodiments, the amount of the peptidomimetic macrocycle administered is about 15 mg/kg.

In some embodiments, the amount of the peptidomimetic macrocycle administered is about 0.16 mg, about 0.32 mg, about 0.64 mg, about 1.28 mg, about 3.56 mg, about 7.12 mg, about 14.24 mg, or about 20 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 0.16 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 0.32 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 0.64 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 1.28 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 3.56 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 7.12 mg per kilogram body weight of the subject. In some examples the amount of the peptidomimetic macrocycle administered is about 14.24 mg per kilogram body weight of the subject.

In some embodiments, a pharmaceutically-acceptable amount of a peptidomimetic macrocycle is administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times a week. In some embodiments about 0.5-about 20 mg or about 0.5-about 10 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered once a week. For example about 0.5-about 1 mg, about 0.5-about 5 mg, about 0.5-about 10 mg, about 0.5-about 15 mg, about 1-about 5 mg, about 1-about 10 mg, about 1-about 15 mg, about 1-about 20 mg, about 5-about 10 mg, about 1-about 15 mg, about 5-about 20 mg, about 10-about 15 mg, about 10-about 20 mg, or about 15-about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered once a week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered once a week. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered once a week. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered once a week.

In some embodiments about 0.5-about 20 mg or about 0.5-about 10 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered two times a week. For example about 0.5-about 1 mg, about 0.5-about 5 mg, about 0.5-about 10 mg, about 0.5-about 15 mg, about 1-about 5 mg, about 1-about 10 mg, about 1-about 15 mg, about 1-about 20 mg, about 5-about 10 mg, about 1-about 15 mg, about 5-about 20 mg, about 10-about 15 mg, about 10-about 20 mg, or about 15-about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered about twice a week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered two times a week. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered two times a week. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered two times a week.

In some embodiments about 0.5-about 20 mg or about 0.5-about 10 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered 3, 4, 5, 6, or 7 times a week. For example, about 0.5-about 1 mg, about 0.5-about 5 mg, about 0.5-about 10 mg, about 0.5-about 15 mg, about 1-about 5 mg, about 1-about 10 mg, about 1-about 15 mg, about 1-about 20 mg, about 5-about 10 mg, about 1-about 15 mg, about 5-about 20 mg, about 10-about 15 mg, about 10-about 20 mg, or about 15-about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered 3, 4, 5, 6, or 7 times a week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered 3, 4, 5, 6, or 7 times a week. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered 3, 4, 5, 6, or 7 times a week. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, or about 10 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered 3, 4, 5, 6, or 7 times a week.

In some embodiments, a pharmaceutically-acceptable amount of a peptidomimetic macrocycle is administered to a subject once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, about 0.5-about 20 mg or about 0.5-about 10 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered once every 2, 3, or 4 weeks. For example, about 0.5-about 1 mg, about 0.5-about 5 mg, about 0.5-about 10 mg, about 0.5-about 15 mg, about 1-about 5 mg, about 1-about 10 mg, about 1-about 15 mg, about 1-about 20 mg, about 5-about 10 mg, about 1-about 15 mg, about 5-about 20 mg, about 10-about 15 mg, about 10-about 20 mg, or about 15-about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administrated 3, 4, 5, 6, or 7 once every 2 or 3 week. In some examples about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, about 5 mg, about 5.25 mg, about 5.5 mg, about 5.75 mg, about 6 mg, about 6.25 mg, about 6.5 mg, about 6.75 mg, about 7 mg, about 7.25 mg, about 7.5 mg, about 7.75 mg, about 8 mg, about 8.25 mg, about 8.5 mg, about 8.75 mg, about 9 mg, about 9.25 mg, about 9.5 mg, about 9.75 mg, about 10 mg, about 10.25 mg, about 10.5 mg, about 10.75 mg, about 11 mg, about 11.25 mg, about 11.5 mg, about 11.75 mg, about 12 mg, about 12.25 mg, about 12.5 mg, about 12.75 mg, about 13 mg, about 13.25 mg, about 13.5 mg, about 13.75 mg, about 14 mg, about 14.25 mg, about 14.5 mg, about 14.75 mg, about 15 mg, about 15.25 mg, about 15.5 mg, about 15.75 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered once every 2 or 3 weeks. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered once every 2 weeks. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered once every 2 weeks. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered once every 3 weeks. In some examples, the amount of the peptidomimetic macrocycle administered is about 1.25 mg, about 2.5 mg, about 5 mg, or about 10 mg per kilogram body weight of the human subject and the peptidomimetic macrocycle is administered once every 3 weeks.

In some embodiments, a pharmaceutically-acceptable amount of a peptidomimetic macrocycle is administered to a subject gradually over a period of time. In some embodiments, an amount of a peptidomimetic macrocycle can be administered to a subject gradually over a period of from about 0.1 h to about 24 h. In some embodiments, an amount of a peptidomimetic macrocycle can be administered to a subject over a period of about 0.1 h, about 0.2 h, about 0.3 h, about 0.4 h, about 0.5 h, about 0.6 h, about 0.7 h, about 0.8 h, about 0.9 h, about 1 h, about 1.5 h, about 2 h, about 2.5 h, about 3 h, about 3.5 h, about 4 h, about 4.5 h, about 5 h, about 5.5 h, about 6 h, about 6.5 h, about 7 h, about 7.5 h, about 8 h, about 8.5 h, about 9 h, about 9.5 h, about 10 h, about 10.5 h, about 11 h, about 11.5 h, about 12 h, about 12.5 h, about 13 h, about 13.5 h, about 14 h, about 14.5 h, about 15 h, about 15.5 h, about 16 h, about 16.5 h, about 17 h, about 17.5 h, about 18 h, about 18.5 h, about 19 h, about 19.5 h, about 20 h, about 20.5 h, about 21 h, about 21.5 h, about 22 h, about 22.5 h, about 23 h, about 23.5 h, or about 24 h. In some embodiments, a pharmaceutically-acceptable amount of a peptidomimetic macrocycle is administered gradually over a period of about 0.5 h. In some embodiments, a pharmaceutically-acceptable amount of a peptidomimetic macrocycle is administered gradually over a period of about 1 h. In some embodiments, a pharmaceutically-acceptable amount of a peptidomimetic macrocycle is administered gradually over a period of about 1.5 h.

Administration of the peptidomimetic macrocycles can continue for as long as clinically necessary. In some embodiments, a peptidomimetic macrocycle of the disclosure can be administered for more than 1 day, more than 1 week, more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, more than 12 months, more than 13 months, more than 14 months, more than 15 months, more than 16 months, more than 17 months, more than 18 months, more than 19 months, more than 20 months, more than 21 months, more than 22 months, more than 23 months, or more than 24 months. In some embodiments, one or more peptidomimetic macrocycle of the disclosure is administered for less than 1 week, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, less than 9 months, less than 10 months, less than 11 months, less than 12 months, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than 23 months, or less than 24 months.

In some embodiments, a peptidomimetic macrocycle can be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times over a treatment cycle. In some embodiments a peptidomimetic macrocycle can be administered to a subject 2, 4, 6, or 8 times over a treatment cycle. In some embodiments, a peptidomimetic macrocycle can be administered to a subject 4 times over a treatment cycle. In some embodiments, a treatment cycle is 7 days, 14 days, 21 days, or 28 days long. In some embodiments, a treatment cycle is 21 days long. In some embodiments, a treatment cycle is 28 days long.

In some embodiments, a peptidomimetic macrocycle is administered on day 1, 8, 15 and 28 of a 28 day cycle. In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 15 and 28 of a 28 day cycle and administration is continued for two cycles. In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 15 and 28 of a 28 day cycle and administration is continued for three cycles. In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 15 and 28 of a 28 day cycle and administration is continued for 4, 5, 6, 7, 8, 9, 10, or more than 10 cycles.

In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 11 and 21 of a 21-day cycle. In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 11 and 21 of a 21-day cycle and administration is continued for two cycles. In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 11 and 21 of a 21-day cycle and administration is continued for three cycles. In some embodiments, the peptidomimetic macrocycle is administered on day 1, 8, 11 and 21 of a 21-day cycle and administration is continued for 4, 5, 6, 7, 8, 9, 10, or more than 10 cycles.

In some embodiments, one or more peptidomimetic macrocycle of the disclosure is administered chronically on an ongoing basis. In some embodiments administration of one or more peptidomimetic macrocycle of the disclosure is continued until documentation of disease progression, unacceptable toxicity, or patient or physician decision to discontinue administration.

In some embodiments, the compounds of the invention can be used to treat one condition. In some embodiments, the compounds of the invention can be used to treat two conditions. In some embodiments, the compounds of the invention can be used to treat three conditions. In some embodiments, the compounds of the invention can be used to treat four conditions. In some embodiments, the compounds of the invention can be used to treat five conditions.

Methods of Use

In one aspect, provided herein are novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled. For example, in the p53/MDMX system, labeled peptidomimetic macrocycles based on p53 can be used in a MDMX binding assay along with small molecules that competitively bind to MDMX. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the p53/MDMX system. Such binding studies can be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners. Further provided are methods for the generation of antibodies against the peptidomimetic macrocycles. In some embodiments, these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as p53, to which the peptidomimetic macrocycles are related. Such antibodies, for example, disrupt the native protein-protein interaction, for example, binding between p53 and MDMX.

In other aspects, provided herein are both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant (e.g., insufficient or excessive) expression or activity of the molecules including p53, MDM2 or MDMX.

In another embodiment, a disorder is caused, at least in part, by an abnormal level of p53 or MDM2 or MDMX, (e.g., over or under expression), or by the presence of p53 or MDM2 or MDMX exhibiting abnormal activity. As such, the reduction in the level and/or activity of p53 or MDM2 or MDMX, or the enhancement of the level and/or activity of p53 or MDM2 or MDMX, by peptidomimetic macrocycles derived from p53, is used, for example, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, provided herein are methods for treating or preventing a disease including hyperproliferative disease and inflammatory disorder by interfering with the interaction or binding between binding partners, for example, between p53 and MDM2 or p53 and MDMX. These methods comprise administering an effective amount of a compound to a warm blooded animal, including a human. In some embodiments, the administration of one or more compounds disclosed herein induces cell growth arrest or apoptosis.

In some embodiments, the peptidomimetic macrocycles can be used to treat, prevent, and/or diagnose cancers and neoplastic conditions. As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states can be categorized as pathologic, i.e., characterizing or constituting a disease state, or can be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of breast, lung, liver, colon and ovarian origin. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. Examples of cellular proliferative and/or differentiation disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. In some embodiments, the peptidomimetic macrocycles are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, lung cancer, metastasis of such cancers and the like.

Examples of cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

In some embodiments, the cancer is head and neck cancer, melanoma, lung cancer, breast cancer, or glioma.

Examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. The diseases can arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), periphieral T-cell lymphoma (PTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.

Examples of cellular proliferative and/or differentiation disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders of the skin include, but are not limited to proliferative skin disease such as melanomas, including mucosal melanoma, superficial spreading melanoma, nodular melanoma, lentigo (e.g. lentigo maligna, lentigo maligna melanoma, or acral lentiginous melanoma), amelanotic melanoma, desmoplastic melanoma, melanoma with features of a Spitz nevus, melanoma with small nevus-like cells, polypoid melanoma, and soft-tissue melanoma; basal cell carcinomas including micronodular basal cell carcinoma, superficial basal cell carcinoma, nodular basal cell carcinoma (rodent ulcer), cystic basal cell carcinoma, cicatricial basal cell carcinoma, pigmented basal cell carcinoma, aberrant basal cell carcinoma, infiltrative basal cell carcinoma, nevoid basal cell carcinoma syndrome, polypoid basal cell carcinoma, pore-like basal cell carcinoma, and fibroepithelioma of Pinkus; squamus cell carcinomas including acanthoma (large cell acanthoma), adenoid squamous cell carcinoma, basaloid squamous cell carcinoma, clear cell squamous cell carcinoma, signet-ring cell squamous cell carcinoma, spindle cell squamous cell carcinoma, Marjolin's ulcer, erythroplasia of Queyrat, and Bowen's disease; or other skin or subcutaneous tumors.

Examples of cellular proliferative and/or differentiation disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders of the ovary include, but are not limited to, ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Combination Treatment

Combination therapy with a peptidomimetic macrocycle of the disclosure and at least one additional therapeutic agent, for example, any additional therapeutic agent described herein, can be used to treat a condition. In some embodiments, the combination therapy can produce a significantly better therapeutic result than the additive effects achieved by each individual constituent when administered alone at a therapeutic dose. In some embodiments, the dosage of the peptidomimetic macrocycle or additional therapeutic agent, for example, any additional therapeutic agent described herein, in combination therapy can be reduced as compared to monotherapy with each agent, while still achieving an overall therapeutic effect. In some embodiments, a peptidomimetic macrocycle and an additional therapeutic agent, for example, any additional therapeutic agent described herein, can exhibit a synergistic effect. In some embodiments, the synergistic effect of a peptidomimetic macrocycle and additional therapeutic agent, for example, any additional therapeutic agent described herein, can be used to reduce the total amount drugs administered to a subject, which decrease side effects experienced by the subject.

The peptidomimetic macrocycles of the disclosure can be used in combination with at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. In some embodiments, the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can modulate the same or a different target as the peptidomimetic macrocycles of the disclosure. In some embodiments, the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can modulate the same target as the peptidomimetic macrocycles of the disclosure, or other components of the same pathway, or overlapping sets of target enzymes. In some embodiments, the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can modulate a different target from the peptidomimetic macrocycles of the disclosure.

Accordingly, in one aspect, the present disclosure provides a method for treating cancer, the method comprising administering to a subject in need thereof (a) an effective amount of a peptidomimetic macrocycle of the disclosure and (b) an effective amount of at least one additional pharmaceutically active agent, for example, any additional therapeutic agent described herein, to provide a combination therapy. In some embodiments, the combination therapy may have an enhanced therapeutic effect compared to the effect of the peptidomimetic macrocycle and the at least one additional pharmaceutically active agent each administered alone. According to certain exemplary embodiments, the combination therapy has a synergistic therapeutic effect. According to this embodiment, the combination therapy produces a significantly better therapeutic result (e.g., anti-cancer, cell growth arrest, apoptosis, induction of differentiation, cell death, etc.) than the additive effects achieved by each individual constituent when administered alone at a therapeutic dose.

Combination therapy includes but is not limited to the combination of peptidomimetic macrocycles of this disclosure with chemotherapeutic agents, therapeutic antibodies, and radiation treatment, to provide a synergistic therapeutic effect. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with one or more anti-cancer (antineoplastic or cytotoxic) chemotherapy drug. Suitable chemotherapeutic agents for use in the combinations of the present disclosure include, but are not limited to, alkylating agents, antibiotic agents, antimetabolic agents, hormonal agents, plant-derived agents, anti-angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, agents affecting cell bioenergetics, biologic agents, e.g., monoclonal antibodies, kinase inhibitors and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, or any combination thereof.

In some embodiments, a method of treating cancer in a subject in need thereof can comprise administering to the subject a therapeutically effective amount of a p53 agent that inhibits the interaction between p53 and MDM2 and/or p53 and MDMX, and/or modulates the activity of p53 and/or MDM2 and/or MDMX; and at least one additional pharmaceutically-active agent. In some examples, the p53 agent is selected from the group consisting of a small organic or inorganic molecule; a saccharine; an oligosaccharide; a polysaccharide; a peptide, a protein, a peptide analog, a peptide derivative; an antibody, an antibody fragment, a peptidomimetic; a peptidomimetic macrocycle of the disclosure; a nucleic acid; a nucleic acid analog, a nucleic acid derivative; an extract made from biological materials; a naturally-occurring or synthetic composition; and any combination thereof.

In some embodiments, the p53 agent is selected from the group consisting of RG7388 (RO5503781, idasanutlin), RG7112 (RO5045337), nutlin3a, nutlin3b, nutlin3, nutlin2, spirooxindole containing small molecules, 1,4-diazepines, 1,4-benzodiazepine-2,5-dione compounds, WK23, WK298, SJ172550, RO2443, RO5963, RO5353, RO2468, MK8242 (SCH900242), M1888, M1773 (SAR405838), NVPCGM097, DS3032b, AM8553, AMG232, NSC207895 (X1006), JNJ26854165 (serdemetan), RITA (NSC652287), YH239EE, or any combination thereof. In some examples, the at least one additional pharmaceutically-active agent is selected from the group consisting of palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; arcyriaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-amino thioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (ryuvidine); and flavopiridol (alvocidib); and any combination thereof.

a. Combination Treatment with Estrogen Receptor Antagonists

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an estrogen receptor antagonist. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with toremifene (Fareston®), fulvestrant (Faslodex®), or tamoxifen citrate (Soltamox®).

Fulvestrant is a selective estrogen receptor degrader (SERD) and is indicated for the treatment of hormone receptor positive metastatic breast cancer in postmenopausal women with disease progression following anti-estrogen therapy. Fulvestrant is a complete estrogen receptor antagonist with little to no agonist effects and accelerates the proteasomal degradation of the estrogen receptor. Fulvestrant has poor oral bioavailability and is administered via intramuscular injection. Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin beta1. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with fulvestrant.

b. Combination Treatment with Aromatase Inhibitors

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an aromatase inhibitor. Aromatase inhibitors are used in the treatment of breast cancer in post-menopausal women and gynecomastia in men. Aromatase inhibitors can be used off-label to reduce estrogen conversion when using external testosterone. Aromatase inhibitors can also be used for chemoprevention in high-risk women.

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a non-selective aromatase inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a non-selective aromatase inhibitor, such as aminoglutethimide or testolactone (Teslac®). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a selective aromatase inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a selective aromatase inhibitor, such as anastrozole (Arimidex®), letrozole (Femara®), exemestane (Aromasin®), vorozole (Rivizor®), formestane (Lentaron®), or fadrozole (Afema®). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with exemestane. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an aromatase inhibitor that has unknown mechanism of action, such as 1,4,6-androstatrien-3,17-dione (ATD) or 4-androstene-3,6,17-trione.

c. Combination Treatment with mTOR Inhibitors

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an mTOR inhibitor. mTOR inhibitors are drugs that inhibit the mechanistic target of rapamycin (mTOR), which is a serine/threonine-specific protein kinase that belongs to the family of phosphatidylinositol-3 kinase (PI3K)-related kinases (PIKKs). mTOR regulates cellular metabolism, growth, and proliferation by forming and signaling through the protein complexes mTORC1 and mTORC2.

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an mTOR inhibitor, such as rapamycin, temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with everolimus (Afinitor®). Everolimus affects the mTORC1 protein complex and can lead to hyper-activation of the kinase AKT, which can lead to longer survival in some cell types. Everolimus binds to FKBP12, a protein receptor which directly interacts with mTORC1 and inhibits downstream signaling. mRNAs that codify proteins implicated in the cell cycle and in the glycolysis process are impaired or altered as a result, inhibiting tumor growth and proliferation.

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a mTOR inhibitor and an aromatase inhibitor. For example, the peptidomimetic macrocycles can be used in combination with everolimus and exemestane.

d. Combination Treatment with Antimetabolites

Antimetabolites are chemotherapy treatments that are similar to normal substances within the cell. When cells incorporate the antimetabolites into the cellular metabolism, the cells are unable to divide. Antimetabolites are cell-cycle specific and attack cells at specific phases in the cell cycle.

In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with one or more antimetabolites, such as a folic acid antagonist, pyrimidine antagonist, purine antagonist, or an adenosine deaminase inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an antimetabolite, such as methotrexate, 5-fluorouracil, foxuridine, cytarabine, capecitabine, gemcitabine, 6-mercaptopurine, 6-thioguanine, cladribine, fludarabine, nelarabine, or pentostatin. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with capecitabine (Xeloda®), gemcitabine (Gemzar®), or cytarabine (Cytosar-U®).

e. Combination Treatment with Plant Alkaloids

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with plant alkaloids. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with plant alkaloids, such as vinca alkaloids, taxanes, podophyllotoxins, or camptothecan analogues. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with plant alkaloids, such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, tenisopide, irinotecan, or topotecan.

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with taxanes, such as paclitaxel (Abraxane® or Taxol®) and docetaxel (Taxotere®). In some embodiments, the peptidomimetic macrocycles of the instant disclosure are used in combination with paclitaxel. In some embodiments, the peptidomimetic macrocycles of the instant disclosure are used in combination with docetaxel.

f. Combination Treatment with Therapeutic Antibodies

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with therapeutic antibodies. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with naked monoclonal antibodies, such as alemtuzumab (Campath®) or trastuzumab (Herceptin®). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with conjugated monoclonal antibodies, such as radiolabeled antibodies or chemolabeled antibodies. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with conjugated monoclonal antibodies, such as ibritumomab tiuxetan (Zevalin®), brentuximab vedotin (Adcetris®), ado-trastuzumab emtansine (Kadcyla®), or denileukin diftitox (Ontak®). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with bispecific monoclonal antibodies, such as blinatumomab (Blincyto®).

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an anti-CD20 antibody, such as rituximab (Mabthera®/Rituxan®), obinutuzumab (Gazyva®), ibritumomab tiuxetan, tositumomab, ofatumumab (Genmab®), ocaratuzumab, ocrelizumab, TRU-015, or veltuzumab. Other antibodies that can be used in combination with the peptidomimetic macrocycles of the disclosure include antibodies against the programed cell death (PD-1) receptor, for example pembrolizumab (Keytruda®) or nivolumba (Opdivo®).

g. Combination Treatment with PD-L1 and/or PD-1 Antagonists

The PD-1 pathway comprises the immune cell co-receptor Programmed Death-1 (PD-1) and the PD-1 ligands PD-L1 and PD-L2. The PD-1 pathway mediates local immunosuppression in the tumor microenvironment. PD-1 and PD-L1 antagonists suppress the immune system. In some embodiments, a PD-1 or PD-L1 antagonist is a monoclonal antibody or antigen binding fragment thereof that specifically binds to, blocks, or downregulates PD-1 or PD-L1, respectively. In some embodiments, a PD-1 or PD-L1 antagonist is a compound or biological molecule that specifically binds to, blocks, or downregulates PD-1 or PD-L1, respectively.

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a PD-1 or PD-L1 antagonist. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a PD-1/PD-L1 antagonist, for example, MK-3475, nivolumab (Opdivo®), pembrolizumab (Keytruda®), humanized antibodies (i.e., h409A1 1, h409A16 and h409A17), AMP-514, BMS-936559, MEDI0680, MEDI4736, MPDL3280A, MSB0010718C, MDX-1105, MDX-1106, or pidilzumab. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a PD-1/PD-L1 antagonist that is an immunoadhesion molecule, such as AMP-224. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a PD-1/PD-L1 antagonist to treat cancer cells or a tumor that overexpresses PD-1 or PD-L1. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a PD-1/PD-L1 antagonist to treat cancer cells or a tumor that overexpresses miR-34.

h. Combination Treatment with Anti-Hormone Therapy

Anti-hormone therapy uses an agent to suppress selected hormones or the effects. Anti-hormone therapy is achieved by antagonizing the function of hormones with a hormone antagonist and/or by preventing the production of hormones. In some embodiments, the suppression of hormones can be beneficial to subjects with certain cancers that grow in response to the presence of specific hormones. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with a hormone antagonist.

In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with anti-androgens, anti-estrogens, aromatase inhibitors, or luteinizing hormone-releasing hormone (LHRH) agonists. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with anti-androgens, such as bicalutamide (Casodex®), cyproterone (Androcur®), flutamide (Euflex®), or nilutamide (Anandron®). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with anti-estrogens, such as fulvestrant (Faslodex®), raloxifene (Evista®), or tamoxifen (Novaladex®, Tamofen®). In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with LHRH agonists, such as buserelin (Suprefact®), goserelin (Zoladex®), or leuprolide (Lupron®, Lupron Depot®, Eligard®).

i. Combination Treatment with Hypomethylating (Demethylating) Agents

Hypomethylating (demethylating) agents inhibit DNA methylation, which affects cellular function through successive generations of cells without changing the underlying DNA sequence. Hypomethylating agents can block the activity of DNA methyltransferase. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with hypomethylating agents, such as azacitidine (Vidaza®, Azadine®) or decitabine (Dacogen®).

j. Combination Treatment with Anti-Inflammatory Agents

In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with nonsteroidal anti-inflammatory drugs (NSAIDs), specific COX-2 inhibitors, or corticosteroids. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with NSAIDs, such as aspirin, ibuprofen, naproxen, celecoxib, ketorolac, or diclofenac. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with specific COX-2 inhibitors, such as celecoxib (Celebrex®), rofecoxib, or etoricoxib. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with corticosteroids, such as dexamethasone or glucosteroids (e.g., hydrocortisone and prednisone).

k. Combination Treatment with HDAC Inhibitors

Histone deacetylase (HDAC) inhibitors are chemical compounds that inhibit histone deacetylase. HDAC inhibitors can induce p21 expression, a regulator of p53 activity. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with an HDAC inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with an HDAC inhibitor, such as vorinostat, romidepsin (Istodax®), chidamide, panobinostat (Farydak®), belinostat (PDX101), panobinostat (LBH589), valproic acid, mocetinostat (MGCD0103), abexinostat (PCI-24781), entinostat (MS-275), SB939, resminostat (4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), HBI-8000, kevetrin, CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, or trichostatin A.

l. Combination Treatment with Platinum-Based Antineoplastic Drugs

Platinum-based antineoplastic drugs are coordinated complex of platinum. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a platinum-based antineoplastic drug, such as cisplatin, oxaliplatin, carboplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with cisplatin or carboplatin. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with cisplatinum, platamin, neoplatin, cismaplat, cis-diamminedichloroplatinum(II), or CDDP; Platinol®) and carboplatin (also known as cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II); tradenames Paraplatin® and Paraplatin-AQ®).

m. Combination Treatment with Kinase Inhibitors

Abnormal activation of protein phosphorylation is frequently either a driver of direct consequence of cancer. Kinase signaling pathways are involved in the phenotypes of tumor biology, including proliferation, survival, motility, metabolism, angiogenesis, and evasion of antitumor immune responses.

MEK Inhibitors:

MEK inhibitors are drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 and/or MEK2. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a MEK1 inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a MEK2 inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with an agent that can inhibit MEK1 and MEK2. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a MEK1/MEK2 inhibitor, such as trametinib (Mekinist®), cobimetinib, binimetinib, selumetinib (AZD6244), pimasertibe (AS-703026), PD-325901, CI-1040, PD035901, or TAK-733. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with trametinib. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with cobimetinib.

BRAF Inhibitors:

BRAF inhibitors are drugs that inhibit the serine/threonine-protein kinase B-raf (BRAF) protein. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BRAF inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BRAF inhibitor that can inhibit wild type BRAF. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BRAF inhibitor that can inhibit mutated BRAF. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BRAF inhibitor that can inhibit V600E mutated BRAF. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BRAF inhibitor, such as vemurafenib (Zelboraf®), dabrafenib (Tafinlar®), C-1, NVP-LGX818, or sorafenib (Nexavar®).

KRAS Inhibitors:

KRAS is a gene that acts as an on/off switch in cell signaling. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a KRAS inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a wild type KRAS inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a mutated KRAS inhibitor.

BTK Inhibitors:

Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase of the Tec kinase family that is involved in B-cell receptor signaling. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BTK inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a BTK inhibitor, such as ibrutinib or acalabrutinib.

CDK Inhibitors:

CDK4 and CDK6 are cyclin-dependent kinases that control the transition between the G1 and S phases of the cell cycle. CDK4/CDK6 activity is deregulated and overactive in cancer cells. Selective CDK4/CDK6 inhibitors can block cell-cycle progression in the mid-G1 phase of the cell cycle, causing arrest and preventing the proliferation of cancer cells. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a CDK4/CDK6 inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a CDK4/CDK6 inhibitor, such as palbociclib (Ibrance®), ribociclib, trilaciclib, seliciclib, dinaciclib, milciclib, roniciclib, atuveciclib, briciclib, riviciclib, voruciclib, or abemaciclib. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with palbociclib. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with ribociclib. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with abemaciclib.

In some examples, the peptidomimetic macrocycles of the disclosure may be used in combination with an inhibitor of CDK4 and/or CDK6 and with an agent that reinforces the cytostatic activity of CDK4/6 inhibitors and/or with an agent that converts reversible cytostasis into irreversible growth arrest or cell death. Exemplary cancer subtypes include NSCLC, melanoma, neuroblastoma, glioblastoma, liposarcoma, and mantle cell lymphoma. In some examples, the peptidomimetic macrocycles of the disclosure may also be used in combination with at least one additional pharmaceutically active agent that alleviates CDKN2A (cyclin-dependent kinase inhibitor 2A) deletion. In some example, the peptidomimetic macrocycles of the disclosure may also be used in combination with at least one additional pharmaceutically active agent that alleviates CDK9 (cyclin-dependent kinase 9) abnormality.

In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a CDK2, CDK7, and/or CDK9 inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a CDK2, CDK7, or CDK9 inhibitor, such as seliciclib, voruciclib, or milciclib. In some embodiments, the peptidomimetic macrocycles of the disclosure can be used in combination with a CDK inhibitor, such as dinaciclib, roniciclib (Kisqali®), or briciclib. In some examples, the peptidomimetic macrocycles of the disclosure may also be used in combination with at least one additional pharmaceutically-active agent that alleviates CDKN2A (cyclin-dependent kinase inhibitor 2A) deletion.

In some embodiments, a method of treating cancer in a subject in need thereof can comprise administering to the subject a therapeutically effective amount of a p53 agent that inhibits the interaction between p53 and MDM2 and/or p53 and MDMX, and/or modulates the activity of p53 and/or MDM2 and/or MDMX; and at least one additional pharmaceutically-active agent, wherein the at least one additional pharmaceutically-active agent modulates the activity of CDK4 and/or CDK6, and/or inhibits CDK4 and/or CDK6.

ATM Regulators:

The peptidomimetic macrocycles of the disclosure may also be used in combination with one or more pharmaceutically-active agent that regulates the ATM (upregulate or downregulate). In some embodiments the compounds described herein can synergize with one or more ATM regulators. In some embodiments one or more of the compounds described herein can synergize with all ATM regulators.

AKT Inhibitors:

In some embodiments, the peptidomimetic macrocycles of the disclosure may be used in combination with one or more pharmaceutically-active agent that inhibits the AKT (protein kinase B (PKB)). In some embodiments the compounds described herein can synergize with one or more AKT inhibitors.

n. Combination Treatment with Other Pharmaceutically-Active Agents

In some examples, the peptidomimetic macrocycles of the disclosure may also be used in combination with at least one additional pharmaceutically-active agent that alleviates PTEN (phosphatase and tensin homolog) deletion.

In some examples, the peptidomimetic macrocycles of the disclosure may also be used in combination with at least one additional pharmaceutically-active agent that alleviates Wip-1Alpha over expression.

In some examples, the peptidomimetic macrocycles of the disclosure may be used in combination with at least one additional pharmaceutically-active agent that is a Nucleoside metabolic inhibitor. Exemplary nucleoside metabolic inhibitors that may be used include capecitabine, gemcitabine and cytarabine (Arac).

The table below lists suitable additional pharmaceutically-active agents for use with the methods described herein.

			Drug works predominately
Cancer Type	Drug name	Brand name	in S or M phase
ALL	ABT-199	none	No
ALL	clofarabine	Clofarex	Yes; S phase
ALL	cyclophosphamide	Clafen, Cytoxan, Neosar	Yes: S phase
ALL	cytarabine	Cytosar-U, Tarabine PFS	Yes: S phase
ALL	doxorubicin	Adriamycin	Yes: S phase
ALL	imatinib mesylate	Gleevec	No
ALL	methotrexate	Abitrexate, Mexate, Folex	Yes: S phase
ALL	prednisone	Deltasone, Medicorten	No
ALL	romidepsin	Istodax
ALL	vincristine	Vincasar	Yes: M phase
AML	ABT-199	none	No
AML	azacitadine	Vidaza	No
AML	cyclophosphamide	Clafen, Cytoxan, Neosar	Yes: S phase
AML	cytarabine	Cytosar-U, Tarabine PFS	Yes: S phase
AML	decitabine	Dacogen	No
AML	doxorubicin	Adriamycin	Yes: S phase
AML	etoposide	Etopophos, Vepesid	Yes: S and M phases
AML	vincristine	Vincasar	Yes: M phase
bone	doxorubicin	Adriamycin	Yes: S phase
bone	methotrexate	Abitrexate, Mexate, Folex	Yes: S phase
breast	capecitabine	Xeloda	Yes: S phase
breast	cyclophosphamide	Clafen, Cytoxan, Neosar	Yes: S phase
breast	docetaxel	Taxotere	Yes: M phase
breast	doxorubicin	Adriamycin	Yes: S phase
breast	eribulin mesylate	Haliben	Yes: M phase
breast	everolimus	Afinitor	No
breast	exemestane	Aromasin	No
breast	fluorouracil	Adrucil, Efudex	Yes: S phase
breast	fulvestrant	Faslofex
breast	gemcitabine	Gemzar	Yes: S phase
breast	goserelin acetate	Zoladex	No
breast	letrozole	Femara	No
breast	megestrol acetate	Megace	No
breast	methotrexate	Abitrexate, Mexate, Folex	Yes: S phase
breast	paclitaxel	Abraxane ®, Taxol	Yes: M phase
breast	palbociclib	Ibrance	Might cause G1 arrest
breast	pertuzumab	Perjeta	No
breast	tamoxifen citrate	Nolvadex	No
breast	trastuzumab	Herceptin, Kadcyla	No
colon	capecitabine	Xeloda	Yes: S phase
colon	cetuximab	Erbitux	No
colon	fluorouracil	Adrucil, Efudex	Yes: S phase
colon	irinotecan	camptosar	Yes: S and M phases
colon	ramucirumab	Cyramza	No
endometrial	carboplatin	Paraplatin, Paraplat	Yes: S phase
endometrial	cisplatin	Platinol	Yes: S phase
endometrial	doxorubicin	Adriamycin	Yes: S phase
endometrial	megestrol acetate	Megace	No
endometrial	paclitaxel	Abraxane ®, Taxol	Yes: M phase
gastric	docetaxel	Taxotere	Yes: M phase
gastric	doxorubicin	Adriamycin	Yes: S phase
gastric	fluorouracil	Adrucil, Efudex	Yes: S phase
gastric	ramucirumab	Cyramza	No
gastric	trastuzumab	Herceptin	No
kidney	axitinib	Inlyta	No
kidney	everolimus	Afinitor	No
kidney	pazopanib	Votrient	No
kidney	sorafenib tosylate	Nexavar	No
liver	sorafenib tosylate	Nexavar	No
melanoma	dacarbazine	DTIC, DTIC-Dome	Yes: S phase
melanoma	paclitaxel	Abraxane ®, Taxol	Yes: M phase
melanoma	trametinib	Mekinist	No
melanoma	vemurafenib	Zelboraf	No
melanoma	dabrafenib	Taflinar
mesothelioma	cisplatin	Platinol	Yes: S phase
mesothelioma	pemetrexed	Alimta	Yes: S phase
NHL	ABT-199	none	No
NHL	bendamustine	Treanda	Causes DNA crosslinking,
			but is also toxic to resting
			cells
NHL	bortezomib	Velcade	No
NHL	brentuximab vedotin	Adcetris	Yes: M phase
NHL	chlorambucil	Ambochlorin, Leukeran, Linfolizin	Yes: S phase
NHL	cyclophosphamide	Clafen, Cytoxan, Neosar	Yes: S phase
NHL	dexamethasone	Decadrone, Dexasone	No
NHL	doxorubicin	Adriamycin	Yes: S phase
NHL	Ibrutinib	Imbruvica	No
NHL	lenalidomide	Revlimid	No
NHL	methotrexate	Abitrexate, Mexate, Folex	Yes: S phase
NHL	obinutuzumab	Gazyva	No
NHL	prednisone	Deltasone, Medicorten	No
NHL	romidepsin	Istodax
NHL	rituximab	Rituxan	No
NHL	vincristine	Vincasar	Yes: M phase
NSCLC	afatinib Dimaleate	Gilotrif	No
NSCLC	carboplatin	Paraplatin, Paraplat	Yes: S phase
NSCLC	cisplatin	Platinol	Yes: S phase
NSCLC	crizotinib	Xalkori	No
NSCLC	docetaxel	Taxotere	Yes: M phase
NSCLC	erlotinib	Tarceva	No
NSCLC	gemcitabine	Gemzar	Yes: S phase
NSCLC	methotrexate	Abitrexate, Mexate, Folex	Yes: S phase
NSCLC	paclitaxel	Abraxane ®, Taxol	Yes: M phase
NSCLC	palbociclib	Ibrance	Might cause G1 arrest
NSCLC	pemetrexed	Alimta	Yes: S phase
NSCLC	ramucirumab	Cyramza	No
ovarian	carboplatin	Paraplatin, Paraplat	Yes: S phase
ovarian	cisplatin	Platinol	Yes; S phase
ovarian	cyclophosphamide	Clafen, Cytoxan, Neosar	Yes: S phase
ovarian	gemcitabine	Gemzar	Yes: S phase
ovarian	olaparib	Lynparza	Yes: G2/M phase arrest
ovarian	paclitaxel	Abraxane ®, Taxol	Yes: M phase
ovarian	topotecan	Hycamtin	Yes: S phase
prostate	abiraterone	Zytiga	No
prostate	cabazitaxel	Jevtana	Yes: M phase
prostate	docetaxel	Taxotere	Yes: M phase
prostate	enzalutamide	Xtandi	No
prostate	goserelin acetate	Zoladex	No
prostate	prednisone	Deltasone, Medicorten	No
soft tissue sarcoma	doxorubicin	Adriamycin	Yes: S phase
soft tissue sarcoma	imatinib mesylate	Gleevec	No
soft tissue sarcoma	pazopanib	Votrient	No
T-cell lymphoma	romidepsin	Istodax

Administration of Combination Treatment

The peptidomimetic macrocycles or a composition comprising same and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, or a composition comprising same can be administered simultaneously (i.e., simultaneous administration) and/or sequentially (i.e., sequential administration).

According to certain embodiments, the peptidomimetic macrocycles and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are administered simultaneously, either in the same composition or in separate compositions. The term “simultaneous administration,” as used herein, means that the peptidomimetic macrocycle and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are administered with a time separation of no more than a few minutes, for example, less than about 15 minutes, less than about 10, less than about 5, or less than about 1 minute. When the drugs are administered simultaneously, the peptidomimetic macrocycle and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, may be contained in the same composition (e.g., a composition comprising both the peptidomimetic macrocycle and the at least additional pharmaceutically-active agent) or in separate compositions (e.g., the peptidomimetic macrocycle is contained in one composition and the at least additional pharmaceutically-active agent is contained in another composition).

According to other embodiments, the peptidomimetic macrocycles and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are administered sequentially, i.e., the peptidomimetic macrocycle is administered either prior to or after the administration of the additional pharmaceutically-active agent. The term “sequential administration” as used herein means that the peptidomimetic macrocycle and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are administered with a time separation of more than a few minutes, for example, more than about 15 minutes, more than about 20 or more minutes, more than about 30 or more minutes, more than about 40 or more minutes, more than about 50 or more minutes, or more than about 60 or more minutes. In some embodiments, the peptidomimetic macrocycle is administered before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. In some embodiments, the pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered before the peptidomimetic macrocycle. The peptidomimetic macrocycle and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are contained in separate compositions, which may be contained in the same or different packages.

In some embodiments, the administration of the peptidomimetic macrocycles and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are concurrent, i.e., the administration period of the peptidomimetic macrocycles and that of the agent overlap with each other. In some embodiments, the administration of the peptidomimetic macrocycles and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are non-concurrent. For example, in some embodiments, the administration of the peptidomimetic macrocycles is terminated before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered. In some embodiments, the administration of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is terminated before the peptidomimetic macrocycle is administered. The time period between these two non-concurrent administrations can range from being days apart to being weeks apart.

The dosing frequency of the peptidomimetic macrocycle and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, may be adjusted over the course of the treatment, based on the judgment of the administering physician. When administered separately, the peptidomimetic macrocycle and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be administered at different dosing frequency or intervals. For example, the peptidomimetic macrocycle can be administered weekly, while the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be administered more or less frequently. Or, the peptidomimetic macrocycle can be administered twice weekly, while the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be administered more or less frequently. In addition, the peptidomimetic macrocycle and the at least one additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be administered using the same route of administration or using different routes of administration.

A therapeutically effective amount of a peptidomimetic macrocycle and/or the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for use in therapy can vary with the nature of the condition being treated, the length of treatment time desired, the age and the condition of the patient, and can be determined by the attending physician. Doses employed for human treatment can be in the range of about 0.01 mg/kg to about 1000 mg/kg per day (e.g., about 0.01 mg/kg to about 100 mg/kg per day, about 0.01 mg/kg to about 10 mg/kg per day, about 0.1 mg/kg to about 100 mg/kg per day, about 0.1 mg/kg to about 50 mg/kg per day, about 0.1 mg/kg to about 10 mg/kg per day) of one or each component of the combinations described herein. In some embodiments, doses of a peptidomimetic macrocycle employed for human treatment are in the range of about 0.01 mg/kg to about 100 mg/kg per day (e.g., about 0.01 mg/kg to about 10 mg/kg per day, about 0.1 mg/kg to about 100 mg/kg per day, about 0.1 mg/kg to about 50 mg/kg per day, about 0.1 mg/kg to about 10 mg/kg per day, about 1 mg/kg per day). In some embodiments, doses of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, employed for human treatment can be in the range of about 0.01 mg/kg to about 100 mg/kg per day (e.g., about 0.1 mg/kg to about 100 mg/kg per day, about 0.1 mg/kg to about 50 mg/kg per day, about 10 mg/kg per day or about 30 mg/kg per day). The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.

In some embodiments, such as when given in combination with the at least one additional pharmaceutically active agent, for example, any additional therapeutic agent described herein, the dosage of a peptidomimetic macrocycle may be given at relatively lower dosages. In some embodiments, the dosage of a peptidomimetic macrocycle may be from about 1 ng/kg to about 100 mg/kg. The dosage of a peptidomimetic macrocycle may be at any dosage including, but not limited to, about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 i g/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.

In some embodiments, the dosage of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, may be from about 1 ng/kg to about 100 mg/kg. The dosage of the additional pharmaceutically-active agent may be at any dosage including, but not limited to, about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 i g/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.

In some embodiments, the dosage of the additional pharmaceutically-active agent is the approved dosage from the label of the additional pharmaceutically-active agent. In some embodiments, the dosage of the additional pharmaceutically-active agent is 600 mg of ribociclib; 150 mg or 200 mg of abemaciclib; 125 mg of palbociclib; 2 mg of trametinib; 175 mg/m², 135 mg/m², or 100 mg/m² of paclitaxel; 1.4 mg/m² of eribulin; 250 mg/m² (breast cancer), 100 mg/m² (non-small cell lung cancer), or 125 mg/m² (pancreatic cancer) of Abraxane®; 200 mg of Keytruda®; or 240 mg or 480 mg of Opdivo®, or a pharmaceutically-acceptable salt of any of the foregoing. In some embodiments, the approved dosages of the additional pharmaceutically-active agents can be reduced to address adverse side effects such as renal impairment or liver impairment.

The peptidomimetic macrocycle and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be provided in a single unit dosage form for being taken together or as separate entities (e.g. in separate containers) to be administered simultaneously or with a certain time difference. This time difference may be between 1 hour and 1 month, e.g., between 1 day and 1 week, e.g., 48 hours and 3 days. In addition, it is possible to administer the peptidomimetic macrocycle via another administration way than the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. For example, it may be advantageous to administer either the peptidomimetic macrocycle or the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, intravenously and the other systemically or orally. For example, the peptidomimetic macrocycle is administered intravenously and the additional pharmaceutically-active agent orally.

In some embodiments, the peptidomimetic macrocycle is administered about 0.1 hour, 0.2 hour, 0.3 hour, 0.4 hour, 0.5 hour, 0.6 hour, 0.7 hour, 0.8 hour, 0.9 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered. In some embodiments, the peptidomimetic macrocycle is administered about 6 hours before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered.

In some embodiments, the peptidomimetic macrocycle is administered about 0.1 hour, 0.2 hour, 0.3 hour, 0.4 hour, 0.5 hour, 0.6 hour, 0.7 hour, 0.8 hour, 0.9 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered. In some embodiments, the peptidomimetic macrocycle is administered about 6 hours after the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered.

In some embodiments, the peptidomimetic macrocycle is administered chronologically before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. In some embodiments, the peptidomimetic macrocycle is administered from 1-24 hours, 2-24 hours, 3-24 hours, 4-24 hours, 5-24 hours, 6-24 hours, 7-24 hours, 8-24 hours, 9-24 hours, 10-24 hours, 11-24 hours, 12-24 hours, 1-30 days, 2-30 days, 3-30 days, 4-30 days, 5-30 days, 6-30 days, 7-30 days, 8-30 days, 9-30 days, 10-30 days, 11-30 days, 12-30 days, 13-30 days, 14-30 days, 15-30 days, 16-30 days, 17-30 days, 18-30 days, 19-30 days, 20-30 days, 21-30 days, 22-30 days, 23-30 days, 24-30 days, 25-30 days, 26-30 days, 27-30 days, 28-30 days, 29-30 days, 1-4 week, 2-4 weeks, 3-4 weeks, 1-12 months, 2-12 months, 3-12 months, 4-12 months, 5-12 months, 6-12 months, 7-12 months, 8-12 months, 9-12 months, 10-12 months, 11-12 months, or any combination thereof, before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered. In some embodiments, the peptidomimetic macrocycle is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered. For example, the peptidomimetic macrocycle can be administered at least 6 hours before a CDKI (e.g., seliciclib, ribociclib, abemaciclib, or palbociclib) is administered.

In some embodiments, the peptidomimetic macrocycle is administered at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the additional pharmaceutically-active agent is administered. For example, the peptidomimetic macrocycle can be administered at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before a CDKI (e.g., seliciclib, ribociclib, abemaciclib, or palbociclib) is administered.

In some embodiments, the peptidomimetic macrocycle is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered. For example, the peptidomimetic macrocycle can be administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before a CDKI (e.g., seliciclib, ribociclib, abemaciclib, or palbociclib) is administered.

In some embodiments, the peptidomimetic macrocycle is administered chronologically at the same time as the at least one additional pharmaceutically active agent, for example, any additional therapeutic agent described herein.

In some embodiments, the peptidomimetic macrocycle is administered chronologically after the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. In some embodiments, the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered from 1-24 hours, 2-24 hours, 3-24 hours, 4-24 hours, 5-24 hours, 6-24 hours, 7-24 hours, 8-24 hours, 9-24 hours, 10-24 hours, 11-24 hours, 12-24 hours, 1-30 days, 2-30 days, 3-30 days, 4-30 days, 5-30 days, 6-30 days, 7-30 days, 8-30 days, 9-30 days, 10-30 days, 11-30 days, 12-30 days, 13-30 days, 14-30 days, 15-30 days, 16-30 days, 17-30 days, 18-30 days, 19-30 days, 20-30 days, 21-30 days, 22-30 days, 23-30 days, 24-30 days, 25-30 days, 26-30 days, 27-30 days, 28-30 days, 29-30 days, 1-4 week, 2-4 weeks, 3-4 weeks, 1-12 months, 2-12 months, 3-12 months, 4-12 months, 5-12 months, 6-12 months, 7-12 months, 8-12 months, 9-12 months, 10-12 months, 11-12 months, or any combination thereof, before the peptidomimetic macrocycle is administered. In some embodiments the additional pharmaceutically-active agent is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the peptidomimetic macrocycle is administered. For example, seliciclib, ribociclib, abemaciclib, or palbociclib can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the peptidomimetic macrocycle is administered.

In some embodiments, a CDKI is administered at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the peptidomimetic macrocycle is administered. For example, seliciclib, ribociclib, abemaciclib, or palbociclib can be administered at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the peptidomimetic macrocycle is administered.

In some embodiments a CDKI is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the peptidomimetic macrocycle is administered. For example, seliciclib, ribociclib, abemaciclib, or palbociclib can be administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the peptidomimetic macrocycle is administered.

Also, contemplated herein is a drug holiday utilized among the administration of a peptidomimetic macrocycle and an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. A drug holiday can be a period of days after the administration of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, and before the administration of a peptidomimetic macrocycle. A drug holiday can be a period of days after the administration of a peptidomimetic macrocycle and before the administration of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. A drug holiday can be a period of days after the sequential administration of one or more of a peptidomimetic macrocycle and an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, and before the administration of the peptidomimetic macrocycle, the additional pharmaceutically-active agent or another therapeutic agent. For example, a drug holiday can be a period of days after the sequential administration of a peptidomimetic macrocycle first, followed administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, and before the administration of the peptidomimetic macrocycle again. For example, a drug holiday can be a period of days after the sequential administration of an additional pharmaceutically-active agent first, followed administration of a peptidomimetic macrocycle and before the administration of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein.

Suitably the drug holiday will be a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days; or from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 days, 1-4, 2-4, or 3-4 weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 months.

In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, will be administered first in the sequence, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, will be administered first in the sequence, followed by administration of a peptidomimetic macrocycle, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent.

In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by an optional drug holiday; followed by administration of a peptidomimetic macrocycle for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months. For example, a cyclin dependent kinase inhibitor is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by a drug holiday of from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by administration of a peptidomimetic macrocycle for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months.

In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by administration of a peptidomimetic macrocycle for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by an optional drug holiday; followed by administration of an additional pharmaceutically-active agent. For example, a cyclin dependent kinase inhibitor is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by administration of a peptidomimetic macrocycle for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by an optional drug holiday of from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by administration of a cyclin dependent kinase inhibitor.

In some embodiments, a peptidomimetic macrocycle will be administered first in the sequence, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. In some embodiments, a peptidomimetic macrocycle will be administered first in the sequence, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle.

In some embodiments, a peptidomimetic macrocycle is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by an optional drug holiday; followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months. For example, a peptidomimetic macrocycle is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by a drug holiday of from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by administration of a cyclin dependent kinase inhibitor for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months

In some embodiments, a peptidomimetic macrocycle is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by an optional drug holiday; followed by administration of a peptidomimetic macrocycle. For example, a peptidomimetic macrocycle is administered for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by administration of a cyclin dependent kinase inhibitor for from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months, followed by an optional drug holiday of from 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, or 12-24 consecutive hours; from 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 26-30, 27-30, 28-30, or 29-30 consecutive days, 1-4, 2-4, or 3-4 consecutive weeks; or from 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 consecutive months; followed by administration of a peptidomimetic macrocycle.

In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, will be administered first in the sequence, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle. In some embodiments, a cyclin dependent kinase inhibitor will be administered first in the sequence, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle, followed by an optional drug holiday, followed by administration of a cyclin dependent kinase inhibitor.

In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 1 to 30 consecutive days, followed by an optional drug holiday, followed by administration of peptidomimetic macrocycle for from 1 to 30 consecutive days. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 1 to 21 consecutive days, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle for from 1 to 21 consecutive days. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 1 to 14 consecutive days, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle for from 1 to 14 consecutive days. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for 14 consecutive days, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle for 7 consecutive days. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for 7 consecutive days, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle for 7 consecutive days.

In some embodiments, a peptidomimetic macrocycle is administered for from 1 to 30 consecutive days, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for from 1 to 30 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for from 1 to 21 consecutive days, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for from 1 to 21 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for from 1 to 14 consecutive days, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for from 1 to 14 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for 14 consecutive days, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for 14 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for 7 consecutive days, followed by an optional drug holiday, followed by administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for 7 consecutive days.

In some embodiments, one of a peptidomimetic macrocycle and an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 2 to 30 consecutive days, followed by an optional drug holiday, followed by administration of the other of a peptidomimetic macrocycle and an additional pharmaceutically-active agent for from 2 to 30 consecutive days. In some embodiments, one of a peptidomimetic macrocycle and an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 2 to 21 consecutive days, followed by an optional drug holiday, followed by administration of the other of a peptidomimetic macrocycle and an additional pharmaceutically-active agent for from 2 to 21 consecutive days. In some embodiments, one of a peptidomimetic macrocycle and an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 2 to 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of the other of a peptidomimetic macrocycle and an additional pharmaceutically-active agent for from 2 to 14 consecutive days. In some embodiments, one of a peptidomimetic macrocycle and an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered for from 3 to 7 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of the other of a peptidomimetic macrocycle and an additional pharmaceutically-active agent for from 3 to 7 consecutive days.

In some embodiments, a cyclin dependent kinase inhibitor will be administered first in the sequence, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle. In some embodiments, a cyclin dependent kinase inhibitor is administered for from 3 to 21 consecutive days, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle for from 3 to 21 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of a peptidomimetic macrocycle for from 3 to 21 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of a peptidomimetic macrocycle for from 3 to 21 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for 21 consecutive days, followed by an optional drug holiday, followed by administration of a peptidomimetic macrocycle for 14 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of a peptidomimetic macrocycle for 14 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for 7 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of a peptidomimetic macrocycle for 7 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for 3 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of a peptidomimetic macrocycle for 7 consecutive days. In some embodiments, a cyclin dependent kinase inhibitor is administered for 3 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of a peptidomimetic macrocycle for 3 consecutive days.

In some embodiments, a peptidomimetic macrocycle will be administered first in the sequence, followed by an optional drug holiday, followed by administration of a cyclin dependent kinase inhibitor. In some embodiments, a peptidomimetic macrocycle is administered for from 3 to 21 consecutive days, followed by an optional drug holiday, followed by administration of a cyclin dependent kinase inhibitor for from 3 to 21 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of a cyclin dependent kinase inhibitor for from 3 to 21 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of a cyclin dependent kinase inhibitor for from 3 to 21 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for 21 consecutive days, followed by an optional drug holiday, followed by administration of a cyclin dependent kinase inhibitor for 14 consecutive days. In some embodiments, a peptidomimetic macrocycle s administered for 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of a cyclin dependent kinase inhibitor for 14 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for 7 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of a cyclin dependent kinase inhibitor for 7 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for 3 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of a cyclin dependent kinase inhibitor for 7 consecutive days. In some embodiments, a peptidomimetic macrocycle is administered for 3 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of a cyclin dependent kinase inhibitor for 3 consecutive days.

In some embodiments, a peptidomimetic macrocycle is administered once, twice, or thrice daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, consecutive days followed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days of rest (e.g., no administration of the peptidomimetic macrocycle/discontinuation of treatment) in a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 day cycle; and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered prior to, concomitantly with, or subsequent to administration of the peptidomimetic macrocycle on one or more days (e.g., on day 1 of cycle 1). In some embodiments, the combination therapy is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 13 cycles of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the combination therapy is administered for 1 to 12 or 13 cycles of 28 days (e.g., about 12 months).

In some embodiments, provided herein is a method of treating a condition or disease comprising administering to a patient in need thereof a therapeutically effective amount of a peptidomimetic macrocycle in combination with a therapeutically effective amount of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, and a secondary active agent, such as a checkpoint inhibitor. In some embodiments, a peptidomimetic macrocycle is administered once, twice, or thrice daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, consecutive days followed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days of rest (e.g., no administration of the peptidomimetic macrocycle/discontinuation of treatment) in a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 day cycle; the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is administered prior to, concomitantly with, or subsequent to administration of the peptidomimetic macrocycle on one or more days (e.g., on day 1 of cycle 1), and the secondary agent is administered daily, weekly, or monthly. In some embodiments, the combination therapy is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 13 cycles of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the combination therapy is administered for 1 to 12 or 13 cycles of 28 days (e.g., about 12 months).

In some embodiments, administration of a combination therapy as described herein modulates expression levels of at least one checkpoint protein (e.g., PD-L1). Thus, provided herein are methods of determining the expression of at least of checkpoint proteins, where the determination of the expression level is performed before, during, and/or after administration of a combination therapy described herein. The checkpoint protein expression levels determined before, during, and/or after administration of a combination therapy as described herein can be compared against each other or standard controls. Such comparisons can translate into determination of the efficacy of the administered treatment where in one embodiment a level of decreased expression of a given checkpoint protein indicates a greater effectiveness of the combination therapy. In some embodiments, treatment using the combination therapies described herein can be monitored or determined using assays to determine expression levels of checkpoint proteins (e.g., PD-L1, TIM-3, LAG-3, CTLA-4, OX40, Treg, CD25, CD127, FoxP3). Determining the expression of such checkpoint proteins can be performed before, during, or after completion of treatment with a combination therapy described herein. Expression can be determined using techniques known in the art, including for example flow-cytometry.

In some embodiments, the components of the combination therapies described herein (e.g., a peptidomimetic macrocycle and a cyclin dependent kinase inhibitor) are cyclically administered to a patient. In some embodiments, a secondary active agent is co-administered in a cyclic administration with the combination therapies provided herein. Cycling therapy involves the administration of an active agent for a period of time, followed by a rest for a period of time, and repeating this sequential administration. Cycling therapy can be performed independently for each active agent (e.g., a peptidomimetic macrocycle and a cyclin dependent kinase inhibitor, and/or a secondary agent) over a prescribed duration of time. In some embodiments, the cyclic administration of each active agent is dependent upon one or more of the active agents administered to the subject. In some embodiments, administration of a peptidomimetic macrocycle or a cyclin dependent kinase inhibitor fixes the day(s) or duration of administration of each agent. In some embodiments, administration of a peptidomimetic macrocycle or a cyclin dependent kinase inhibitor fixes the days(s) or duration of administration of a secondary active agent.

In some embodiments, a peptidomimetic macrocycle, a cyclin dependent kinase inhibitor, and/or a secondary active agent is administered continually (e.g., daily, weekly, monthly) without a rest period. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid, or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment or therapeutic agent.

In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In some embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, a compound for use in combination therapies described herein is administered once a day. In some embodiments, a compound for use in combination therapies described herein is administered twice a day. In some embodiments, a compound for use in combination therapies described herein is administered three times a day. In some embodiments, a compound for use in combination therapies described herein is administered four times a day.

In some embodiments, the frequency of administration of a peptidomimetic macrocycle is in the range of about a daily dose to about a monthly dose. In some embodiments, administration of a peptidomimetic macrocycle is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, a peptidomimetic macrocycle for use in combination therapies described herein is administered once a day. In some embodiments, a peptidomimetic macrocycle for use in combination therapies described herein is administered twice a day. In some embodiments, a peptidomimetic macrocycle for use in combination therapies described herein is administered three times a day. In some embodiments, a peptidomimetic macrocycle for use in combination therapies described herein is administered four times a day.

In some embodiments, the frequency of administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is in the range of about a daily dose to about a monthly dose. In some embodiments, administration of an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for use in combination therapies described herein is administered once a day. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for use in combination therapies described herein is administered twice a day. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for use in combination therapies described herein is administered three times a day. In some embodiments, an additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, for use in combination therapies described herein is administered four times a day.

In some embodiments, a compound for use in combination therapies described herein is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. In some embodiments, a compound for use in combination therapies described herein is administered once per day for one week, two weeks, three weeks, or four weeks. In some embodiments, a compound for use in combination therapies described herein is administered once per day for one week. In some embodiments, a compound for use in combination therapies described herein is administered once per day for two weeks. In some embodiments, a compound for use in combination therapies described herein is administered once per day for three weeks. In some embodiments, a compound for use in combination therapies described herein is administered once per day for four weeks.

Therapeutic compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

In some embodiments, the periodic administration of a peptidomimetic macrocycle and/or the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is effected daily. In some embodiments, the periodic administration of a peptidomimetic macrocycle and/or the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is effected twice daily at one half the amount.

In some embodiments, the periodic administration of a peptidomimetic macrocycle and/or the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is effected once every 3 to 11 days; or once every 5 to 9 days; or once every 7 days; or once every 24 hours. In some embodiments, the periodic administration of a peptidomimetic macrocycle and/or the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, is effected once every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 6 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days.

In some embodiments, the periodic administration of a peptidomimetic macrocycle and/or additional pharmaceutically-active agent is effected one, twice, or thrice daily.

For each administration schedule of a peptidomimetic macrocycle, the periodic administration of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, may be effected once every 16-32 hours; or once every 18-30 hours; or once every 20-28 hours; or once every 22-26 hours. In some embodiments, the administration of a peptidomimetic macrocycle substantially precedes the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein. In some embodiments, the administration of the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, substantially precedes the administration of a peptidomimetic macrocycle.

In some embodiments, a peptidomimetic macrocycle and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, may be administered for a period of time of at least 4 days. In some embodiments, the period of time may be 5 days to 5 years; or 10 days to 3 years; or 2 weeks to 1 year; or 1 month to 6 months; or 3 months to 4 months. In some embodiments, a peptidomimetic macrocycle and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, may be administered for the lifetime of the subject.

Pharmaceutical Compositions for Combination Treatment

According to certain embodiments, the peptidomimetic macrocycles and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are administered within a single pharmaceutical composition. In some embodiments, the peptidomimetic macrocycles of the invention and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be provided in a single unit dosage form for being taken together. According to some embodiments, the pharmaceutical composition further comprises pharmaceutically-acceptable diluents or carrier. According to certain embodiments, the peptidomimetic macrocycles and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, are administered within different pharmaceutical composition. In some embodiments, the peptidomimetic macrocycles of the invention and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be provided in a single unit dosage as separate entities (e.g., in separate containers) to be administered simultaneously or with a certain time difference. In some embodiments, the peptidomimetic macrocycles of the disclosure and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be administered via the same route of administration. In some embodiments, the peptidomimetic macrocycles of the disclosure and the additional pharmaceutically-active agent, for example, any additional therapeutic agent described herein, can be administered via the different route of administration.

In some embodiments, the at least one additional pharmaceutical agent, for example, any additional therapeutic agent described herein, is administered at the therapeutic amount known to be used for treating the specific type of cancer. In some embodiments, the at least one additional pharmaceutical agent, for example, any additional therapeutic agent described herein, is administered in an amount lower than the therapeutic amount known to be used for treating the disease, i.e. a sub-therapeutic amount of the at least one additional pharmaceutical agent is administered.

A peptidomimetic macrocycle of the disclosure and at least one additional pharmaceutical agent, for example, any additional therapeutic agent described herein, administered to the subject can each be from about 0.01 mg/kg to about 100 mg/kg per body weight of the subject. In some embodiments, a peptidomimetic macrocycle of the disclosure and the at least one additional pharmaceutical agent, for example, any additional therapeutic agent described herein, administered to the subject can each be from about 0.01 mg/kg to about 1 mg/kg, 0.01 mg/kg to about 10 mg/kg, 0.01 mg/kg to about 100 mg/kg, 0.1 mg to about 1 mg/kg, 0.1 mg/kg to about 10 mg/kg, or 0.1 mg/kg to about 100 mg/kg per body weight of the subject. In some embodiments, the doses of a peptidomimetic macrocycle and additional therapeutic agent, for example, any additional therapeutic agent described herein, can be administered as a single dose or as multiple doses.

Sequence Homology

Two or more peptides can share a degree of homology. A pair of peptides can have, for example, up to about 20% pairwise homology, up to about 25% pairwise homology, up to about 30% pairwise homology, up to about 35% pairwise homology, up to about 40% pairwise homology, up to about 45% pairwise homology, up to about 50% pairwise homology, up to about 55% pairwise homology, up to about 60% pairwise homology, up to about 65% pairwise homology, up to about 70% pairwise homology, up to about 75% pairwise homology, up to about 80% pairwise homology, up to about 85% pairwise homology, up to about 90% pairwise homology, up to about 95% pairwise homology, up to about 96% pairwise homology, up to about 97% pairwise homology, up to about 98% pairwise homology, up to about 99% pairwise homology, up to about 99.5% pairwise homology, or up to about 99.9% pairwise homology. A pair of peptides can have, for example, at least about 20% pairwise homology, at least about 25% pairwise homology, at least about 30% pairwise homology, at least about 35% pairwise homology, at least about 40% pairwise homology, at least about 45% pairwise homology, at least about 50% pairwise homology, at least about 55% pairwise homology, at least about 60% pairwise homology, at least about 65% pairwise homology, at least about 70% pairwise homology, at least about 75% pairwise homology, at least about 80% pairwise homology, at least about 85% pairwise homology, at least about 90% pairwise homology, at least about 95% pairwise homology, at least about 96% pairwise homology, at least about 97% pairwise homology, at least about 98% pairwise homology, at least about 99% pairwise homology, at least about 99.5% pairwise homology, at least about 99.9% pairwise homology.

Methods of Detecting Wild Type p53 and/or p53 Mutations

In some embodiments, a subject lacking p53-deactivating mutations is a candidate for cancer treatment with a compound of the invention. Cancer cells from patient groups should be assayed in order to determine p53-deactivating mutations and/or expression of wild type p53 prior to treatment with a compound of the invention.

The activity of the p53 pathway can be determined by the mutational status of genes involved in the p53 pathways, including, for example, AKT1, AKT2, AKT3, ALK, BRAF, CDK4, CDKN2A, DDR2, EGFR, ERBB2 (HER2), FGFR1, FGFR3, GNA11, GNQ, GNAS, KDR, KIT, KRAS, MAP2K1 (MEK1), MET, HRAS, NOTCH1, NRAS, NTRK2, PIK3CA, NF1, PTEN, RAC1, RB1, NTRK3, STK11, PIK3R1, TSC1, TSC2, RET, TP53, and VHL. Genes that modulate the activity of p53 can also be assessed, including, for example, kinases: ABL1, JAK1, JAAK2, JAK3; receptor tyrosine kinases: FLT3 and KIT; receptors: CSF3R, IL7R, MPL, and NOTCH1; transcription factors: BCOR, CEBPA, CREBBP, ETV6, GATA1, GATA2. MLL, KZF1, PAX5, RUNX1, STAT3, WT1, and TP53; epigenetic factors: ASXL1, DNMT3A, EZH2, KDM6A (UTX), SUZ12, TET2, PTPN11, SF3B1, SRSF2, U2AF35, ZRSR2; RAS proteins: HRAS, KRAS, and NRAS; adaptors CBL and CBL-B; FBXW7, IDH1, IDH2, and NPM1.

Cancer cell samples can be obtained, for example, from solid or liquid tumors via primary or metastatic tumor resection (e.g. pneumonectomy, lobetomy, wedge resection, and craniotomy) primary or metastatic disease biopsy (e.g. transbronchial or needle core), pleural or ascites fluid (e.g. FFPE cell pellet), bone marrow aspirate, bone marrow clot, and bone marrow biopsy, or macro-dissection of tumor rich areas (solid tumors).

To detect the p53 wild type gene and/or lack of p53 deactivation mutation in a tissue, cancerous tissue can be isolated from surrounding normal tissues. For example, the tissue can be isolated from paraffin or cryostat sections. Cancer cells can also be separated from normal cells by flow cytometry. If the cancer cells tissue is highly contaminated with normal cells, detection of mutations can be more difficult.

Various methods and assays for analyzing wild type p53 and/or p53 mutations are suitable for use in the invention. Non-limiting examples of assays include polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), microarray, Southern Blot, Northern Blot, Western Blot, Eastern Blot, HandE staining, microscopic assessment of tumors, next-generation DNA sequencing (NGS) (e.g. extraction, purification, quantification, and amplification ofDNA, library preparation) immunohistochemistry, and fluorescent in situ hybridization (FISH).

A microarray allows a researcher to investigate multiple DNA sequences attached to a surface, for example, a DNA chip made of glass or silicon, or a polymeric bead or resin. The DNA sequences are hybridized with fluorescent or luminescent probes. The microarray can indicate the presence of oligonucleotide sequences in a sample based on hybridization of sample sequences to the probes, followed by washing and subsequent detection of the probes. Quantification of the fluorescent or luminescent signal indicates the presence of known oligonucleotide sequences in the sample.

PCR allows amplification of DNA oligomers rapidly, and can be used to identify an oligonucleotide sequence in a sample. PCR experiments involve contacting an oligonucleotide sample with a PCR mixture containing primers complementary to a target sequence, one or more DNA polymerase enzymes, deoxnucleotide triphosphate (dNTP) building blocks, including dATP, dGTP, dTTP, and dCTP, and suitable buffers, salts, and additives. If a sample contains an oligonucleotide sequence complementary to a pair of primers, the experiment amplifies the sample sequence, which can be collected and identified.

In some embodiments, an assay comprises amplifying a biomolecule from the cancer sample. The biomolecule can be a nucleic acid molecule, such as DNA or RNA. In some embodiments, the assay comprises circularization of a nucleic acid molecule, followed by digestion of the circularized nucleic acid molecule.

In some embodiments, the assay comprises contacting an organism, or a biochemical sample collected from an organism, such as a nucleic acid sample, with a library of oligonucleotides, such as PCR primers. The library can contain any number of oligonucleotide molecules. The oligonucleotide molecules can bind individual DNA or RNA motifs, or any combination of motifs described herein. The motifs can be any distance apart, and the distance can be known or unknown. In some embodiments, two or more oligonucleotides in the same library bind motifs a known distance apart in a parent nucleic acid sequence. Binding of the primers to the parent sequence can take place based on the complementarity of the primers to the parent sequence. Binding can take place, for example, under annealing, or under stringent conditions.

In some embodiments, the results of an assay are used to design a new oligonucleotide sequence for future use. In some embodiments, the results of an assay are used to design a new oligonucleotide library for future use. In some embodiments, the results of an assay are used to revise, refine, or update an existing oligonucleotide library for future use. For example, an assay can reveal that a previously-undocumented nucleic acid sequence is associated with the presence of a target material. This information can be used to design or redesign nucleic acid molecules and libraries.

In some embodiments, one or more nucleic acid molecules in a library comprise a barcode tag. In some embodiments, one or more of the nucleic acid molecules in a library comprise type I or type II restriction sites suitable for circularization and cutting an amplified sample nucleic acid sequence. Such primers can be used to circularize a PCR product and cut the PCR product to provide a product nucleic acid sequence with a sequence that is organized differently from the nucleic acid sequence native to the sample organism.

After a PCR experiment, the presence of an amplified sequence can be verified. Non-limiting examples of methods for finding an amplified sequence include DNA sequencing, whole transcriptome shotgun sequencing (WTSS, or RNA-seq), mass spectrometry (MS), microarray, pyrosequencing, column purification analysis, polyacrylamide gel electrophoresis, and index tag sequencing of a PCR product generated from an index-tagged primer.

In some embodiments, more than one nucleic acid sequence in the sample organism is amplified. Non-limiting examples of methods of separating different nucleic acid sequences in a PCR product mixture include column purification, high performance liquid chromatography (HPLC), HPLC/MS, polyacrylamide gel electrophoresis, size exclusion chromatography.

The amplified nucleic acid molecules can be identified by sequencing. Nucleic acid sequencing can be done on automated instrumentation. Sequencing experiments can be done in parallel to analyze tens, hundreds, or thousands of sequences simultaneously. Non-limiting examples of sequencing techniques follow.

In pyrosequencing, DNA is amplified within a water droplet containing a single DNA template bound to a primer-coated bead in an oil solution. Nucleotides are added to a growing sequence, and the addition of each base is evidenced by visual light.

Ion semiconductor sequencing detects the addition of a nucleic acid residue as an electrical signal associated with a hydrogen ion liberated during synthesis. A reaction well containing a template is flooded with the four types of nucleotide building blocks, one at a time. The timing of the electrical signal identifies which building block was added, and identifies the corresponding residue in the template.

DNA nanoball uses rolling circle replication to amplify DNA into nanoballs. Unchained sequencing by ligation of the nanoballs reveals the DNA sequence.

In a reversible dyes approach, nucleic acid molecules are annealed to primers on a slide and amplified. Four types of fluorescent dye residues, each complementary to a native nucleobase, are added, the residue complementary to the next base in the nucleic acid sequence is added, and unincorporated dyes are rinsed from the slide. Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Fluorescence indicates the addition of a dye residue, thus identifying the complementary base in the template sequence. The dye residue is chemically removed, and the cycle repeats.

Detection of point mutations can be accomplished by molecular cloning of the p53 allele(s) present in the cancer cell tissue and sequencing that allele(s). Alternatively, the polymerase chain reaction can be used to amplify p53 gene sequences directly from a genomic DNA preparation from the cancer cell tissue. The DNA sequence of the amplified sequences can then be determined. Specific deletions of p53 genes can also be detected. For example, restriction fragment length polymorphism (RFLP) probes for the p53 gene or surrounding marker genes can be used to score loss of a p53 allele.

Loss of wild type p53 genes can also be detected on the basis of the loss of a wild type expression product of the p53 gene. Such expression products include both the mRNA as well as the p53 protein product itself. Point mutations can be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques. The cDNA can also be sequenced via the polymerase chain reaction (PCR).

Alternatively, mismatch detection can be used to detect point mutations in the p53 gene or the mRNA product. The method can involve the use of a labeled riboprobe that is complementary to the human wild type p53 gene. The riboprobe and either mRNA or DNA isolated from the cancer cell tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, the enzyme cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product is seen that is smaller than the full-length duplex RNA for the riboprobe and the p53 mRNA or DNA. The riboprobe need not be the full length of the p53 mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the p53 mRNA or gene it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR before hybridization.

DNA sequences of the p53 gene from the cancer cell tissue which have been amplified by use of polymerase chain reaction can also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the p53 gene sequence harboring a known mutation. For example, one oligomer can be about 30 nucleotides in length, corresponding to a portion of the p53 gene sequence. At the position coding for the 175th codon of p53 gene the oligomer encodes an alanine, rather than the wild type codon valine. By use of a battery of such allele-specific probes, the PCR amplification products can be screened to identify the presence of a previously identified mutation in the p53 gene. Hybridization of allele-specific probes with amplified p53 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe indicates the presence of the same mutation in the cancer cell tissue as in the allele-specific probe.

The identification of p53 gene structural changes in cancer cells can be facilitated through the application of a diverse series of high resolution, high throughput microarray platforms. Essentially two types of array include those that carry PCR products from cloned nucleic acids (e.g. cDNA, BACs, cosmids) and those that use oligonucleotides. The methods can provide a way to survey genome wide DNA copy number abnormalities and expression levels to allow correlations between losses, gains and amplifications in cancer cells with genes that are over- and under-expressed in the same samples. The gene expression arrays that provide estimates of mRNA levels in cancer cells have given rise to exon-specific arrays that can identify both gene expression levels, alternative splicing events and mRNA processing alterations.

Oligonucleotide arrays can be used to interrogate single nucleotide polymorphisms (SNPs) throughout the genome for linkage and association studies and these have been adapted to quantify copy number abnormalities and loss of heterozygosity events. DNA sequencing arrays can allow resequencing of chromosome regions, exomes, and whole genomes.

SNP-based arrays or other gene arrays or chips can determine the presence of wild type p53 allele and the structure of mutations. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. For example, there are an estimated 5-10 million SNPs in the human genome. SNPs can be synonymous or nonsynonymous substitutions. Synonymous SNP substitutions do not result in a change of amino acid in the protein due to the degeneracy of the genetic code, but can affect function in other ways. For example, a seemingly silent mutation in a gene that codes for a membrane transport protein can slow down translation, allowing the peptide chain to misfold, and produce a less functional mutant membrane transport protein. Nonsynonymous SNP substitutions can be missense substitutions or nonsense substitutions. Missense substitutions occur when a single base change results in change in amino acid sequence of the protein and malfunction thereof leads to disease. Nonsense substitutions occur when a point mutation results in a premature stop codon, or a nonsense codon in the transcribed mRNA, which results in a truncated and usually, nonfunctional, protein product. As SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research. SNP array is a useful tool to study the whole genome.

In addition, SNP array can be used for studying the Loss Of Heterozygosity (LOH). LOH is a form of allelic imbalance that can result from the complete loss of an allele or from an increase in copy number of one allele relative to the other. While other chip-based methods (e.g., comparative genomic hybridization can detect only genomic gains or deletions), SNP array has the additional advantage of detecting copy number neutral LOH due to uniparental disomy (UPD). In UPD, one allele or whole chromosome from one parent are missing leading to reduplication of the other parental allele (uni-parental=from one parent, disomy=duplicated). In a disease setting this occurrence can be pathologic when the wild type allele (e.g., from the mother) is missing and instead two copies of the heterozygous allele (e.g., from the father) are present. This usage of SNP array has a huge potential in cancer diagnostics as LOH is a prominent characteristic of most human cancers. SNP array technology have shown that cancers (e.g. gastric cancer, liver cancer, etc.) and hematologic malignancies (ALL, MDS, CML etc) have a high rate of LOH due to genomic deletions or UPD and genomic gains. In the present disclosure, using high density SNP array to detect LOH allows identification of pattern of allelic imbalance to determine the presence of wild type p53 allele.

Mutations of wild type p53 genes can also be detected on the basis of the mutation of a wild type expression product of the p53 gene. Such expression products include both the mRNA as well as the p53 protein product itself. Point mutations can be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques. The cDNA can also be sequenced via the polymerase chain reaction (PCR). A panel of monoclonal antibodies could be used in which each of the epitopes involved in p53 functions are represented by a monoclonal antibody. Loss or perturbation of binding of a monoclonal antibody in the panel can indicate mutational alteration of the p53 protein and thus of the p53 gene itself. Mutant p53 genes or gene products can also be detected in body samples, including, for example, serum, stool, urine, and sputum. The same techniques discussed above for detection of mutant p53 genes or gene products in tissues can be applied to other body samples.

Loss of wild type p53 genes can also be detected by screening for loss of wild type p53 protein function. Although all of the functions which the p53 protein undoubtedly possesses have yet to be elucidated, at least two specific functions are known. Protein p53 binds to the SV40 large T antigen as well as to the adenovirus E1B antigen. Loss of the ability of the p53 protein to bind to either or both of these antigens indicates a mutational alteration in the protein which reflects a mutational alteration of the gene itself. Alternatively, a panel of monoclonal antibodies could be used in which each of the epitopes involved in p53 functions are represented by a monoclonal antibody. Loss or perturbation of binding of a monoclonal antibody in the panel would indicate mutational alteration of the p53 protein and thus of the p53 gene itself. Any method for detecting an altered p53 protein can be used to detect loss of wild type p53 genes.

Assays

The properties of peptidomimetic macrocycles are assayed, for example, by using the methods described below. In some embodiments, a peptidomimetic macrocycle has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.

a. Assays to Determine α-Helicity

In solution, the secondary structure of polypeptides with α-helical domains will reach a dynamic equilibrium between random coil structures and α-helical structures, often expressed as a “percent helicity”. Thus, for example, alpha-helical domains are predominantly random coils in solution, with α-helical content usually under 25%. Peptidomimetic macrocycles with optimized linkers, on the other hand, possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide. In some embodiments, macrocycles will possess an alpha-helicity of greater than 50%. To assay the helicity of peptidomimetic macrocycles, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H₂O, to concentrations of 25-50 μM). Circular dichroism (CD) spectra are obtained on a spectropolarimeter using standard measurement parameters (e.g. temperature, 20° C.; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [Φ]222obs) by the reported value for a model helical decapeptide.

b. Assay to Determine Melting Temperature (T_m)

A peptidomimetic macrocycle comprising a secondary structure such as an α-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Peptidomimetic macrocycles exhibit T_m of >60° C. representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on melting temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H₂O (e.g. at a final concentration of 50 μM) and the T_m is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95° C.) on a spectropolarimeter using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

c. Protease Resistance Assay

The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, buries the amide backbone and therefore can shield it from proteolytic cleavage. The peptidomimetic macrocycles can be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (S/E ˜125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln [S] versus time (k=−1×slope).

d. Ex Vivo Stability Assay

Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more. For ex vivo serum stability studies, a variety of assays can be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 mcg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8, and 24 hours. To determine the level of intact compound, the following procedure can be used: The samples are extracted by transferring 100 μL of sera to 2 ml centrifuge tubes followed by the addition of 10 μL of 50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPM for 10 min at 4±2° C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N₂<10 psi, 37° C. The samples are reconstituted in 100 μL of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.

e. In Vitro Binding Assays

To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25-1000 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer. Kd values can be determined by nonlinear regression analysis using, for example, GraphPad Prism software. A peptidomimetic macrocycle shows, In some embodiments, similar or lower Kd than a corresponding uncrosslinked polypeptide.

f. In Vitro Displacement Assays to Characterize Antagonists of Peptide-Protein Interactions

To assess the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.

For example, putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer. Kd values can be determined by nonlinear regression analysis. Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.

g. Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry

To assess the binding and affinity of test compounds for proteins, an affinity-selection mass spectrometry assay is used, for example. Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system-wide control experiment using 1 μM peptidomimetic macrocycle plus 5 μM hMDM2. A 1 μL DMSO aliquot of a 40 μM stock solution of peptidomimetic macrocycle is dissolved in 19 μL of PBS (50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl). The resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To a 4 μL aliquot of the resulting supernatant is added 4 μL of 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 1 μM peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4° C. prior to size-exclusion chromatography-LC-MS analysis of 5.0 μL injections. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step. The SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. The (M+3H)³⁺ ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.

h. Assay for Protein-Ligand K_d Titration Experiments

To assess the binding and affinity of test compounds for proteins, a protein-ligand Kd titration experiment is performed, for example. Protein-ligand K_d titrations experiments are conducted as follows: 2 μL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared then dissolved in 38 μL of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS, varying concentrations (125, 62.5, . . . , 0.24 μM) of the titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections. The (M+H)¹⁺, (M+2H)²⁺, (M+3H)³⁺, and/or (M+Na)¹⁺ ion is observed by ESI-MS; extracted ion chromatograms are quantified, then fit to equations to derive the binding affinity K_d.

i. Assay for Competitive Binding Experiments by Affinity Selection-Mass Spectrometry

To determine the ability of test compounds to bind competitively to proteins, an affinity selection mass spectrometry assay is performed, for example. A mixture of ligands at 40 μM per component is prepared by combining 2 μL aliquots of 400 μM stocks of each of the three compounds with 14 μL of DMSO. Then, 1 μL aliquots of this 40 μM per component mixture are combined with 1 μL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078 mM). These 2 μL samples are dissolved in 38 μL of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2 protein in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO, and varying concentrations (125, 62.5, . . . , 0.98 μM) of the titrant peptidomimetic macrocycle. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections.

j. Binding Assays in Intact Cells

It is possible to measure binding of peptides or peptidomimetic macrocycles to their natural acceptors in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluoresceinated (FITC-labeled) compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs. Cells are then pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 μL goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed by further 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μL of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.

k. Cellular Penetrability Assays

A peptidomimetic macrocycle is, for example, more cell penetrable compared to a corresponding uncrosslinked macrocycle. Peptidomimetic macrocycles with optimized linkers possess, for example, cell penetrability that is at least two-fold greater than a corresponding uncrosslinked macrocycle, and often 20% or more of the applied peptidomimetic macrocycle will be observed to have penetrated the cell after 4 hours. To measure the cell penetrability of peptidomimetic macrocycles and corresponding uncrosslinked macrocycle, intact cells are incubated with fluorescently-labeled (e.g. fluoresceinated) peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 μM) for 4 hrs in serum free media at 37° C., washed twice with media and incubated with trypsin (0.25%) for 10 min at 37° C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed.

l. Cellular Efficacy Assays

The efficacy of certain peptidomimetic macrocycles is determined, for example, in cell-based killing assays using a variety of tumorigenic and non-tumorigenic cell lines and primary cells derived from human or mouse cell populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) to identify those that kill at EC₅₀<10 μM. Several standard assays that measure cell viability are commercially available and are optionally used to assess the efficacy of the peptidomimetic macrocycles. In addition, assays that measure Annexin V and caspase activation are optionally used to assess whether the peptidomimetic macrocycles kill cells by activating the apoptotic machinery. For example, the Cell Titer-glo assay is used which determines cell viability as a function of intracellular ATP concentration.

m. In Vivo Stability Assay

To investigate the in vivo stability of the peptidomimetic macrocycles, the compounds are, for example, administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 μL of fresh serum are then measured by LC-MS/MS as above.

n. In Vivo Efficacy in Animal Models

To determine the anti-oncogenic activity of peptidomimetic macrocycles in vivo, the compounds are, for example, given alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In one example, 5×10⁶ RS4;11 cells (established from the bone marrow of a patient with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID mice 3 hrs after they have been subjected to total body irradiation. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals. Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software. Peptidomimetic macrocycles alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment. Expired mice are optionally subjected to necropsy at the end of the experiment. Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from human follicular lymphoma that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.

o. Clinical Trials

To determine the suitability of the peptidomimetic macrocycles for treatment of humans, clinical trials are performed. For example, patients diagnosed with cancer and in need of treatment can be selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle, while the control groups receive a placebo or a known anti-cancer drug. The treatment safety and efficacy of the peptidomimetic macrocycles can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life. In this example, the patient group treated with a peptidomimetic macrocycle can show improved long-term survival compared to a patient control group treated with a placebo.

EXAMPLES

Example 1: Synthesis of 6-chlorotryptophan Fmoc Amino Acids

Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1

To a stirred solution of dry DMF (12 mL) was added dropwise POCl₃ (3.92 mL, 43 mmol, 1.3 equiv) at 0° C. under argon. The solution was stirred at 0° C. for 20 min before a solution of 6-chloroindole (5.0 g, 33 mmol, 1 eq.) in dry DMF (30 mL) was added dropwise. The resulting mixture was warmed to room temperature and stirred for an additional 2.5 h. Water (50 mL) was added to the reaction mixture, and the solution was neutralized with 4M aqueous NaOH (pH ˜8). The resulting solid was filtered off, washed with water, and dried under vacuum. This material was used in the next step without additional purification.

To a stirred solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150 mL) was added successively Boc₂O (7.91 g, 36.3 mmol, 1.1 equiv) and DMAP (0.4 g, 3.3 mmol, 0.1 equiv) at room temperature under N₂. The resulting mixture was stirred at room temperature for 1.5 h, and the solvent was evaporated under reduced pressure. The residue was taken up in EtOAc and washed with 1N HCl, dried, and concentrated to afford formyl indole 1 (9 g, 98% over 2 steps) as a white solid. ¹H NMR (CDCl₃) δ: 1.70 (s, Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).

Tert-butyl 6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2

To a solution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL) was added NaBH₄ (2.4 g, 63 mmol, 2 eq.). The reaction was stirred for 3 h at room temperature. The reaction mixture was concentrated, and the residue was poured into diethyl ether and water. The organic layer was separated, dried over magnesium sulfate, and concentrated to give a white solid (8.7 g, 98%). This material was directly used in the next step without additional purification. ¹H NMR (CDCl₃) δ: 1.65 (s, Boc, 9H); 4.80 (s, 2H, CH₂); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs, 1H).

Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate, 3

To a solution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in dichloromethane (50 mL) under argon was added a solution of triphenylphosphine (4.59 g, 17.5 mmol, 1.2 eq.) in dichloromethane (50 mL) at −40° C. The reaction was stirred for 30 min at 40° C. NBS (3.38 g, 19 mmol, 1.3 eq.) was then added to the reaction mixture. The resulting mixture was warmed to room temperature and stirred overnight. Dichloromethane was evaporated, carbon tetrachloride (100 mL) was added, and the mixture was stirred for 1 h and filtrated. The filtrate was concentrated, loaded on a silica plug, and quickly eluted with 25% EtOAc in hexanes. The solution was concentrated to afford a white foam (3.84 g, 77%). ¹H NMR (CDCl₃) δ: 1.66 (s, Boc, 9H); 4.63 (s, 2H, CH₂); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs, 1H); 8.18 (bs, 1H).

αMe-6Cl-Trp(Boc)-Ni—S-BPB, 4

To S-Ala-Ni—S-BPB (2.66 g, 5.2 mmol, 1 eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.) was added 50 mL of DMF under argon. The bromide derivative compound 3 (2.68 g, 7.8 mmol, 1.5 eq.) was dissolved in DMF (5.0 mL) and added to the reaction mixture using a syringe. The reaction mixture was stirred at ambient temperature for 1 h. The solution was then quenched with 5% aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried, and concentrated. The oily product 4 was purified by flash chromatography (solid loading) on normal phase using EtOAc and hexanes as eluents to give a red solid (1.78 g, 45% yield). M+H calc. 775.21, M+H obs. 775.26; ¹H NMR (CDCl₃) δ: 1.23 (s, 3H, cMe); 1.56 (m, 11H, Boc+CH₂); 1.82-2.20 (m, 4H, 2CH₂); 3.03 (m, 1H, CH_α); 3.24 (m, 2H, CH₂); 3.57 and 4.29 (AB system, 2H, CH₂ (benzyl), J=12.8 Hz); 6.62 (d, 2H); 6.98 (d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m, 2H); 7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).

6Cl-Trp(Boc)-Ni—S-BPB, 5

To Gly-Ni—S-BPB (4.6 g, 9.2 mmol, 1 eq.) and KO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95 mL of DMF under argon. The bromide derivative compound 3 (3.5 g, 4.6 mmol, 1.1 eq.) was dissolved in DMF (10 mL) and added to the reaction mixture using a syringe. The reaction mixture was stirred at ambient temperature for 1 h. The solution was then quenched with 5% aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 5 was purified by flash chromatography (solid loading) on normal phase using EtOAc and hexanes as eluents to give a red solid (5 g, 71% yield). M+H calc. 761.20, M+H obs. 761.34; ¹H NMR (CDCl₃) δ: 1.58 (m, 11H, Boc+CH₂); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m, 2H, CH₂); 3.00 (m, 1H, CH_α); 3.22 (m, 2H, CH₂); 3.45 and 4.25 (AB system, 2H, CH₂ (benzyl), J=12.8 Hz); 4.27 (m, 1H, CH_α); 6.65 (d, 2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m, 3H); 7.35-7.39 (m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m, 2H).

Fmoc-αMe-6Cl-Trp(Boc)-OH, 6

To a solution of 3N HCl/MeOH (1/3, 15 mL) at 50° C. was added a solution of compound 4 (1.75 g, 2.3 mmol, 1 eq.) in MeOH (5 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0° C. with an ice bath and quenched with an aqueous solution of Na₂CO₃ (1.21 g, 11.5 mmol, 5 eq.). Methanol was removed and 8 eq. of Na₂CO₃ (1.95 g, 18.4 mmol) were added to the suspension. EDTA disodium salt dihydrate (1.68 g, 4.5 mmol, 2 eq.) was then added, and the resulting suspension was stirred for 2 h. A solution of Fmoc-OSu (0.84 g, 2.5 mmol, 1.1 eq.) in acetone (50 mL) was added, and the reaction was stirred overnight. The reaction was diluted with diethyl ether and 1N HCl. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 6 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (0.9 g, 70% yield). M+H calc. 575.19, M+H obs. 575.37; ¹H NMR (CDCl₃) 1.59 (s, 9H, Boc); 1.68 (s, 3H, Me); 3.48 (bs, 2H, CH₂); 4.22 (m, 1H, CH); 4.39 (bs, 2H, CH₂); 5.47 (s, 1H, NH); 7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H); 7.75 (d, 2H); 8.12 (bs, 1H).

Fmoc-6Cl-Trp(Boc)-OH, 7

To a solution of 3N HCl/MeOH (1/3, 44 mL) at 50° C. was added a solution of compound 5 (5 g, 6.6 mmol, 1 eq.) in MeOH (10 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0° C. with an ice bath and quenched with an aqueous solution of Na₂CO₃ (3.48 g, 33 mmol, 5 eq.). Methanol was removed and 8 eq. of Na₂CO₃ (5.57 g, 52 mmol) were added to the suspension. EDTA disodium salt dihydrate (4.89 g, 13.1 mmol, 2 eq.) was added to the suspension, and the resulting suspension was stirred for 2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in acetone (100 mL) was added, and the reaction was stirred overnight. The reaction was diluted with diethyl ether and 1N HCl. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 7 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (2.6 g, 69% yield). M+H calc. 561.17, M+H obs. 561.37; ¹H NMR (CDCl₃) 1.63 (s, 9H, Boc); 3.26 (m, 2H, CH₂); 4.19 (m, 1H, CH); 4.39 (m, 2H, CH₂); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H); 7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).

Example 2: Peptidomimetic Macrocycles

Peptidomimetic macrocycles were designed by replacing two or more naturally-occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions. Peptide synthesis was performed manually or using an automated peptide synthesizer under solid phase conditions using rink amide AM resin and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids, 10 eq. of amino acid and a 1:1:2 molar ratio of coupling reagents HBTU/HOBt/DIEA were employed. Non-natural amino acids (4 eq.) were coupled with a 1:1:2 molar ratio of HATU/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, and the C-termini were amidated.

Purification of crosslinked compounds was achieved by HPLC on a reverse phase C18 column to yield the pure compounds. The chemical compositions of the pure products were confirmed by LC/MS mass spectrometry and amino acid analysis.

Synthesis of Dialkyne-Crosslinked Peptidomimetic Macrocycles, Including SP662, SP663 and SP664.

Fully protected resin-bound peptides were synthesized on a PEG-PS resin (loading 0.45 mmol/g) on a 0.2 mmol scale. Deprotection of the temporary Fmoc group was achieved by 3×10 min treatments of the resin bound peptide with 20% (v/v) piperidine in DMF. After washing with NMP (3×), dichloromethane (3×) and NMP (3×), coupling of each successive amino acid was achieved with 1×60 min incubation with the appropriate pre-activated Fmoc-amino acid derivative. All protected amino acids (0.4 mmol) were dissolved in NMP and activated with HCTU (0.4 mmol) and DIEA (0.8 mmol) prior to transfer of the coupling solution to the de-protected resin-bound peptide. After coupling was completed, the resin was washed in preparation for the next deprotection/coupling cycle.

Acetylation of the amino terminus was carried out in the presence of acetic anhydride/DIEA in NMP. The LC-MS analysis of a cleaved and de-protected sample obtained from an aliquot of the fully assembled resin-bound peptide was accomplished in order to verifying the completion of each coupling. In a typical example, tetrahydrofuran (4 ml) and triethylamine (2 ml) were added to the peptide resin (0.2 mmol) in a 40 ml glass vial and shaken for 10 minutes. Pd(PPh₃)₂Cl₂ (0.014 g, 0.02 mmol) and copper iodide (0.008 g, 0.04 mmol) were then added and the resulting reaction mixture was mechanically shaken 16 hours while open to atmosphere. The diyne-cyclized resin-bound peptides were de-protected and cleaved from the solid support by treatment with TFA/H₂O/TIS (95/5/5 v/v) for 2.5 h at room temperature. After filtration of the resin the TFA solution was precipitated in cold diethyl ether and centrifuged to yield the desired product as a solid. The crude product was purified by preparative HPLC.

Synthesis of Single Alkyne-Crosslinked Peptidomimetic Macrocycles, Including SP665.

Fully protected resin-bound peptides were synthesized on a Rink amide MBHA resin (loading 0.62 mmol/g) on a 0.1 mmol scale. Deprotection of the temporary Fmoc group was achieved by 2×20 min treatments of the resin bound peptide with 25% (v/v) piperidine in NMP. After extensive flow washing with NMP and dichloromethane, coupling of each successive amino acid was achieved with 1×60 min incubation with the appropriate pre-activated Fmoc-amino acid derivative. All protected amino acids (1 mmol) were dissolved in NMP and activated with HCTU (1 mmol) and DIEA (1 mmol) prior to transfer of the coupling solution to the de-protected resin-bound peptide. After coupling was completed, the resin was extensively flow washed in preparation for the next deprotection/coupling cycle.

Acetylation of the amino terminus was carried out in the presence of acetic anhydride/DIEA in NMP/NMM. The LC-MS analysis of a cleaved and de-protected sample obtained from an aliquot of the fully assembled resin-bound peptide was accomplished to verify the completion of each coupling reaction. In a typical example, the peptide resin (0.1 mmol) was washed with DCM. Resin was loaded into a microwave vial. The vessel was evacuated and purged with nitrogen. Molybdenum hexacarbonyl (0.01 eq.) was added. Anhydrous chlorobenzene was added to the reaction vessel. Then 2-fluorophenol (1 eq.) was added. The reaction was then loaded into the microwave and held at 130° C. for 10 minutes. The reaction pushed for a longer period time when needed to complete the reaction. The alkyne-metathesized resin-bound peptides were de-protected and cleaved from the solid support by treating the solid support with TFA/H₂O/TIS (94/3/3 v/v) for 3 h at room temperature. After filtration of the resin, the TFA solution was precipitated in cold diethyl ether and centrifuged to yield the desired product as a solid. The crude product was purified by preparative HPLC.

TABLE 1
TABLE 1 shows a list of peptidomimetic macrocycles prepared.
		SEQ				Calc	Calc	Calc
		ID	Iso-	Exact	Found	(M +	(M +	(M +
SP	Sequence	NO:	mer	Mass	Mass	1)/1	2)/2	3)/3
1	Ac-F$r8AYWEAc3cL$AAA-NH2	10		1456.78	729.44	1457.79	729.4	486.6
2	Ac-F$r8AYWEAc3cL$AAibA-NH2	11		1470.79	736.4	1471.8	736.4	491.27
3	Ac-LTF$r8AYWAQL$SANle-NH2	12		1715.97	859.02	1716.98	858.99	573
4	Ac-LTF$r8AYWAQL$SAL-NH2	13		1715.97	859.02	1716.98	858.99	573
5	Ac-LTF$r8AYWAQL$SAM-NH2	14		1733.92	868.48	1734.93	867.97	578.98
6	Ac-LTF$r8AYWAQL$SAhL-NH2	15		1729.98	865.98	1730.99	866	577.67
7	Ac-LTF$r8AYWAQL$SAF-NH2	16		1749.95	876.36	1750.96	875.98	584.32
8	Ac-LTF$r8AYWAQL$SAI-NH2	17		1715.97	859.02	1716.98	858.99	573
9	Ac-LTF$r8AYWAQL$SAChg-NH2	18		1741.98	871.98	1742.99	872	581.67
10	Ac-LTF$r8AYWAQL$SAAib-NH2	19		1687.93	845.36	1688.94	844.97	563.65
11	Ac-LTF$r8AYWAQL$SAA-NH2	20		1673.92	838.01	1674.93	837.97	558.98
12	Ac-LTF$r8AYWA$L$S$Nle-NH2	21		1767.04	884.77	1768.05	884.53	590.02
13	Ac-LTF$r8AYWA$L$S$A-NH2	22		1724.99	864.23	1726	863.5	576
14	Ac-F$r8AYWEAc3cL$AANle-NH2	23		1498.82	750.46	1499.83	750.42	500.61
15	Ac-F$r8AYWEAc3cL$AAL-NH2	24		1498.82	750.46	1499.83	750.42	500.61
16	Ac-F$r8AYWEAc3cL$AAM-NH2	25		1516.78	759.41	1517.79	759.4	506.6
17	Ac-F$r8AYWEAc3cL$AAhL-NH2	26		1512.84	757.49	1513.85	757.43	505.29
18	Ac-F$r8AYWEAc3cL$AAF-NH2	27		1532.81	767.48	1533.82	767.41	511.94
19	Ac-F$r8AYWEAc3cL$AAI-NH2	28		1498.82	750.39	1499.83	750.42	500.61
20	Ac-F$r8AYWEAc3cL$AAChg-NH2	29		1524.84	763.48	1525.85	763.43	509.29
21	Ac-F$r8AYWEAc3cL$AACha-NH2	30		1538.85	770.44	1539.86	770.43	513.96
22	Ac-F$r8AYWEAc3cL$AAAib-NH2	31		1470.79	736.84	1471.8	736.4	491.27
23	Ac-LTF$r8AYWAQL$AAAibV-NH2	32		1771.01	885.81	1772.02	886.51	591.34
24	Ac-LTF$r8AYWAQL$AAAibV-NH2	33	iso2	1771.01	886.26	1772.02	886.51	591.34
25	Ac-LTF$r8AYWAQL$SAibAA-NH2	34		1758.97	879.89	1759.98	880.49	587.33
26	Ac-LTF$r8AYWAQL$SAibAA-NH2	35	iso2	1758.97	880.34	1759.98	880.49	587.33
27	Ac-HLTF$r8HHWHQL$AANleNle-NH2	36		2056.15	1028.86	2057.16	1029.08	686.39
28	Ac-DLTF$r8HHWHQL$RRLV-NH2	37		2190.23	731.15	2191.24	1096.12	731.08
29	Ac-HHTF$r8HHWHQL$AAML-NH2	38		2098.08	700.43	2099.09	1050.05	700.37
30	Ac-F$r8HHWHQL$RRDCha-NH2	39		1917.06	959.96	1918.07	959.54	640.03
31	Ac-F$r8HHWHQL$HRFV-NH2	40		1876.02	938.65	1877.03	939.02	626.35
32	Ac-HLTF$r8HHWHQL$AAhLA-NH2	41		2028.12	677.2	2029.13	1015.07	677.05
33	Ac-DLTF$r8HHWHQL$RRChgl-NH2	42		2230.26	1115.89	2231.27	1116.14	744.43
34	Ac-DLTF$r8HHWHQL$RRChgl-NH2	43	iso2	2230.26	1115.96	2231.27	1116.14	744.43
35	Ac-HHTF$r8HHWHQL$AAChav-NH2	44		2106.14	1053.95	2107.15	1054.08	703.05
36	Ac-F$r8HHWHQL$RRDa-NH2	45		1834.99	918.3	1836	918.5	612.67
37	Ac-F$r8HHWHQL$HRAibG-NH2	46		1771.95	886.77	1772.96	886.98	591.66
38	Ac-F$r8AYWAQL$HHNleL-NH2	47		1730.97	866.57	1731.98	866.49	578
39	Ac-F$r8AYWSAL$HQANle-NH2	48		1638.89	820.54	1639.9	820.45	547.3
40	Ac-F$r8AYWVQL$QHChgl-NH2	49		1776.01	889.44	1777.02	889.01	593.01
41	Ac-F$r8AYWTAL$QQNlev-NH2	50		1671.94	836.97	1672.95	836.98	558.32
42	Ac-F$r8AYWYQL$HAibAa-NH2	51		1686.89	844.52	1687.9	844.45	563.3
43	Ac-LTF$r8AYWAQL$HHLa-NH2	52		1903.05	952.27	1904.06	952.53	635.36
44	Ac-LTF$r8AYWAQL$HHLa-NH2	53	iso2	1903.05	952.27	1904.06	952.53	635.36
45	Ac-LTF$r8AYWAQL$HQNlev-NH2	54		1922.08	962.48	1923.09	962.05	641.7
46	Ac-LTF$r8AYwAQL$HQNlev-NH2	55	iso2	1922.08	962.4	1923.09	962.05	641.7
47	Ac-LTF$r8AYWAQL$QQM1-NH2	56		1945.05	973.95	1946.06	973.53	649.36
48	Ac-LTF$r8AYWAQL$QQM1-NH2	57	iso2	1945.05	973.88	1946.06	973.53	649.36
49	Ac-LTF$r8AYWAQL$HAibhLV-NH2	58		1893.09	948.31	1894.1	947.55	632.04
50	Ac-LTF$r8AYWAQL$AHFA-NH2	59		1871.01	937.4	1872.02	936.51	624.68
51	Ac-HLTF$r8HHWHQL$AANlel-NH2	60		2056.15	1028.79	2057.16	1029.08	686.39
52	Ac-DLTF$r8HHWHQL$RRLa-NH2	61		2162.2	721.82	2163.21	1082.11	721.74
53	Ac-HHTF$r8HHWHQL$AAMv-NH2	62		2084.07	1042.92	2085.08	1043.04	695.7
54	Ac-F$r8HHWHQL$RRDA-NH2	63		1834.99	612.74	1836	918.5	612.67
55	Ac-F$r8HHWHQL$HRFCha-NH2	64		1930.06	966.47	1931.07	966.04	644.36
56	Ac-F$r8AYWEAL$AA-NHAm	65		1443.82	1445.71	1444.83	722.92	482.28
57	Ac-F$r8AYWEAL$AA-NHiAm	66		1443.82	723.13	1444.83	722.92	482.28
58	Ac-F$r8AYWEAL$AA-NHnPr3Ph	67		1491.82	747.3	1492.83	746.92	498.28
59	Ac-F$r8AYWEAL$AA-NHnBu33Me	68		1457.83	1458.94	1458.84	729.92	486.95
60	Ac-F$r8AYWEAL$AA-NHnPr	69		1415.79	709.28	1416.8	708.9	472.94
61	Ac-F$r8AYWEAL$AA-NHnEt2Ch	70		1483.85	1485.77	1484.86	742.93	495.62
62	Ac-F$r8AYWEAL$AA-NHnEt2Cp	71		1469.83	1470.78	1470.84	735.92	490.95
63	Ac-F$r8AYWEAL$AA-NHHex	72		1457.83	730.19	1458.84	729.92	486.95
64	Ac-LTF$r8AYWAQL$AAIA-NH2	73		1771.01	885.81	1772.02	886.51	591.34
65	Ac-LTF$r8AYWAQL$AAIA-NH2	74	iso2	1771.01	866.8	1772.02	886.51	591.34
66	Ac-LTF$r8AYWAAL$AAMA-NH2	75		1731.94	867.08	1732.95	866.98	578.32
67	Ac-LTF$r8AYWAAL$AAMA-NH2	76	iso2	1731.94	867.28	1732.95	866.98	578.32
68	Ac-LTF$r8AYwAQL$AANleA-NH2	77		1771.01	867.1	1772.02	886.51	591.34
69	Ac-LTF$r8AYWAQL$AANleA-NH2	78	iso2	1771.01	886.89	1772.02	886.51	591.34
70	Ac-LTF$r8AYWAQL$AAIa-NH2	79		1771.01	886.8	1772.02	886.51	591.34
71	Ac-LTF$r8AYWAQL$AAIa-NH2	80	iso2	1771.01	887.09	1772.02	886.51	591.34
72	Ac-LTF$r8AYWAAL$AAMa-NH2	81		1731.94	867.17	1732.95	866.98	578.32
73	Ac-LTF$r8AYWAAL$AAMa-NH2	82	iso2	1731.94	867.37	1732.95	866.98	578.32
74	Ac-LTF$r8AYWAQL$AANlea-NH2	83		1771.01	887.08	1772.02	886.51	591.34
75	Ac-LTF$r8AYWAQL$AANlea-NH2	84	iso2	1771.01	887.08	1772.02	886.51	591.34
76	Ac-LTF$r8AYWAAL$AAIv-NH2	85		1742.02	872.37	1743.03	872.02	581.68
77	Ac-LTF$r8AYWAAL$AAIv-NH2	86	iso2	1742.02	872.74	1743.03	872.02	581.68
78	Ac-LTF$r8AYWAQL$AAMv-NH2	87		1817	910.02	1818.01	909.51	606.67
79	Ac-LTF$r8AYWAAL$AANlev-NH2	88		1742.02	872.37	1743.03	872.02	581.68
80	Ac-LTF$r8AYWAAL$AANlev-NH2	89	iso2	1742.02	872.28	1743.03	872.02	581.68
81	Ac-LTF$r8AYWAQL$AAI1-NH2	90		1813.05	907.81	1814.06	907.53	605.36
82	Ac-LTF$r8AYWAQL$AAIl-NH2	91	iso2	1813.05	907.81	1814.06	907.53	605.36
83	Ac-LTF$r8AYWAAL$AAMl-NH2	92		1773.99	887.37	1775	888	592.34
84	Ac-LTF$r8AYWAQL$AANlel-NH2	93		1813.05	907.61	1814.06	907.53	605.36
85	Ac-LTF$r8AYWAQL$AANlel-NH2	94	iso2	1813.05	907.71	1814.06	907.53	605.36
86	Ac-F$r8AYWEAL$AAMA-NH2	95		1575.82	789.02	1576.83	788.92	526.28
87	Ac-F$r8AYWEAL$AANleA-NH2	96		1557.86	780.14	1558.87	779.94	520.29
88	Ac-F$r8AYWEAL$AAIa-NH2	97		1557.86	780.33	1558.87	779.94	520.29
89	Ac-F$r8AYWEAL$AAMa-NH2	98		1575.82	789.3	1576.83	788.92	526.28
90	Ac-F$r8AYWEAL$AANlea-NH2	99		1557.86	779.4	1558.87	779.94	520.29
91	Ac-F$r8AYWEAL$AAIv-NH2	100		1585.89	794.29	1586.9	793.95	529.64
92	Ac-F$r8AYwEAL$AAmv-NH2	101		1603.85	803.08	1604.86	802.93	535.62
93	Ac-F$r8AYWEAL$AANlev-NH2	102		1585.89	793.46	1586.9	793.95	529.64
94	Ac-F$r8AYWEAL$AAIl-NH2	103		1599.91	800.49	1600.92	800.96	534.31
95	Ac-F$r8AYWEAL$AAMl-NH2	104		1617.86	809.44	1618.87	809.94	540.29
96	Ac-F$r8AYWEAL$AANlel-NH2	105		1599.91	801.7	1600.92	800.96	534.31
97	Ac-F$r8AYWEAL$AANlel-NH2	106	iso2	1599.91	801.42	1600.92	800.96	534.31
98	Ac-LTF$r8AY6clWAQLSAA-NH2	107		1707.88	855.72	1708.89	854.95	570.3
99	Ac-LTF$r8AY6clWAQLSAA-NH2	108	iso2	1707.88	855.35	1708.89	854.95	570.3
100	Ac-WTF$r8FYWSQL$AVAa-NH2	109		1922.01	962.21	1923.02	962.01	641.68
101	Ac-WTF$r8FYWSQL$AVAa-NH2	110	iso2	1922.01	962.49	1923.02	962.01	641.68
102	Ac-WTF$r8VYWSQL$AVA-NH2	111		1802.98	902.72	1803.99	902.5	602
103	Ac-WTF$r8VYWSQL$AVA-NH2	112	iso2	1802.98	903	1803.99	902.5	602
104	Ac-WTF$r8FYWSQL$SAAa-NH2	113		1909.98	956.47	1910.99	956	637.67
105	Ac-WTF$r8FYwSQL$SAAa-NH2	114	iso2	1909.98	956.47	1910.99	956	637.67
106	Ac-WTF$r8VYWSQL$AVAaa-NH2	115		1945.05	974.15	1946.06	973.53	649.36
107	Ac-WTF$r8VYWSQL$AVAaa-NH2	116	iso2	1945.05	973.78	1946.06	973.53	649.36
108	Ac-LTF$r8AYWAQL$AVG-NH2	117		1671.94	837.52	1672.95	836.98	558.32
109	Ac-LTF$r8AYWAQL$AVG-NH2	118	iso2	1671.94	837.21	1672.95	836.98	558.32
110	Ac-LTF$r8AYWAQL$AVQ-NH2	119		1742.98	872.74	1743.99	872.5	582
111	Ac-LTF$r8AYWAQL$AVQ-NH2	120	iso2	1742.98	872.74	1743.99	872.5	582
112	Ac-LTF$r8AYWAQL$SAa-NH2	121		1673.92	838.23	1674.93	837.97	558.98
113	Ac-LTF$r8AYWAQL$SAa-NH2	122	iso2	1673.92	838.32	1674.93	837.97	558.98
114	Ac-LTF$r8AYWAQhL$SAA-NH2	123		1687.93	844.37	1688.94	844.97	563.65
115	Ac-LTF$r8AYWAQhL$SAA-NH2	124	iso2	1687.93	844.81	1688.94	844.97	563.65
116	Ac-LTF$r8AYWEQLStSA$-NH2	125		1826	905.27	1827.01	914.01	609.67
117	Ac-LTF$r8AYWAQL$SLA-NH2	126		1715.97	858.48	1716.98	858.99	573
118	Ac-LTF$r8AYWAQL$SLA-NH2	127	iso2	1715.97	858.87	1716.98	858.99	573
119	Ac-LTF$r8AYWAQL$SWA-NH2	128		1788.96	895.21	1789.97	895.49	597.33
120	Ac-LTF$r8AYWAQL$SWA-NH2	129	iso2	1788.96	895.28	1789.97	895.49	597.33
121	Ac-LTF$r8AYWAQL$SVS-NH2	130		1717.94	859.84	1718.95	859.98	573.65
122	Ac-LTF$r8AYWAQL$SAS-NH2	131		1689.91	845.85	1690.92	845.96	564.31
123	Ac-LTF$r8AYWAQL$SVG-NH2	132		1687.93	844.81	1688.94	844.97	563.65
124	Ac-ETF$r8VYWAQL$SAa-NH2	133		1717.91	859.76	1718.92	859.96	573.64
125	Ac-ETF$r8VYWAQL$SAA-NH2	134		1717.91	859.84	1718.92	859.96	573.64
126	Ac-ETF$r8VYWAQL$SVA-NH2	135		1745.94	873.82	1746.95	873.98	582.99
127	Ac-ETF$r8VYWAQL$SLA-NH2	136		1759.96	880.85	1760.97	880.99	587.66
128	Ac-ETF$r8VYWAQL$SWA-NH2	137		1832.95	917.34	1833.96	917.48	611.99
129	Ac-ETF$r8KYWAQL$SWA-NH2	138		1861.98	931.92	1862.99	932	621.67
130	Ac-ETF$r8VYWAQL$SVS-NH2	139		1761.93	881.89	1762.94	881.97	588.32
131	Ac-ETF$r8VYWAQL$SAS-NH2	140		1733.9	867.83	1734.91	867.96	578.97
132	Ac-ETF$r8VYWAQL$SVG-NH2	141		1731.92	866.87	1732.93	866.97	578.31
133	Ac-LTF$r8VYWAQL$SSa-NH2	142		1717.94	859.47	1718.95	859.98	573.65
134	Ac-ETF$r8VYWAQL$SSa-NH2	143		1733.9	867.83	1734.91	867.96	578.97
135	Ac-LTF$r8VYWAQL$SNa-NH2	144		1744.96	873.38	1745.97	873.49	582.66
136	Ac-ETF$r8VYWAQL$SNa-NH2	145		1760.91	881.3	1761.92	881.46	587.98
137	Ac-LTF$r8VYWAQL$SAa-NH2	146		1701.95	851.84	1702.96	851.98	568.32
138	Ac-LTF$r8VYWAQL$SVA-NH2	147		1729.98	865.53	1730.99	866	577.67
139	Ac-LTF$r8VYWAQL$SVA-NH2	148	iso2	1729.98	865.9	1730.99	866	577.67
140	Ac-LTF$r8VYWAQL$SWA-NH2	149		1816.99	909.42	1818	909.5	606.67
141	Ac-LTF$r8VYWAQL$SVS-NH2	150		1745.98	873.9	1746.99	874	583
142	Ac-LTF$r8VYWAQL$SVS-NH2	151	iso2	1745.98	873.9	1746.99	874	583
143	Ac-LTF$r8VYWAQL$SAS-NH2	152		1717.94	859.84	1718.95	859.98	573.65
144	Ac-LTF$r8VYWAQL$SAS-NH2	153	iso2	1717.94	859.91	1718.95	859.98	573.65
145	Ac-LTF$r8VYWAQL$SVG-NH2	154		1715.97	858.87	1716.98	858.99	573
146	Ac-LTF$r8VYWAQL$SVG-NH2	155	iso2	1715.97	858.87	1716.98	858.99	573
147	Ac-LTF$r8EYWAQCha$SAA-NH2	156		1771.96	886.85	1772.97	886.99	591.66
148	Ac-LTF$r8EYWAQCha$SAA-NH2	157	iso2	1771.96	886.85	1772.97	886.99	591.66
149	Ac-LTF$r8EYWAQCpg$SAA-NH2	158		1743.92	872.86	1744.93	872.97	582.31
150	Ac-LTF$r8EYWAQCpg$SAA-NH2	159	iso2	1743.92	872.86	1744.93	872.97	582.31
151	Ac-LTF$r8EYWAQF$SAA-NH2	160		1765.91	883.44	1766.92	883.96	589.64
152	Ac-LTF$r8EYWAQF$SAA-NH2	161	iso2	1765.91	883.89	1766.92	883.96	589.64
153	Ac-LTF$r8EYWAQCba$SAA-NH2	162		1743.92	872.42	1744.93	872.97	582.31
154	Ac-LTF$r8EYWAQCba$SAA-NH2	163	iso2	1743.92	873.39	1744.93	872.97	582.31
155	Ac-LTF3Cl$r8EYWAQL$SAA-NH2	164		1765.89	883.89	1766.9	883.95	589.64
156	Ac-LTF3Cl$r8EYWAQL$SAA-NH2	165	iso2	1765.89	883.96	1766.9	883.95	589.64
157	Ac-LTF34F2$r8EYWAQL$SAA-NH2	166		1767.91	884.48	1768.92	884.96	590.31
158	Ac-LTF34F2$r8EYWAQL$SAA-NH2	167	iso2	1767.91	884.48	1768.92	884.96	590.31
159	Ac-LTF34F2$r8EYWAQhL$SAA-NH2	168		1781.92	891.44	1782.93	891.97	594.98
160	Ac-LTF34F2$r8EYWAQhL$SAA-NH2	169	iso2	1781.92	891.88	1782.93	891.97	594.98
161	Ac-ETF$r8EYWAQL$SAA-NH2	170		1747.88	874.34	1748.89	874.95	583.63
162	Ac-LTF$r8AYWVQL$SAA-NH2	171		1701.95	851.4	1702.96	851.98	568.32
163	Ac-LTF$r8AHWAQL$SAA-NH2	172		1647.91	824.83	1648.92	824.96	550.31
164	Ac-LTF$r8AEWAQL$SAA-NH2	173		1639.9	820.39	1640.91	820.96	547.64
165	Ac-LTF$r8ASWAQL$SAA-NH2	174		1597.89	799.38	1598.9	799.95	533.64
166	Ac-LTF$r8AEWAQL$SAA-NH2	175	iso2	1639.9	820.39	1640.91	820.96	547.64
167	Ac-LTF$r8ASWAQL$SAA-NH2	176	iso2	1597.89	800.31	1598.9	799.95	533.64
168	Ac-LTF$r8AF4coohWAQL$SAA-NH2	177		1701.91	851.4	1702.92	851.96	568.31
169	Ac-LTF$r8AF4coohWAQL$SAA-NH2	178	iso2	1701.91	851.4	1702.92	851.96	568.31
170	Ac-LTF$r8AHWAQL$AAIa-NH2	179		1745	874.13	1746.01	873.51	582.67
171	Ac-ITF$r8FYWAQL$AAIa-NH2	180		1847.04	923.92	1848.05	924.53	616.69
172	Ac-ITF$r8EHWAQL$AAIa-NH2	181		1803.01	903.17	1804.02	902.51	602.01
173	Ac-ITF$r8EHWAQL$AAIa-NH2	182	iso2	1803.01	903.17	1804.02	902.51	602.01
174	Ac-ETF$r8EHWAQL$AAIa-NH2	183		1818.97	910.76	1819.98	910.49	607.33
175	Ac-ETF$r8EHWAQL$AAIa-NH2	184	iso2	1818.97	910.85	1819.98	910.49	607.33
176	Ac-LTF$r8AHWVQL$AAIa-NH2	185		1773.03	888.09	1774.04	887.52	592.02
177	Ac-ITF$r8FYWVQL$AAIa-NH2	186		1875.07	939.16	1876.08	938.54	626.03
178	Ac-ITF$r8EYWVQL$AAIa-NH2	187		1857.04	929.83	1858.05	929.53	620.02
179	Ac-ITF$r8EHWVQL$AAIa-NH2	188		1831.04	916.86	1832.05	916.53	611.35
180	Ac-LTF$r8AEWAQL$AAIa-NH2	189		1736.99	869.87	1738	869.5	580
181	Ac-LTF$r8AF4coohWAQL$AAIa-NH2	190		1799	900.17	1800.01	900.51	600.67
182	Ac-LTF$r8AF4coohWAQL$AAIa-NH2	191	iso2	1799	900.24	1800.01	900.51	600.67
183	Ac-LTF$r8AHWAQL$AHFA-NH2	192		1845.01	923.89	1846.02	923.51	616.01
184	Ac-ITF$r8FYWAQL$AHFA-NH2	193		1947.05	975.05	1948.06	974.53	650.02
185	Ac-ITF$r8FYWAQL$AHFA-NH2	194	iso2	1947.05	976.07	1948.06	974.53	650.02
186	Ac-ITF$r8FHWAQL$AEFA-NH2	195		1913.02	958.12	1914.03	957.52	638.68
187	Ac-ITF$r8FHWAQL$AEFA-NH2	196	iso2	1913.02	957.86	1914.03	957.52	638.68
188	Ac-ITF$r8EHWAQL$AHFA-NH2	197		1903.01	952.94	1904.02	952.51	635.34
189	Ac-ITF$r8EHWAQL$AHFA-NH2	198	iso2	1903.01	953.87	1904.02	952.51	635.34
190	Ac-LTF$r8AHWVQL$AHFA-NH2	199		1873.04	937.86	1874.05	937.53	625.35
191	Ac-ITF$r8FYWVQL$AHFA-NH2	200		1975.08	988.83	1976.09	988.55	659.37
192	Ac-ITF$r8EYWVQL$AHFA-NH2	201		1957.05	979.35	1958.06	979.53	653.36
193	Ac-ITF$r8EHWVQL$AHFA-NH2	202		1931.05	967	1932.06	966.53	644.69
194	Ac-ITF$r8EHWVQL$AHFA-NH2	203	iso2	1931.05	967.93	1932.06	966.53	644.69
195	Ac-ETF$r8EYWAAL$SAA-NH2	204		1690.86	845.85	1691.87	846.44	564.63
196	Ac-LTF$r8AYWVAL$SAA-NH2	205		1644.93	824.08	1645.94	823.47	549.32
197	Ac-LTF$r8AHWAAL$SAA-NH2	206		1590.89	796.88	1591.9	796.45	531.3
198	Ac-LTF$r8AEWAAL$SAA-NH2	207		1582.88	791.9	1583.89	792.45	528.63
199	Ac-LTF$r8AEWAAL$SAA-NH2	208	iso2	1582.88	791.9	1583.89	792.45	528.63
200	Ac-LTF$r8ASWAAL$SAA-NH2	209		1540.87	770.74	1541.88	771.44	514.63
201	Ac-LTF$r8ASWAAL$SAA-NH2	210	iso2	1540.87	770.88	1541.88	771.44	514.63
202	Ac-LTF$r8AYwAAL$AAIa-NH2	211		1713.99	857.39	1715	858	572.34
203	Ac-LTF$r8AYWAAL$AAIa-NH2	212	iso2	1713.99	857.84	1715	858	572.34
204	Ac-LTF$r8AYWAAL$AHFA-NH2	213		1813.99	907.86	1815	908	605.67
205	Ac-LTF$r8EHWAQL$AHIa-NH2	214		1869.03	936.1	1870.04	935.52	624.02
206	Ac-LTF$r8EHWAQL$AHIa-NH2	215	iso2	1869.03	937.03	1870.04	935.52	624.02
207	Ac-LTF$r8AHWAQL$AHIa-NH2	216		1811.03	906.87	1812.04	906.52	604.68
208	Ac-LTF$r8EYWAQL$AHIa-NH2	217		1895.04	949.15	1896.05	948.53	632.69
209	Ac-LTF$r8AYWAQL$AAFa-NH2	218		1804.99	903.2	1806	903.5	602.67
210	Ac-LTF$r8AYWAQL$AAFa-NH2	219	iso2	1804.99	903.28	1806	903.5	602.67
211	Ac-LTF$r8AYWAQL$AAWa-NH2	220		1844	922.81	1845.01	923.01	615.67
212	Ac-LTF$r8AYWAQL$AAVa-NH2	221		1756.99	878.86	1758	879.5	586.67
213	Ac-LTF$r8AYWAQL$AAVa-NH2	222	iso2	1756.99	879.3	1758	879.5	586.67
214	Ac-LTF$r8AYWAQL$AALa-NH2	223		1771.01	886.26	1772.02	886.51	591.34
215	Ac-LTF$r8AYWAQL$AALa-NH2	224	iso2	1771.01	886.33	1772.02	886.51	591.34
216	Ac-LTF$r8EYWAQL$AAIa-NH2	225		1829.01	914.89	1830.02	915.51	610.68
217	Ac-LTF$r8EYWAQL$AAIa-NH2	226	iso2	1829.01	915.34	1830.02	915.51	610.68
218	Ac-LTF$r8EYWAQL$AAFa-NH2	227		1863	932.87	1864.01	932.51	622.01
219	Ac-LTF$r8EYWAQL$AAFa-NH2	228	iso2	1863	932.87	1864.01	932.51	622.01
220	Ac-LTF$r8EYWAQL$AAVa-NH2	229		1815	908.23	1816.01	908.51	606.01
221	Ac-LTF$r8EYWAQL$AAVa-NH2	230	iso2	1815	908.31	1816.01	908.51	606.01
222	Ac-LTF$r8EHWAQL$AAIa-NH2	231		1803.01	903.17	1804.02	902.51	602.01
223	Ac-LTF$r8EHWAQL$AAIa-NH2	232	iso2	1803.01	902.8	1804.02	902.51	602.01
224	Ac-LTF$r8EHWAQL$AAWa-NH2	233		1876	939.34	1877.01	939.01	626.34
225	Ac-LTF$r8EHWAQL$AAWa-NH2	234	iso2	1876	939.62	1877.01	939.01	626.34
226	Ac-LTF$r8EHWAQL$AALa-NH2	235		1803.01	902.8	1804.02	902.51	602.01
227	Ac-LTF$r8EHWAQL$AALa-NH2	236	iso2	1803.01	902.9	1804.02	902.51	602.01
228	Ac-ETF$r8EHWVQL$AALa-NH2	237		1847	924.82	1848.01	924.51	616.67
229	Ac-LTF$r8AYWAQL$AAAa-NH2	238		1728.96	865.89	1729.97	865.49	577.33
230	Ac-LTF$r8AYWAQL$AAAa-NH2	239	iso2	1728.96	865.89	1729.97	865.49	577.33
231	Ac-LTF$r8AYWAQL$AAAibA-NH2	240		1742.98	872.83	1743.99	872.5	582
232	Ac-LTF$r8AYWAQL$AAAibA-NH2	241	iso2	1742.98	872.92	1743.99	872.5	582
233	Ac-LTF$r8AYWAQL$AAAAa-NH2	242		1800	901.42	1801.01	901.01	601.01
234	Ac-LTF$r5AYWAQL$s8AAIa-NH2	243		1771.01	887.17	1772.02	886.51	591.34
235	Ac-LTF$r5AYWAQL$s8SAA-NH2	244		1673.92	838.33	1674.93	837.97	558.98
236	Ac-LTF$r8AYWAQCba$AANleA-NH2	245		1783.01	892.64	1784.02	892.51	595.34
237	Ac-ETF$r8AYWAQCba$AANleA-NH2	246		1798.97	900.59	1799.98	900.49	600.66
238	Ac-LTF$r8EYWAQCba$AANleA-NH2	247		1841.01	922.05	1842.02	921.51	614.68
239	Ac-LTF$r8AYWAQCba$AWNleA-NH2	248		1898.05	950.46	1899.06	950.03	633.69
240	Ac-ETF$r8AYWAQCba$AWNleA-NH2	249		1914.01	958.11	1915.02	958.01	639.01
241	Ac-LTF$r8EYWAQCba$AWNleA-NH2	250		1956.06	950.62	1957.07	979.04	653.03
242	Ac-LTF$r8EYWAQCba$SAFA-NH2	251		1890.99	946.55	1892	946.5	631.34
243	Ac-LTF34F2$r8EYWAQCba$SANleA-	252		1892.99	947.57	1894	947.5	632
	NH2
244	Ac-LTF$r8EF4coohWAQCba$SANleA-	253		1885	943.59	1886.01	943.51	629.34
	NH2
245	Ac-LTF$r8EYWSQCba$SANleA-NH2	254		1873	937.58	1874.01	937.51	625.34
246	Ac-LTF$r8EYWWQCba$SANleA-NH2	255		1972.05	987.61	1973.06	987.03	658.36
247	Ac-LTF$r8EYWAQCba$AAIa-NH2	256		1841.01	922.05	1842.02	921.51	614.68
248	Ac-LTF34F2$r8EYWAQCba$AAIa-NH2	257		1876.99	939.99	1878	939.5	626.67
249	Ac-LTF$r8EF4coohWAQCba$AAIa-	258		1869.01	935.64	1870.02	935.51	624.01
	NH2
250	Pam-ETF$r8EYWAQCba$SAA-NH2	259		1956.1	979.57	1957.11	979.06	653.04
251	Ac-LThF$r8EFWAQCba$SAA-NH2	260		1741.94	872.11	1742.95	871.98	581.65
252	Ac-LTA$r8EYWAQCba$SAA-NH2	261		1667.89	835.4	1668.9	834.95	556.97
253	Ac-LTF$r8EYAAQCba$SAA-NH2	262		1628.88	815.61	1629.89	815.45	543.97
254	Ac-LTF$r8EY2NalAQCba$SAA-NH2	263		1754.93	879.04	1755.94	878.47	585.98
255	Ac-LTF$r8AYWAQCba$SAA-NH2	264		1685.92	844.71	1686.93	843.97	562.98
256	Ac-LTF$r8EYWAQCba$SAF-NH2	265		1819.96	911.41	1820.97	910.99	607.66
257	Ac-LTF$r8EYWAQCba$SAFa-NH2	266		1890.99	947.41	1892	946.5	631.34
258	Ac-LTF$r8AYWAQCba$SAF-NH2	267		1761.95	882.73	1762.96	881.98	588.32
259	Ac-LTF34F2$r8AYWAQCba$SAF-NH2	268		1797.93	900.87	1798.94	899.97	600.32
260	Ac-LTF$r8AF4coohWAQCba$SAF-NH2	269		1789.94	896.43	1790.95	895.98	597.65
261	Ac-LTF$r8EY6clWAQCba$SAF-NH2	270		1853.92	929.27	1854.93	927.97	618.98
262	Ac-LTF$r8AYWSQCba$SAF-NH2	271		1777.94	890.87	1778.95	889.98	593.65
263	Ac-LTF$r8AYWWQCba$SAF-NH2	272		1876.99	939.91	1878	939.5	626.67
264	Ac-LTF$r8AYWAQCba$AAIa-NH2	273		1783.01	893.19	1784.02	892.51	595.34
265	Ac-LTF34F2$r8AYWAQCba$AAIa-NH2	274		1818.99	911.23	1820	910.5	607.34
266	Ac-LTF$r8AY6clWAQCba$AAIa-NH2	275		1816.97	909.84	1817.98	909.49	606.66
267	Ac-LTF$r8AF4coohWAQCba$AAIa-	276		1811	906.88	1812.01	906.51	604.67
	NH2
268	Ac-LTF$r8EYWAQCba$AAFa-NH2	277		1875	938.6	1876.01	938.51	626.01
269	Ac-LTF$r8EYWAQCba$AAFa-NH2	278	iso2	1875	938.6	1876.01	938.51	626.01
270	Ac-ETF$r8AYWAQCba$AWNlea-NH2	279		1914.01	958.42	1915.02	958.01	639.01
271	Ac-LTF$r8EYWAQCba$AWNlea-NH2	280		1956.06	979.42	1957.07	979.04	653.03
272	Ac-ETF$r8EYWAQCba$AWNlea-NH2	281		1972.01	987.06	1973.02	987.01	658.34
273	Ac-ETF$r8EYWAQCba$AWNlea-NH2	282	iso2	1972.01	987.06	1973.02	987.01	658.34
274	Ac-LTF$r8AYWAQCba$SAFa-NH2	283		1832.99	917.89	1834	917.5	612
275	Ac-LTF$r8AYWAQCba$SAFa-NH2	284	iso2	1832.99	918.07	1834	917.5	612
276	Ac-ETF$r8AYWAQL$AWNlea-NH2	285		1902.01	952.22	1903.02	952.01	635.01
277	Ac-LTF$r8EYWAQL$AWNlea-NH2	286		1944.06	973.5	1945.07	973.04	649.03
278	Ac-ETF$r8EYWAQL$AWNlea-NH2	287		1960.01	981.46	1961.02	981.01	654.34
279	Dmaac-LTF$r8EYWAQhL$SAA-NH2	288		1788.98	896.06	1789.99	895.5	597.33
280	Hexac-LTF$r8EYWAQhL$SAA-NH2	289		1802	902.9	1803.01	902.01	601.67
281	Napac-LTF$r8EYWAQhL$SAA-NH2	290		1871.99	937.58	1873	937	625
282	Decac-LTF$r8EYWAQhL$SAA-NH2	291		1858.06	930.55	1859.07	930.04	620.36
283	Admac-LTF$r8EYWAQhL$SAA-NH2	292		1866.03	934.07	1867.04	934.02	623.02
284	Tmac-LTF$r8EYWAQhL$SAA-NH2	293		1787.99	895.41	1789	895	597
285	Pam-LTF$r8EYWAQhL$SAA-NH2	294		1942.16	972.08	1943.17	972.09	648.39
286	Ac-LTF$r8AYWAQCba$AANleA-NH2	295	iso2	1783.01	892.64	1784.02	892.51	595.34
287	Ac-LTF34F2$r8EYWAQCba$AAIa-NH2	296	iso2	1876.99	939.62	1878	939.5	626.67
288	Ac-LTF34F2$r8EYWAQCba$SAA-NH2	297		1779.91	892.07	1780.92	890.96	594.31
289	Ac-LTF34F2$r8EYWAQCba$SAA-NH2	298	iso2	1779.91	891.61	1780.92	890.96	594.31
290	Ac-LTF$r8EF4coohWAQCba$SAA-NH2	299		1771.92	887.54	1772.93	886.97	591.65
291	Ac-LTF$r8EF4coohWAQCba$SAA-NH2	300	iso2	1771.92	887.63	1772.93	886.97	591.65
292	Ac-LTF$r8EYWSQCba$SAA-NH2	301		1759.92	881.9	1760.93	880.97	587.65
293	Ac-LTF$r8EYWSQCba$SAA-NH2	302	iso2	1759.92	881.9	1760.93	880.97	587.65
294	Ac-LTF$r8EYWAQhL$SAA-NH2	303		1745.94	875.05	1746.95	873.98	582.99
295	Ac-LTF$r8AYWAQhL$SAF-NH2	304		1763.97	884.02	1764.98	882.99	589
296	Ac-LTF$r8AYWAQhL$SAF-NH2	305	iso2	1763.97	883.56	1764.98	882.99	589
297	Ac-LTF34F2$r8AYWAQhL$SAA-NH2	306		1723.92	863.67	1724.93	862.97	575.65
298	Ac-LTF34F2$r8AYWAQhL$SAA-NH2	307	iso2	1723.92	864.04	1724.93	862.97	575.65
299	Ac-LTF$r8AF4coohWAQhL$SAA-NH2	308		1715.93	859.44	1716.94	858.97	572.98
300	Ac-LTF$r8AF4coohWAQhL$SAA-NH2	309	iso2	1715.93	859.6	1716.94	858.97	572.98
301	Ac-LTF$r8AYWSQhL$SAA-NH2	310		1703.93	853.96	1704.94	852.97	568.98
302	Ac-LTF$r8AYWSQhL$SAA-NH2	311	iso2	1703.93	853.59	1704.94	852.97	568.98
303	Ac-LTF$r8EYWAQL$AANleA-NH2	312		1829.01	915.45	1830.02	915.51	610.68
304	Ac-LTF34F2$r8AYWAQL$AANleA-NH2	313		1806.99	904.58	1808	904.5	603.34
305	Ac-LTF$r8AF4coohWAQL$AANleA-	314		1799	901.6	1800.01	900.51	600.67
	NH2
306	Ac-LTF$r8AYWSQL$AANleA-NH2	315		1787	894.75	1788.01	894.51	596.67
307	Ac-LTF34F2$r8AYWAQhL$AANleA-	316		1821	911.79	1822.01	911.51	608.01
	NH2
308	Ac-LTF34F2$r8AYWAQhL$AANleA-	317	iso2	1821	912.61	1822.01	911.51	608.01
	NH2
309	Ac-LTF$r8AF4coohWAQhL$AANleA-	318		1813.02	907.95	1814.03	907.52	605.35
	NH2
310	Ac-LTF$r8AF4coohWAQhL$AANleA-	319	iso2	1813.02	908.54	1814.03	907.52	605.35
	NH2
311	Ac-LTF$r8AYWSQhL$AANleA-NH2	320		1801.02	901.84	1802.03	901.52	601.35
312	Ac-LTF$r8AYWSQhL$AANleA-NH2	321	iso2	1801.02	902.62	1802.03	901.52	601.35
313	Ac-LTF$r8AYWAQhL$AAAAa-NH2	322		1814.01	908.63	1815.02	908.01	605.68
314	Ac-LTF$r8AYWAQhL$AAAAa-NH2	323	iso2	1814.01	908.34	1815.02	908.01	605.68
315	Ac-LTF$r8AYWAQL$AAAAAa-NH2	324		1871.04	936.94	1872.05	936.53	624.69
316	Ac-LTF$r8AYWAQL$AAAAAAa-NH2	325	iso2	1942.07	972.5	1943.08	972.04	648.37
317	Ac-LTF$r8AYWAQL$AAAAAAa-NH2	326	iso1	1942.07	972.5	1943.08	972.04	648.37
318	Ac-LTF$r8EYWAQhL$AANleA-NH2	327		1843.03	922.54	1844.04	922.52	615.35
319	Ac-AATF$r8AYWAQL$AANleA-NH2	328		1800	901.39	1801.01	901.01	601.01
320	Ac-LTF$r8AYWAQL$AANleAA-NH2	329		1842.04	922.45	1843.05	922.03	615.02
321	Ac-ALTF$r8AYWAQL$AANleAA-NH2	330		1913.08	957.94	1914.09	957.55	638.7
322	Ac-LTF$r8AYWAQCba$AANleAA-NH2	331		1854.04	928.43	1855.05	928.03	619.02
323	Ac-LTF$r8AYWAQhL$AANleAA-NH2	332		1856.06	929.4	1857.07	929.04	619.69
324	Ac-LTF$r8EYWAQCba$SAAA-NH2	333		1814.96	909.37	1815.97	908.49	605.99
325	Ac-LTF$r8EYWAQCba$SAAA-NH2	334	iso2	1814.96	909.37	1815.97	908.49	605.99
326	Ac-LTF$r8EYWAQCba$SAAAA-NH2	335		1886	944.61	1887.01	944.01	629.67
327	Ac-LTF$r8EYWAQCba$SAAAA-NH2	336	iso2	1886	944.61	1887.01	944.01	629.67
328	Ac-ALTF$r8EYWAQCba$SAA-NH2	337		1814.96	909.09	1815.97	908.49	605.99
329	Ac-ALTF$r8EYWAQCba$SAAA-NH2	338		1886	944.61	1887.01	944.01	629.67
330	Ac-ALTF$r8EYWAQCba$SAA-NH2	339	iso2	1814.96	909.09	1815.97	908.49	605.99
331	Ac-LTF$r8EYWAQL$AAAAAa-NH2	340	iso2	1929.04	966.08	1930.05	965.53	644.02
332	Ac-LTF$r8EY6clWAQCba$SAA-NH2	341		1777.89	890.78	1778.9	889.95	593.64
333	Ac-	342		1918.96	961.27	1919.97	960.49	640.66
	LTF$r8EF4cooh6clWAQCba$SANleA-
	NH2
334	Ac-	343	iso2	1918.96	961.27	1919.97	960.49	640.66
	LTF$r8EF4cooh6clWAQCba$SANleA-
	NH2
335	Ac-	344		1902.97	953.03	1903.98	952.49	635.33
	LTF$r8EF4cooh6clWAQCba$AAIa-
	NH2
336	Ac-	345	iso2	1902.97	953.13	1903.98	952.49	635.33
	LTF$r8EF4cooh6clWAQCba$AAIa-
	NH2
337	Ac-LTF$r8AY6cLWAQL$AAAAAa-NH2	346		1905	954.61	1906.01	953.51	636.01
338	Ac-LTF$r8AY6clWAQL$AAAAAa-NH2	347	iso2	1905	954.9	1906.01	953.51	636.01
339	Ac-F$r8AY6clWEAL$AAAAAAa-NH2	348		1762.89	883.01	1763.9	882.45	588.64
340	Ac-ETF$r8EYWAQL$AAAAAa-NH2	349		1945	974.31	1946.01	973.51	649.34
341	Ac-ETF$r8EYWAQL$AAAAAa-NH2	350	iso2	1945	974.49	1946.01	973.51	649.34
342	Ac-LTF$r8EYWAQL$AAAAAAa-NH2	351		2000.08	1001.6	2001.09	1001.05	667.7
343	Ac-LTF$r8EYWAQL$AAAAAAa-NH2	352	iso2	2000.08	1001.6	2001.09	1001.05	667.7
344	Ac-LTF$r8AYWAQL$AANleAAa-NH2	353		1913.08	958.58	1914.09	957.55	638.7
345	Ac-LTF$r8AYWAQL$AANleAAa-NH2	354	iso2	1913.08	958.58	1914.09	957.55	638.7
346	Ac-LTF$r8EYWAQCba$AAAAAa-NH2	355		1941.04	972.55	1942.05	971.53	648.02
347	Ac-LTF$r8EYWAQCba$AAAAAa-NH2	356	iso2	1941.04	972.55	1942.05	971.53	648.02
348	Ac-LTF$r8EF4coohWAQCba$AAAAAa-	357		1969.04	986.33	1970.05	985.53	657.35
	NH2
349	Ac-LTF$r8EF4coohWAQCba$AAAAAa-	358	iso2	1969.04	986.06	1970.05	985.53	657.35
	NH2
350	Ac-LTF$r8EYWSQCba$AAAAAa-NH2	359		1957.04	980.04	1958.05	979.53	653.35
351	Ac-LTF$r8EYWSQCba$AAAAAa-NH2	360	iso2	1957.04	980.04	1958.05	979.53	653.35
352	Ac-LTF$r8EYWAQCba$SAAa-NH2	361		1814.96	909	1815.97	908.49	605.99
353	Ac-LTF$r8EYWAQCba$SAAa-NH2	362	iso2	1814.96	909	1815.97	908.49	605.99
354	Ac-ALTF$r8EYWAQCba$SAAa-NH2	363		1886	944.52	1887.01	944.01	629.67
355	Ac-ALTF$r8EYWAQCba$SAAa-NH2	364	iso2	1886	944.98	1887.01	944.01	629.67
356	Ac-ALTF$r8EYWAQCba$SAAAa-NH2	365		1957.04	980.04	1958.05	979.53	653.35
357	Ac-ALTF$r8EYWAQCba$SAAAa-NH2	366	iso2	1957.04	980.04	1958.05	979.53	653.35
358	Ac-AALTF$r8EYWAQCba$SAAAa-NH2	367		2028.07	1016.1	2029.08	1015.04	677.03
359	Ac-AALTF$r8EYWAQCba$SAAAa-NH2	368	iso2	2028.07	1015.57	2029.08	1015.04	677.03
360	Ac-RTF$r8EYWAQCba$SAA-NH2	369		1786.94	895.03	1787.95	894.48	596.65
361	Ac-LRF$r8EYWAQCba$SAA-NH2	370		1798.98	901.51	1799.99	900.5	600.67
362	Ac-LTF$r8EYWRQCba$SAA-NH2	371		1828.99	916.4	1830	915.5	610.67
363	Ac-LTF$r8EYWARCba$SAA-NH2	372		1771.97	887.63	1772.98	886.99	591.66
364	Ac-LTF$r8EYWAQCba$RAA-NH2	373		1812.99	908.08	1814	907.5	605.34
365	Ac-LTF$r8EYWAQCba$SRA-NH2	374		1828.99	916.12	1830	915.5	610.67
366	Ac-LTF$r8EYWAQCba$SAR-NH2	375		1828.99	916.12	1830	915.5	610.67
367	5-FAM-BaLTF$r8EYWAQCba$SAA-NH2	376		2131	1067.09	2132.01	1066.51	711.34
368	5-FAM-BaLTF$r8AYWAQL$AANleA-	377		2158.08	1080.6	2159.09	1080.05	720.37
	NH2
369	Ac-LAF$r8EYWAQL$AANleA-NH2	378		1799	901.05	1800.01	900.51	600.67
370	Ac-ATF$r8EYWAQL$AANleA-NH2	379		1786.97	895.03	1787.98	894.49	596.66
371	Ac-AAF$r8EYWAQL$AANleA-NH2	380		1756.96	880.05	1757.97	879.49	586.66
372	Ac-AAAF$r8EYWAQL$AANleA-NH2	381		1827.99	915.57	1829	915	610.34
373	Ac-AAAAF$r8EYWAQL$AANleA-NH2	382		1899.03	951.09	1900.04	950.52	634.02
374	Ac-AATF$r8EYWAQL$AANleA-NH2	383		1858	930.92	1859.01	930.01	620.34
375	Ac-AALTF$r8EYWAQL$AANleA-NH2	384		1971.09	987.17	1972.1	986.55	658.04
376	Ac-AAALTF$r8EYWAQL$AANleA-NH2	385		2042.12	1023.15	2043.13	1022.07	681.71
377	Ac-LTF$r8EYWAQL$AANleAA-NH2	386		1900.05	952.02	1901.06	951.03	634.36
378	Ac-ALTF$r8EYWAQL$AANleAA-NH2	387		1971.09	987.63	1972.1	986.55	658.04
379	Ac-AALTF$r8EYWAQL$AANleAA-NH2	388		2042.12	1022.69	2043.13	1022.07	681.71
380	Ac-LTF$r8EYWAQCba$AANleAA-NH2	389		1912.05	958.03	1913.06	957.03	638.36
381	Ac-LTF$r8EYWAQhL$AANleAA-NH2	390		1914.07	958.68	1915.08	958.04	639.03
382	Ac-ALTF$r8EYWAQhL$AANleAA-NH2	391		1985.1	994.1	1986.11	993.56	662.71
383	Ac-LTF$r8ANmYWAQL$AANleA-NH2	392		1785.02	894.11	1786.03	893.52	596.01
384	Ac-LTF$r8ANmYWAQL$AANleA-NH2	393	iso2	1785.02	894.11	1786.03	893.52	596.01
385	Ac-LTF$r8AYNmWAQL$AANleA-NH2	394		1785.02	894.11	1786.03	893.52	596.01
386	Ac-LTF$r8AYNmWAQL$AANleA-NH2	395	iso2	1785.02	894.11	1786.03	893.52	596.01
387	Ac-LTF$r8AYAmwAQL$AANleA-NH2	396		1785.02	894.01	1786.03	893.52	596.01
388	Ac-LTF$r8AYAmwAQL$AANleA-NH2	397	iso2	1785.02	894.01	1786.03	893.52	596.01
389	Ac-LTF$r8AYWAibQL$AANleA-NH2	398		1785.02	894.01	1786.03	893.52	596.01
390	Ac-LTF$r8AYWAibQL$AANleA-NH2	399	iso2	1785.02	894.01	1786.03	893.52	596.01
391	Ac-LTF$r8AYWAQL$AAibNleA-NH2	400		1785.02	894.38	1786.03	893.52	596.01
392	Ac-LTF$r8AYWAQL$AAibNleA-NH2	401	iso2	1785.02	894.38	1786.03	893.52	596.01
393	Ac-LTF$r8AYWAQL$AaNleA-NH2	402		1771.01	887.54	1772.02	886.51	591.34
394	Ac-LTF$r8AYWAQL$AaNleA-NH2	403	iso2	1771.01	887.54	1772.02	886.51	591.34
395	Ac-LTF$r8AYWAQL$ASarNleA-NH2	404		1771.01	887.35	1772.02	886.51	591.34
396	Ac-LTF$r8AYWAQL$ASarNleA-NH2	405	iso2	1771.01	887.35	1772.02	886.51	591.34
397	Ac-LTF$r8AYWAQL$AANleAib-NH2	406		1785.02	894.75	1786.03	893.52	596.01
398	Ac-LTF$r8AYWAQL$AANleAib-NH2	407	iso2	1785.02	894.75	1786.03	893.52	596.01
399	Ac-LTF$r8AYWAQL$AANleNmA-NH2	408		1785.02	894.6	1786.03	893.52	596.01
400	Ac-LTF$r8AYWAQL$AANleNmA-NH2	409	iso2	1785.02	894.6	1786.03	893.52	596.01
401	Ac-LTF$r8AYWAQL$AANleSar-NH2	410		1771.01	886.98	1772.02	886.51	591.34
402	Ac-LTF$r8AYWAQL$AANleSar-NH2	411	iso2	1771.01	886.98	1772.02	886.51	591.34
403	Ac-LTF$r8AYWAQL$AANleAAib-NH2	412		1856.06		1857.07	929.04	619.69
404	Ac-LTF$r8AYWAQL$AANleAAib-NH2	413	iso2	1856.06		1857.07	929.04	619.69
405	Ac-LTF$r8AYWAQL$AANleANmA-NH2	414		1856.06	930.37	1857.07	929.04	619.69
406	Ac-LTF$r8AYWAQL$AANleANmA-NH2	415	iso2	1856.06	930.37	1857.07	929.04	619.69
407	Ac-LTF$r8AYWAQL$AANleAa-NH2	416		1842.04	922.69	1843.05	922.03	615.02
408	Ac-LTF$r8AYWAQL$AANleAa-NH2	417	iso2	1842.04	922.69	1843.05	922.03	615.02
409	Ac-LTF$r8AYWAQL$AANleASar-NH2	418		1842.04	922.6	1843.05	922.03	615.02
410	Ac-LTF$r8AYWAQL$AANleASar-NH2	419	iso2	1842.04	922.6	1843.05	922.03	615.02
411	Ac-LTF$/r8AYWAQL$/AANleA-NH2	420		1799.04	901.14	1800.05	900.53	600.69
412	Ac-LTFAibAYWAQLAibAANleA-NH2	421		1648.9	826.02	1649.91	825.46	550.64
413	Ac-LTF$r8Cou4YWAQL$AANleA-NH2	422		1975.05	989.11	1976.06	988.53	659.36
414	Ac-LTF$r8Cou4YWAQL$AANleA-NH2	423	iso2	1975.05	989.11	1976.06	988.53	659.36
415	Ac-LTF$r8AYWCou4QL$AANleA-NH2	424		1975.05	989.11	1976.06	988.53	659.36
416	Ac-LTF$r8AYWAQL$Cou4ANleA-NH2	425		1975.05	989.57	1976.06	988.53	659.36
417	Ac-LTF$r8AYWAQL$Cou4ANleA-NH2	426	iso2	1975.05	989.57	1976.06	988.53	659.36
418	Ac-LTF$r8AYWAQL$ACou4NleA-NH2	427		1975.05	989.57	1976.06	988.53	659.36
419	Ac-LTF$r8AYWAQL$ACou4NleA-NH2	428	iso2	1975.05	989.57	1976.06	988.53	659.36
420	Ac-LTF$r8AYWAQL$AANleA-OH	429		1771.99	887.63	1773	887	591.67
421	Ac-LTF$r8AYWAQL$AANleA-OH	430	iso2	1771.99	887.63	1773	887	591.67
422	Ac-LTF$r8AYWAQL$AANleA-NHnPr	431		1813.05	908.08	1814.06	907.53	605.36
423	Ac-LTF$r8AYWAQL$AANleA-NHnPr	432	iso2	1813.05	908.08	1814.06	907.53	605.36
424	Ac-LTF$r8AYWAQL$AANleA-	433		1855.1	929.17	1856.11	928.56	619.37
	NHnBu33me
425	Ac-LTF$r8AYWAQL$AANleA-	434	iso2	1855.1	929.17	1856.11	928.56	619.37
	NHnBu33Me
426	Ac-LTF$r8AYWAQL$AANleA-NHHex	435		1855.1	929.17	1856.11	928.56	619.37
427	Ac-LTF$r8AYWAQL$AANleA-NHHex	436	iso2	1855.1	929.17	1856.11	928.56	619.37
428	Ac-LTA$r8AYWAQL$AANleA-NH2	437		1694.98	849.33	1695.99	848.5	566
429	Ac-LThL$r8AYWAQL$AANleA-NH2	438		1751.04	877.09	1752.05	876.53	584.69
430	Ac-LTF$r8AYAAQL$AANleA-NH2	439		1655.97	829.54	1656.98	828.99	553
431	Ac-LTF$r8AY2NalAQL$AANleA-NH2	440		1782.01	892.63	1783.02	892.01	595.01
432	Ac-LTF$r8EYWCou4QCba$SAA-NH2	441		1947.97	975.8	1948.98	974.99	650.33
433	Ac-LTF$r8EYWCou7QCba$SAA-NH2	442		16.03	974.9	17.04	9.02	6.35
434	Ac-LTF%r8EYWAQCba%SAA-NH2	443		1745.94	874.8	1746.95	873.98	582.99
435	Dmaac-LTF$r8EYWAQCba$SAA-NH2	444		1786.97	894.8	1787.98	894.49	596.66
436	Dmaac-LTF$r8AYWAQL$AAAAAa-NH2	445		1914.08	958.2	1915.09	958.05	639.03
437	Dmaac-LTF$r8AYWAQL$AAAAAa-NH2	446	iso2	1914.08	958.2	1915.09	958.05	639.03
438	Dmaac-LTF$r8EYWAQL$AAAAAa-NH2	447		1972.08	987.3	1973.09	987.05	658.37
439	Dmaac-LTF$r8EYWAQL$AAAAAa-NH2	448	iso2	1972.08	987.3	1973.09	987.05	658.37
440	Dmaac-	449		1912.05	957.4	1913.06	957.03	638.36
	LTF$r8EF4coohWAQCba$AAIa-NH2
441	Dmaac-	450	iso2	1912.05	957.4	1913.06	957.03	638.36
	LTF$r8EF4coohWAQCba$AAIa-NH2
442	Dmaac-LTF$r8AYWAQL$AANleA-NH2	451		1814.05	908.3	1815.06	908.03	605.69
443	Dmaac-LTF$r8AYWAQL$AANleA-NH2	452	iso2	1814.05	908.3	1815.06	908.03	605.69
444	Ac-LTF%r8AYWAQL%AANleA-NH2	453		1773.02	888.37	1774.03	887.52	592.01
445	Ac-LTF%r8EYWAQL%AAAAAa-NH2	454		1931.06	966.4	1932.07	966.54	644.69
446	Cou6BaLTF$r8EYWAQhL$SAA-NH2	455		2018.05	1009.9	2019.06	1010.03	673.69
447	Cou8BaLTF$r8EYWAQhL$SAA-NH2	456		1962.96	982.34	1963.97	982.49	655.32
448	Ac-LTF4I$r8EYWAQL$AAAAAa-NH2	457		2054.93	1028.68	2055.94	1028.47	685.98
449	Ac-LTF$r8EYWAQL$AAAAAa-NH2	458		1929.04	966.17	1930.05	965.53	644.02
550	Ac-LTF$r8EYWAQL$AAAAAa-OH	459		1930.02	966.54	1931.03	966.02	644.35
551	Ac-LTF$r8EYWAQL$AAAAAa-OH	460	iso2	1930.02	965.89	1931.03	966.02	644.35
552	Ac-LTF$r8EYwAEL$AAAAAa-NH2	461		1930.02	966.82	1931.03	966.02	644.35
553	Ac-LTF$r8EYWAEL$AAAAAa-NH2	462	iso2	1930.02	966.91	1931.03	966.02	644.35
554	Ac-LTF$r8EYWAEL$AAAAAa-OH	463		1931.01	967.28	1932.02	966.51	644.68
555	Ac-LTF$r8EY6clWAQL$AAAAAa-NH2	464		1963	983.28	1964.01	982.51	655.34
556	Ac-LTF$r8EF4bOH2wAQL$AAAAAa-	465		1957.05	980.04	1958.06	979.53	653.36
	NH2
557	Ac-AAALTF$r8EYWAQL$AAAAAa-NH2	466		2142.15	1072.83	2143.16	1072.08	715.06
558	Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2	467		1965.02	984.3	1966.03	983.52	656.01
559	Ac-RTF$r8EYWAQL$AAAAAa-NH2	468		1972.06	987.81	1973.07	987.04	658.36
560	Ac-LTA$r8EYWAQL$AAAAAa-NH2	469		1853.01	928.33	1854.02	927.51	618.68
561	Ac-LTF$r8EYWAibQL$AAAAAa-NH2	470		1943.06	973.48	1944.07	972.54	648.69
562	Ac-LTF$r8EYWAQL$AAibAAAa-NH2	471		1943.06	973.11	1944.07	972.54	648.69
563	Ac-LTF$r8EYWAQL$AAAibAAa-NH2	472		1943.06	973.48	1944.07	972.54	648.69
564	Ac-LTF$r8EYWAQL$AAAAibAa-NH2	473		1943.06	973.48	1944.07	972.54	648.69
565	Ac-LTF$r8EYWAQL$AAAAAiba-NH2	474		1943.06	973.38	1944.07	972.54	648.69
566	Ac-LTF$r8EYWAQL$AAAAAiba-NH2	475	iso2	1943.06	973.38	1944.07	972.54	648.69
567	Ac-LTF$r8EYWAQL$AAAAAAib-NH2	476		1943.06	973.01	1944.07	972.54	648.69
568	Ac-LTF$r8EYWAQL$AaAAAa-NH2	477		1929.04	966.54	1930.05	965.53	644.02
569	Ac-LTF$r8EYWAQL$AAaAAa-NH2	478		1929.04	966.35	1930.05	965.53	644.02
570	Ac-LTF$r8EYWAQL$AAAaAa-NH2	479		1929.04	966.54	1930.05	965.53	644.02
571	Ac-LTF$r8EYWAQL$AAAaAa-NH2	480	iso2	1929.04	966.35	1930.05	965.53	644.02
572	Ac-LTF$r8EYWAQL$AAAAaa-NH2	481		1929.04	966.35	1930.05	965.53	644.02
573	Ac-LTF$r8EYWAQL$AAAAAA-NH2	482		1929.04	966.35	1930.05	965.53	644.02
574	Ac-LTF$r8EYWAQL$ASarAAAa-NH2	483		1929.04	966.54	1930.05	965.53	644.02
575	Ac-LTF$r8EYWAQL$AASarAAa-NH2	484		1929.04	966.35	1930.05	965.53	644.02
576	Ac-LTF$r8EYWAQL$AAASarAa-NH2	485		1929.04	966.35	1930.05	965.53	644.02
577	Ac-LTF$r8EYWAQL$AAAASara-NH2	486		1929.04	966.35	1930.05	965.53	644.02
578	Ac-LTF$r8EYWAQL$AAAAASar-NH2	487		1929.04	966.08	1930.05	965.53	644.02
579	Ac-7LTF$r8EYWAQL$AAAAAa-NH2	488		1918.07	951.99	1919.08	960.04	640.37
581	Ac-TF$r8EYWAQL$AAAAAa-NH2	489		1815.96	929.85	1816.97	908.99	606.33
582	Ac-F$r8EYWAQL$AAAAAa-NH2	490		1714.91	930.92	1715.92	858.46	572.64
583	Ac-LVF$r8EYWAQL$AAAAAa-NH2	491		1927.06	895.12	1928.07	964.54	643.36
584	Ac-AAF$r8EYWAQL$AAAAAa-NH2	492		1856.98	859.51	1857.99	929.5	620
585	Ac-LTF$r8EYWAQL$AAAAa-NH2	493		1858	824.08	1859.01	930.01	620.34
586	Ac-LTF$r8EYWAQL$AAAa-NH2	494		1786.97	788.56	1787.98	894.49	596.66
587	Ac-LTF$r8EYWAQL$AAa-NH2	495		1715.93	1138.57	1716.94	858.97	572.98
588	Ac-LTF$r8EYWAQL$Aa-NH2	496		1644.89	1144.98	1645.9	823.45	549.3
589	Ac-LTF$r8EYWAQL$a-NH2	497		1573.85	1113.71	1574.86	787.93	525.62
590	Ac-LTF$r8EYWAQL$AAA-OH	498		1716.91	859.55	1717.92	859.46	573.31
591	Ac-LTF$r8EYWAQL$A-OH	499		1574.84	975.14	1575.85	788.43	525.95
592	Ac-LTF$r8EYWAQL$AAA-NH2	500		1715.93	904.75	1716.94	858.97	572.98
593	Ac-LTF$r8EYWAQCba$SAA-OH	501		1744.91	802.49	1745.92	873.46	582.64
594	Ac-LTF$r8EYWAQCba$S-OH	502		1602.83	913.53	1603.84	802.42	535.28
595	Ac-LTF$r8EYWAQCba$S-NH2	503		1601.85	979.58	1602.86	801.93	534.96
596	4-FBzl-LTF$r8EYWAQL$AAAAAa-NH2	504		2009.05	970.52	2010.06	1005.53	670.69
597	4-FBzl-LTF$r8EYWAQCba$SAA-NH2	505		1823.93	965.8	1824.94	912.97	608.98
598	Ac-LTF$r8RYWAQL$AAAAAa-NH2	506		1956.1	988.28	1957.11	979.06	653.04
599	Ac-LTF$r8HYWAQL$AAAAAa-NH2	507		1937.06	1003.54	1938.07	969.54	646.69
600	Ac-LTF$r8QYWAQL$AAAAAa-NH2	508		1928.06	993.92	1929.07	965.04	643.69
601	Ac-LTF$r8CitYWAQL$AAAAAa-NH2	509		1957.08	987	1958.09	979.55	653.37
602	Ac-LTF$r8GlaYWAQL$AAAAAa-NH2	510		1973.03	983	1974.04	987.52	658.68
603	Ac-LTF$r8F4gYWAQL$AAAAAa-NH2	511		2004.1	937.86	2005.11	1003.06	669.04
604	Ac-LTF$r82mRYWAQL$AAAAAa-NH2	512		1984.13	958.58	1985.14	993.07	662.38
605	Ac-LTF$r8ipKYWAQL$AAAAAa-NH2	513		1970.14	944.52	1971.15	986.08	657.72
606	Ac-LTF$r8F4NH2YWAQL$AAAAAa-NH2	514		1962.08	946	1963.09	982.05	655.03
607	Ac-LTF$r8EYWAAL$AAAAAa-NH2	515		1872.02	959.32	1873.03	937.02	625.01
608	Ac-LTF$r8EYWALL$AAAAAa-NH2	516		1914.07	980.88	1915.08	958.04	639.03
609	Ac-LTF$r8EYWAAibL$AAAAAa-NH2	517		1886.03	970.61	1887.04	944.02	629.68
610	Ac-LTF$r8EYWASL$AAAAAa-NH2	518		1888.01	980.51	1889.02	945.01	630.34
611	Ac-LTF$r8EYWANL$AAAAAa-NH2	519		1915.02	1006.41	1916.03	958.52	639.35
612	Ac-LTF$r8EYWACitL$AAAAAa-NH2	520		1958.07		1959.08	980.04	653.7
613	Ac-LTF$r8EYWAHL$AAAAAa-NH2	521		1938.04	966.24	1939.05	970.03	647.02
614	Ac-LTF$r8EYWARL$AAAAAa-NH2	522		1957.08		1958.09	979.55	653.37
615	Ac-LTF$r8EpYWAQL$AAAAAa-NH2	523		2009.01		2010.02	1005.51	670.68
616	Cbm-LTF$r8EYWAQCba$SAA-NH2	524		1590.85		1591.86	796.43	531.29
617	Cbm-LTF$r8EYWAQL$AAAAAa-NH2	525		1930.04		1931.05	966.03	644.35
618	Ac-LTF$r8EYWAQL$SAAAAa-NH2	526		1945.04	1005.11	1946.05	973.53	649.35
619	Ac-LTF$r8EYWAQL$AAAASa-NH2	527		1945.04	986.52	1946.05	973.53	649.35
620	Ac-LTF$r8EYWAQL$SAAASa-NH2	528		1961.03	993.27	1962.04	981.52	654.68
621	Ac-LTF$r8EYWAQTba$AAAAAa-NH2	529		1943.06	983.1	1944.07	972.54	648.69
622	Ac-LTF$r8EYWAQAdm$AAAAAa-NH2	530		2007.09	990.31	2008.1	1004.55	670.04
623	Ac-LTF$r8EYWAQCha$AAAAAa-NH2	531		1969.07	987.17	1970.08	985.54	657.36
624	Ac-LTF$r8EYWAQhCha$AAAAAa-NH2	532		1983.09	1026.11	1984.1	992.55	662.04
625	Ac-LTF$r8EYWAQF$AAAAAa-NH2	533		1963.02	957.01	1964.03	982.52	655.35
626	Ac-LTF$r8EYWAQhF$AAAAAa-NH2	534		1977.04	1087.81	1978.05	989.53	660.02
627	Ac-LTF$r8EYWAQL$AANleAAa-NH2	535		1971.09	933.45	1972.1	986.55	658.04
628	Ac-LTF$r8EYWAQAdm$AANleAAa-NH2	536		2049.13	1017.97	2050.14	1025.57	684.05
629	4-FBz-BaLTF$r8EYWAQL$AAAAAa-	537		2080.08		2081.09	1041.05	694.37
	NH2
630	4-FBz-BaLTF$r8EYWAQCba$SAA-NH2	538		1894.97		1895.98	948.49	632.66
631	Ac-LTF$r5EYWAQL$s8AAAAAa-NH2	539		1929.04	1072.68	1930.05	965.53	644.02
632	Ac-LTF$r5EYWAQCba$s8SAA-NH2	540		1743.92	1107.79	1744.93	872.97	582.31
633	Ac-LTF$r8EYWAQL$AAhhLAAa-NH2	541		1999.12		2000.13	1000.57	667.38
634	Ac-LTF$r8EYWAQL$AAAAAAAa-NH2	542		2071.11		2072.12	1036.56	691.38
635	Ac-LTF$r8EYWAQL$AAAAAAAAa-NH2	543		2142.15	778.1	2143.16	1072.08	715.06
636	Ac-LTF$r8EYWAQL$AAAAAAAAAa-NH2	544		2213.19	870.53	2214.2	1107.6	738.74
637	Ac-LTA$r8EYAAQCba$SAA-NH2	545		1552.85		1553.86	777.43	518.62
638	Ac-LTA$r8EYAAQL$AAAAAa-NH2	546		1737.97	779.45	1738.98	869.99	580.33
639	Ac-LTF$r8EPmpWAQL$AAAAAa-NH2	547		2007.03	779.54	2008.04	1004.52	670.02
640	Ac-LTF$r8EPmpWAQCba$SAA-NH2	548		1821.91	838.04	1822.92	911.96	608.31
641	Ac-ATF$r8HYWAQL$S-NH2	549		1555.82	867.83	1556.83	778.92	519.61
642	Ac-LTF$r8HAWAQL$S-NH2	550		1505.84	877.91	1506.85	753.93	502.95
643	Ac-LTF$r8HYWAQA$S-NH2	551		1555.82	852.52	1556.83	778.92	519.61
644	Ac-LTF$r8EYWAQCba$SA-NH2	552		1672.89	887.18	1673.9	837.45	558.64
645	Ac-LTF$r8EYWAQL$SAA-NH2	553		1731.92	873.32	1732.93	866.97	578.31
646	Ac-LTF$r8HYWAQCba$SAA-NH2	554		1751.94	873.05	1752.95	876.98	584.99
647	Ac-LTF$r8SYWAQCba$SAA-NH2	555		1701.91	844.88	1702.92	851.96	568.31
648	Ac-LTF$r8RYWAQCba$SAA-NH2	556		1770.98	865.58	1771.99	886.5	591.33
649	Ac-LTF$r8KYWAQCba$SAA-NH2	557		1742.98	936.57	1743.99	872.5	582
650	Ac-LTF$r8QYWAQCba$SAA-NH2	558		1742.94	930.93	1743.95	872.48	581.99
651	Ac-LTF$r8EYWAACba$SAA-NH2	559		1686.9	1032.45	1687.91	844.46	563.31
652	Ac-LTF$r8EYWAQCba$AAA-NH2	560		1727.93	895.46	1728.94	864.97	576.98
653	Ac-LTF$r8EYWAQL$AAAAA-OH	561		1858.99	824.54	1860	930.5	620.67
654	Ac-LTF$r8EYWAQL$AAAA-OH	562		1787.95	894.48	1788.96	894.98	596.99
655	Ac-LTF$r8EYWAQL$AA-OH	563		1645.88	856	1646.89	823.95	549.63
656	Ac-LTF$r8AF4bOH2WAQL$AAAAAa-	564
	NH2
657	Ac-LTF$r8AF4bOH2WAAL$AAAAAa-	565
	NH2
658	Ac-LTF$r8EF4bOH2WAQCba$SAA-NH2	566
659	Ac-LTF$r8ApYWAQL$AAAAAa-NH2	567
660	Ac-LTF$r8ApYWAAL$AAAAAa-NH2	568
661	Ac-LTF$r8EpYWAQCba$SAA-NH2	569
662	Ac-LTF$rda6AYWAQL$da5AAAAAa-	570		1974.06	934.44
	NH2
663	Ac-LTF$rda6EYWAQCba$da5SAA-NH2	571		1846.95	870.52		869.94
664	Ac-LTF$rda6EYWAQL$da5AAAAAa-	572
	NH2
665	Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2	573		936.57		935.51
666	Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2	574
667	Ac-LTF$ra9EYWAQCba$a6SAA-NH2	575
668	Ac-LTA$ra9EYWAQCba$a6SAA-NH2	576
669	5-FAM-BaLTF$ra9EYWAQCba$a6SAA-	577
	NH2
670	5-FAM-BaLTF$r8EYWAQL$AAAAAa-	578		2316.11
	NH2
671	5-FAM-BaLTF$/r8EYWAQL$/AAAAAa-	579		2344.15
	NH2
672	5-FAM-BaLTA$r8EYWAQL$AAAAAa-	580		2240.08
	NH2
673	5-FAM-BaLTF$r8AYWAQL$AAAAAa-	581		2258.11
	NH2
674	5-FAM-BaATF$r8EYWAQL$AAAAAa-	582		2274.07
	NH2
675	5-FAM-BaLAF$r8EYWAQL$AAAAAa-	583		2286.1
	NH2
676	5-FAM-BaLTF$r8EAWAQL$AAAAAa-	584		2224.09
	NH2
677	5-FAM-BaLTF$r8EYAAQL$AAAAAa-	585		2201.07
	NH2
678	5-FAM-BaLTA$r8EYAAQL$AAAAAa-	586		2125.04
	NH2
679	5-FAM-BaLTF$r8EYWAAL$AAAAAa-	587		2259.09
	NH2
680	5-FAM-BaLTF$r8EYWAQA$AAAAAa-	588		2274.07
	NH2
681	5-FAM-BaLTF$/r8EYWAQCba$/SAA-	589		2159.03
	NH2
682	5-FAM-BaLTA$r8EYWAQCba$SAA-NH2	590		2054.97
683	5-FAM-BaLTF$r8EYAAQCba$SAA-NH2	591		2015.96
684	5-FAM-BaLTA$r8EYAAQCba$SAA-NH2	592		1939.92
685	5-FAM-BaQSQQTF$r8NLWRLL$QN-NH2	593		2495.23
686	5-TAMRA-BaLTF$r8EYWAQCba$SAA-	594		2186.1
	NH2
687	5-TAMRA-BaLTA$r8EYWAQCba$SAA-	595		2110.07
	NH2
688	5-TAMRA-BaLTF$r8EYAAQCba$SAA-	596		2071.06
	NH2
689	5-TAMRA-BaLTA$r8EYAAQCba$SAA-	597		1995.03
	NH2
690	5-TAMRA-	598		2214.13
	BaLTF$/r8EYWAQCba$/SAA-NH2
691	5-TAMRA-BaLTF$r8EYWAQL$AAAAAa-	599		2371.22
	NH2
692	5-TAMRA-BaLTA$r8EYWAQL$AAAAAa-	600		2295.19
	NH2
693	5-TAMRA-	601		2399.25
	BaLTF$/r8EYWAQL$/AAAAAa-NH2
694	Ac-LTF$r8EYWCou7QCba$SAA-OH	602		1947.93
695	Ac-LTF$r8EYWCou7QCba$S-OH	603		1805.86
696	Ac-LTA$r8EYWCou7QCba$SAA-NH2	604		1870.91
697	Ac-LTF$r8EYACou7QCba$SAA-NH2	605		1831.9
698	Ac-LTA$r8EYACou7QCba$SAA-NH2	606		1755.87
699	Ac-LTF$/r8EYWCou7QCba$/SAA-NH2	607		1974.98
700	Ac-LTF$r8EYWCou7QL$AAAAAa-NH2	608		2132.06
701	Ac-LTF$/r8EYWCou7QL$/AAAAAa-	609		2160.09
	NH2
702	Ac-LTF$r8EYWCou7QL$AAAAA-OH	610		2062.01
703	Ac-LTF$r8EYwCou7QL$AAAA-OH	611		1990.97
704	Ac-LTF$r8EYwCou7QL$AAA-OH	612		1919.94
705	Ac-LTF$r8EYWCou7QL$AA-OH	613		1848.9
706	Ac-LTF$r8EYWCou7QL$A-OH	614		1777.86
707	Ac-LTF$r8EYWAQL$AAAASa-NH2	615	iso2		974.4		973.53
708	Ac-LTF$r8AYWAAL$AAAAAa-NH2	616	iso2	1814.01	908.82	1815.02	908.01	605.68
709	Biotin-BaLTF$r8EYWAQL$AAAAAa-	617		2184.14	1093.64	2185.15	1093.08	729.05
	NH2
710	Ac-LTF$r8HAWAQL$S-NH2	618	iso2	1505.84	754.43	1506.85	753.93	502.95
711	Ac-LTF$r8EYWAQCba$SA-NH2	619	iso2	1672.89	838.05	1673.9	837.45	558.64
712	Ac-LTF$r8HYWAQCba$SAA-NH2	620	iso2	1751.94	877.55	1752.95	876.98	584.99
713	Ac-LTF$r8SYWAQCba$SAA-NH2	621	iso2	1701.91	852.48	1702.92	851.96	568.31
714	Ac-LTF$r8RYWAQCba$SAA-NH2	622	iso2	1770.98	887.45	1771.99	886.5	591.33
715	Ac-LTF$r8KYWAQCba$SAA-NH2	623	iso2	1742.98	872.92	1743.99	872.5	582
716	Ac-LTF$r8EYWAQCba$AAA-NH2	624	iso2	1727.93	865.71	1728.94	864.97	576.98
717	Ac-LTF$r8EYWAQL$AAAAAaBaC-NH2	625		2103.09	1053.12	2104.1	1052.55	702.04
718	Ac-LTF$r8EYWAQL$AAAAAadPeg4C-	626		2279.19	1141.46	2280.2	1140.6	760.74
	NH2
719	Ac-LTA$r8AYWAAL$AAAAAa-NH2	627		1737.98	870.43	1738.99	870	580.33
720	Ac-LTF$r8AYAAAL$AAAAAa-NH2	628		1698.97	851	1699.98	850.49	567.33
721	5-FAM-BaLTF$r8AYWAAL$AAAAAa-	629		2201.09	1101.87	2202.1	1101.55	734.7
	NH2
722	Ac-LTA$r8AYWAQL$AAAAAa-NH2	630		1795	898.92	1796.01	898.51	599.34
723	Ac-LTF$r8AYAAQL$AAAAAa-NH2	631		1755.99	879.49	1757	879	586.34
724	Ac-LTF$rda6AYWAAL$da5AAAAAa-	632		1807.97		1808.98	904.99	603.66
	NH2
725	FITC-BaLTF$r8EYWAQL$AAAAAa-NH2	633		2347.1	1174.49	2348.11	1174.56	783.37
726	FITC-BaLTF$r8EYWAQCba$SAA-NH2	634		2161.99	1082.35	2163	1082	721.67
733	Ac-LTF$r8EYWAQL$EAAAAa-NH2	635		1987.05	995.03	1988.06	994.53	663.36
734	Ac-LTF$r8AYWAQL$EAAAAa-NH2	636		1929.04	966.35	1930.05	965.53	644.02
735	Ac-LTF$r8EYWAQL$AAAAAaBaKbio-	637		2354.25	1178.47	2355.26	1178.13	785.76
	NH2
736	Ac-LTF$r8AYWAAL$AAAAAa-NH2	638		1814.01	908.45	1815.02	908.01	605.68
737	Ac-LTF$r8AYAAAL$AAAAAa-NH2	639	iso2	1698.97	850.91	1699.98	850.49	567.33
738	Ac-LTF$r8AYAAQL$AAAAAa-NH2	640	iso2	1755.99	879.4	1757	879	586.34
739	Ac-LTF$r8EYWAQL$EAAAAa-NH2	641	iso2	1987.05	995.21	1988.06	994.53	663.36
740	Ac-LTF$r8AYWAQL$EAAAAa-NH2	642	iso2	1929.04	966.08	1930.05	965.53	644.02
741	Ac-LTF$r8EYWAQCba$SAAAAa-NH2	643		1957.04	980.04	1958.05	979.53	653.35
742	Ac-LTF$r8EYWAQLStAAA$r5AA-NH2	644		2023.12	1012.83	2024.13	1012.57	675.38
743	Ac-LTF$r8EYWAQL$A$AAA$A-NH2	645		2108.17	1055.44	2109.18	1055.09	703.73
744	Ac-LTF$r8EYWAQL$AA$AAA$A-NH2	646		2179.21	1090.77	2180.22	1090.61	727.41
745	Ac-LTF$r8EYWAQL$AAA$AAA$A-NH2	647		2250.25	1126.69	2251.26	1126.13	751.09
746	Ac-AAALTF$r8EYWAQL$AAA-OH	648		1930.02		1931.03	966.02	644.35
747	Ac-AAALTF$r8EYWAQL$AAA-NH2	649		1929.04	965.85	1930.05	965.53	644.02
748	Ac-AAAALTF$r8EYWAQL$AAA-NH2	650		2000.08	1001.4	2001.09	1001.05	667.7
749	Ac-AAAAALTF$r8EYwAQL$AAA-NH2	651		2071.11	1037.13	2072.12	1036.56	691.38
750	Ac-AAAAAALTF$r8EYwAQL$AAA-NH2	652		2142.15		2143.16	1072.08	715.06
751	Ac-LTF$rda6EYWAQCba$da6SAA-NH2	653	iso2	1751.89	877.36	1752.9	876.95	584.97
752	Ac-t$r5wya$r5f4CF3ekllr-NH2	654			844.25
753	Ac-tawy$r5nf4CF3e$r5llr-NH2	655			837.03
754	Ac-tawya$r5f4CF3ek$r5lr-NH2	656			822.97
755	Ac-tawyanf4CF3e$r5llr$r5a-NH2	657			908.35
756	Ac-t$s8wyanf4CF3e$r5llr-NH2	658			858.03
757	Ac-tawy$s8nf4CF3ekll$r5a-NH2	659			879.86
758	Ac-tawya$s8f4CF3ekllr$r5a-NH2	660			936.38
759	Ac-tawy$s8naekll$r5a-NH2	661			844.25
760	5-FAM-Batawy$s8nf4CF3ekll$r5a-	662
	NH2
761	5-FAM-Batawy$s8naekll$r5a-NH2	663
762	Ac-tawy$s8nf4CF3eall$r5a-NH2	664
763	Ac-tawy$s8nf4CF3ekll$r5aaaaa-	665
	NH2
764	Ac-tawy$s8nf4CF3eall$r5aaaaa-	666
	NH2

TABLE 1a shows a selection of peptidomimetic macrocycles.

TABLE 1a
TABLE 1a shows a selection of peptidomimetic macrocycles.
		SEQ				Calc	Calc	Calc
		ID		Exact	Found	(M +	(M +	(M +
SP	Sequence	NO:	Isomer	Mass	Mass	1)/1	2)/2	3)/3
244	Ac-LTF$r8EF4coohWAQCba$SANleA-	667		1885	943.59	1886.01	943.51	629.34
	NH2
331	Ac-LTF$r8EYWAQL$AAAAAa-NH2	668	iso2	1929.04	966.08	1930.05	965.53	644.02
555	Ac-LTF$r8EY6clWAQL$AAAAAa-NH2	669		1963	983.28	1964.01	982.51	655.34
557	Ac-AAALTF$r8EYWAQL$AAAAAa-NH2	670		2142.15	1072.83	2143.16	1072.08	715.06
558	Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2	671		1965.02	984.3	1966.03	983.52	656.01
562	Ac-LTF$r8EYWAQL$AAibAAAa-NH2	672		1943.06	973.11	1944.07	972.54	648.69
564	Ac-LTF$r8EYWAQL$AAAAibAa-NH2	673		1943.06	973.48	1944.07	972.54	648.69
566	Ac-LTF$r8EYWAQL$AAAAAiba-NH2	674	iso2	1943.06	973.38	1944.07	972.54	648.69
567	Ac-LTF$r8EYWAQL$AAAAAAib-NH2	675		1943.06	973.01	1944.07	972.54	648.69
572	Ac-LTF$r8EYWAQL$AAAAaa-NH2	676		1929.04	966.35	1930.05	965.53	644.02
573	Ac-LTF$r8EYWAQL$AAAAAA-NH2	677		1929.04	966.35	1930.05	965.53	644.02
578	Ac-LTF$r8EYWAQL$AAAAASar-NH2	678		1929.04	966.08	1930.05	965.53	644.02
551	Ac-LTF$r8EYWAQL$AAAAAa-OH	679	iso2	1930.02	965.89	1931.03	966.02	644.35
662	Ac-LTF$rda6AYWAQL$da5AAAAAa-	680		1974.06	934.44		933.49
	NH2
367	5-FAM-BaLTF$r8EYWAQCba$SAA-NH2	681		2131	1067.09	2132.01	1066.51	711.34
349	Ac-LTF$r8EF4coohWAQCba$AAAAAa-	682	iso2	1969.04	986.06	1970.05	985.53	657.35
	NH2
347	Ac-LTF$r8EYWAQCba$AAAAAa-NH2	683	iso2	1941.04	972.55	1942.05	971.53	648.02

TABLE 1b shows a further selection of peptidomimetic macrocycles.

TABLE 1b
TABLE 1b shows a further selection of peptidomimetic macrocycles.
		SEQ				Calc	Calc	Calc
		ID		Exact	Found	(M +	(M +	(M +
SP	Sequence	NO:	Isomer	Mass	Mass	1)/1	2)/2	3)/3
581	Ac-TF$r8EYWAQL$AAAAAa-NH2	684		1815.96	929.85	1816.97	908.99	606.33
582	Ac-F$r8EYWAQL$AAAAAa-NH2	685		1714.91	930.92	1715.92	858.46	572.64
583	Ac-LVF$r8EYWAQL$AAAAAa-	686		1927.06	895.12	1928.07	964.54	643.36
	NH2
584	Ac-AAF$r8EYWAQL$AAAAAa-	687		1856.98	859.51	1857.99	929.5	620
	NH2
585	Ac-LTF$r8EYWAQL$AAAAa-NH2	688		1858	824.08	1859.01	930.01	620.34
586	Ac-LTF$r8EYWAQL$AAAa-NH2	689		1786.97	788.56	1787.98	894.49	596.66
587	Ac-LTF$r8EYWAQL$AAa-NH2	690		1715.93	1138.57	1716.94	858.97	572.98
588	Ac-LTF$r8EYwAQL$Aa-NH2	691		1644.89	1144.98	1645.9	823.45	549.3
589	Ac-LTF$r8EYWAQL$a-NH2	692		1573.85	1113.71	1574.86	787.93	525.62

In the sequences shown above and elsewhere, the following abbreviations are used: “Nle” represents norleucine, “Aib” represents 2-aminoisobutyric acid, “Ac” represents acetyl, and “Pr” represents propionyl. Amino acids represented as “$” are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an all-carbon crosslinker comprising one double bond. Amino acids represented as “$r5” are alpha-Me R5-pentenyl-alanine olefin amino acids connected by an all-carbon comprising one double bond. Amino acids represented as “$s8” are alpha-Me S8-octenyl-alanine olefin amino acids connected by an all-carbon crosslinker comprising one double bond. Amino acids represented as “$r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon crosslinker comprising one double bond. “Ahx” represents an aminocyclohexyl linker.

The crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid. Amino acids represented as “$/” are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/r5” are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/s8” are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as “$/r8” are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker.

Amino acids represented as “Amw” are alpha-Me tryptophan amino acids. Amino acids represented as “Aml” are alpha-Me leucine amino acids. Amino acids represented as “Amf” are alpha-Me phenylalanine amino acids. Amino acids represented as “2ff” are 2-fluoro-phenylalanine amino acids. Amino acids represented as “3ff” are 3-fluoro-phenylalanine amino acids. Amino acids represented as “St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated. Amino acids represented as “St//” are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked. Amino acids represented as “% St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks. Amino acids represented as “Ba” are beta-alanine. The lower-case character “e” or “z” within the designation of a crosslinked amino acid (e.g. “$er8” or “$zr8”) represents the configuration of the double bond (E or Z, respectively). In other contexts, lower-case letters such as “a” or “f” represent D amino acids (e.g. D-alanine, or D-phenylalanine, respectively).

Amino acids designated as “NmW” represent N-methyltryptophan. Amino acids designated as “NmY” represent N-methyltyrosine. Amino acids designated as “NmA” represent N-methylalanine. “Kbio” represents a biotin group attached to the side chain amino group of a lysine residue. Amino acids designated as “Sar” represent sarcosine. Amino acids designated as “Cha” represent cyclohexyl alanine. Amino acids designated as “Cpg” represent cyclopentyl glycine. Amino acids designated as “Chg” represent cyclohexyl glycine. Amino acids designated as “Cba” represent cyclobutyl alanine. Amino acids designated as “F4I” represent 4-iodo phenylalanine. “7L” represents N15 isotopic leucine. Amino acids designated as “F3Cl” represent 3-chloro phenylalanine. Amino acids designated as “F4cooh” represent 4-carboxy phenylalanine. Amino acids designated as “F34F2” represent 3,4-difluoro phenylalanine. Amino acids designated as “6clW” represent 6-chloro tryptophan. Amino acids designated as “$rda6” represent alpha-Me R6-hexynyl-alanine alkynyl amino acids, crosslinked via a dialkyne bond to a second alkynyl amino acid.

Amino acids designated as “$da5” represent alpha-Me S5-pentynyl-alanine alkynyl amino acids, wherein the alkyne forms one half of a dialkyne bond with a second alkynyl amino acid. Amino acids designated as “$ra9” represent alpha-Me R9-nonynyl-alanine alkynyl amino acids, crosslinked via an alkyne metathesis reaction with a second alkynyl amino acid. Amino acids designated as “$a6” represent alpha-Me S6-hexynyl-alanine alkynyl amino acids, crosslinked via an alkyne metathesis reaction with a second alkynyl amino acid. The designation “iso1” or “iso2” indicates that the peptidomimetic macrocycle is a single isomer.

Amino acids designated as “Cit” represent citrulline. Amino acids designated as “Cou4”, “Cou6”, “Cou7” and “Cou8”, respectively, represent the following structures:

In some embodiments, a peptidomimetic macrocycle is obtained in more than one isomer, for example due to the configuration of a double bond within the structure of the crosslinker (E vs Z). Such isomers can or cannot be separable by conventional chromatographic methods. In some embodiments, one isomer has improved biological properties relative to the other isomer. In one embodiment, an E crosslinker olefin isomer of a peptidomimetic macrocycle has better solubility, better target affinity, better in vivo or in vitro efficacy, higher helicity, or improved cell permeability relative to its Z counterpart. In another embodiment, a Z crosslinker olefin isomer of a peptidomimetic macrocycle has better solubility, better target affinity, better in vivo or in vitro efficacy, higher helicity, or improved cell permeability relative to its E counterpart.

TABLE 1c shows exemplary peptidomimetic macrocycles.

TABLE 1c
	SEQ
	ID
SP#	NO:	Structure
154	163
115	124
114	123
99	108
388	397
331	340
445	454
351	360
71	80
69	78
7	16
160	169
315	324
249	258
437	446
349	358
555	464
557	466
558	467
367	376
562	471
564	473
566	475
567	476
572	481
573	482
578	487
664	572
662	572
	1500

In some embodiments, peptidomimetic macrocycles exclude peptidomimetic macrocycles shown in TABLE 2a:

TABLE 2a
Sequence             SEQ ID NO:
L$r5QETESD$s8WKLLPEN 693
LSQ$r5TESDLW$s8LLPEN 694
LSQE$r5FSDLWK$s8LPEN 695
LSQET$r5SDLWKL$s8PEN 696
LSQETF$r5DLWKLL$s8EN 697
LXQETES$r5LWKLLP$s8N 698
LSQETESD$r5WKLLPE$s8 699
LSQQTF$r5DLWKLL$s8EN 700
LSQETF$r5DLWKLL$s8QN 701
LSQQTF$r5DLWKLL$s8QN 702
LSQETF$r5NLWKLL$s8QN 703
LSQQTF$r5NLWKLL$s8QN 704
LSQQTF$r5NLWRLL$s8QN 705
QSQQTF$r5NLWKLL$s8QN 706
QSQQTF$r5NLWRLL$s8QN 707
QSQQTA$r5NLWRLL$s8QN 708
L$r8QETFSD$WKLLPEN   709
LSQ$r8TFSDLW$LLPEN   710
LSQE$r8FSDLWK$LPEN   711
LSQET$r8SDLWKL$PEN   712
LSQETF$r8DLWKLL$EN   713
LXQETFS$r8LWKLLP$N   714
LSQETFSD$r8WKLLPE$   715
LSQQTF$r8DLWKLL$EN   716
LSQETF$r8DLWKLL$QN   717
LSQQTF$r8DLWKLL$QN   718
LSQETF$r8NLWKLL$QN   719
LSQQTF$r8NLWKLL$QN   720
LSQQTF$r8NLWRLL$QN   721
QSQQTF$r8NLWKLL$QN   722
QSQQTF$r8NLWRLL$QN   723
QSQQTA$r8NLWRLL$QN   724
QSQQTF$r8NLWRKK$QN   725
QQTF$r8DLWRLL$EN     726
QQTF$r8DLWRLL$       727
LSQQTF$DLW$LL        728
QQTF$DLW$LL          729
QQTA$r8DLWRLL$EN     730
QSQQTF$r5NLWRLL$s8QN 731
(dihydroxylated olefin)
QSQQTA$r5NLWRLL$s8QN 732
(dihydroxylated olefin)
QSQQTF$r8DLWRLL$QN   733
QTF$r8NLWRLL$        734
QSQQTF$NLW$LLPQN     735
QS$QTF$NLWRLLPQN     736
$TFS$LWKLL           737
ETF$DLW$LL           738
QTF$NLW$LL           739
$SQE$FSNLWKLL        740

In TABLE 2a, the peptides can comprise an N-terminal capping group such as acetyl or an additional linker such as beta-alanine between the capping group and the start of the peptide sequence.

In some embodiments, peptidomimetic macrocycles do not comprise a peptidomimetic macrocycle structure as shown in TABLE 2a.

In some embodiments, peptidomimetic macrocycles exclude those shown in TABLE 2b:

TABLE 2b
		SEQ			Observed mass
SP	Sequence	ID NO:	Exact Mass	M + 2	(m/e)
1	Ac-LSQETF$r8DLWKLL$EN-NH2	741	2068.13	1035.07	1035.36
2	Ac-LSQETF$r8NLWKLL$QN-NH2	742	2066.16	1034.08	1034.31
3	Ac-LSQQTF$r8NLWRLL$QN-NH2	743	2093.18	1047.59	1047.73
4	Ac-QSQQTF$r8NLWKLL$QN-NH2	744	2080.15	1041.08	1041.31
5	Ac-QSQQTF$r8NLWRLL$QN-NH2	745	2108.15	1055.08	1055.32
6	Ac-QSQQTA$r8NLWRLL$QN-NH2	746	2032.12	1017.06	1017.24
7	Ac-QAibQQTF$r8NLWRLL$QN-NH2	747	2106.17	1054.09	1054.34
8	Ac-QSQQTFSNLWRLLPQN-NH2	748	2000.02	1001.01	1001.26
9	Ac-QSQQTF$/r8NLwRLLVQN-NH2	749	2136.18	1069.09	1069.37
10	Ac-QSQAibTF$r8NLWRLL$QN-NH2	750	2065.15	1033.58	1033.71
11	Ac-QSQQTF$r8NLWRLL$AN-NH2	751	2051.13	1026.57	1026.70
12	Ac-ASQQTF$r8NLWRLL$QN-NH2	752	2051.13	1026.57	1026.90
13	Ac-QSQQTF$r8ALWRLL$QN-NH2	753	2065.15	1033.58	1033.41
14	Ac-QSQETF$r8NLWRLL$QN-NH2	754	2109.14	1055.57	1055.70
15	Ac-RSQQTF$r8NLWRLL$QN-NH2	755	2136.20	1069.10	1069.17
16	Ac-RSQQTF$r8NLWRLL$EN-NH2	756	2137.18	1069.59	1069.75
17	Ac-LSQETFSDLWKLLPEN-NH2	757	1959.99	981.00	981.24
18	Ac-QSQ$TFS$LWRLLPQN-NH2	758	2008.09	1005.05	1004.97
19	Ac-QSQQ$FSN$WRLLPQN-NH2	759	2036.06	1019.03	1018.86
20	Ac-QSQQT$SNL$RLLPQN-NH2	760	1917.04	959.52	959.32
21	Ac-QSQQTF$NLW$LLPQN-NH2	761	2007.06	1004.53	1004.97
22	Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2	762	2310.26	1156.13	1156.52
23	Ac-QSQQTF$r8NLWRLL$RN-NH2	763	2136.20	1069.10	1068.94
24	Ac-QSQRTF$r8NLWRLL$QN-NH2	764	2136.20	1069.10	1068.94
25	Ac-QSQQTF$r8NNleWRLL$QN-NH2	765	2108.15	1055.08	1055.44
26	Ac-QSQQTF$r8NLWRNleL$QN-NH2	766	2108.15	1055.08	1055.84
27	Ac-QSQQTF$r8NLWRLNle$QN-NH2	767	2108.15	1055.08	1055.12
28	Ac-QSQQTY$r8NLWRLL$QN-NH2	768	2124.15	106108	1062.92
29	Ac-RAibQQTF$r8NLWRLL$QN-NH2	769	2134.22	1068.11	1068.65
30	Ac-MPRFMDYWEGLN-NH2	770	1598.70	800.35	800.45
31	Ac-RSQQRF$r8NLwRLL$QN-NH2	771	2191.25	1096.63	1096.83
32	Ac-QSQQRF$r8NLWRLL$QN-NH2	772	2163.21	1082.61	1082.87
33	Ac-RAibQQRF$r8NLWRLL$QN-NH2	773	2189.27	1095.64	1096.37
34	Ac-RSQQRF$r8NFwRLL$QN-NH2	774	2225.23	1113.62	1114.37
35	Ac-RSQQRF$r8NYWRLL$QN-NH2	775	2241.23	1121.62	1122.37
36	Ac-RSQQTF$r8NLWQLL$QN-NH2	776	2108.15	1055.08	1055.29
37	Ac-QSQQTF$r8NLWQAmlL$QN-NH2	777	2094.13	1048.07	1048.32
38	Ac-QSQQTF$r8NAmlWRLL$QN-NH2	778	2122.17	1062.09	1062.35
39	Ac-NlePRF$r8DYWEGL$QN-NH2	779	1869.98	935.99	936.20
40	Ac-NlePRF$r8NYWRLL$QN-NH2	780	1952.12	977.06	977.35
41	Ac-RF$r8NLWRLL$Q-NH2	781	1577.96	789.98	790.18
42	Ac-QSQQTF$r8N2ffWRLL$QN-NH2	782	2160.13	1081.07	1081.40
43	Ac-QSQQTF$r8N3ffWRLL$QN-NH2	783	2160.13	1081.07	1081.34
44	Ac-QSQQTF#r8NLWRLL#QN-NH2	784	2080.12	1041.06	1041.34
45	Ac-RSQQTA$r8NLWRLL$QN-NH2	785	2060.16	1031.08	1031.38
46	Ac-QSQQTF%r8NLWRLL%QN-NH2	786	2110.17	1056.09	1056.55
47	HepQSQ$TFSNLWRLLPQN-NH2	787	2051.10	1026.55	1026.82
48	HepQSQ$TF$r8NLWRLL$QN-NH2	788	2159.23	1080.62	1080.89
49	Ac-QSQQTF$r8NL6clWRLL$QN-NH2	789	2142.11	1072.06	1072.35
50	Ac-QSQQTF$r8NLMe6c1wRLL$QN-NH2	790	2156.13	1079.07	1079.27
51	Ac-LTFEHYWAQLTS-NH2	791	1535.74	768.87	768.91
52	Ac-LTF$HYW$QLTS-NH2	792	1585.83	791.92	761.67
53	Ac-LTFE$YWA$LTS-NH2	793	1520.79	761.40	761.67
54	Ac-LTF$zr8HYWAQL$zS-NH2	794	1597.87	799.94	800.06
55	Ac-LTF$r8HYWRQL$S-NH2	795	1682.93	842.47	842.72
56	Ac-QS$QTFStNLWRLL$s8QN-NH2	796	2145.21	1071.61	1073.90
57	Ac-QSQQTASNLWRLLPQN-NH2	797	1923.99	961.00	961.26
58	Ac-QSQQTA$/r8NLwRLL$NN-NH2	798	2060.15	1031.08	1031.24
59	Ac-ASQQTF$/r8NLwRLL$NN-NH2	799	2079.16	1040.58	1040.89
60	Ac-$SQQ$FSNLWRLLAibQN-NH2	800	2009.09	1005.55	1005.86
61	Ac-QS$QTF$NLwRLLAibQN-NH2	801	2023.10	1012.55	1012.79
62	Ac-QSQQ$FSN$WRLLAibQN-NH2	802	2024.06	1013.03	1011.31
63	Ac-QSQQTF$NLW$LLAibQN-NH2	803	1995.06	998.53	998.87
64	Ac-QSQQTFS$LWR$LAibQN-NH2	804	2011.06	1006.53	1006.83
65	Ac-QSQQTFSNLW$LLA$N-NH2	805	1940.02	971.01	971.29
66	Ac-$/SQQ$/FSNLWRLLAibQN-NH2	806	2037.12	1019.56	1019.78
67	Ac-QS$NTFVNLwRLLAibQN-NH2	807	2051.13	1026.57	1026.90
68	Ac-QSQQ$/FSN$/WRLLAibQN-NH2	808	2052.09	1027.05	1027.36
69	Ac-QSQQTFVNLWVLLAibQN-NH2	809	2023.09	1012.55	1013.82
70	Ac-QSQ$TFS$LWRLLAibQN-NH2	810	1996.09	999.05	999.39
71	Ac-QSQ$/TFS$/LWRLLAibQN-NH2	811	2024.12	1011.06	1011.37
72	Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2	812	2201.27	1101.64	1101.00
73	Ac-$r8SQQTFS$LWRLLAibQN-NH2	813	2038.14	1020.07	1020.23
74	Ac-QSQ$r8TFSNLW$LLAibQN-NH2	814	1996.08	999.04	999.32
75	Ac-QSQQTFS$r8LWRLLA$N-NH2	815	2024.12	1011.06	1011.37
76	Ac-QS$r5QTFStNLW$LLAibQN-NH2	816	2032.12	1017.06	1017.39
77	Ac-$/r8SQQTFS$/LWRLLAibQN-NH2	817	2066.17	1034.09	1034.80
78	Ac-QSQ$/r8TFSNLW$/LLAibQN-NH2	818	2024.11	1011.06	1014.34
79	Ac-QSQQTFS$/r8LWRLLA$/N-NH2	819	2052.15	1027.08	1027.16
80	Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2	820	2088.18	1045.09	1047.10
81	Ac-QSQQTFSNLWRLLAibQN-NH2	821	1988.02	995.01	995.31
82	Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2	822	2215.29	1108.65	1108.93
83	Ac-ASQQTF$r8NLRWLL$QN-NH2	823	2051.13	1026.57	1026.90
84	Ac-QSQQTF$/r8NLWRLLS/Q-NH2	824	2022.14	1012.07	1012.66
85	Ac-QSQQTF$r8NLWRLL$Q-NH2	825	1994.11	998.06	998.42
86	Ac-AAARAA$r8AAARAA$AA-NH2	826	1515.90	758.95	759.21
87	Ac-LTFEHYWAQLTSA-NH2	827	1606.78	804.39	804.59
88	Ac-LTF$r8HYWAQL$SA-NH2	828	1668.90	835.45	835.67
89	Ac-ASQQTFSNLWRLLPQN-NH2	829	1943.00	972.50	971.27
90	Ac-QS$QTFStNLW$r5LLAibQN-NH2	830	2032.12	1017.06	1017.30
91	Ac-QSQQTFAibNLWRLLAibQN-NH2	831	1986.04	994.02	994.19
92	Ac-QSQQTFNleNLWRLLNleQN-NH2	832	2042.11	1022.06	1022.23
93	Ac-QSQQTF$/r8NLWRLLAibQN-NH2	833	2082.14	1042.07	1042.23
94	Ac-QSQQTF$/r8NLWRLLNleQN-NH2	834	2110.17	1056.09	1056.29
95	Ac-QSQQTFAibNLWRLLS/QN-NH2	835	2040.09	1021.05	1021.25
96	Ac-QSQQTFNleNLWRLL$/QN-NH2	836	2068.12	1035.06	1035.31
97	Ac-QSQQTF%r8NL6clWRNleL%QN-NH2	837	2144.13	1073.07	1071.32
98	Ac-QSQQTF%r8NLMe6clWRLL%QN-NH2	838	2158.15	1080.08	1080.31
101	Ac-FNle$YWE$L-NH2	839	1160.63	—	1161.70
102	Ac-F$r8AYWELL$A-NH2	840	1344.75	—	1345.90
103	Ac-F$r8AYWQLL$A-NH2	841	1341.76	—	1344.83
104	Ac-NlePRF$r8NYWELL$QN-NH2	842	1925.06	96153	961.69
105	Ac-NlePRF$r8DYWRLL$QN-NH2	843	1953.10	977.55	977.68
106	Ac-NlePRFSr8NYWRLLSQ-NH2	844	1838.07	920.04	920.18
107	Ac-NlePRF$r8NYWRLL$-NH2	845	1710.01	856.01	856.13
108	Ac-QSQQTFSr8DLWRLLSQN-NH2	846	2109.14	1055.57	1055.64
109	Ac-QSQQTFSr8NLWRLLSEN-NH2	847	2109.14	1055.57	1055.70
110	Ac-QSQQTFSr8NLWRLLSQD-NH2	848	2109.14	1055.57	1055.64
111	Ac-QSQQTF$r8NLWRLL$S-NH2	849	1953.08	977.54	977.60
112	Ac-ESQQTFSr8NLWRLLSQN-NH2	850	2109.14	1055.57	1055.70
113	Ac-LTFSr8NLWRNleLSQ-NH2	851	1635.99	819.00	819.10
114	Ac-LRFSr8NLWRNleLSQ-NH2	852	1691.04	846.52	846.68
115	Ac-QSQQTFSr8NWWRNleLSQN-NH2	853	2181.15	1091.58	1091.64
116	Ac-QSQQTFSr8NLWRNleLSQ-NH2	854	1994.11	998.06	998.07
117	Ac-QTFSr8NLWRNleLSQN-NH2	855	1765.00	883.50	883.59
118	Ac-NlePRFSr8NWWRLLSQN-NH2	856	1975.13	988.57	988.75
119	Ac-NlePRFSr8NWWRLLSA-NH2	857	1804.07	903.04	903.08
120	Ac-TSFAEYWNLLNH2	858	1467.70	734.85	734.90
121	Ac-QTF$r8HWWSQL$S-NH2	859	1651.85	826.93	827.12
122	Ac-FM$YWE$L-NH2	860	1178.58	—	1179.64
123	Ac-QTFEHWWSQLLS-NH2	861	1601.76	801.88	801.94
124	Ac-QSQQTFSr8NLAmwRLNleSQN-NH2	862	2122.17	1062.09	1062.24
125	Ac-FMAibY6clWEAc3cL-NH2	863	1130.47	—	1131.53
126	Ac-FNle$Y6clWE$L-NH2	864	1194.59	—	1195.64
127	Ac-FSzr8AY6clWEAc3cLSz-NH2	865	1277.63	639.82	1278.71
128	Ac-FSr8AY6clWEAc3cLSA-NH2	866	1348.66	—	1350.72
129	Ac-NlePRFSr8NY6clWRLLSQN-NH2	867	1986.08	994.04	994.64
130	Ac-AF$r8AAWALA$A-NH2	868	1223.71	—	1224.71
131	Ac-TFSr8AAWRLASQ-NH2	869	1395.80	698.90	399.04
132	Pr-TFSr8AAWRLASQ-NH2	870	1409.82	705.91	706.04
133	Ac-QSQQTF%r8NLWRNleL%QN-NH2	871	2110.17	1056.09	1056.22
134	Ac-LTF%r8HYwAQL%sA-NH2	872	1670.92	836.46	836.58
135	Ac-NlePRF%r8NYWRLL%QN-NH2	873	1954.13	978.07	978.19
136	Ac-NlePRF%r8NY6clWRLL%QN-NH2	874	1988.09	995.05	995.68
137	Ac-LTF%r8HY6clWAQL%S-NH2	875	1633.84	817.92	817.93
138	Ac-QS%QTF%StNLWRLL%s8QN-NH2	876	2149.24	1075.62	1075.65
139	Ac-LTF%r8HY6clWRQL%S-NH2	877	1718.91	860.46	860.54
140	Ac-QSQQTF%r8NL6clWRLL%QN-NH2	878	2144.13	1073.07	1073.64
141	Ac-%r8SQQTFS%LWRLLAibQN-NH2	879	2040.15	1021.08	1021.13
142	Ac-LTF%r8HYWAQL%S-NH2	880	1599.88	800.94	801.09
143	Ac-TSF%r8QYWNLL%P-NH2	881	1602.88	802.44	802.58
147	Ac-LTFEHYWAQLTS-NH2	882	1535.74	768.87	769.5
152	Ac-F$er8AY6clWEAc3cL$e-NH2	883	1277.63	639.82	1278.71
153	Ac-AFSr8AAWALASA-NH2	884	1277.63	639.82	1277.84
154	Ac-TF$r8AAWRLA$Q-NH2	885	1395.80	698.90	699.04
155	Pr-TF$r8AAWRLA$Q-NH2	886	1409.82	705.91	706.04
156	Ac-LTF$er8HYWAQL$eS-NH2	887	1597.87	799.94	800.44
159	Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2	888	2745.30	1373.65	1372.99
160	Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-NH2	889	2669.27	1335.64	1336.09
161	Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2	890	2589.26	1295.63	1296.2
162	Ac-LTF$/r8HYWAQL$/S-NH2	891	1625.90	813.95	814.18
163	Ac-F%r8HY6clWRAc3cL%-NH2	892	1372.72	687.36	687.59
164	Ac-QTF%r8HWWSQL%S-NH2	893	1653.87	827.94	827.94
165	Ac-LTA$r8HYWRQL$S-NH2	894	1606.90	804.45	804.66
166	Ac-Q$r8QQTFSN$WRLLAibQN-NH2	895	2080.12	1041.06	1041.61
167	Ac-QSQQ$r8FSNLWR$LAibQN-NH2	896	2066.11	1034.06	1034.58
168	Ac-F$r8AYWEAc3cL$A-NH2	897	1314.70	658.35	1315.88
169	Ac-F$r8AYWEAc3cL$S-NH2	898	1330.70	666.35	1331.87
170	Ac-F$r8AYWEAc3cL$Q-NH2	899	1371.72	686.86	1372.72
171	Ac-F$r8AYWEAibL$S-NH2	900	1332.71	667.36	1334.83
172	Ac-F$r8AYWEAL$S-NH2	901	1318.70	660.35	1319.73
173	Ac-F$r8AYWEQL$S-NH2	902	1375.72	688.86	1377.53
174	Ac-F$r8HYWEQL$S-NH2	903	1441.74	721.87	1443.48
175	Ac-F$r8HYWAQL$S-NH2	904	1383.73	692.87	1385.38
176	Ac-F$r8HYWAAc3cL$S-NH2	905	1338.71	670.36	1340.82
177	Ac-F$r8HYWRAc3cL$S-NH2	906	1423.78	712.89	713.04
178	Ac-F$r8AYWEAc3cL#A-NH2	907	1300.69	651.35	1302.78
179	Ac-NlePTF%r8NYWRLL%QN-NH2	908	1899.08	950.54	950.56
180	Ac-TF$r8AAWRAL$Q-NH2	909	1395.80	698.90	699.13
181	Ac-TSF%r8HYWAQL%S-NH2	910	1573.83	787.92	787.98
184	Ac-F%r8AY6clWEAc3cL%A-NH2	911	1350.68	676.34	676.91
185	Ac-LTF$r8HYWAQI$S-NH2	912	1597.87	799.94	800.07
186	Ac-LTF$r8HYWAQNle$S-NH2	913	1597.87	799.94	800.07
187	Ac-LTF$r8HYWAQL$A-NH2	914	1581.87	791.94	792.45
188	Ac-LTF$r8HYWAQL$Abu-NH2	915	1595.89	798.95	799.03
189	Ac-LTF$r8HYWAbuQL$S-NH2	916	1611.88	806.94	807.47
190	Ac-LTF$er8AYWAQL$eS-NH2	917	1531.84	766.92	766.96
191	Ac-LAF$r8HYWAQL$S-NH2	918	1567.86	784.93	785.49
192	Ac-LAF$r8AYWAQL$S-NH2	919	1501.83	751.92	752.01
193	Ac-LTF$er8AYWAQL$eA-NH2	920	1515.85	758.93	758.97
194	Ac-LAF$r8AYWAQL$A-NH2	921	1485.84	743.92	744.05
195	Ac-LTF$r8NLWANleL$Q-NH2	922	1550.92	776.46	776.61
196	Ac-LTF$r8NLWANleL$A-NH2	923	1493.90	747.95	1495.6
197	Ac-LTF$r8ALWANleL$Q-NH2	924	1507.92	754.96	755
198	Ac-LAF$r8NLWANleL$Q-NH2	925	1520.91	761.46	761.96
199	Ac-LAF$r8ALWANleL$A-NH2	926	1420.89	711.45	1421.74
200	Ac-A$r8AYwEAc3cL$A-NH2	927	1238.67	620.34	1239.65
201	Ac-F$r8AYWEAc3cL$AA-NH2	928	1385.74	693.87	1386.64
202	Ac-F$r8AYWEAc3cL$Abu-NH2	929	1328.72	665.36	1330.17
203	Ac-F$r8AYWEAc3cL$Nle-NH2	930	1356.75	679.38	1358.22
204	Ac-F$r5AYWEAc3cL$s8A-NH2	931	1314.70	658.35	1315.51
205	Ac-F$AYWEAc3cL$r8A-NH2	932	1314.70	658.35	1315.66
206	Ac-F$r8AYWEAc3cI$A-NH2	933	1314.70	658.35	1316.18
207	Ac-F$r8AYWEAc3cNle$A-NH2	934	1314.70	658.35	1315.66
208	Ac-F$r8AYWEAmlL$A-NH2	935	1358.76	680.38	1360.21
209	Ac-F$r8AYWENleL$A-NH2	936	1344.75	673.38	1345.71
210	Ac-F$r8AYWQAc3cL$A-NH2	937	1313.72	657.86	1314.7
211	Ac-F$r8AYwAAc3cL$A-NH2	938	1256.70	629.35	1257.56
212	Ac-F$r8AYWAbuAc3cL$A-NH2	939	1270.71	636.36	1272.14
213	Ac-F$r8AYWNleAc3cL$A-NH2	940	1298.74	650.37	1299.67
214	Ac-F$r8AbuYWEAc3cL$A-NH2	941	1328.72	665.36	1329.65
215	Ac-F$r8NleYWEAc3cL$A-NH2	942	1356.75	679.38	1358.66
216	5-FAM-BaLTFEHYWAQLTS-NH2	943	1922.82	962.41	962.87
217	5-FAM-BaLTF%r8HYWAQL%S-NH2	944	1986.96	994.48	994.97
218	Ac-LTF$r8HYWAQhL$S-NH2	945	1611.88	806.94	807
219	Ac-LTF$r8HYWAQTle$S-NH2	946	1597.87	799.94	799.97
220	Ac-LTF$r8HYWAQAdm$S-NH2	947	1675.91	838.96	839.09
221	Ac-LTF$r8HYWAQhCha$S-NH2	948	1651.91	826.96	826.98
222	Ac-LTF$r8HYWAQCha$S-NH2	949	1637.90	819.95	820.02
223	Ac-LTF$r8HYWAc6cQL$S-NH2	950	1651.91	826.96	826.98
224	Ac-LTF$r8HYWAc5cQL$S-NH2	951	1637.90	819.95	820.02
225	Ac-LThF$r8HYWAQL$S-NH2	952	1611.88	806.94	807
226	Ac-LTIg1$r8HYWAQL$S-NH2	953	1625.90	813.95	812.99
227	Ac-LTF$r8HYwAQChg-S S-NH2	954	1623.88	812.94	812.99
228	Ac-LTF$r8HYWAQF$S-NH2	955	1631.85	816.93	816.99
229	Ac-LTF$r8HYWAQIgl$S-NH2	956	1659.88	830.94	829.94
230	Ac-LTF$r8HYWAQCba$S-NH2	957	1609.87	805.94	805.96
231	Ac-LTF$r8HYWAQCpg$S-NH2	958	1609.87	805.94	805.96
232	Ac-LTF$r8HhYWAQL$S-NH2	959	1611.88	806.94	807
233	Ac-F$r8AYWEAc3chL$A-NH2	960	1328.72	665.36	665.43
234	Ac-F$r8AYWEAc3cTle$A-NH2	961	1314.70	658.35	1315.62
235	Ac-F$r8AYWEAc3cAdm$A-NH2	962	1392.75	697.38	697.47
236	Ac-F$r8AYWEAc3chCha$A-NH2	963	1368.75	685.38	685.34
237	Ac-F$r8AYWEAc3cCha$A-NH2	964	1354.73	678.37	678.38
238	Ac-F$r8AYWEAc6cL$A-NH2	965	1356.75	679.38	679.42
239	Ac-F$r8AYWEAc5cL$A-NH2	966	1342.73	672.37	672.46
240	Ac-hF$r8AYWEAc3cL$A-NH2	967	1328.72	665.36	665.43
241	Ac-Ig1$r8AYWEAc3cL$A-NH2	968	1342.73	672.37	671.5
243	Ac-F$r8AYWEAc3cF$A-NH2	969	1348.69	675.35	675.35
244	Ac-F$r8AYWEAc3cIg1$A-NH2	970	1376.72	689.36	688.37
245	Ac-F$r8AYWEAc3cCba$A-NH2	971	1326.70	664.35	664.47
246	Ac-F$r8AYWEAc3cCpg$A-NH2	972	1326.70	664.35	664.39
247	Ac-F$r8AhYWEAc3cL$A-NH2	973	1328.72	665.36	665.43
248	Ac-F$r8AYWEAc3cL$Q-NH2	974	1371.72	686.86	1372.87
249	Ac-F$r8AYWEAibL$A-NH2	975	1316.72	659.36	1318.18
250	Ac-F$r8AYWEAL$A-NH2	976	1302.70	652.35	1303.75
251	Ac-LAF$r8AYWAAL$A-NH2	977	1428.82	715.41	715.49
252	Ac-LTF$r8HYWAAc3cL$S-NH2	978	1552.84	777.42	777.5
253	Ac-NleTF$r8HYWAQL$S-NH2	979	1597.87	799.94	800.04
254	Ac-VTF$r8HYWAQL$S-NH2	980	1583.85	792.93	793.04
255	Ac-FTF$r8HYWAQL$S-NH2	981	1631.85	816.93	817.02
256	Ac-WTF$r8HYWAQL$S-NH2	982	1670.86	836.43	836.85
257	Ac-RTF$r8HYWAQL$S-NH2	983	1640.88	821.44	821.9
258	Ac-KTF$r8HYWAQL$S-NH2	984	1612.88	807.44	807.91
259	Ac-LNleF$r8HYWAQL$S-NH2	985	1609.90	805.95	806.43
260	Ac-LVF$r8HYWAQL$S-NH2	986	1595.89	798.95	798.93
261	Ac-LFF$r8HYWAQL$S-NH2	987	1643.89	822.95	823.38
262	Ac-LWF$r8HYWAQL$S-NH2	988	1682.90	842.45	842.55
263	Ac-LRF$r8HYWAQL$S-NH2	989	1652.92	827.46	827.52
264	Ac-LKF$r8HYWAQL$S-NH2	990	1624.91	813.46	813.51
265	Ac-LTF$r8NleYWAQL$S-NH2	991	1573.89	787.95	788.05
266	Ac-LTF$r8VYWAQL$S-NH2	992	1559.88	780.94	780.98
267	Ac-LTF$r8FYWAQL$S-NH2	993	1607.88	804.94	805.32
268	Ac-LTF$r8WYWAQL$S-NH2	994	1646.89	824.45	824.86
269	Ac-LTF$r8RYWAQL$S-NH2	995	1616.91	809.46	809.51
270	Ac-LTF$r8KYWAQL$S-NH2	996	1588.90	795.45	795.48
271	Ac-LTF$r8HNleWAQL$S-NH2	997	1547.89	774.95	774.98
272	Ac-LTF$r8HVWAQL$S-NH2	998	1533.87	767.94	767.95
273	Ac-LTF$r8HFWAQL$S-NH2	999	1581.87	791.94	792.3
274	Ac-LTF$r8HWWAQL$S-NH2	1000	1620.88	811.44	811.54
275	Ac-LTF$r8HRWAQL$S-NH2	1001	1590.90	796.45	796.52
276	Ac-LTF$r8HKWAQL$S-NH2	1002	1562.90	782.45	782.53
277	Ac-LTF$r8HYWNleQL$S-NH2	1003	1639.91	820.96	820.98
278	Ac-LTF$r8HYWVQL$S-NH2	1004	1625.90	813.95	814.03
279	Ac-LTF$r8HYWFQL$S-NH2	1005	1673.90	837.95	838.03
280	Ac-LTF$r8HYWWQL$S-NH2	1006	1712.91	857.46	857.5
281	Ac-LTF$r8HYWKQL$S-NH2	1007	1654.92	828.46	828.49
282	Ac-LTF$r8HYWANleL$S-NH2	1008	1582.89	792.45	792.52
283	Ac-LTF$r8HYWAVL$S-NH2	1009	1568.88	785.44	785.49
284	Ac-LTF$r8HYWAFL$S-NH2	1010	1616.88	809.44	809.47
285	Ac-LTF$r8HYWAWL$S-NH2	1011	1655.89	828.95	829
286	Ac-LTF$r8HYWARL$S-NH2	1012	1625.91	813.96	813.98
287	Ac-LTF$r8HYWAQL$Nle-NH2	1013	1623.92	812.96	813.39
288	Ac-LTF$r8HYWAQL$V-NH2	1014	1609.90	805.95	805.99
289	Ac-LTF$r8HYWAQL$F-NH2	1015	1657.90	829.95	830.26
290	Ac-LTF$r8HYWAQL$W-NH2	1016	1696.91	849.46	849.5
291	Ac-LTF$r8HYWAQL$R-NH2	1017	1666.94	834.47	834.56
292	Ac-LTF$r8HYWAQL$K-NH2	1018	1638.93	820.47	820.49
293	Ac-Q$r8QQTFSN$wRLLAibQN-NH2	1019	2080.12	1041.06	1041.54
294	Ac-QSQQ$r8FSNLWR$LAibQN-NH2	1020	2066.11	1034.06	1034.58
295	Ac-LT2Pal$r8HYWAQL$S-NH2	1021	1598.86	800.43	800.49
296	Ac-LT3Pal$r8HYWAQL$S-NH2	1022	1598.86	800.43	800.49
297	Ac-LT4Pal$r8HYWAQL$S-NH2	1023	1598.86	800.43	800.49
298	Ac-LTF2CF3$r8HYWAQL$S-NH2	1024	1665.85	833.93	834.01
299	Ac-LTF2CN$r8HYWAQL$S-NH2	1025	1622.86	812.43	812.47
300	Ac-LTF2Me$r8HYWAQL$S-NH2	1026	1611.88	806.94	807
301	Ac-LTF3Cl$r8HYWAQL$S-NH2	1027	1631.83	816.92	816.99
302	Ac-LTF4CF3$r8HYwAQL$S-NH2	1028	1665.85	833.93	833.94
303	Ac-LTF4tBu$r8HYWAQL$S-NH2	1029	1653.93	827.97	828.02
304	Ac-LTF5F$r8HYWAQL$S-NH2	1030	1687.82	844.91	844.96
305	Ac-LTF$r8HY3BthAAQL$S-NH2	1031	1614.83	808.42	808.48
306	Ac-LTF2Br$r8HYWAQL$S-NH2	1032	1675.78	838.89	838.97
307	Ac-LTF4Br$r8HYWAQL$S-NH2	1033	1675.78	838.89	839.86
308	Ac-LTF2Cl$r8HYWAQL$S-NH2	1034	1631.83	816.92	816.99
309	Ac-LTF4Cl$r8HYWAQL$S-NH2	1035	1631.83	816.92	817.36
310	Ac-LTF3CN$r8HYWAQL$S-NH2	1036	1622.86	812.43	812.47
311	Ac-LTF4CN$r8HYWAQL$S-NH2	1037	1622.86	812.43	812.47
312	Ac-LTF34Cl2$r8HYWAQL$S-NH2	1038	1665.79	833.90	833.94
313	Ac-LTF34F2$r8HYWAQL$S-NH2	1039	1633.85	817.93	817.95
314	Ac-LTF35F2$r8HYWAQL$S-NH2	1040	1633.85	817.93	817.95
315	Ac-LTDip$r8HYWAQL$S-NH2	1041	1673.90	837.95	838.01
316	Ac-LTF2F$r8HYWAQL$S-NH2	1042	1615.86	808.93	809
317	Ac-LTF3F$r8HYWAQL$S-NH2	1043	1615.86	808.93	809
318	Ac-LTF4F$r8HYWAQL$S-NH2	1044	1615.86	808.93	809
319	Ac-LTF4I$r8HYWAQL$S-NH2	1045	1723.76	862.88	862.94
320	Ac-LTF3Me$r8HYWAQL$S-NH2	1046	1611.88	806.94	807.07
321	Ac-LTF4Me$r8HYWAQL$S-NH2	1047	1611.88	806.94	807
322	Ac-LT1Nal$r8HYWAQL$S-NH2	1048	1647.88	824.94	824.98
323	Ac-LT2Nal$r8HYWAQL$S-NH2	1049	1647.88	824.94	825.06
324	Ac-LTF3CF3$r8HYWAQL$S-NH2	1050	1665.85	833.93	834.01
325	Ac-LTF4NO2$r8HYWAQL$S-NH2	1051	1642.85	822.43	822.46
326	Ac-LTF3NO2$r8HYWAQL$S-NH2	1052	1642.85	822.43	822.46
327	Ac-LTF$r82ThiYWAQL$S-NH2	1053	1613.83	807.92	807.96
328	Ac-LTF$r8HBipWAQL$S-NH2	1054	1657.90	829.95	830.01
329	Ac-LTF$r8HF4tBuWAQL$S-NH2	1055	1637.93	819.97	820.02
330	Ac-LTF$r8HF4CF3WAQL$S-NH2	1056	1649.86	825.93	826.02
331	Ac-LTF$r8HF4C1WAQL$S-NH2	1057	1615.83	808.92	809.37
332	Ac-LTF$r8HF4MeWAQL$S-NH2	1058	1595.89	798.95	799.01
333	Ac-LTF$r8HF4BrWAQL$S-NH2	1059	1659.78	830.89	830.98
334	Ac-LTF$r8HF4CNWAQL$S-NH2	1060	1606.87	804.44	804.56
335	Ac-LTF$r8HF4NO2WAQL$S-NH2	1061	1626.86	814.43	814.55
336	Ac-LTF$r8H1NalWAQL$S-NH2	1062	1631.89	816.95	817.06
337	Ac-LTF$r8H2NalWAQL$S-NH2	1063	1631.89	816.95	816.99
338	Ac-LTF$r8HWAQL$S-NH2	1064	1434.80	718.40	718.49
339	Ac-LTF$r8HY1NalAQL$S-NH2	1065	1608.87	805.44	805.52
340	Ac-LTF$r8HY2NalAQL$S-NH2	1066	1608.87	805.44	805.52
341	Ac-LTF$r8HYWAQI$S-NH2	1067	1597.87	799.94	800.07
342	Ac-LTF$r8HYWAQNle$S-NH2	1068	1597.87	799.94	800.44
343	Ac-LTF$er8HYWAQL$eA-NH2	1069	1581.87	791.94	791.98
344	Ac-LTF$r8HYWAQL$Abu-NH2	1070	1595.89	798.95	799.03
345	Ac-LTF$r8HYWAbuQL$S-NH2	1071	1611.88	806.94	804.47
346	Ac-LAF$r8HYWAQL$S-NH2	1072	1567.86	784.93	785.49
347	Ac-LTF$r8NLWANleL$Q-NH2	1073	1550.92	776.46	777.5
348	Ac-LTF$r8ALWANleL$Q-NH2	1074	1507.92	754.96	755.52
349	Ac-LAF$r8NLWANleL$Q-NH2	1075	1520.91	761.46	762.48
350	Ac-F$r8AYWAAc3cL$A-NH2	1076	1256.70	629.35	1257.56
351	Ac-LTF$r8AYWAAL$S-NH2	1077	1474.82	738.41	738.55
352	Ac-LVF$r8AYWAQL$S-NH2	1078	1529.87	765.94	766
353	Ac-LTF$r8AYWAbuQL$S-NH2	1079	1545.86	773.93	773.92
354	Ac-LTF$r8AYWNleQL$S-NH2	1080	1573.89	787.95	788.17
355	Ac-LTF$r8AbuYWAQL$S-NH2	1081	1545.86	773.93	773.99
356	Ac-LTF$r8AYWHQL$S-NH2	1082	1597.87	799.94	799.97
357	Ac-LTF$r8AYWKQL$S-NH2	1083	1588.90	795.45	795.53
358	Ac-LTF$r8AYWOQL$S-NH2	1084	1574.89	788.45	788.5
359	Ac-LTF$r8AYWRQL$S-NH2	1085	1616.91	809.46	809.51
360	Ac-LTF$r8AYWSQL$S-NH2	1086	1547.84	774.92	774.96
361	Ac-LTF$r8AYWRAL$S-NH2	1087	1559.89	780.95	780.95
362	Ac-LTF$r8AYwRQL$A-NH2	1088	1600.91	801.46	801.52
363	Ac-LTF$r8AYWRAL$A-NH2	1089	1543.89	772.95	773.03
364	Ac-LTF$r5HYWAQL$s8S-NH2	1090	1597.87	799.94	799.97
365	Ac-LTF$HYWAQL$r8S-NH2	1091	1597.87	799.94	799.97
366	Ac-LTF$r8HYWAAL$S-NH2	1092	1540.84	771.42	771.48
367	Ac-LTF$r8HYWAAbuL$S-NH2	1093	1554.86	778.43	778.51
368	Ac-LTF$r8HYWALL$S-NH2	1094	1582.89	792.45	792.49
369	Ac-F$r8AYWHAL$A-NH2	1095	1310.72	656.36	656.4
370	Ac-F$r8AYWAAL$A-NH2	1096	1244.70	623.35	1245.61
371	Ac-F$r8AYWSAL$A-NH2	1097	1260.69	631.35	1261.6
372	Ac-F$r8AYWRAL$A-NH2	1098	1329.76	665.88	1330.72
373	Ac-F$r8AYWKAL$A-NH2	1099	1301.75	651.88	1302.67
374	Ac-F$r8AYWOAL$A-NH2	1100	1287.74	644.87	1289.13
375	Ac-F$r8VYWEAc3cL$A-NH2	1101	1342.73	672.37	1343.67
376	Ac-F$r8FYWEAc3cL$A-NH2	1102	1390.73	696.37	1392.14
377	Ac-F$r8WYWEAc3cL$A-NH2	1103	1429.74	715.87	1431.44
378	Ac-F$r8RYWEAc3cL$A-NH2	1104	1399.77	700.89	700.95
379	Ac-F$r8KYWEAc3cL$A-NH2	1105	1371.76	686.88	686.97
380	Ac-F$r8ANleWEAc3cL$A-NH2	1106	1264.72	633.36	1265.59
381	Ac-F$r8AVWEAc3cL$A-NH2	1107	1250.71	626.36	1252.2
382	Ac-F$r8AFWEAc3cL$A-NH2	1108	1298.71	650.36	1299.64
383	Ac-F$r8AWWEAc3cL$A-NH2	1109	1337.72	669.86	1338.64
384	Ac-F$r8ARWEAc3cL$A-NH2	1110	1307.74	654.87	655
385	Ac-F$r8AKWEAc3cL$A-NH2	1111	1279.73	640.87	641.01
386	Ac-F$r8AYWVAc3cL$A-NH2	1112	1284.73	643.37	643.38
387	Ac-F$r8AYWFAc3cL$A-NH2	1113	1332.73	667.37	667.43
388	Ac-F$r8AYWWAc3cL$A-NH2	1114	1371.74	686.87	686.97
389	Ac-F$r8AYWRAc3cL$A-NH2	1115	1341.76	671.88	671.94
390	Ac-F$r8AYWKAc3cL$A-NH2	1116	1313.75	657.88	657.88
391	Ac-F$r8AYWEVL$A-NH2	1117	1330.73	666.37	666.47
392	Ac-F$r8AYWEFL$A-NH2	1118	1378.73	690.37	690.44
393	Ac-F$r8AYWEWL$A-NH2	1119	1417.74	709.87	709.91
394	Ac-F$r8AYWERL$A-NH2	1120	1387.77	694.89	1388.66
395	Ac-F$r8AYWEKL$A-NH2	1121	1359.76	680.88	1361.21
396	Ac-F$r8AYWEAc3cL$V-NH2	1122	1342.73	672.37	1343.59
397	Ac-F$r8AYWEAc3cL$F-NH2	1123	1390.73	696.37	1392.58
398	Ac-F$r8AYWEAc3cL$W-NH2	1124	1429.74	715.87	1431.29
399	Ac-F$r8AYWEAc3cL$R-NH2	1125	1399.77	700.89	700.95
400	Ac-F$r8AYWEAc3cL$K-NH2	1126	1371.76	686.88	686.97
401	Ac-F$r8AYWEAc3cL$AV-NH2	1127	1413.77	707.89	707.91
402	Ac-F$r8AYWEAc3cL$AF-NH2	1128	1461.77	731.89	731.96
403	Ac-F$r8AYWEAc3cL$Aw-NH2	1129	1500.78	751.39	751.5
404	Ac-F$r8AYWEAc3cL$AR-NH2	1130	1470.80	736.40	736.47
405	Ac-F$r8AYWEAc3cL$AK-NH2	1131	1442.80	722.40	722.41
406	Ac-F$r8AYWEAc3cL$AH-NH2	1132	1451.76	726.88	726.93
407	Ac-LTF2NO2$r8HYWAQL$S-NH2	1133	1642.85	822.43	822.54
408	Ac-LTA$r8HYAAQL$S-NH2	1134	1406.79	704.40	704.5
409	Ac-LTF$r8HYAAQL$S-NH2	1135	1482.82	742.41	742.47
410	Ac-QSQQTF$r8NLWALL$AN-NH2	1136	1966.07	984.04	984.38
411	Ac-QAibQQTF$r8NLWALL$AN-NH2	1137	1964.09	983.05	983.42
412	Ac-QAibQQTF$r8ALWALL$AN-NH2	1138	1921.08	961.54	961.59
413	Ac-AAAATF$r8AAWAAL$AA-NH2	1139	1608.90	805.45	805.52
414	Ac-F$r8AAWRAL$Q-NH2	1140	1294.76	648.38	648.48
415	Ac-TF$r8AAWAAL$Q-NH2	1141	1310.74	656.37	1311.62
416	Ac-TF$r8AAWRAL$A-NH2	1142	1338.78	670.39	670.46
417	Ac-VF$r8AAWRAL$Q-NH2	1143	1393.82	697.91	697.99
418	Ac-AF$r8AAWAAL$A-NH2	1144	1223.71	612.86	1224.67
420	Ac-TF$r8AAWKAL$Q-NH2	1145	1367.80	684.90	684.97
421	Ac-TF$r8AAWOAL$Q-NH2	1146	1353.78	677.89	678.01
422	Ac-TF$r8AAWSAL$Q-NH2	1147	1326.73	664.37	664.47
423	Ac-LTF$r8AAWRAL$Q-NH2	1148	1508.89	755.45	755.49
424	Ac-F$r8AYWAQL$A-NH2	1149	1301.72	651.86	651.96
425	Ac-F$r8AWWAAL$A-NH2	1150	1267.71	634.86	634.87
426	Ac-F$r8AWWAQL$A-NH2	1151	1324.73	663.37	663.43
427	Ac-F$r8AYWEAL$-NH2	1152	1231.66	616.83	1232.93
428	Ac-F$r8AYWAAL$-NH2	1153	1173.66	587.83	1175.09
429	Ac-F$r8AYWKAL$-NH2	1154	1230.72	616.36	616.44
430	Ac-F$r8AYWOAL$-NH2	1155	1216.70	609.35	609.48
431	Ac-F$r8AYWQAL$-NH2	1156	1230.68	616.34	616.44
432	Ac-F$r8AYWAQL$-NH2	1157	1230.68	616.34	616.37
433	Ac-F$r8HYWDQL$S-NH2	1158	1427.72	714.86	714.86
434	Ac-F$r8HFWEQL$S-NH2	1159	1425.74	713.87	713.98
435	Ac-F$r8AYWHQL$S-NH2	1160	1383.73	692.87	692.96
436	Ac-F$r8AYWKQL$S-NH2	1161	1374.77	688.39	688.45
437	Ac-F$r8AYWOQL$S-NH2	1162	1360.75	681.38	681.49
438	Ac-F$r8HYWSQL$S-NH2	1163	1399.73	700.87	700.95
439	Ac-F$r8HWWEQL$S-NH2	1164	1464.76	733.38	733.44
440	Ac-F$r8HWWAQL$S-NH2	1165	1406.75	704.38	704.43
441	Ac-F$r8AWWHQL$S-NH2	1166	1406.75	704.38	704.43
442	Ac-F$r8AWWKQL$S-NH2	1167	1397.79	699.90	699.92
443	Ac-F$r8AWWOQL$S-NH2	1168	1383.77	692.89	692.96
444	Ac-F$r8HWWSQL$S-NH2	1169	1422.75	712.38	712.42
445	Ac-LTF$r8NYWANleL$Q-NH2	1170	1600.90	801.45	801.52
446	Ac-LTF$r8NLWAQL$Q-NH2	1171	1565.90	783.95	784.06
447	Ac-LTF$r8NYWANleL$A-NH2	1172	1543.88	772.94	773.03
448	Ac-LTF$r8NLWAQL$A-NH2	1173	1508.88	755.44	755.49
449	Ac-LTF$r8AYWANleL$Q-NH2	1174	1557.90	779.95	780.06
450	Ac-LTF$r8ALWAQL$Q-NH2	1175	1522.89	762.45	762.45
451	Ac-LAF$r8NYWANleL$Q-NH2	1176	1570.89	786.45	786.5
452	Ac-LAF$r8NLWAQL$Q-NH2	1177	1535.89	768.95	769.03
453	Ac-LAF$r8AYWANleL$A-NH2	1178	1470.86	736.43	736.47
454	Ac-LAF$r8ALWAQL$A-NH2	1179	1435.86	718.93	719.01
455	Ac-LAF$r8AYWAAL$A-NH2	1180	1428.82	715.41	715.41
456	Ac-F$r8AYWEAc3cL$AAib-NH2	1181	1399.75	700.88	700.95
457	Ac-F$r8AYWAQL$AA-NH2	1182	1372.75	687.38	687.78
458	Ac-F$r8AYWAAc3cL$AA-NH2	1183	1327.73	664.87	664.84
459	Ac-F$r8AYWSAc3cL$AA-NH2	1184	1343.73	672.87	672.9
460	Ac-F$r8AYWEAc3cL$AS-NH2	1185	1401.73	701.87	701.84
461	Ac-F$r8AYWEAc3cL$AT-NH2	1186	1415.75	708.88	708.87
462	Ac-F$r8AYWEAc3cL$AL-NH2	1187	1427.79	714.90	714.94
463	Ac-F$r8AYWEAc3cL$AQ-NH2	1188	1442.76	722.38	722.41
464	Ac-F$r8AFWEAc3cL$AA-NH2	1189	1369.74	685.87	685.93
465	Ac-F$r8AWWEAc3cL$AA-NH2	1190	1408.75	705.38	705.39
466	Ac-F$r8AYWEAc3cL$SA-NH2	1191	1401.73	701.87	701.99
467	Ac-F$r8AYWEAL$AA-NH2	1192	1373.74	687.87	687.93
468	Ac-F$r8AYWENleL$AA-NH2	1193	1415.79	708.90	708.94
469	Ac-F$r8AYWEAc3cL$AbuA-NH2	1194	1399.75	700.88	700.95
470	Ac-F$r8AYWEAc3cL$NleA-NH2	1195	1427.79	714.90	714.86
471	Ac-F$r8AYWEAibL$NleA-NH2	1196	1429.80	715.90	715.97
472	Ac-F$r8AYWEAL$NleA-NH2	1197	1415.79	708.90	708.94
473	Ac-F$r8AYWENleL$NleA-NH2	1198	1457.83	729.92	729.96
474	Ac-F$r8AYWEAibL$Abu-NH2	1199	1330.73	666.37	666.39
475	Ac-F$r8AYWENleL$Abu-NH2	1200	1358.76	680.38	680.39
476	Ac-F$r8AYWEAL$Abu-NH2	1201	1316.72	659.36	659.36
477	Ac-LTF$r8AFWAQL$S-NH2	1202	1515.85	758.93	759.12
478	Ac-LTF$r8AWWAQL$S-NH2	1203	1554.86	778.43	778.51
479	Ac-LTF$r8AYWAQI$S-NH2	1204	1531.84	766.92	766.96
480	Ac-LTF$r8AYWAQNle$S-NH2	1205	1531.84	766.92	766.96
481	Ac-LTF$r8AYWAQL$SA-NH2	1206	1602.88	802.44	802.48
482	Ac-LTF$r8AWWAQL$A-NH2	1207	1538.87	770.44	770.89
483	Ac-LTFSr8AYWAQISA-NH2	1208	1515.85	758.93	759.42
484	Ac-LTF$r8AYWAQNle$A-NH2	1209	1515.85	758.93	759.42
485	Ac-LTFSr8AYWAQLSAA-NH2	1210	1586.89	794.45	794.94
486	Ac-LTF$r8HWWAQL$S-NH2	1211	1620.88	811.44	811.47
487	Ac-LTFSr8HRWAQLSS-NH2	1212	1590.90	796.45	796.52
488	Ac-LTF$r8HKWAQL$S-NH2	1213	1562.90	782.45	782.53
489	Ac-LTFSr8HYWAQLSW-NH2	1214	1696.91	849.46	849.5
491	Ac-F$r8AYWAbuAL$A-NH2	1215	1258.71	630.36	630.5
492	Ac-FSr8AbuYWEALSA-NH2	1216	1316.72	659.36	659.51
493	Ac-NlePRF%r8NYWRLL%QN-NH2	1217	1954.13	978.07	978.54
494	Ac-TSF%r8HYWAQL%S-NH2	1218	1573.83	787.92	787.98
495	Ac-LTF%r8AYWAQL%S-NH2	1219	1533.86	767.93	768
496	Ac-HTFSr8HYWAQLSS-NH2	1220	1621.84	811.92	811.96
497	Ac-LHFSr8HYWAQLSS-NH2	1221	1633.88	817.94	818.02
498	Ac-LTFSr8HHWAQLSS-NH2	1222	1571.86	786.93	786.94
499	Ac-LTFSr8HYWHQLSS-NH2	1223	1663.89	832.95	832.38
500	Ac-LTFSr8HYWAHLSS-NH2	1224	1606.87	804.44	804.48
501	Ac-LTFSr8HYWAQLSH-NH2	1225	1647.89	824.95	824.98
502	Ac-LTF$r8HYWAQL$S-NHPr	1226	1639.91	820.96	820.98
503	Ac-LTF$r8HYWAQL$S-NHsBu	1227	1653.93	827.97	828.02
504	Ac-LTF$r8HYWAQL$S-NHiBu	1228	1653.93	827.97	828.02
505	Ac-LTF$r8HYWAQL$S-NHBn	1229	1687.91	844.96	844.44
506	Ac-LTF$r8HYWAQL$S-NHPe	1230	1700.92	851.46	851.99
507	Ac-LTF$r8HYWAQL$S-NHChx	1231	1679.94	840.97	841.04
508	Ac-ETFSr8AYWAQLSS-NH2	1232	1547.80	774.90	774.96
509	Ac-STFSr8AYWAQLSS-NH2	1233	1505.79	753.90	753.94
510	Ac-LEFSr8AYWAQLSS-NH2	1234	1559.84	780.92	781.25
511	Ac-LSFSr8AYWAQLSS-NH2	1235	1517.83	759.92	759.93
512	Ac-LTFSr8EYWAQLSS-NH2	1236	1589.85	795.93	795.97
513	Ac-LTFSr8SYWAQLSS-NH2	1237	1547.84	774.92	774.96
514	Ac-LTFSr8AYWEQLSS-NH2	1238	1589.85	795.93	795.9
515	Ac-LTFSr8AYWAELSS-NH2	1239	1532.83	767.42	766.96
516	Ac-LTFSr8AYWASLSS-NH2	1240	1490.82	746.41	746.46
517	Ac-LTFSr8AYWAQLSE-NH2	1241	1573.85	787.93	787.98
518	Ac-LTF2CNSr8HYWAQLSS-NH2	1242	1622.86	812.43	812.47
519	Ac-LTF3ClSr8HYWAQLSS-NH2	1243	1631.83	816.92	816.99
520	Ac-LTDipSr8HYWAQLSS-NH2	1244	1673.90	837.95	838.01
521	Ac-LTFSr8HYWAQTle$S-NH2	1245	1597.87	799.94	800.04
522	Ac-F$r8AY6clWEAL$A-NH2	1246	1336.66	669.33	1338.56
523	Ac-F$r8AYdl6brWEAL$A-NH2	1247	1380.61	691.31	692.2
524	Ac-F$r8AYdl6fWEAL$A-NH2	1248	1320.69	661.35	1321.61
525	Ac-F$r8AYdl4mWEAL$A-NH2	1249	1316.72	659.36	659.36
526	Ac-F$r8AYdl5clWEAL$A-NH2	1250	1336.66	669.33	669.35
527	Ac-F$r8AYdl7mWEAL$A-NH2	1251	1316.72	659.36	659.36
528	Ac-LTF%r8HYWAQL%A-NH2	1252	1583.89	792.95	793.01
529	Ac-LTF$r8HCouWAQL$S-NH2	1253	1679.87	840.94	841.38
530	Ac-LTFEHCouWAQLTS-NH2	1254	1617.75	809.88	809.96
531	Ac-LTA$r8HCouWAQL$S-NH2	1255	1603.84	802.92	803.36
532	Ac-F$r8AYWEAL$AbuA-NH2	1256	1387.75	694.88	694.88
533	Ac-F$r8AYWEAI$AA-NH2	1257	1373.74	687.87	687.93
534	Ac-F$r8AYWEANle$AA-NH2	1258	1373.74	687.87	687.93
535	Ac-F$r8AYWEAmlL$AA-NH2	1259	1429.80	715.90	715.97
536	Ac-F$r8AYWQAL$AA-NH2	1260	1372.75	687.38	687.48
537	Ac-F$r8AYWAAL$AA-NH2	1261	1315.73	658.87	658.92
538	Ac-F$r8AYWAbuAL$AA-NH2	1262	1329.75	665.88	665.95
539	Ac-F$r8AYWNleAL$AA-NH2	1263	1357.78	679.89	679.94
540	Ac-F$r8AbuYWEAL$AA-NH2	1264	1387.75	694.88	694.96
541	Ac-F$r8NleYWEAL$AA-NH2	1265	1415.79	708.90	708.94
542	Ac-F$r8FYWEAL$AA-NH2	1266	1449.77	725.89	725.97
543	Ac-LTF$r8HYWAQhL$S-NH2	1267	1611.88	806.94	807
544	Ac-LTF$r8HYWAQAdm$S-NH2	1268	1675.91	838.96	839.04
545	Ac-LTF$r8HYWAQIgl$S-NH2	1269	1659.88	830.94	829.94
546	Ac-F$r8AYWAQL$AA-NH2	1270	1372.75	687.38	687.48
547	Ac-LTF$r8ALWAQL$Q-NH2	1271	1522.89	762.45	762.52
548	Ac-F$r8AYWEAL$AA-NH2	1272	1373.74	687.87	687.93
549	Ac-F$r8AYWENleL$AA-NH2	1273	1415.79	708.90	708.94
550	Ac-F$r8AYWEAibL$Abu-NH2	1274	1330.73	666.37	666.39
551	Ac-F$r8AYWENleL$Abu-NH2	1275	1358.76	680.38	680.38
552	Ac-F$r8AYWEAL$Abu-NH2	1276	1316.72	659.36	659.36
553	Ac-F$r8AYWEAc3cL$AbuA-NH2	1277	1399.75	700.88	700.95
554	Ac-F$r8AYWEAc3cL$NleA-NH2	1278	1427.79	714.90	715.01
555	H-LTF$r8AYWAQL$S-NH2	1279	1489.83	745.92	745.95
556	mdPEG3-LTF$r8AYWAQL$S-NH2	1280	1679.92	840.96	840.97
557	mdPEG7-LTF$r8AYWAQL$S-NH2	1281	1856.02	929.01	929.03
558	Ac-F$r8ApmpEt6clWEAL$A-NH2	1282	1470.71	736.36	788.17
559	Ac-LTF3Cl$r8AYWAQL$S-NH2	1283	1565.81	783.91	809.18
560	Ac-LTF3Cl$r8HYWAQL$A-NH2	1284	1615.83	808.92	875.24
561	Ac-LTF3Cl$r8HYWWQL$S-NH2	1285	1746.87	874.44	841.65
562	Ac-LTF3Cl$r8AYWWQL$S-NH2	1286	1680.85	841.43	824.63
563	Ac-LTF$r8AYWWQL$S-NH2	1287	1646.89	824.45	849.98
564	Ac-LTF$r8HYWWQL$A-NH2	1288	1696.91	849.46	816.67
565	Ac-LTF$r8AYWWQL$A-NH2	1289	1630.89	816.45	776.15
566	Ac-LTF4F$r8AYWAQL$S-NH2	1290	1549.83	775.92	776.15
567	Ac-LTF2F$r8AYWAQL$S-NH2	1291	1549.83	775.92	776.15
568	Ac-LTF3F$r8AYWAQL$S-NH2	1292	1549.83	775.92	785.12
569	Ac-LTF34F2$r8AYWAQL$S-NH2	1293	1567.83	784.92	785.12
570	Ac-LTF35F2$r8AYWAQL$S-NH2	1294	1567.83	784.92	1338.74
571	Ac-F3Cl$r8AYWEAL$A-NH2	1295	1336.66	669.33	705.28
572	Ac-F3Cl$r8AYWEAL$AA-NH2	1296	1407.70	704.85	680.11
573	Ac-F$r8AY6clWEAL$AA-NH2	1297	1407.70	704.85	736.83
574	Ac-F$r8AY6clWEAL$-NH2	1298	1265.63	633.82	784.1
575	Ac-LTF$r8HYWAQLSt/S-NH2	1299	16.03	9.02	826.98
576	Ac-LTF$r8HYWAQL$S-NHsBu	1300	1653.93	827.97	828.02
577	Ac-STF$r8AYWAQL$S-NH2	1301	1505.79	753.90	753.94
578	Ac-LTF$r8AYWAEL$S-NH2	1302	1532.83	767.42	767.41
579	Ac-LTF$r8AYWAQL$E-NH2	1303	1573.85	787.93	787.98
580	mdPEG3-LTF$r8AYWAQL$S-NH2	1304	1679.92	840.96	840.97
581	Ac-LTF$r8AYWAQhL$S-NH2	1305	1545.86	773.93	774.31
583	Ac-LTF$r8AYWAQCha$S-NH2	1306	1571.88	786.94	787.3
584	Ac-LTF$r8AYWAQChg$S-NH2	1307	1557.86	779.93	780.4
585	Ac-LTF$r8AYWAQCba$S-NH2	1308	1543.84	772.92	780.13
586	Ac-LTF$r8AYWAQF$S-NH2	1309	1565.83	783.92	784.2
587	Ac-LTF4F$r8HYWAQhL$S-NH2	1310	1629.87	815.94	815.36
588	Ac-LTF4F$r8HYWAQCha$S-NH2	1311	1655.89	828.95	828.39
589	Ac-LTF4F$r8HYWAQChg$S-NH2	1312	1641.87	821.94	821.35
590	Ac-LTF4F$r8HYWAQCba$S-NH2	1313	1627.86	814.93	814.32
591	Ac-LTF4F$r8AYWAQhL$S-NH2	1314	1563.85	782.93	782.36
592	Ac-LTF4F$r8AYWAQCha$S-NH2	1315	1589.87	795.94	795.38
593	Ac-LTF4F$r8AYWAQChg$S-NH2	1316	1575.85	788.93	788.35
594	Ac-LTF4F$r8AYWAQCba$S-NH2	1317	1561.83	781.92	781.39
595	Ac-LTF3Cl$r8AYWAQhL$S-NH2	1318	1579.82	790.91	790.35
596	Ac-LTF3Cl$r8AYWAQCha$S-NH2	1319	1605.84	803.92	803.67
597	Ac-LTF3Cl$r8AYWAQChg$S-NH2	1320	1591.82	796.91	796.34
598	Ac-LTF3Cl$r8AYWAQCba$S-NH2	1321	1577.81	789.91	789.39
599	Ac-LTF$r8AYWAQhF$S-NH2	1322	1579.84	790.92	791.14
600	Ac-LTF$r8AYWAQF3CF3$S-NH2	1323	1633.82	817.91	818.15
601	Ac-LTF$r8AYWAQF3Me$S-NH2	1324	1581.86	791.93	791.32
602	Ac-LTF$r8AYWAQ1Nal$S-NH2	1325	1615.84	808.92	809.18
603	Ac-LTF$r8AYWAQBip$S-NH2	1326	1641.86	821.93	822.13
604	Ac-LTF$r8FYWAQL$A-NH2	1327	1591.88	796.94	797.33
605	Ac-LTF$r8HYWAQL$S-NHAm	1328	1667.94	834.97	835.92
606	Ac-LTF$r8HYWAQL$S-NHiAm	1329	1667.94	834.97	835.55
607	Ac-LTF$r8HYWAQL$S-NHnPr3Ph	1330	1715.94	858.97	859.79
608	Ac-LTF$r8HYWAQL$S-NHnBu3, 3Me	1331	1681.96	841.98	842.49
610	Ac-LTF$r8HYWAQL$S-NHnPr	1332	1639.91	820.96	821.58
611	Ac-LTF$r8HYWAQL$S-NHnEt2Ch	1333	1707.98	854.99	855.35
612	Ac-LTF$r8HYWAQL$S-NHHex	1334	1681.96	841.98	842.4
613	Ac-LTF$r8AYWAQL$S-NHmdPeg2	1335	1633.91	817.96	818.35
614	Ac-LTF$r8AYWAQL$A-NHmdPeg2	1336	1617.92	809.96	810.3
615	Ac-LTF$r8AYwAQL$A-NHmdPeg4	1337	1705.97	853.99	854.33
616	Ac-F$r8AYd14mwEAL$A-NH2	1338	1316.72	659.36	659.44
617	Ac-F$r8AYdl5clWEAL$A-NH2	1339	1336.66	669.33	669.43
618	Ac-LThF$r8AYWAQL$S-NH2	1340	1545.86	773.93	774.11
619	Ac-LT2Nal$r8AYwAQL$S-NH2	1341	1581.86	791.93	792.43
620	Ac-LTA$r8AYWAQL$S-NH2	1342	1455.81	728.91	729.15
621	Ac-LTF$r8AYWVQL$S-NH2	1343	1559.88	780.94	781.24
622	Ac-LTF$r8HYWAAL$A-NH2	1344	1524.85	763.43	763.86
623	Ac-LTF$r8VYWAQL$A-NH2	1345	1543.88	772.94	773.37
624	Ac-LTF$r8IYWAQL$S-NH2	1346	1573.89	787.95	788.17
625	Ac-FTF$r8VYWSQL$S-NH2	1347	1609.85	805.93	806.22
626	Ac-ITF$r8FYWAQL$S-NH2	1348	1607.88	804.94	805.2
627	Ac-2NalTF$r8VYWSQL$S-NH2	1349	1659.87	830.94	831.2
628	Ac-ITF$r8LYWSQL$S-NH2	1350	1589.89	795.95	796.13
629	Ac-FTF$r8FYWAQL$S-NH2	1351	1641.86	821.93	822.13
630	Ac-WTF$r8VYWAQL$S-NH2	1352	1632.87	817.44	817.69
631	Ac-WTF$r8WYWAQL$S-NH2	1353	1719.88	860.94	861.36
632	Ac-VTF$r8AYWSQL$S-NH2	1354	1533.82	767.91	768.19
633	Ac-WTF$r8FYWSQL$S-NH2	1355	1696.87	849.44	849.7
634	Ac-FTF$r8IYWAQL$S-NH2	1356	1607.88	804.94	805.2
635	Ac-WTF$r8VYWSQL$S-NH2	1357	1648.87	825.44	824.8
636	Ac-FTF$r8LYWSQL$S-NH2	1358	1623.87	812.94	812.8
637	Ac-YTF$r8FYWSQL$S-NH2	1359	1673.85	837.93	837.8
638	Ac-LTF$r8AY6clWEAL$A-NH2	1360	1550.79	776.40	776.14
639	Ac-LTF$r8AY6clWSQL$S-NH2	1361	1581.80	791.90	791.68
640	Ac-F$r8AY6clWSAL$A-NH2	1362	1294.65	648.33	647.67
641	Ac-F$r8AY6clWQAL$AA-NH2	1363	1406.72	704.36	703.84
642	Ac-LHF$r8AYWAQL$S-NH2	1364	1567.86	784.93	785.21
643	Ac-LTF$r8AYWAQL$S-NH2	1365	1531.84	766.92	767.17
644	Ac-LTF$r8AHWAQL$S-NH2	1366	1505.84	753.92	754.13
645	Ac-LTF$r8AYWAHL$S-NH2	1367	1540.84	771.42	771.61
646	Ac-LTF$r8AYWAQL$H-NH2	1368	1581.87	791.94	792.15
647	H-LTF$r8AYWAQL$A-NH2	1369	1473.84	737.92	737.29
648	Ac-HHF$r8AYWAQL$S-NH2	1370	1591.83	796.92	797.35
649	Ac-aAibWTF$r8VYWSQL$S-NH2	1371	1804.96	903.48	903.64
650	Ac-AibWTF$r8HYWAQL$S-NH2	1372	1755.91	878.96	879.4
651	Ac-AibAWTF$r8HYWAQL$S-NH2	1373	1826.95	914.48	914.7
652	Ac-fWTF$r8HYWAQL$S-NH2	1374	1817.93	909.97	910.1
653	Ac-AibWWTF$r8HYWAQL$S-NH2	1375	1941.99	972.00	972.2
654	Ac-WTF$r8LYWSQL$S-NH2	1376	1662.88	832.44	832.8
655	Ac-WTF$r8NleYWSQL$S-NH2	1377	1662.88	832.44	832.6
656	Ac-LTF$r8AYWSQL$a-NH2	1378	1531.84	766.92	767.2
657	Ac-LTF$r8EYWARL$A-NH2	1379	1601.90	801.95	802.1
658	Ac-LTF$r8EYWAHL$A-NH2	1380	1582.86	792.43	792.6
659	Ac-aTF$r8AYWAQL$S-NH2	1381	1489.80	745.90	746.08
660	Ac-AibTF$r8AYWAQL$S-NH2	1382	1503.81	752.91	753.11
661	Ac-AmfTF$r8AYWAQL$S-NH2	1383	1579.84	790.92	791.14
662	Ac-AmwTF$r8AYWAQL$S-NH2	1384	1618.86	810.43	810.66
663	Ac-NmLTF$r8AYWAQL$S-NH2	1385	1545.86	773.93	774.11
664	Ac-LNmTF$r8AYWAQL$S-NH2	1386	1545.86	773.93	774.11
665	Ac-LSarF$r8AYWAQL$S-NH2	1387	1501.83	751.92	752.18
667	Ac-LGF$r8AYWAQL$S-NH2	1388	1487.82	744.91	745.15
668	Ac-LTNmF$r8AYWAQL$S-NH2	1389	1545.86	773.93	774.2
669	Ac-TF$r8AYWAQL$S-NH2	1390	1418.76	710.38	710.64
670	Ac-ETF$r8AYWAQL$A-NH2	1391	1531.81	766.91	767.2
671	Ac-LTF$r8EYWAQL$A-NH2	1392	1573.85	787.93	788.1
672	Ac-LT2Nal$r8AYWSQL$S-NH2	1393	1597.85	799.93	800.4
673	Ac-LTF$r8AYWAAL$S-NH2	1394	1474.82	738.41	738.68
674	Ac-LTF$r8AYWAQhCha$S-NH2	1395	1585.89	793.95	794.19
675	Ac-LTF$r8AYWAQChg$S-NH2	1396	1557.86	779.93	780.97
676	Ac-LTF$r8AYWAQCba$S-NH2	1397	1543.84	772.92	773.19
677	Ac-LTF$r8AYWAQF3CF3$S-NH2	1398	1633.82	817.91	818.15
678	Ac-LTF$r8AYWAQ1Nal$S-NH2	1399	1615.84	808.92	809.18
679	Ac-LTF$r8AYWAQBip$S-NH2	1400	1641.86	821.93	822.32
680	Ac-LT2Nal$r8AYWAQL$S-NH2	1401	1581.86	791.93	792.15
681	Ac-LTF$r8AYWVQL$S-NH2	1402	1559.88	780.94	781.62
682	Ac-LTF$r8AWWAQL$S-NH2	1403	1554.86	778.43	778.65
683	Ac-FTF$r8VYWSQL$S-NH2	1404	1609.85	805.93	806.12
684	Ac-ITF$r8FYWAQL$S-NH2	1405	1607.88	804.94	805.2
685	Ac-ITF$r8LYWSQL$S-NH2	1406	1589.89	795.95	796.22
686	Ac-FTF$r8FYWAQL$S-NH2	1407	1641.86	821.93	822.41
687	Ac-VTF$r8AYWSQL$S-NH2	1408	1533.82	767.91	768.19
688	Ac-LTF$r8AHWAQL$S-NH2	1409	1505.84	753.92	754.31
689	Ac-LTF$r8AYWAQL$H-NH2	1410	1581.87	791.94	791.94
690	Ac-LTF$r8AYWAHL$S-NH2	1411	1540.84	771.42	771.61
691	Ac-aAibWTF$r8VYWSQL$S-NH2	1412	1804.96	903.48	903.9
692	Ac-AibWTF$r8HYWAQL$S-NH2	1413	1755.91	878.96	879.5
693	Ac-AibAWTF$r8HYWAQL$S-NH2	1414	1826.95	914.48	914.7
694	Ac-fWTF$r8HYWAQL$S-NH2	1415	1817.93	909.97	910.2
695	Ac-AibWWTF$r8HYWAQL$S-NH2	1416	1941.99	972.00	972.7
696	Ac-WTF$r8LYWSQL$S-NH2	1417	1662.88	832.44	832.7
697	Ac-WTF$r8NleYWSQL$S-NH2	1418	1662.88	832.44	832.7
698	Ac-LTF$r8AYWSQL$a-NH2	1419	1531.84	766.92	767.2
699	Ac-LTF$r8EYWARL$A-NH2	1420	1601.90	801.95	802.2
700	Ac-LTF$r8EYWAHL$A-NH2	1421	1582.86	792.43	792.6
701	Ac-aTF$r8AYWAQL$S-NH2	1422	1489.80	745.90	746.1
702	Ac-AibTF$r8AYWAQL$S-NH2	1423	1503.81	752.91	753.2
703	Ac-AmfTF$r8AYWAQL$S-NH2	1424	1579.84	790.92	791.2
704	Ac-AmwTF$r8AYWAQL$S-NH2	1425	1618.86	810.43	810.7
705	Ac-NmLTF$r8AYWAQL$S-NH2	1426	1545.86	773.93	774.1
706	Ac-LNmTF$r8AYWAQL$S-NH2	1427	1545.86	773.93	774.4
707	Ac-LSarF$r8AYWAQL$S-NH2	1428	1501.83	751.92	752.1
708	Ac-TF$r8AYWAQL$S-NH2	1429	1418.76	710.38	710.8
709	Ac-ETF$r8AYWAQL$A-NH2	1430	1531.81	766.91	767.4
710	Ac-LTF$r8EYWAQL$A-NH2	1431	1573.85	787.93	788.2
711	Ac-WTF$r8VYWSQL$S-NH2	1432	1648.87	825.44	825.2
713	Ac-YTF$r8FYWSQL$S-NH2	1433	1673.85	837.93	837.3
714	Ac-F$r8AY6clWSAL$A-NH2	1434	1294.65	648.33	647.74
715	Ac-ETF$r8EYWVQL$S-NH2	1435	1633.84	817.92	817.36
716	Ac-ETF$r8EHWAQL$A-NH2	1436	1563.81	782.91	782.36
717	Ac-ITF$r8EYWAQL$S-NH2	1437	1589.85	795.93	795.38
718	Ac-ITF$r8EHWVQL$A-NH2	1438	1575.88	788.94	788.42
719	Ac-ITF$r8EHWAQL$S-NH2	1439	1563.85	782.93	782.43
720	Ac-LTF4F$r8AYWAQCba$S-NH2	1440	1561.83	781.92	781.32
721	Ac-LTF3Cl$r8AYWAQhL$S-NH2	1441	1579.82	790.91	790.64
722	Ac-LTF3Cl$r8AYWAQCha$S-NH2	1442	1605.84	803.92	803.37
723	Ac-LTF3Cl$r8AYWAQChg$S-NH2	1443	1591.82	796.91	796.27
724	Ac-LTF3Cl$r8AYWAQCba$S-NH2	1444	1577.81	789.91	789.83
725	Ac-LTF$r8AY6clWSQL$S-NH2	1445	1581.80	791.90	791.75
726	Ac-LTF4F$r8HYWAQhL$S-NH2	1446	1629.87	815.94	815.36
727	Ac-LTF4F$r8HYWAQCba$S-NH2	1447	1627.86	814.93	814.32
728	Ac-LTF4F$r8AYWAQhL$S-NH2	1448	1563.85	782.93	782.36
729	Ac-LTF4F$r8AYWAQChg$S-NH2	1449	1575.85	788.93	788.35
730	Ac-ETF$r8EYWVAL$S-NH2	1450	1576.82	789.41	788.79
731	Ac-ETF$r8EHWAAL$A-NH2	1451	1506.79	754.40	754.8
732	Ac-ITF$r8EYWAAL$S-NH2	1452	1532.83	767.42	767.75
733	Ac-ITF$r8EHWVAL$A-NH2	1453	1518.86	760.43	760.81
734	Ac-ITF$r8EHWAAL$S-NH2	1454	1506.82	754.41	754.8
735	Pam-LTF$r8EYWAQL$S-NH2	1455	1786.07	894.04	894.48
736	Pam-ETF$r8EYWAQL$S-NH2	1456	1802.03	902.02	902.34
737	Ac-LTF$r8AYWLQL$S-NH2	1457	1573.89	787.95	787.39
738	Ac-LTF$r8EYWLQL$S-NH2	1458	1631.90	816.95	817.33
739	Ac-LTF$r8EHWLQL$S-NH2	1459	1605.89	803.95	804.29
740	Ac-LTF$r8VYWAQL$S-NH2	1460	1559.88	780.94	781.34
741	Ac-LTF$r8AYWSQL$S-NH2	1461	1547.84	774.92	775.33
742	Ac-ETF$r8AYWAQL$S-NH2	1462	1547.80	774.90	775.7
743	Ac-LTF$r8EYWAQL$S-NH2	1463	1589.85	795.93	796.33
744	Ac-LTF$r8HYWAQL$S-NHAm	1464	1667.94	834.97	835.37
745	Ac-LTF$r8HYWAQL$S-NHiAm	1465	1667.94	834.97	835.27
746	Ac-LTF$r8HYWAQL$S-NHnPr3Ph	1466	1715.94	858.97	859.42
747	Ac-LTF$r8HYWAQL$S-NHnBu3, 3Me	1467	1681.96	841.98	842.67
748	Ac-LTF$r8HYWAQL$S-NHnBu	1468	1653.93	827.97	828.24
749	Ac-LTF$r8HYWAQL$S-NHnPr	1469	1639.91	820.96	821.31
750	Ac-LTF$r8HYWAQL$S-NHnEt2Ch	1470	1707.98	854.99	855.35
751	Ac-LTF$r8HYWAQL$S-NHHex	1471	1681.96	841.98	842.4
752	Ac-LTF$r8AYWAQL$S-NHmdPeg2	1472	1633.91	817.96	855.35
753	Ac-LTF$r8AYWAQL$A-NHmdPeg2	1473	1617.92	809.96	810.58
754	Ac-LTF$r5AYWAAL$s8S-NH2	1474	1474.82	738.41	738.79
755	Ac-LTF$r8AYWCouQL$S-NH2	1475	1705.88	853.94	854.61
756	Ac-LTF$r8CouYWAQL$S-NH2	1476	1705.88	853.94	854.7
757	Ac-CouTF$r8AYWAQL$S-NH2	1477	1663.83	832.92	833.33
758	H-LTF$r8AYWAQL$A-NH2	1478	1473.84	737.92	737.29
759	Ac-HHF$r8AYWAQL$S-NH2	1479	1591.83	796.92	797.72
760	Ac-LT2Nal$r8AYWSQL$S-NH2	1480	1597.85	799.93	800.68
761	Ac-LTF$r8HCouWAQL$S-NH2	1481	1679.87	840.94	841.38
762	Ac-LTF$r8AYWCou2QL$S-NH2	1482	1789.94	895.97	896.51
763	Ac-LTF$r8Cou2YWAQL$S-NH2	1483	1789.94	895.97	896.5
764	Ac-Cou2TF$r8AYWAQL$S-NH2	1484	1747.90	874.95	875.42
765	Ac-LTF$r8ACou2WAQL$S-NH2	1485	1697.92	849.96	850.82
766	Dmaac-LTF$r8AYWAQL$S-NH2	1486	1574.89	788.45	788.82
767	Hexac-LTF$r8AYWAQL$S-NH2	1487	1587.91	794.96	795.11
768	Napac-LTF$r8AYWAQL$S-NH2	1488	1657.89	829.95	830.36
769	Pam-LTF$r8AYWAQL$S-NH2	1489	1728.06	865.03	865.45
770	Ac-LT2Nal$r8HYAAQL$S-NH2	1490	1532.84	767.42	767.61
771	Ac-LT2Nal$/r8HYWAQL$/S-NH2	1491	1675.91	838.96	839.1
772	Ac-LT2Nal$r8HYFAQL$S-NH2	1492	1608.87	805.44	805.9
773	Ac-LT2Nal$r8HWAAQL$S-NH2	1493	1555.86	778.93	779.08
774	Ac-LT2Nal$r8HYAWQL$S-NH2	1494	1647.88	824.94	825.04
775	Ac-LT2Nal$r8HYAAQW$S-NH2	1495	1605.83	803.92	804.05
776	Ac-LTW$r8HYWAQL$S-NH2	1496	1636.88	819.44	819.95
777	Ac-LT1Nal$r8HYWAQL$S-NH2	1497	1647.88	824.94	825.41

In some embodiments, a peptidomimetic macrocycles disclosed herein does not comprise a peptidomimetic macrocycle structure as shown in TABLE 2b.

TABLE 2c shows examples of non-crosslinked polypeptides comprising D-amino acids.

TABLE 2C
		SEQ		Exact	Found	Calc	Calc	Calc
SP	Sequence	ID NO:	Isomer	Mass	Mass	(M + 1)/1	(M + 2)/2	(M + 3)/3
765	Ac-tawyanfekllr-NH2	1498	777.46
766	Ac-tawyanf4CF3ekllr-NH2	1499	811.41

Example 3: Preparation of Peptidomimetic Macrocycles Using a Boc-Protected Amino Acid

Peptidomimetic macrocycle precursors comprising an R8 amino acid at position “i” and an S5 amino acid at position “i+7” were prepared. The amino acid at position “i+3” was a Boc-protected tryptophan, which was incorporated during solid-phase synthesis. Specifically, the Boc-protected tryptophan amino acid shown below was used during solid phase synthesis:

Metathesis was performed using a ruthenium catalyst prior to the cleavage and deprotection steps. The composition obtained following cyclization was determined by HPLC analysis, and was found to contain primarily peptidomimetic macrocycles having a crosslinker comprising a trans olefin (“iso2”, comprising the double bond in an E configuration). Unexpectedly, a ratio of 90:10 was observed for the trans and cis products, respectively.

Example 4: Preparation of Peptidomimetic Macrocycles Using a Boc-Protected Amino Acid

Peptidomimetic macrocycles were first dissolved in neat N, N-dimethylacetamide (DMA) to make 20× stock solutions over a concentration range of 20-140 mg/mL. The DMA stock solutions were diluted 20-fold in an aqueous vehicle containing 2% Solutol-HS-15, 25 mM histidine, and 45 mg/mL mannitol to obtain final concentrations of 1-7 mg/ml of the peptidomimetic macrocycles in 5% DMA, 2% Solutol-HS-15, 25 mM histidine, and 45 mg/mL mannitol. The final solutions were mixed gently by repeat pipetting or light vortexing. The final solutions were sonicated for 10 min at room temperature in an ultrasonic water bath. Careful visual observations were performed under a hood light using a 7× visual amplifier to determine if precipitates existed on the bottom of the flasks or as a suspension. Additional concentration ranges were tested as needed to determine the maximum solubility limit for each peptidomimetic macrocycle.

Example 5: X-Ray Co-Crystallography of Peptidomimetic Macrocycles in Complex with MDMX

For co-crystallization with peptide 46 (TABLE 2b), a stoichiometric amount of compound from a 100 mM stock solution in DMSO was added to a zebrafish MDMX protein solution. The solution was allowed to sit overnight at 4° C. before setting up crystallization experiments. Protein (residues 15-129, L46V/V95L) was obtained from an E. coli BL21 (DE3) expression system using the pET15b vector. Cells were grown at 37° C. and induced with 1 mM IPTG at an OD₆₀₀ of 0.7. Cells were allowed to grow an additional 18 hr at 23° C. The protein was purified using Ni-NT Agarose followed by Superdex 75 buffered with 50 mM NaPO₄, pH 8.0, 150 mM NaCl, and 2 mM TCEP, and concentrating to 24 mg/ml. The buffer was exchanged to 20 mM Tris, pH 8.0, 50 mM NaCl, and 2 mM DTT for crystallization experiments. Initial crystals were obtained with the Nextal AMS screen #94, and the final optimized reservoir was 2.6 M AMS, 75 mM Hepes, pH 7.5. Crystals grew routinely as thin plates at 4° C. and were cryo-protected by pulling the crystals through a solution containing concentrated (3.4 M) malonate followed by flash cooling, storage, and shipment in liquid nitrogen.

Data collection was performed at the APS at beamline 31-ID (SGX-CAT) at 100° K and wavelength 0.97929 Å. The beamline was equipped with a Rayonix 225-HE detector. For data collection, crystals were rotated through 180° in 1° increments using 0.8 second exposure times. Data were processed and reduced using Mosflm/scala (CCP4) in space group C2 (unit cell: a=109.2786, b=81.0836, c=30.9058 Å, α=90, β=89.8577, γ=90°). Molecular replacement with program Molrep (CCP4) was performed with the MDMX component of the structure, and two molecules were identified in the asymmetric unit. Initial refinement of just the two molecules of the zebrafish MDMX with program Refmac (CCP4) resulted in an R-factor of 0.3424 (R_free=0.3712) and rmsd values for bonds (0.018 Å) and angles (1.698°). The electron densities of the stapled peptide components, starting with Gln¹⁹ and including the entire aliphatic staple, were very clear. Further refinement with CNX using data to 2.3 Å resolution resulted in a model (comprised of 1448 atoms from MDMX, 272 atoms from the stapled peptides and 46 water molecules) that was well refined (R_f=0.2601, R_free=0.3162, rmsd bonds=0.007 Å and rmsd angles=0.916°).

Example 6: Circular Dichroism (CD) Analysis of Alpha-Helicity

Peptide solutions were analyzed by CD spectroscopy using a spectropolarimeter. A temperature controller was used to maintain temperature control of the optical cell. Results are expressed as mean molar ellipticity [0] (deg cm² dmol⁻¹) as calculated from the equation [θ]=θobs·MRW/10*l*c where θobs is the observed ellipticity in millidegrees, MRW is the mean residue weight of the peptide (peptide molecular weight/number of residues), l is the optical path length of the cell in centimeters, and c is the peptide concentration in mg/ml. Peptide concentrations were determined by amino acid analysis. Stock solutions of peptides were prepared in benign CD buffer (20 mM phosphoric acid, pH 2). The stock solutions were used to prepare peptide solutions of 0.05 mg/ml in either benign CD buffer or CD buffer with 50% trifluoroethanol (TFE) for analyses in a 10 mm path length cell. Variable wavelength measurements of peptide solutions were scanned at 4° C. from 195 to 250 nm, in 0.2 nm increments, and a scan rate 50 nm per minute. The average of six scans is reported.

TABLE 3 shows CD data for selected peptidomimetic macrocycles:

TABLE 3
	Molar	Molar	Molar	% Helix	% Helix
	Ellipticity	Ellipticity	Ellipticity	50% TFE	benign
	Benign	50% TFE	TFE − Molar	compared	compared
	(222 in	(222 in	Ellipticity	to 50% TFE	to 50% TFE
SP#	0% TFE)	50% TFE)	Benign	parent (CD)	parent (CD)
7	124	−19921.4	−20045.4	137.3	−0.9
11	−398.2	−16623.4	16225.2	106.1	2.5
41	−909	−21319.4	20410.4	136	5.8
43	−15334.5	−18247.4	2912.9	116.4	97.8
69	−102.6	−21509.7	−21407.1	148.2	0.7
71	−121.2	−17957	−17835.9	123.7	0.8
154	−916.2	−30965.1	−30048.9	213.4	6.3
230	−213.2	−17974	−17760.8	123.9	1.5
233	−477.9	−19032.6	−18554.7	131.2	3.3

Example 7: Direct Binding Assay MDM2 with Fluorescence Polarization (FP)

The assay was performed according to the following general protocol:

1. Dilute MDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5) to make 10 μM working stock solution.2. Add 30 μl of 10 μM of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices).3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12.4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM concentration at the last dilution point.5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10 μM with water (dilution 1:10) and then dilute with FP buffer from 10 μM to 40 nM (dilution 1:250). This is the working solution which will be a 10 nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use.6. Add 10 μl of 10 nM of FAM labeled peptide into each well and incubate, and read at different time points. K_D with 5-FAM-BaLTFEHYWAQLTS-NH₂ (SEQ ID NO: 943) is ˜13.38 nM.

Example 8: Competitive Fluorescence Polarization Assay for MDM2

MDM2 (41 kD) was diluted into FP buffer (high-salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5) to make a 84 nM (2×) working stock solution. 20 μl of the 84 nM (2×) protein stock solution was added into each well of a 96-well black microplate. 1 mM of FAM-labeled linear peptide (in 100% DMSO) was diluted to 100 μM with DMSO (dilution 1:10). Then, diluted solution was further diluted from 100 μM to 10 μM with water (dilution 1:10), and diluted again with FP buffer from 10 μM to 40 nM (dilution 1:250). The resulting working solution resulted in a 10 nM concentration in each well (dilution 1:4). The diluted FAM-labeled peptides were kept in the dark until use.

Unlabeled peptide dose plates were prepared with FP buffer starting with 1 μM (final) of the peptide. 5-fold serial dilutions were made for 6 points using the following dilution scheme. 10 mM of the solution (in 100% DMSO) with DMSO to 5 mM (dilution 1:2); dilution from 5 mM to 500 μM with H₂O (dilution 1:10); and dilution with FP buffer from 500 μM to 20 μM (dilution 1:25). 5-fold serial dilutions from 4 μM (4×) were made for 6 points. 10 l of the serial diluted unlabeled peptides were transferred to each well, which was filled with 20 μl of 84 nM of protein. 10 μl of 10 nM (4×) of FAM-labeled peptide was added into each well, and the wells were incubated for 3 h before being read.

Example 9: Direct Binding Assay MDMX with Fluorescence Polarization (FP)

MDMX (40 kD) was diluted into FP buffer (high-salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5) to make a 10 μM working stock solution. 30 μl of the 10 μM of protein stock solution was added into the A1 and B1 wells of a 96-well black microplate. 30 μl of FP buffer was added to columns A2 to A12, B2 to B12, C1 to C12, and D1 to D12. 2-fold or 3-fold series dilutions of protein stocks were created from A1, B1 into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM concentration at the last dilution point. 1 mM (in 100% DMSO) of a FAM-labeled linear peptide was diluted with DMSO to 100 μM (dilution 1:10). The resulting solution was diluted from 100 μM to 10 μM with water (dilution 1:10), and diluted again with FP buffer from 10 μM to 40 nM (dilution 1:250). The working solution resulted in 10 nM concentration in each well (dilution 1:4). The FAM-labeled peptides were kept in the dark until use. 10 μl of the 10 nM FAM-labeled peptide was added into each well, and the plate was incubated and read at different time points. The K_D with 5-FAM-BaLTFEHYWAQLTS-NH₂ (SEQ ID NO: 943) was −51 nM.

Example 10: Competitive Fluorescence Polarization Assay for MDMX

MDMX (40 kD) was diluted into FP buffer (high-salt buffer 200 mM NaCl, 5 mM CHAPS, pH 7.5) to make a 300 nM (2×) working stock solution. 20 μl of the 300 nM (2×) of protein stock solution was added into each well of 96-well black microplate. 1 mM (in 100% DMSO) of a FAM-labeled linear peptide was diluted with DMSO to a concentration of 100 μM (dilution 1:10). The solution was diluted from 100 μM to 10 μM with water (dilution 1:10), and diluted further with FP buffer from 10 μM to 40 nM (dilution 1:250). The final working solution resulted in a concentration of 10 nM per well (dilution 1:4). The diluted FAM-labeled peptide was kept in the dark until use. An unlabeled peptide dose plate was prepared with FP buffer starting with a concentration of 5 μM (final) of a peptide. 5-fold serial dilutions were prepared for 6 points using the following dilution scheme. 10 mM (in 100% DMSO) of the solution was diluted with DMSO to prepare a 5 mM (dilution 1:2) solution. The solution was diluted from 5 mM to 500 μM with H₂O (dilution 1:10), and diluted further with FP buffer from 500 μM to 20 μM (dilution 1:25). 5-fold serial dilutions from 20 μM (4×) were prepared for 6 points. 10 μl of the serially diluted unlabeled peptides were added to each well, which was filled with 20 μl of the 300 nM protein solution. 10 μl of the 10 nM (4×) FAM-labeled peptide solution was added into each well, and the wells were incubated for 3 h before reading.

Results from EXAMPLE 7-EXAMPLE 10 are shown in TABLE 4. The following scale is used: “+” represents a value greater than 1000 nM, “++” represents a value greater than 100 and less than or equal to 1000 nM, “+++” represents a value greater than 10 nM and less than or equal to 100 nM, and “++++” represents a value of less than or equal to 10 nM.

TABLE 4
SP#	IC50 (MDM2)	IC50 (MDMX)	Ki (MDM2)	Ki (MDMX)
3	++	++	+++	+++
4	+++	++	++++	+++
5	+++	++	++++	+++
6	++	++	+++	+++
7	+++	+++	++++	+++
8	++	++	+++	+++
9	++	++	+++	+++
10	++	++	+++	+++
11	+++	++	++++	+++
12	+	+	+++	++
13	++	++	+++	++
14	+++	+++	++++	++++
15	+++	++	+++	+++
16	+++	+++	++++	+++
17	+++	+++	++++	+++
18	+++	+++	++++	++++
19	++	+++	+++	+++
20	++	++	+++	+++
21	++	+++	+++	+++
22	+++	+++	++++	+++
23	++	++	+++	+++
24	+++	++	++++	+++
26	+++	++	++++	+++
28	+++	+++	++++	+++
30	++	++	+++	+++
32	+++	++	++++	+++
38	+	++	++	+++
39	+	++	++	++
40	++	++	++	+++
41	++	+++	+++	+++
42	++	++	+++	++
43	+++	+++	++++	+++
45	+++	+++	++++	++++
46	+++	+++	++++	+++
47	++	++	+++	+++
48	++	++	+++	+++
49	++	++	+++	+++
50	+++	++	++++	+++
52	+++	+++	++++	++++
54	++	++	+++	+++
55	+	+	++	++
65	+++	++	++++	+++
68	++	++	+++	+++
69	+++	++	++++	+++
70	++	++	++++	+++
71	+++	++	++++	+++
75	+++	++	++++	+++
77	+++	++	++++	+++
80	+++	++	++++	+++
81	++	++	+++	+++
82	++	++	+++	+++
85	+++	++	++++	+++
99	++++	++	++++	+++
100	++	++	++++	+++
101	+++	++	++++	+++
102	++	++	++++	+++
103	++	++	++++	+++
104	+++	++	++++	+++
105	+++	++	++++	+++
106	++	++	+++	+++
107	++	++	+++	+++
108	+++	++	++++	+++
109	+++	++	++++	+++
110	++	++	++++	+++
111	++	++	++++	+++
112	++	++	+++	+++
113	++	++	+++	+++
114	+++	++	++++	+++
115	++++	++	++++	+++
116	+	+	++	++
118	++++	++	++++	+++
120	+++	++	++++	+++
121	++++	++	++++	+++
122	++++	++	++++	+++
123	++++	++	++++	+++
124	++++	++	++++	+++
125	++++	++	++++	+++
126	++++	++	++++	+++
127	++++	++	++++	+++
128	++++	++	++++	+++
129	++++	++	++++	+++
130	++++	++	++++	+++
133	++++	++	++++	+++
134	++++	++	++++	+++
135	++++	++	++++	+++
136	++++	++	++++	+++
137	++++	++	++++	+++
139	++++	++	++++	+++
142	++++	+++	++++	+++
144	++++	++	++++	+++
146	++++	++	++++	+++
148	++++	++	++++	+++
150	++++	++	++++	+++
153	++++	+++	++++	+++
154	++++	+++	++++	++++
156	++++	++	++++	+++
158	++++	++	++++	+++
160	++++	++	++++	+++
161	++++	++	++++	+++
166	++++	++	++++	+++
167	+++	++	++++	++
169	++++	+++	++++	+++
170	++++	++	++++	+++
173	++++	++	++++	+++
175	++++	++	++++	+++
177	+++	++	++++	+++
180	+++	++	++++	+++
182	++++	++	++++	+++
185	+++	+	++++	++
186	+++	++	++++	+++
189	+++	++	++++	+++
192	+++	++	++++	+++
194	+++	++	++++	++
196	+++	++	++++	+++
197	++++	++	++++	+++
199	+++	++	++++	++
201	+++	++	++++	++
203	+++	++	++++	+++
204	+++	++	++++	+++
206	+++	++	++++	+++
207	++++	++	++++	+++
210	++++	++	++++	+++
211	++++	++	++++	+++
213	++++	++	++++	+++
215	+++	++	++++	+++
217	++++	++	++++	+++
218	++++	++	++++	+++
221	++++	+++	++++	+++
227	++++	++	++++	+++
230	++++	+++	++++	++++
232	++++	++	++++	+++
233	++++	+++	++++	+++
236	+++	++	++++	+++
237	+++	++	++++	+++
238	+++	+++	++++	+++
239	+++	++	+++	+++
240	+++	++	++++	+++
241	+++	++	++++	+++
242	+++	++	++++	+++
243	+++	+++	++++	+++
244	+++	+++	++++	++++
245	+++	+++	++++	+++
246	+++	++	++++	+++
247	+++	+++	++++	+++
248	+++	+++	++++	+++
249	+++	+++	++++	++++
250	++	+	++	+
252	++	+	++	+
254	+++	++	++++	+++
255	+++	+++	++++	+++
256	+++	+++	++++	+++
257	+++	+++	++++	+++
258	+++	++	++++	+++
259	+++	+++	++++	+++
260	+++	+++	++++	+++
261	+++	++	++++	+++
262	+++	++	++++	+++
263	+++	++	++++	+++
264	+++	+++	++++	+++
266	+++	++	++++	+++
267	+++	+++	++++	++++
270	++++	+++	++++	+++
271	++++	+++	++++	++++
272	++++	+++	++++	++++
276	+++	+++	++++	++++
277	+++	+++	++++	++++
278	+++	+++	++++	++++
279	++++	+++	++++	+++
280	+++	++	++++	+++
281	+++	+	+++	++
282	++	+	+++	+
283	+++	++	+++	++
284	+++	++	++++	+++
289	+++	+++	++++	+++
291	+++	+++	++++	++++
293	++++	+++	++++	+++
306	++++	++	++++	+++
308	++	++	+++	+++
310	+++	+++	++++	+++
312	+++	++	+++	+++
313	++++	++	++++	+++
314	++++	+++	++++	++++
315	+++	+++	++++	+++
316	++++	++	++++	+++
317	+++	++	+++	+++
318	+++	++	+++	+++
319	+++	++	+++	++
320	+++	++	+++	++
321	+++	++	++++	+++
322	+++	++	+++	++
323	+++	+	+++	++
328	+++	+++	++++	+++
329	+++	+++	++++	+++
331	++++	+++	++++	++++
332	++++	+++	++++	++++
334	++++	+++	++++	++++
336	++++	+++	++++	++++
339	++++	++	++++	+++
341	+++	+++	++++	++++
343	+++	+++	++++	++++
347	+++	+++	++++	+++
349	++++	+++	++++	++++
351	++++	+++	++++	++++
353	++++	+++	++++	++++
355	++++	+++	++++	++++
357	++++	+++	++++	++++
359	++++	+++	++++	+++
360	++++	++++	++++	++++
363	+++	+++	++++	++++
364	+++	+++	++++	++++
365	+++	+++	++++	++++
366	+++	+++	++++	+++
369	++	++	+++	+++
370	+++	+++	++++	+++
371	++	++	+++	+++
372	++	++	+++	+++
373	+++	+++	+++	+++
374	+++	+++	++++	++++
375	+++	+++	++++	++++
376	+++	+++	++++	++++
377	+++	+++	++++	+++
378	+++	+++	++++	+++
379	+++	+++	++++	+++
380	+++	+++	++++	+++
381	+++	+++	++++	+++
382	+++	+++	++++	++++
384	++	+	++	+
386	++	+	++	+
388	++	+++	+++	++++
390	+++	+++	++++	+++
392	+++	+++	++++	++++
394	++++	+++	++++	++++
396	++++	++++	++++	++++
398	+++	+++	++++	+++
402	++++	++++	++++	++++
404	+++	+++	++++	++++
408	+++	+++	++++	+++
410	++++	++++	++++	++++
411	++	+	++	+
412	++++	+++	++++	++++
415	++++	++++	++++	++++
416	+++	+++	++++	+++
417	+++	+++	++++	+++
418	++++	+++	++++	++++
419	+++	+++	+++	++++
421	++++	++++	++++	++++
423	+++	+++	++++	+++
425	+++	+++	+++	+++
427	++	++	+++	+++
432	++++	+++	++++	++++
434	+++	+++	++++	+++
435	++++	+++	++++	++++
437	+++	+++	++++	+++
439	++++	+++	++++	++++
441	++++	++++	++++	++++
443	+++	+++	++++	+++
445	+++	++	++++	+++
446	+++	+	++++	+
447	++	+	++	+
551	N/A	N/A	++++	+++
555	N/A	N/A	++++	+++
556	N/A	N/A	++++	+++
557	N/A	N/A	+++	+++
558	N/A	N/A	+++	+++
559	N/A	N/A	+++	+++
560	N/A	N/A	+	+
561	N/A	N/A	++++	+++
562	N/A	N/A	+++	+++
563	N/A	N/A	+++	+++
564	N/A	N/A	++++	+++
565	N/A	N/A	+++	+++
566	N/A	N/A	++++	+++
567	N/A	N/A	++++	+++
568	N/A	N/A	++++	++++
569	N/A	N/A	++++	+++
570	N/A	N/A	++++	+++
571	N/A	N/A	++++	+++
572	N/A	N/A	+++	+++
573	N/A	N/A	+++	+++
574	N/A	N/A	++++	+++
575	N/A	N/A	++++	+++
576	N/A	N/A	++++	+++
577	N/A	N/A	++++	+++
578	N/A	N/A	++++	+++
585	N/A	N/A	+++	+++
586	N/A	N/A	++++	+++
587	N/A	N/A	++++	++++
589	N/A	N/A	++++
594	N/A	N/A	++++	++++
596	N/A	N/A	++++	+++
597	N/A	N/A	++++	+++
598	N/A	N/A	++++	+++
600	N/A	N/A	++++	++++
602	N/A	N/A	++++	++++
603	N/A	N/A	++++	++++
604	N/A	N/A	+++	+++
608	N/A	N/A	++++	+++
609	N/A	N/A	++++	+++
610	N/A	N/A	++++	+++
611	N/A	N/A	++++	+++
612	N/A	N/A	++++	+++
613	N/A	N/A	++++	+++
615	N/A	N/A	++++	++++
433	N/A	N/A	++++	+++
686	N/A	N/A	++++	+++
687	N/A	N/A	++	++
595	N/A	N/A	+	N/A
665	N/A	N/A	+++	N/A
708	N/A	N/A	+++	+++
710	N/A	N/A	+++	+++
711	N/A	N/A	+++	++
712	N/A	N/A	++++	++++
713	N/A	N/A	++++	++++
716	N/A	N/A	++++	++++
765	+	+
766	+++	+
752	++	+
753	+++	+
754	++	+
755	++++	+
756	+++	+
757	++++	+
758	+++	+

Example 11: Competition Binding ELISA Assay for MDM2 and MDMX

p53-His6 protein (30 nM/well) was coated overnight at room temperature in the wells of 96-well plates. On the day of the experiment, the plates were washed with 1×PBS-Tween 20 (0.05%) using an automated ELISA plate washer, and blocked with ELISA microwell blocking buffer for 30 minutes at room temperature. The excess blocking agent was washed off by washing the plates with 1×PBS-Tween 20 (0.05%). The peptides were diluted from 10 mM DMSO stock solutions to 500 μM working stock solutions using sterile water. Further dilutions were made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The peptide solutions were added to the wells at 2× the desired concentrations in 50 μL volumes, followed by addition of diluted GST-MDM2 or GST-HMDX protein (final concentration: 10 nM). The samples were incubated at room temperature for 2 h, and the plates were washed with PBS-Tween 20 (0.05%) prior to adding 100 μL of HRP-conjugated anti-GST antibody diluted to 0.5 μg/ml in HRP-stabilizing buffer. The plates were incubated with a detection antibody for 30 min, and the plates were washed and incubated with 100 μL per well of TMB-E substrate solution for up to 30 minutes. The reactions were stopped using 1M HCL, and absorbance was measured at 450 nm using a micro plate reader. The data were analyzed using Graph Pad PRISM software.

Example 12: Cell Viability Assay

Cells were trypsinized, counted, and seeded at pre-determined densities in 96-well plates one day prior to conducting the cell viability assay. The following cell densities were used for each cell line: SJSA-1: 7500 cells/well; RKO: 5000 cells/well; RKO-E6: 5000 cells/well; HCT-116: 5000 cells/well; SW-480: 2000 cells/well; and MCF-7: 5000 cells/well. On the day of cell viability assay, the media was replaced with fresh media containing 11% FBS (assay media) at room temperature. 180 μL of the assay media was added to each well. Control wells were prepared with no cells, and the control wells received 200 μL of media.

Peptide dilutions were made at room temperature, and the diluted peptide solutions were added to the cells at room temperature. 10 mM stock solutions of the peptides were prepared in DMSO. The stock solutions were serially diluted using a 1:3 dilution scheme to obtain 10 mM, 3.3 mM, 1.1 mM, 0.33 mM, 0.11 mM, 0.03 mM, and 0.01 mM solutions in DMSO. The serially DMSO-diluted peptides were diluted 33.3 times using sterile water, resulting in a range of 10× working stock solutions. A DMSO/sterile water (3% DMSO) solution was prepared for use in the control well. The working stock solution concentrations ranges were 300 μM, 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0 μM. The solutions were mixed well at each dilution step using a multichannel pipette.

Row H of the plate contained the controls. Wells H1-H3 received 20 μL of assay media. Rows H4-H9 received 20 μL of the 3% DMSO-water vehicle. Wells H10-H12 received media alone control with no cells. The MDM2 small molecule inhibitor Nutlin-3a (10 mM) was used as a positive control. Nutlin-3a was diluted using the same dilution scheme used for the peptides.

20 μL of a 10× concentration peptide stock solution was added to the appropriate well to achieve the final concentration in 200 μL in each well. For example, 20 μL of 300 μM peptide solution+180 μL of cells in media=30 μM final concentration in 200 μL volume in wells. The solution was mixed gently a few times using a pipette. The final concentration range was 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, and 0 μM. Further dilutions were used for potent peptides. Controls included wells that received no peptides, but contained the same concentration of DMSO as the wells containing peptides and wells containing no cells. The plates were incubated for 72 hours at 37° C. in a humidified 5% CO₂ atmosphere.

The viability of the cells was determined using MTT reagent. The viability of SJSA-1, RKO, RKO-E6, HCT-116 cells was determined on day 3. The viability of MCF-7 cells was determined on day 5. The viability of SW-480 cells was determined on on day 6. At the end of the designated incubation time, the plates were cooled to room temperature. 80 μL of assay media was removed from each well. 15 μL of thawed MTT reagent was then added to each well. The plate was incubated for 2 h at 37° C. in a humidified 5% CO₂ atmosphere. 100 μL of the solubilization reagent was added to each well. The plates were incubated with agitation for 1 h at room temperature, and read using a multiplate reader for absorbance at 570 nM. Cell viability was analyzed against the DMSO controls.

Results from cell viability assays are shown in TABLE 5 and TABLE 6. “+” represents a value greater than 30 μM, “++” represents a value greater than 15 μM and less than or equal to 30 M, “+++” represents a value greater than 5 μM and less than or equal to 15 μM, and “++++” represents a value of less than or equal to 5 μM. “IC₅₀ ratio” represents the ratio of average IC₅₀ in p53+/+ cells relative to average IC₅₀ in p53−/− cells.

TABLE 5
    SJSA-1 EC50
SP# (72 h)
3   +++
4   +++
5   ++++
6   ++
7   ++++
8   +++
9   +++
10  +++
11  ++++
12  ++
13  +++
14  +
15  ++
16  +
17  +
18  +
19  ++
20  +
21  +
22  +
24  +++
26  ++++
28  +
29  +
30  +
32  ++
38  +
39  +
40  +
41  +
42  +
43  ++
45  +
46  +
47  +
48  +
49  +++
50  ++++
52  +
54  +
55  +
65  ++++
68  ++++
69  ++++
70  ++++
71  ++++
72  ++++
74  ++++
75  ++++
77  ++++
78  ++
80  ++++
81  +++
82  +++
83  +++
84  +
85  +++
99  ++++
102 +++
103 +++
104 +++
105 +++
108 +++
109 +++
110 +++
111 ++
114 ++++
115 ++++
118 ++++
120 ++++
121 ++++
122 ++++
123 ++++
124 +++
125 ++++
126 ++++
127 ++++
128 +++
129 ++
130 ++++
131 +++
132 ++++
133 +++
134 +++
135 +++
136 ++
137 +++
139 ++++
142 +++
144 ++++
147 ++++
148 ++++
149 ++++
150 ++++
152 +++
153 ++++
154 ++++
155 ++
156 +++
157 +++
158 +++
160 ++++
161 ++++
162 +++
163 +++
166 ++
167 +++
168 ++
169 ++++
170 ++++
171 ++
173 +++
174 ++++
175 +++
176 +++
177 ++++
179 +++
180 +++
181 +++
182 ++++
183 ++++
184 +++
185 +++
186 ++
188 ++
190 ++++
192 +++
193 ++
194 +
195 ++++
196 ++++
197 ++++
198 ++
199 +++
200 +++
201 ++++
202 +++
203 ++++
204 ++++
205 ++
206 ++
207 +++
208 +++
209 ++++
210 +++
211 ++++
213 ++++
214 ++++
215 ++++
216 ++++
217 ++++
218 ++++
219 ++++
220 +++
221 ++++
222 +++
223 ++++
224 ++
225 +++
226 ++
227 +++
228 ++++
229 ++++
230 ++++
231 ++++
232 ++++
233 ++++
234 ++++
235 ++++
236 ++++
237 ++++
238 ++++
239 +++
240 ++
241 +++
242 ++++
243 ++++
244 ++++
245 ++++
246 +++
247 ++++
248 ++++
249 ++++
250 ++
251 +
252 +
253 +
254 +++
255 +++
256 ++
257 +++
258 +++
259 ++
260 ++
261 ++
262 +++
263 ++
264 ++++
266 +++
267 ++++
270 ++
271 ++
272 ++
276 ++
277 ++
278 ++
279 ++++
280 +++
281 ++
282 ++
283 ++
284 ++++
289 ++++
290 +++
291 ++++
292 ++++
293 ++++
294 ++++
295 +++
296 ++++
297 +++
298 ++++
300 ++++
301 ++++
302 ++++
303 ++++
304 ++++
305 ++++
306 ++++
307 +++
308 ++++
309 +++
310 ++++
312 ++++
313 ++++
314 ++++
315 ++++
316 ++++
317 ++++
318 ++++
319 ++++
320 ++++
321 ++++
322 ++++
323 ++++
324 ++++
326 ++++
327 ++++
328 ++++
329 ++++
330 ++++
331 ++++
332 ++++
333 ++
334 +++
335 ++++
336 ++++
337 ++++
338 ++++
339 ++++
340 ++++
341 ++++
342 ++++
343 ++++
344 ++++
345 ++++
346 ++++
347 ++++
348 ++++
349 ++++
350 ++++
351 ++++
352 ++++
353 ++++
355 ++++
357 ++++
358 ++++
359 ++++
360 ++++
361 +++
362 ++++
363 ++++
364 ++++
365 +++
366 ++++
367 ++++
368 +
369 ++++
370 ++++
371 ++++
372 +++
373 +++
374 ++++
375 ++++
376 ++++
377 ++++
378 ++++
379 ++++
380 ++++
381 ++++
382 ++++
386 +++
388 ++
390 ++++
392 +++
394 +++
396 +++
398 +++
402 +++
404 +++
408 ++++
410 +++
411 +++
412 +
421 +++
423 ++++
425 ++++
427 ++++
434 +++
435 ++++
436 ++++
437 ++++
438 ++++
439 ++++
440 ++++
441 ++++
442 ++++
443 ++++
444 +++
445 ++++
449 ++++
551 ++++
552 ++++
554 +
555 ++++
586 ++++
587 ++++
588 ++++
589 +++
432 ++++
672 +
673 ++
682 +
686 +
557 ++++
558 ++++
560 +
561 ++++
562 ++++
563 ++++
564 ++++
566 ++++
567 ++++
568 +++
569 ++++
571 ++++
572 ++++
573 ++++
574 ++++
575 ++++
576 ++++
577 ++++
578 ++++
585 ++++
687 +
662 ++++
663 ++++
553 +++
559 ++++
579 ++++
581 ++++
582 ++
582 ++++
584 +++
675 ++++
676 ++++
677 +
679 ++++
700 +++
704 +++
591 +
706 ++
695 ++
595 ++++
596 ++++
597 +++
598 +++
599 ++++
600 ++++
601 +++
602 +++
603 +++
604 +++
606 ++++
607 ++++
608 ++++
610 ++++
611 ++++
612 ++++
613 +++
614 +++
615 ++++
618 ++++
619 ++++
707 ++++
620 ++++
621 ++++
622 ++++
623 ++++
624 ++++
625 ++++
626 +++
631 ++++
633 ++++
634 ++++
635 +++
636 +++
638 +
641 +++
665 ++++
708 ++++
709 +++
710 +
711 ++++
712 ++++
713 ++++
714 +++
715 +++
716 ++++
765 +
753 +
754 +
755 +
756 +
757 ++++
758 +++

TABLE 6
				SW480
	HCT-116 EC50	RKO EC50	RKO-E6 EC50	EC50	IC50
SP#	(72 h)	(72 h)	(72 h)	(6 days)	Ratio
4	++++	++++	+++	++++
5	++++	++++	+++	++++
7	++++	++++	+++	++++
10	++++	+++	+++	+++
11	++++	++++	++	+++
50	++++	++++	++	+++
65	+++	+++	+++	+++
69	++++	++++	+	++++
70	++++	++++	++	+++
71	++++	++++	+++	+++
81	+++	+++	+++	+++
99	++++	++++	+++	++++
109	++++	++++	++	+++
114		+++	+	+++
115		+++	+	+++	1-29
118	+++	++++	+	++++
120	++++	++++	+	++++
121	++++	++++	+	++++
122		+++	+	+++	1-29
125	+++	+++	+	+
126	+	+	+	+
148		++	+	+
150		++	+	+
153	+++		+
154	+++	+++	+	+	30-49
158	+	+	+	+
160	+++	+	+	+	1-29
161	+++	+	+	+
175	+	+	+	+
196	++++	++++	+++	++++
219	++++	+++	+	+	1-29
233	++++
237	++++		+	+
238	++++		+	+
243	++++		+	+
244	++++		+	+	≥50
245	++++		+	+
247	++++		+	+
249	++++	++++	+	+	≥50
255	++++		+
291			+
293	+++		+
303	+++		+		1-29
305			+
306	++++		+
310	++++		+
312	++++
313	++++		++
314			+
315	++++	++++	++	++++	≥50
316	++++	++++	+	+++	≥50
317	+++		+	++
321	++++		+
324	+++		+
325	+++
326	+++		+
327	+++		+
328	+++		++
329	++++		+
330			+
331	++++	++++	+	+	≥50
338	++++	++++	++	+++
341	+++	++	+	+
343	+++		+	+
346	++++		+	+
347	+++		+	+
349	++++	+++	+	+	30-49
350	++++		+	+
351	++++	+++	+	+	30-49
353	++	++	+	+
355	++++	++	+	+	1-29
357	++++	++++	+	+
358	++++	++	+	+
359	++++	++	+	+
367	++++		+	+	30-49
386	++++	++++	++++	++++
388	++	++	+	+++	1-29
390	++++	++++	+++	++++
435	+++	++	+
436	++++	++++	++
437	++++	++++	++	++++	30-49
440	++	++	+
442	++++	++++	++
444	++++	++++	+++
445	++++	+++	+	+	≥50
555					≥50
557					≥50
558					30-49
562					30-49
564					30-49
566					30-49
567					≥50
572					≥50
573					30-49
578					30-49
662					≥50
379					1-29
375					1-29
559					≥50
561					1-29
563					1-29
568					1-29
569					1-29
571					1-29
574					1-29
575					1-29
576					1-29
577					30-49
433					1-29
551					30-49
553					1-29
710				+
711				+
712				++
713				++
714				+++
715				+++
716				+

Example 13: p21 ELISA Assay

SJSA-1 cells were trypsinized, counted, and seeded at a density of 7500 cells/100 μL/well in 96-well plates one day prior to running the assay. On the day of the assay, the media was replaced with fresh RPMI-11% FBS assay media. 90 μL of the assay media was added to each well. The control wells contained no cells and received 100 μL of the assay media.

10 mM stock solutions of the peptides were prepared in DMSO. The stock solutions were serially diluted using a 1:3 dilution scheme to obtain 10 mM, 3.3 mM, 1.1 mM, 0.33 mM, 0.11 mM, 0.03 mM, and 0.01 mM solutions in DMSO. The solutions were serially diluted 33.3 times using sterile water to provide a range of 10× working stock solutions. A DMSO/sterile water (3% DMSO) solution was prepared for use in the control wells. The working stock solution concentration range was 300 μM, 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0 μM. Each solution was mixed well at each dilution step using a multichannel pipette. Row H contained the control wells. Wells H1-H3 received 10 μL of the assay media. Wells H4-H9 received 10 μL of the 3% DMSO-water solution. Wells H10-H12 received media alone and contained no cells. The MDM2 small molecule inhibitor Nutlin-3a (10 mM) was used as a positive control. Nutlin-3a was diluted using the same dilution scheme used for the peptides.

10 μL of a 10× peptide solution was added to the appropriate well to achieve a final concentration in a volume of 100 μL. For example, 10 μL of 300 μM peptide+90 μL of cells in media=30 μM final concentration in 100 μL volume in wells. The final concentration range used was 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0 μM. Control wells included wells that did not receive peptides but contained the same concentration of DMSO as the wells containing the peptides and wells containing no cells.

20 h after incubation, the media was aspirated from the wells. The cells were washed with 1×PBS (without Ca⁺⁺/Mg⁺⁺) and lysed in 60 μL of 1× cell lysis buffer (10× buffer diluted to 1× and supplemented with protease inhibitors and phosphatase inhibitors) on ice for 30 min. The plates were centrifuged at 5000 rpm at 4° C. for 8 min. The clear supernatants were collected and frozen at −80° C. until further use. The total protein contents of the lysates were measured using a BCA protein detection kit and BSA standards. Each well provided about 6-7 μg of protein. 50 μL of the lysate was used per well to set up the p21 ELISA assay. For the human total p21 ELISA assay, 50 μL of lysate was used for each well, and each well was set up in triplicate.

Example 14: Caspase 3 Detection Assay

SJSA-1 cells were trypsinized, counted, and seeded at a density of 7500 cells/100 μL/well in 96-well plates one day prior to conducting the assay. One the day of the assay, the media was replaced with fresh RPMI-11% FBS assay media. 180 μL of the assay media was added to each well. Control wells contained no cells, and received 200 μL of the assay media.

10 mM stock solutions of the peptides were prepared in DMSO. The stock solutions were serially diluted using a 1:3 dilution scheme to obtain 10 mM, 3.3 mM, 1.1 mM, 0.33 mM, 0.11 mM, 0.03 mM, and 0.01 mM solutions in DMSO. The solutions were serially diluted 33.3 times using sterile water to provide a range of 10× working stock solutions. A DMSO/sterile water (3% DMSO) solution was prepared for use in the control wells. The working stock solution concentration range was 300 μM, 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0 μM. Each well was mixed well at each dilution step using a multichannel pipette. 20 μL of the 10× working stock solutions were added to the appropriate wells. Row H of the plates had control wells. Wells H1-H3 received 20 μL of the assay media. Wells H4-H9 received 20 μL of the 3% DMSO-water solutions. Wells H10-H12 received media and had no cells. The MDM2 small molecule inhibitor Nutlin-3a (10 mM) was used as a positive control. Nutlin-3a was diluted using the same dilution scheme as the peptides.

10 μL of the 10× stock solutions were added to the appropriate wells to achieve the final concentrations in a total volume of 100 μL. For example, 10 μL of 300 μM peptide+90 μL of cells in media=30 μM final concentration in 100 μL volume in wells. The final concentration range used was 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0 μM. Control wells contained no peptides but contained the same concentration of DMSO as the wells containing the peptides and well containing no cells. 48 h after incubation, 80 μL of the media was aspirated from each well. 100 μL of Caspase 3/7Glo assay reagent was added to each well. The plates were incubated with gentle shaking for 1 h at room temperature and read using a multi-plate reader for luminescence. Data were analyzed as Caspase 3 activation over DMSO-treated cells. Results from EXAMPLE 13 and EXAMPLE 14 are shown in TABLE 7.

TABLE 7
	caspase	caspase	caspase	caspase	caspase	p21	p21	p21	p21	p21
SP#	0.3 μM	1 μM	3 μM	10 μM	30 μM	0.3 μM	1 μM	3 μM	10 μM	30 μM
4			9	37	35			317	3049	3257
7	0.93	1.4	5.08	21.7	23.96		18	368	1687	2306
8			1	19	25			34	972	2857
10	1		1	17	32		10	89	970	2250
11	1		5	23	33.5		140	350	2075.5	3154
26	1		1	3	14
50			8	29	29		44	646	1923	1818
65	1		6	28	34	−69	−24	122	843	1472
69	4.34	9.51	16.39	26.59	26.11	272	458.72	1281.39	2138.88	1447.22
70			1	9	26		−19	68	828	1871
71	0.95	1.02	3.68	14.72	23.52		95	101	1204	2075
72	1		1	4	10	−19	57	282	772	1045
77	1		2	19	23
80	1		2	13	20
81	1		1	6	21		0	0	417	1649
99	1		7	31	33	−19	117	370	996	1398
109			4	16	25		161	445	1221	1680
114	1		6	28	34	−21	11	116	742	910
115	1		10	26	32	−10	36	315	832	1020
118	1		2	18	27	−76	−62	−11	581	1270
120	2		11	20	30	−4	30	164	756	1349
121	1		5	19	30	9	33	81	626	1251
122	1		2	15	30	−39	−18	59	554	1289
123	1		1	6	14
125	1		3	9	29	50	104	196	353	1222
126	1		1	6	30	−47	−10	90	397	1443
127	1		1	4	13
130	1		2	6	17
139	1		2	9	18
142	1		2	15	20
144	1		4	10	16
148	1		11	23	31	−23	55	295	666	820
149	1		2	4	10	35	331	601	1164	1540
150	2		11	19	35	−37	24	294	895	906
153	2		10	15	20
154	2.68	4	13.93	19.86	30.14	414.04	837.45	1622.42	2149.51	2156.98
158	1		1.67	5	16.33	−1.5	95	209.5	654	1665.5
160	2		10	16	31	−43	46	373	814	1334
161	2		8	14	22	13	128	331	619	1078
170	1		1	16	20
175	1		5	12	21	−65	1	149	543	1107
177	1		1	8	20
183	1		1	4	8	−132	−119	−14	1002	818
196	1		4	33	26	−49	−1	214	1715	687
197	1		1	10	20
203	1		3	12	10	77	329	534	1805	380
204	1		4	10	10	3	337	928	1435	269
218	1		2	8	18
219	1		5	17	34	28	53	289	884	1435
221	1		3	6	12	127	339	923	1694	1701
223	1		1	5	18
230	1		2	3	11	245.5	392	882	1549	2086
233	6	8	17	22	23	2000	2489	3528	3689	2481
237	1		5	9	15	0	0	2	284	421
238	1		2	4	21	0	149	128	825	2066
242	1		4	5	18	0	0	35	577	595
243	1		2	5	23	0	0	0	456	615
244	1		2	7	17	0	178	190	708	1112
245	1		3	9	16	0	0	0	368	536
247	1		3	11	24	0	0	49	492	699
248						0	50	22	174	1919
249	2		5	11	23	0	0	100	907	1076
251						0	0	0	0	0
252						0	0	0	0	0
253						0	0	0	0	0
254	1	3	7	14	22	118	896	1774	3042	3035
286	1	4	11	20	22	481	1351	2882	3383	2479
287	1	1	3	11	23	97	398	986	2828	3410
315	11	14.5	25.5	32	34	2110	2209	2626	2965	2635
316	6.5	10.5	21	32	32.5	1319	1718	2848	2918	2540
317	3	4	9	26	35	551	624	776	1367	1076
331	4.5	8	11	14.5	30.5	1510	1649	2027	2319	2509
338	1	5	23	20	29	660.37	1625.38	3365.87	2897.62	2727
341	3	8	11	14	21	1325.62	1873	2039.75	2360.75	2574
343	1	1	2	5	29	262	281	450	570	1199
346						235.86	339.82	620.36	829.32	1695.78
347	2	3	5	8	29	374	622	659	905	1567
349	1	8	11	16	24	1039.5	1598.88	1983.75	2191.25	2576.38
351	3	9	13	15	24	1350.67	1710.67	2030.92	2190.67	2668.54
353	1	2	5	7	30	390	490	709	931	1483
355	1	4	11	13	30	191	688	1122	1223	1519
357	2	7	11	15	23	539	777	1080	1362	1177
358	1	2	3	6	24	252	321	434	609	1192
359	3	9	11	13	23	1163.29	1508.79	1780.29	2067.67	2479.29
416						33.74	39.82	56.57	86.78	1275.28
417						0	0	101.13	639.04	2016.58
419						58.28	97.36	221.65	1520.69	2187.94
432						54.86	68.86	105.11	440.28	1594.4

Example 15: Cell Lysis by Peptidomimetic Macrocycles

SJSA-1 cells were plated out one day in advance in clear, flat-bottom plates at a density of 7500 cells/well with 100 μL/well of growth media. Row H columns 10-12 were left empty to be treated with media alone. On the day of the assay, the media was exchanged with RPMI 1% FBS media to result in 90 μL of media per well. 10 mM stock solutions of the peptidomimetic macrocycles were prepared in 100% DMSO. The peptidomimetic macrocycles were diluted serially in 100% DMSO, and further diluted 20-fold in sterile water to prepare working stock solutions in 5% DMSO/water. The concentrations of the peptidomimetic macrocycles ranged from 500 μM to 62.5 μM.

10 μL of each compound solution was added to the 90 μL of SJSA-1 cells to yield final concentration of 50 μM to 6.25 μM in 0.5% DMSO-containing media. The negative control (non-lytic sample) was 0.5% of DMSO alone. The positive control (lytic) samples included 10 μM of Melittin and 1% Triton X-100. The cell plates were incubated for 1 h at 37° C. After incubation for 1 h, the morphology of the cells was examined by microscope. The plates were then centrifuged at 1200 rpm for 5 min at room temperature. 40 μL of the supernatant for each peptidomimetic macrocycle and control sample was transferred to clear assay plates. LDH release was measured using an LDH cytotoxicity assay kit. The results of the cell lysis assay are shown in TABLE 8:

TABLE 8
	6.25 μM	12.5 μM	25 μM	50 μM
	% Lysed	% Lysed	% Lysed	% Lysed
SP#	cells (1 h LDH)	cells (1 h LDH)	cells (1 h LDH)	cells (1 h LDH)
3	1	0	1	3
4	−2	1	1	2
6	1	1	1	1
7	0	0	0	0
8	−1	0	1	1
9	−3	0	0	2
11	−2	1	2	3
15	1	2	2	5
18	0	1	2	4
19	2	2	3	21
22	0	−1	0	0
26	2	5	−1	0
32	0	0	2	0
39	0	−1	0	3
43	0	0	−1	−1
55	1	5	9	13
65	0	0	0	2
69	1	0.5	−0.5	5
71	0	0	0	0
72	2	1	0	3
75	−1	3	1	1
77	−2	−2	1	−1
80	0	1	1	5
81	1	1	0	0
82	0	0	0	1
99	1.5	3	2	3.5
108	0	0	0	1
114	3	−1	4	9
115	0	1	−1	6
118	4	2	2	4
120	0	−1	0	6
121	1	0	1	7
122	1	3	0	6
123	−2	2	5	3
125	0	1	0	2
126	1	2	1	1
130	1	3	0	−1
139	−2	−3	−1	−1
142	1	0	1	3
144	1	2	−1	2
147	8	9	16	55
148	0	1	−1	0
149	6	7	7	21
150	−1	−2	0	2
153	4	3	2	3
154	−1	−1.5	−1	−1
158	0	−6	−2
160	−1	0	−1	1
161	1	1	−1	0
169	2	3	3	7
170	2	2	1	−1
174	5	3	2	5
175	3	2	1	0
177	−1	−1	0	1
182	0	2	3	6
183	2	1	0	3
190	−1	−1	0	1
196	0	−2	0	3
197	1	−4	−1	−2
203	0	−1	2	2
204	4	3	2	0
211	5	4	3	1
217	2	1	1	2
218	0	−3	−4	1
219	0	0	−1	2
221	3	3	3	11
223	−2	−2	−4	−1
230	0.5	−0.5	0	3
232	6	6	5	5
233	2.5	4.5	3.5	6
237	0	3	7	55
243	4	23	39	64
244	0	1	0	4
245	1	14	11	56
247	0	0	0	4
249	0	0	0	0
254	11	34	60	75
279	6	4	5	6
280	5	4	6	18
284	5	4	5	6
286	0	0	0	0
287	0	6	11	56
316	0	1	0	1
317	0	1	0	0
331	0	0	0	0
335	0	0	0	1
336	0	0	0	0
338	0	0	0	1
340	0	2	0	0
341	0	0	0	0
343	0	1	0	0
347	0	0	0	0
349	0	0	0	0
351	0	0	0	0
353	0	0	0	0
355	0	0	0	0
357	0	0	0	0
359	0	0	0	0
413	5	3	3	3
414	3	3	2	2
415	4	4	2	2

Example 16: p53 GRIP Assay

The p53 GRIP assay monitors the protein interaction of p53 and MDM2, and the cellular translocation of GFP-tagged p53 in response to drug compounds or other stimuli. Recombinant CHO-hIR cells stably express human p53 (1-312) fused to the C-terminus of enhanced green fluorescent protein (EGFP) and PDE4A4-MDM2 (1-124), a fusion protein between PDE4A4 and MDM2 (1-124). The effects of experimental conditions on the interaction of p53 and MDM2 can be measured.

CHO-hIR cells were regularly maintained in Ham's F12 media supplemented with penicillin-streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin, and 10% FBS. Cells were seeded into 96-well plates at a density of 7000 cells/100 μL/well using culture media 18-24 h prior to running the assay. On the day of the assay, the media was refreshed, and PD-177 was added to cells to reach a final concentration of 3 μM to activate foci formation. Control wells were kept without PD-177. 24 h post-stimulation with PD-177, the cells were washed once with reduced-serum media. 50 μL of the reduced-serum media supplemented with PD-177 (6 μM) was added to the cells. The peptides were diluted from 10 mM DMSO stock solutions to 500 μM working stock solutions in sterile water. Further dilutions were made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final highest DMSO concentration was 0.5%, and 0.5% DMSO was used as the negative control. (−)-Nutlin-3 (10 mM) was used as a positive control. Nutlin was diluted using the same dilution scheme used for the peptides.

50 μL of the 2× desired concentration peptide solutions were added to the appropriate wells to achieve desired final concentrations. Cells were then incubated with the peptides for 6 h at 37° C. in a humidified 5% CO₂ atmosphere. After incubation, the cells were fixed by gently aspirating out the media and adding 150 μL of fixing solution per well for 20 minutes at room temperature. The fixed cells were washed 4 times with 200 μL PBS per well each time. At the end of last wash, 100 L of 1 M Hoechst staining solution was added. The sealed plates were incubated for at least 30 min in the dark, and washed with PBS to remove excess staining solution. PBS was added to each well. The plates could be stored at 4° C. in the dark for up to 3 days. The translocation of p53/MDM2 was imaged using a molecular translocation module using 10× objective and XF-100 filter sets for Hoechst and GFP. The minimal acceptable number of cells per well used for image analysis was set to 500 cells.

Example 17: MCF-7 Breast Cancer Study Using SP315, SP249 and SP154

A xenograft study was performed to test the efficacy of SP315, SP249 and SP154 in inhibiting tumor growth in athymic mice in the MCF-7 breast cancer xenograft model. A negative control stapled peptide (SP252) and a point mutation of SP154 (F to A at position 19) were tested in one group. The negative control stapled peptide exhibited no activity in the SJSA-1 in vitro viability assay. Slow release 90 day 0.72 mg 17β-estradiol pellets were implanted subcutaneously (sc) on the nape of the neck one day prior to tumor cell implantation (Day −1). On Day 0, MCF-7 tumor cells were implanted sc in the flank of female nude (Cr1:NU-Foxn1nu) mice. On Day 18, the resultant sc tumors were measured using calipers to determine their length and width, and the mice were weighed. The tumor sizes were calculated using the formula (length×width²)/2, and expressed as cubic millimeters (mm³). Mice with tumors smaller than 85.3 mm³ or larger than 417.4 mm³ were excluded from the subsequent group formation. Thirteen groups of mice, 10 mice per group, were formed by randomization such that the group mean tumor sizes were essentially equivalent (mean of groups±standard deviation of groups=180.7±17.5 mm³).

SP315, SP249, SP154 and SP252 dosing solutions were prepared from peptides formulated in a vehicle containing MPEG(2K)-DSPE at 50 mg/mL concentration in a 10 mM histidine-buffered saline solution at pH 7. The peptide formulations were prepared once for the duration of the study. The vehicle was used as the vehicle control in the subsequent study.

Each group was assigned to a different treatment regimen. Group 1, as the vehicle negative control group, received the vehicle administered at 8 mL/kg body weight intravenously (I.V.) three times per week from Days 18-39. Group 2 received SP154 as an I.V. injection at 30 mg/kg three times per week. Group 3 received SP154 as an I.V. injection at 40 mg/kg twice a week. Group 4 received 6.7 mg/kg SP249 as an I.V. injection three times per week. Group 5 received SP315 as an I.V. injection of 26.7 mg/kg three times per week. Group 6 received SP315 as an I.V. injection of 20 mg/kg twice per week. Group 7 received SP315 as an I.V. injection of 30 mg/kg twice per week. Group 8 received SP315 as an I.V. injection of 40 mg/kg twice per week. Group 9 received 30 mg/kg SP252 as an I.V. injection three times per week.

During the dosing period, the mice were weighed and the tumors were measured 1-2 times per week. Tumor growth inhibition was compared with the vehicle group. Changes in body weight and number of mice with ≥20% body weight loss or death is shown in TABLE 9. Tumor growth inhibition (TGI) was calculated as

% TGI=100−[(TuVol^{Treated-day x}−TuVol^{Treated-day 18})/(TuVol^{Vehicle negative control-day x}−TuVol^{vehicle negative control-day 18})*100,

where x=day that effect of treatment is being assessed. Group 1, the vehicle negative control group, showed good tumor growth rates.

2 mice died during treatment with SP154 when dosed with 40 mg/kg twice a week. The dosing regimen of 30 mg/kg of SP154 three times per week yielded a TGI of 84%. 4 mice died in the group dosed with SP249 6.7 mg/kg three times. No body weight loss or deaths were noted for all groups treated with SP315. Dosing with 40 mg/kg of SP315 twice per week produced the highest TGI (92%). The dosing regimens of SP315 of 26.7 mg/kg three times per week, 20 mg/kg twice per week, 30 mg/kg twice per week produced TGI of 86, 82, and 85%, respectively. No body weight loss or deaths were noted for the group treated with SP252 30 mg/kg three times per week. The TGI was 88% on day 32, and reduced to 41% by day 39.

TABLE 9
Group		% BW	No. with ≥10%	No. with ≥20%
Number	Treatment Group	Change	BW Loss	BW Loss or death	% TGI
1	Vehicle	+8.6	0/10	0/10	—
2	SP154 30 mg/kg	+5.7	0/10	0/10	*84
	3x/wk I.V.
3	SP154 40 mg/kg	N/A	0/10	2/10 (2 deaths)	Regimen not
	2x/wk I.V.				tolerated
4	SP249 6.7 mg/kg	N/A	6/10	4/10	Regimen not
	3x/wk I.V.				tolerated
5	SP315 26.7 mg/kg	+3.7	0/10	0/10	*86
	3x/wk I.V.
6	SP315 20 mg/kg	+3.9	0/10	0/10	*82
	2x/wk I.V.
7	SP315 30 mg/kg	+8.0	0/10	0/10	*85
	2x/wk I.V.
8	SP315 40 mg/kg	+2.1	0/10	0/10	*92
	2x/wk I.V.
9	SP252 30 mg/kg	+3.3	0/10	0/10	*41
	3x/wk I.V.
*p ≤ 0.05 Vs Vehicle Control

Example 18: Testing of Peptidomimetic Macrocycles for Ability to Reduce Immune Checkpoint Protein Expression or Inhibit Immune Checkpoint Protein Activity

Assays were performed to determine whether the peptidomimetic macrocycles can diminish PD-L1 activity or expression. HCT-116 p53^+/+ cells and HCT-116 p53^−/− cells were treated with DMSO or 10 μM SP or 20 μM SP. FIG. 1 shows that treatment with SP262 and SP154 resulted in decreased PD-L1 expression in HCT-116 p53^+/+ cells, but not HCT-116 p53^−/− cells. Similar assays are performed in cell lines that express higher levels of PD-L1, such as A549 cells, H460 cells, and syngeneic mouse cell lines.

Assays are performed to determine whether the peptidomimetic macrocycles can diminish PD-L1 activity or expression via miR-34a to enhance immune response against tumors. Assays are performed to determine whether the peptidomimetic macrocycles of the invention mimic the immune-enhancing effects of anti-PD-1 and/or anti-PD-L1 agents, with the added benefit of inducing cell cycle arrest and apoptosis.

Cancer cells from different lineages MCF-7 (breast), HCT-116 (large intestine), MV4-11 (leukemia), DOHH2, and A375 (melanoma) are dosed with peptidomimetic macrocycles. These cell lines and others are selected to include cell lines that have high levels of PD-L1 expression and others that have low levels of PD-L1 expression. Changes in protein and mRNA levels of PD-1, PD-L1 and miR-34a are measured, for example, using flow cytometry. p53 and p21 are used as controls. RT-PCR assays are performed to quantify miR-34a, miR-34b, and/or miR-34c levels in samples in parallel with flow cytometry measurements. Full dose-response curves are taken 24, 48, and 72 hours after dosing. Additionally, apoptosis measurements are made in parallel.

Example 19: Phase I Dose-Escalation Clinical Trial for AP1

A dose-escalation study was conducted in a Phase 1 open-label, multi-center, two-arm trial of the compound AP1. AP1 was administered by I.V. infusion to patients with advanced solid tumors or lymphomas expressing WT p53 that was refractory to or intolerant of standard therapy or for which no standard therapy existed. The trial established a 3.1 mg/kg dose of AP1 as the MTD (i.e., the highest dose of the drug that did not cause unacceptable side effects) when dosed once a week by I.V. administration. The trial also evaluated the safety, tolerability, and the pharmacokinetics of AP1 and provided a preliminary assessment of activity using pharmacodynamic biomarkers and imaging assessments.

71 patients were enrolled in the dose-escalation trial. The trial used a “3+3” dose-escalation design. Patients in the first two dose levels received AP1 once a week for three consecutive weeks over a 28-day cycle or a lower dose level twice a week for two consecutive weeks over a 21-day cycle. The dose-escalation study was used to evaluate different benefit-risk ratios and provide supporting evidence for dose selection during the clinical development of AP1.

Starting with dose level 4, patients who had cancers associated with HPV were excluded from enrollment because HPV is known to destroy WT p53. Dosing started at relatively low dose levels, and the protocol did not require patients in the first three dose levels to have WT p53 or cancers unassociated with HPV because the trial focused primarily on the safety and tolerability of AP1.

To identify specific WT p53 patients for the clinical trials, commercially available third-party assays and a central laboratory were used to conduct next generation sequencing on archived tumor tissue samples or fresh biopsy samples from patients taken prior to enrollment.

WT p53 status was not required of the patients for the initial three dose levels prior to enrollment. The patients' WT p53 statuses were established through testing after enrollment. Seven of the 13 patients enrolled in those dose levels who completed at least one cycle were confirmed to have WT p53 status, the status of four was unknown, and two patients tested positive for mutated p53. Starting with dose level 4, WT p53 status was a mandatory eligibility criterion.

Clinical activity or a patient's response to AP1 was determined using pharmacodynamics (PD) biomarkers and imaging assessments. PD biomarkers provided information on on-target activity, specific patient type responses, and provided an early insight on AP1's effect on tumors. The effect of AP1 on potential PD biomarkers was determined for different sources of biological samples, such as tumor biopsies, circulating tumor cells where detectable, mononuclear blood cells, and blood and bone marrow samples. Depending on the sample type, the PD biomarkers included measures of MDMX, MDM2, p21, p53, apoptosis, macrophage inhibitory cytokine 1, or MIC-1. Standard imaging assessments, such as computed tomography (CT) or positron emission tomography (PET), were used to obtain images from patients.

Anti-tumor activity was measured using Response Evaluation Criteria in Solid Tumors (RECIST) criteria for patients with solid tumors, and 2015 International Working Group (IWG) criteria for patients with lymphomas. The RECIST and IWG criteria enabled the objective evaluation of whether a tumor had progressed, stabilized, or decreased in size. Anti-tumor effects were also determined through physical examinations or clinically validated blood or serum tumor markers.

FIG. 2 illustrates the dosing regiments (DRs) used in the “3+3” dose escalation trial. DR-A depicts patients that received AP1 once a week for three consecutive weeks over a 28-day cycle. DR-B depicts patients who received lower doses of AP1 twice a week for two consecutive weeks over a 21-day cycle. The MTD of AP1 was 3.1 mg/kg, and the multiple-ascending dose (MAD) ended at 4.4 mg/kg.

Example 20: Pharmacokinetic Profile of AP1

AP1 was delivered systematically using I.V. administration because of the potential advantage of avoiding metabolic impact from hepatic and gastrointestinal enzymes as well as reproducible systemic bioavailability.

FIG. 3 shows drug concentration levels in patient plasma at all dose levels tested in Arm A (left panel) and Arm B (right panel). AP1 consistently produced a dose-related increase in maximum drug serum concentrations observed (C_max) in patients, and longer corresponding half-lives of between five and seven hours at higher dose levels. Data were collected at different time points after the start of infusion (SOI) and the end of infusion (EOI).

Example 21: Safety Results for AP1

AP1 has been considered by the dose escalation trial's investigators to be generally well tolerated. The most frequently reported drug-related events to date reported by ≥10% of the patients were nausea, fatigue, vomiting, decreased appetite, anemia, headache, and constipation. Transient decreases in lymphocytes post-dosing and primarily Grade 1 and 2 abnormalities were observed in approximately 50% of patients with full recovery within a few days.

Seven patients experienced infusion-related reactions, and administration of AP1 was discontinued for three patients. Eight patients experienced dose reductions, including two patients who had been on study treatment for over 1 year, and another patient who had been on study treatment for 11 months. One dose limiting toxicity (DLT) of Grade 3 fatigue was reported at 3.1 mg/kg once weekly dosing, and four DLTs (Grade 3 elevated alkaline phosphatase, Grade 3 hypotension, Grade 3 anemia, Grade 4 neutropenia) were reported at 4.4 mg/kg once weekly. Nine severe adverse events (SAEs) were reported, two of which were related to AP1. Both events were Grade 3 hypotension and were at the 3.1 mg/kg and the 4.4 mg/kg once-weekly dosing levels. Grade 3/4 events that were at least possibly related to AP1 occurred in fifteen patients, and included anemia (n=2), an increase in blood alkaline phosphatase levels, diarrhea, fatigue (n=3), hyponatremia, hypotension (n=2), hypoxia, nausea, neutropenia (n=3), and vomiting. Five patients discontinued treatment with AP1 due to these events.

Example 22: Biomarker Assessments for the Biological Activity of AP1

Several exploratory biomarkers were used to confirm the pharmacological or on-target biological activity of AP1, aid patient recruitment, and help inform dose selection. In the Phase 1 dose-escalation study, plasma MIC-1 levels were measured at several time points after initial infusion.

FIG. 4 shows fold-increase levels from baseline levels of plasma MIC-1 on cycle one, day one, two, or three (C1D1, C1D2, C1D3) at dose levels at or above 0.83 mg/kg. The results demonstrated that dose-related, on-mechanism increased in MIC-1 blood levels after the end of AP1 infusion (EOI) achieved a maximal 24 hr MIC-1 increase above baseline at doses ≥2.1 mg/kg. Prolonged p53 activation of MIC-1 was observed 48 hours after the start of infusion (SOI).

Example 23: Clinical Activity of AP1

Clinical activity or responses to AP1 were assessed using imaging methods. Anti-tumor activity was measured using RECIST criteria for patients with solid tumors and the IWG criteria for patients with lymphomas to objectively evaluate whether a tumor progressed, stabilized, or shrunk. In the dose-escalation Phase 1 trial patients in Arm A (28-day cycle group) of the pharmacokinetic study (EXAMPLE 20), plasma AP1 levels were measured at baseline and again after two cycles of study medication, or approximately within 56 days following initial dosing and every 2 cycles thereafter. Patients in Arm B (21-day cycle group of the pharmacokinetic study (EXAMPLE 20), plasmas AP1 levels were measured at baseline and again after three cycles of study medication, or approximately within 63 days following initial dosing and every three cycles thereafter.

FIG. 5 shows a waterfall plot that illustrates the anti-tumor activity of AP1 in patients of the Phase 1 dose-escalation trial. The percent change in tumor volume for each evaluable patient (i.e., having measurable disease by CT or PET-CT scan) is plotted from the highest to lowest value, or low to high response, and each bar of the histogram is colored by the best overall response measured for that patient per RECIST or IWG criteria.

57 patients were evaluated using RECIST or IWG guidelines, including the 52 with CT- or PET-CT scans shown in FIG. 5, and five with clinical or objective evidence of disease progression without scans. Of the evaluable patients, 25 (44%) patients demonstrated disease control in at least one scan following the start of AP1 therapy. There were two responses (CRs), two partial responses (PRs), and 21 responses with stabilization of tumor size (SDs). The latter included 7 stable diseased patients that exhibited tumor shrinkage.

The anti-tumor activity of the Phase 1 dose-escalation trial compared favorably to results of Phase 1 trials using valuable oncology agents, such as nivolumab and pembrolizumab. The results for AP1 in 57 patients included 2 R₅, 2 PRs, and 21 (7 shrinkages). For 39 patients treated with nivolumab, the results were 1 R, 2 PRs, and 12 SDs (2 shrinkages). For 30 patients treated with pembrolizumab, the results were 2 R₅, 3 PRs, and 11 SDs (3 shrinkages).

AP1 yielded a disease control rate of 20/35 (56%) when considering the anti-tumor activity of the Phase 1 dose-escalation trial at doses most relevant to continued clinical development (≥3.2 mg/kg/cycle) and limiting analyses to patients with WT p53. FIG. 6 shows results of the anti-tumor activity study for 33 patients. The study also included results for three additional patients with clinical or objective evidence of disease progression without imaging scans.

The duration of time a patient continued treatment with AP1 served as an additional measure of anti-tumor activity and continued response to AP1 therapy. FIG. 7 shows the time-on-drug for evaluable p53-WT patients who had CRs, PRs, and SDs when dosed with AP1 at ≥3.2 mg/kg/cycle. The median time a patient received AP1 was 147 days, with an average of 192 days, and a max for one patient of 613 days. Three patients received AP1 for over a year, and all patients who achieved R₅ or PRs since inception of the trial remained on AP1 therapy.

FIG. 8 PANEL A-FIG. 8 PANEL D shows patient scans from two CR patients observed in the dose-escalation Phase 1 trial. FIG. 8 PANEL A shows a 50-year-old patient with peripheral T-Cell Lymphoma (PTCL), a highly aggressive form of non-Hodgkin's lymphoma. The images showed a strong signal for aberrant cellular metabolism, which indicated cancer in a lymph node of the patient's chest. After six cycles of AP1 treatment, the lymph node returned to its normal size and no was longer detected by the PET tracer as being cancerous (FIG. 8 PANEL B).

FIG. 8 PANEL C shows images of a 73-year-old patient with Merkel Cell Carcinoma (MCC), a highly aggressive skin cancer. The patient exhibited skin lesions consistent with MCC. After one cycle of AP1 therapy, the skin lesions diminished in size and left only mild scar tissue (FIG. 8 PANEL D). After further treatment with AP1, a biopsy sample from the formerly tumorous skin areas and PET-CT scans showed no trace of cancer in the patient.

Example 24: Phase 2a Clinical Trials with AP1 in Patients with Peripheral T-Cell Lymphoma

Based on the results of the dose-escalation study and the complete response observed in a patient with PTCL, a Phase 2a clinical trial was conducted in patients with PTCL. The first patient enrolled in the Phase 2 study in PTCL achieved a partial response. FIG. 9 LEFT PANEL shows PET scans from the first patient enrolled in the Phase 2 study prior to treatment with AP1. FIG. 9 RIGHT PANEL shows PET scans from the first patient enrolled in the Phase 2 study after 2 cycles of treatment with AP1. Before beginning treatment with AP1, a 39-year-old male patient exhibited strong signals for aberrant cellular metabolism indicative of cancer in the lymph nodes of his neck, under his arms, in his chest, and in his groin (FIG. 9 LEFT PANEL). Following two cycles of treatment with AP1, the lymph nodes picked up a substantially reduced amount of the PET tracer that would indicate the lymph nodes were cancerous (FIG. 9 RIGHT PANEL).

TABLE 10 shows Phase 2a study results of seven PTCL patients on AP1 therapy, with details on the status, days on AP1 treatment and best overall response of each patient.

TABLE 10
Patient			Days on	Best overall
#	Study	Status	treatment	response
1	Dose escalation	Ongoing	487	CR
2	Phase 2a	Disease progression	122	PR
3	Phase 2a	Ongoing	134	SD
4	Phase 2a	Disease progression	66	ODP
5	Phase 2a	Ongoing	53	Tbd
6	Phase 2a	Ongoing	32	Tbd
7	Phase 2a	Ongoing	1	Tbd

Example 25: Survival in an In Vivo Xenotransplantation Model

AP1 was tested for overall survival in an in vivo xenotransplantation model. Engraftment of human CD45 leukemic cells after 5 weeks ranged from 1% to 73% in vehicle and 0% to 0.05% in AP1 treated animals. Mice treated with AP1 lived significantly longer than vehicle treated counterparts. The median survival for group 1 and group 2 was 34 days and 83 days, respectively (p<0.0001). Long term survival was assessed at 130 days post start of treatment, with 22% of mice in group 2 and 60% of mice in group 3 still alive.

Treatment with AP1 doubled the overall survival in an in vivo implantation model. FIG. 10 TOP PANEL shows percentage of human CD45 engraftment in bone marrow for the vehicle, and treatment with 20 mg/kg AP1. FIG. 10 BOTTOM PANEL shows the percentage survival of mice upon treatment with the vehicle or administration of AP1.

Example 26: WST-1 Cell Proliferation Assays

The WST-1 variant of the MTT assay was used to measure cell viability. WST-1 is a cell-impermeable, sulfonated tetrazolium salt that can be used to examine cell viability without killing the cells. The human tumor cell lines MCF-7 and MOLT-3 were grown in EMEM and RPMI1640, respectively. All media were supplemented with 10% (v/v) fetal calf serum, 100 units of penicillin, and 100 μg/mL of streptomycin at 37° C. and 5% CO₂. Prior to dosing, MCF-7 cells were switched to serum-free medium and grown at 37° C. overnight. One day prior to assaying, the cells were trypsinized, counted, and seeded at pre-determined densities in 96-well plates as follows: MCF-7, 5000 cells/well/200 μL; MOLT-3, 30,000 cells/well/200 μL.

FIG. 11 shows a graph of MCF-7 cell proliferation determined using a WST-1 assay measured at the indicated time points after different numbers of MCF-7 cells were grown at 37° C. for a 24 hour growth period. The MCF-7 cells were not treated with any peptides or compounds.

Example 27: Combination Therapy with AP1 and CDK4/CDK6 Inhibitors

a. Combination Therapy with AP1 and Ribociclib

MCF-7 cell proliferation was measured using the WST-1 assay described in EXAMPLE 26. MCF-7 cells were treated with ribociclib at concentrations of 0 μM, 0.0003 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM. FIG. 12 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of ribociclib. MCF-7 cells were treated with AP1 or a combination of AP1 and ribociclib at concentrations of 0.1 μM, 0.3 μM, 1 μM, and 3 μM. The concentration of AP1 was kept constant. FIG. 13 shows MCF-7 cell proliferation when the cells were treated with AP1 or AP1 with varying concentrations of ribociclib.

MCF-7 cells were treated with AP1 at concentrations of 0 μM, 0.0003 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM. FIG. 14 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of AP1. MCF-7 cells were treated with ribociclib or a combination of ribociclib and AP1 at concentrations of 0.1 μM, 0.3 μM, and 1 μM. The concentration of ribociclib was kept constant. FIG. 15 shows MCF-7 cell proliferation when the cells were treated with ribociclib or ribociclib with varying concentrations of AP1. FIG. 16 shows a combination index plot of ribociclib in MCF-7 cells.

b. Combination Therapy with AP1 and Abemaciclib

MCF-7 cell proliferation was measured using the WST-1 assay described in EXAMPLE 26. MCF-7 cells were treated with abemaciclib at concentrations of 0 μM, 0.0003 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM. FIG. 17 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of abemaciclib. MCF-7 cells were treated with AP1 or a combination of AP1 and abemaciclib at concentrations of 0.1 μM, 0.3 μM, 1 μM, and 3 μM. The concentration of AP1 was kept constant. FIG. 18 shows MCF-7 cell proliferation when the cells were treated with AP1 or AP1 with varying concentrations of abemaciclib.

MCF-7 cells were treated with AP1 at concentrations of 0 μM, 0.0003 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM. FIG. 19 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of AP1. MCF-7 cells were treated with abemaciclib or a combination of abemaciclib and AP1 at concentrations of 0.1 μM, 0.3 μM, and 1 μM. The concentration of abemaciclib was kept constant. FIG. 20 shows MCF-7 cell proliferation when the cells were treated with abemaciclib or abemaciclib with varying concentrations of AP1.

c. Combination Therapy with AP1 and Palbociclib

The combination of AP1 and palbociclib was tested at various drug doses on MCF-7 cells. Various MCF-7 cell numbers were plated and evaluated 3-7 days after plating to determine the optimal number of cells to be plated and to determine the treatment duration. The optimal number of cells were plated and treated with various concentrations of AP1 alone or with palbociclib alone. MCF-7 cells were evaluated for viability using the WST-1 assay or the CyQUANT method 3-7 days or 120 h after beginning treatment. FIG. 21 shows cell proliferation of MCF-7 cells when the cells were treated with palbociclib alone. FIG. 22 shows cell proliferation of MCF-7 cells when the cells were treated with AP1 alone. A number of concentrations around the IC₅₀ of AP1, and a number of concentrations around the IC₅₀ of palbociclib were determined. The EC₅₀ of AP1 on MCF-7 cells was determined to be 410 nM. The concentrations used to obtain the EC₅₀ value were tested on MCF-7 cells to test the effect of treatment with the combination of AP1 and palbociclib.

The optimal number of MCF-7 cells were plated and treated with AP1 and palbociclib. The MCF-7 cells were incubated for 3-5 days or 3-7 days. AP1 was added to the cells simultaneously with palbociclib, before adding palbociclib, or after the addition of palbociclib. The cells were evaluated for viability using the CyQUANT method after beginning the simultaneous treatments. FIG. 23 shows MCF-7 cell proliferation when the cells were treated simultaneously with a fixed amount of AP1 and varying amounts of palbociclib. FIG. 24 shows MCF-7 cell proliferation when the cells were treated simultaneously with a fixed amount of palbociclib and varying amounts of AP1.

The cells were evaluated for viability using a WST-1 assay or MTT assay 3-7 days after beginning the treatments. The effects of adding AP1 and palbociclib in different orders was evaluated using various concentrations of AP1 using the CyQUANT method. FIG. 25 shows MCF-7 cell proliferation when the cells were treated with varying concentrations of AP1 and palbociclib in different orders over a period of 72 h. FIG. 26 shows MCF-7 cell proliferation when the cells were pre-treated with AP1 for 24 h and subsequently treated with varying concentrations of palbociclib; and when the cells were pre-treated with varying concentrations of palbociclib for 24 h and subsequently treated with a fixed amount of AP1. AP1 suppressed MCF-7 cell growth with or without treatment with palbociclib. FIG. 27 shows MCF-7 cell proliferation when the cells were pre-treated with varying concentrations of AP1 for 24 h and subsequently treated with fixed amounts of palbociclib; and when the cells were pre-treated with fixed amounts of palbociclib and subsequently treated with varying concentrations of AP1. Palbociclib suppressed MCF-7 cell growth with or without treatment with AP1.

The combination of AP1 and palbociclib was tested at various drug doses on MOLT-3 cells. Various MOLT-3 cell numbers were plated and evaluated 3-7 days after plating to determine the optimal number of cells to be plated and to determine the treatment duration. The optimal number of cells were plated and treated with various concentrations of AP1 alone or with palbociclib alone. The MOLT-3 cells were evaluated for viability using the WST-1 assay or the CyQUANT method 3-7 days or 120 h after beginning treatment. FIG. 28 shows MOLT-3 cell proliferation when the cells were treated with palbociclib alone. FIG. 29 shows MOLT-3 cell proliferation when the cells were treated with AP1 alone.

Combination Index Plots of AP1 and Palbociclib Using WST-1 and CyQUANT Assays in MCF-7 Cells.

Combination index plots of treatment with AP1 and palbociclib in MCF-7 cells showed additive or increased complementarity. FIG. 30 shows the combination index plot of the treatment of MCF-7 cells with AP1 and palbociclib using a WST-1 assay. FIG. 31 shows the combination index plot of the treatment of MCF-7 cells with AP1 and palbociclib using CyQUANT. Example cooperativity index calculations are shown in TABLE 14. The data are expressed as log(CI). CI values: 0-0.1, very strong synergism; 0.1-0.3, strong synergism; 0.3-0.7, synergism; 0.7-0.85, moderate synergism; 0.85-0.90, slight synergism; 0.90-1.10, nearly additive; 1.10-1.20, slight antagonism; 1.20-1.45, moderate antagonism; 1.45-3.3, antagonism; 3.3-10, strong antagonism; 10, very strong antagonism.

	TABLE 14
	Dose AP1	Dose palbociclib
	(μM)	(μM)	Effect	CI
	0.001	0.3	0.178	0.59570
	0.003	0.3	0.184	0.59898
	0.01	0.3	0.223	0.54530
	0.03	0.3	0.25	0.62998
	0.1	0.3	0.325	0.79278
	0.3	0.3	0.532	0.68885
	1.0	0.3	0.65	1.13080
	3.0	0.3	0.743	1.92593
	10.0	0.3	0.924	1.17267
	30.0	0.3	0.945	2.32597
	0.4	0.001	0.585	0.57898
	0.4	0.003	0.553	0.67550
	0.4	0.01	0.55	0.68802
	0.4	0.03	0.545	0.71276
	0.4	0.1	0.556	0.70459
	0.4	0.3	0.608	0.61579
	0.4	1.0	0.592	0.90805
	0.4	3.0	0.614	1.46501
	0.4	10.0	0.698	2.61449
	0.4	30.0	0.999	0.02893

The efficacy of AP1 alone and in combination with palbociclib was tested in the SJSA-1 osteosarcoma xenograft model using female athymic nude mice. Charles River NCr nu/nu mice with 5×10⁶ SJSA-1 tumor cells in 0% Matrigel® were injected subcutaneously into the flank of the mice. The cell injection volume was 0.1 mL/mouse. The mice were 8-12 weeks of age at the beginning of the study. A pair match was performed when tumors reached an average size of 100 mm³-150 mm³, and the treatment regimen was started. Body weight and caliper measurements were made biweekly to the end of the study.

Any individual animal with a single observation of >30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized. Any group with a mean body weight loss of >20% or >10% mortality was removed from the study. The group was not euthanized, and the mice were allowed to recover. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint was euthanized. If the group treatment-related body weight loss was recovered to within 10% of the original weights, dosing was resumed at a lower dose or less frequent dosing schedule. Animals were monitored individually. The end point of the experiment was a tumor volume of 2000 mm³ or 60 days, whichever came first. Responders were followed for a longer period of time. When the endpoint was reached, the animals were euthanized.

Palbociclib was prepared as a solution in sodium lactate buffer (50 mM, pH 4.0). An aqueous phosphate-buffered saline solution or sodium lactate (50 mM, pH 4.0) solution was used as the vehicle. The dosing volume was 10 mL/kg (0.2 mL/20 g mouse), and the volume was adjusted according to the body weight of the mouse.

FIG. 32 shows the effects of AP1, palbociclib, or combination treatment with AP1+palbociclib on the median tumor volumes in the SJSA-1 osteosarcoma xenograft model. The data show that mice treated with a combination of AP1 and palbociclib required a longer duration to reach the same median tumor volume as mice treated with vehicle alone, AP1 alone, or palbociclib alone. Mice first treated with AP1 and treated with palbociclib 6 h after administration of AP1 required a longer duration to reach the same median tumor volume as mice first treated with palbociclib and treated with AP1 6 h after administration of palbociclib.

TABLE 11 shows the results of the CDK inhibitor efficacy test using combination treatment with AP1 and palbociclib in the SJSA-1 osteosarcoma xenograft model.

	TABLE 11
	Median
	tumor	% TGI		Median time	Median time
	volume	(±SEM) on	Animals	to tumor	to tumor
	(mm3)	d221 (end	with disease	volume > 1000	volume > 2000
Treatment	D1	D22	of dosing)	progression2	mm3 (days)	mm3 (days)
Vehicle	117	2500	—	10/10	12	16
AP1 20 mg/kg	117	2150	17 (6)	10/10	16	21
qwkx4
Palbociclib 75	126	1418	51 (9)	9/10	18	24
mg/kg qdx22
AP1 +	126	550	82 (2)	10/10	27	34
Palbociclib
(dose 6 h post
AP1)
Palbociclib +	126	727	71 (3)	10/10	25	32
AP1 (6 h post
palbociclib
1Calculated using vehicle median volumes on d0 and d22
2Defined as three consecutive measurements > 150% of d1 volume

The efficacy of AP1 alone and in combination with palbociclib was tested in the MCF-7.1 human breast carcinoma xenograft model using female athymic nude mice. Female athymic nude mice were provided with drinking water with 10 μg/mL with 17 beta estradiol supplementation 3 days prior to cell implantation and for the duration of the study. Charles River NCI athymic nude mice were treated with 1×10⁷ MCF-7.1 tumor cells in 0% Matrigel® subcutaneously in the flank. The cell injection volume was 0.1 mL/mouse. The mice were between 8-12 weeks of age at the beginning of the study. A pair match was performed when tumors reached an average size of 100 mm³-150 mm³, and the treatment regimen was started. Body weight and caliper measurements were made biweekly to the end of the study.

Any individual animal with a single observation of >30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized. Any group with a mean body weight loss of >20% or >10% mortality was removed from the study. The group was not euthanized, and the mice were allowed to recover. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint was euthanized. If the group treatment-related body weight loss was recovered to within 10% of the original weights, dosing was resumed at a lower dose or less frequent dosing schedule. Animals were monitored individually. The end point of the experiment was a tumor volume of 1000 mm³ or 60 days, whichever came first. Responders were followed for a longer period of time. When the endpoint was reached, the animals were euthanized.

Palbociclib was prepared as a solution in sodium lactate buffer (50 mM, pH 4.0). An aqueous phosphate-buffered saline solution or sodium lactate (50 mM, pH 4.0) solution was used as the vehicle. The dosing volume was 10 mL/kg (0.2 mL/20 g mouse), and the volume was adjusted according to the body weight of the mouse.

FIG. 33 shows the effects of AP1, palbociclib, or combination treatment with AP1+palbociclib on the median tumor volumes in the MCF-7.1 human breast carcinoma xenograft model. The data show that mice treated with a combination of AP1 and palbociclib required a longer duration to reach the same median tumor volume as mice treated with vehicle alone, AP1 alone, or palbociclib alone. FIG. 34 shows individual tumor volumes of mice treated with MCF-7.1 human breast carcinoma xenografts treated with the vehicle. FIG. 35 PANEL A shows the individual tumor volumes of mice treated with AP1 20 mg/kg qwk×4. FIG. 35 PANEL B shows the individual tumor volumes of mice treated with palbociclib 75 mg/kg qd×22. FIG. 35 PANEL C shows the individual tumor volumes of mice treated with AP1, and treated with palbociclib 6 h after administration of AP1. FIG. 35 PANEL D shows the individual tumor volumes of mice treated with palbociclib, and treated with AP1 6 h after administration of AP1. The data show that mice treated with a combination of AP1 and palbociclib required a longer duration to reach the same median tumor volume as mice treated with AP1 alone or palbociclib alone.

TABLE 12 shows the results of the CDK inhibitor efficacy test using the MCF-7.1 human breast carcinoma xenograft model.

	TABLE 12
	Median
	tumor	% TGI		Median time	Median time
	volume	(±SEM) on	Animals	to tumor	to tumor
	(mm3)	d221 (end	with disease	volume > 500	volume > 1000
Treatment	D1	D22	of dosing)	progression2	mm3 (days)	mm3 (days)
Vehicle	108	666	—	10/10	19	27
						9/10 animals
						have reached
						endpoint
AP1 20 mg/kg	126	473	30 (9)	10/10	22	35
qwkx4						10/10 animals
						have reached
						endpoint
Palbociclib 75	108	196	84 (4)	8/8	37	48
mg/kg qdx22						10/10 animals
						have reached
						endpoint
AP1 +	126	126	95 (3)	10/10	42	53
Palbociclib						7/10 animals
(dose 6 h post						have reached
AP1)						endpoint
Palbociclib +	126	196	88 (2)	9/9	37	49
AP1 (6 h post						6/7 animals
palbociclib						have reached
						endpoint
1Calculated using vehicle median volumes on d0 and d22
2Defined as three consecutive measurements > 150% of d1 volume

The efficacy of AP1 alone and in combination with palbociclib was tested in the A549 xenograft model using female athymic nude mice with the methods described above. FIG. 36 shows the effects of AP1, palbociclib, or combination treatment with AP1+palbociclib on the median tumor volumes in the A549 xenograft model. FIG. 37 PANEL A shows the effect of vehicle treatment on median tumor volumes in the A549 xenograft model. FIG. 37 PANEL B shows the effect of vehicle treatment on median tumor volumes in the A549 xenograft model. The arrows indicate spontaneous tumor shrinkage in vehicle controls. The arrows with * indicate poor growth of tumors late in the study.

TABLE 13 shows the CDK inhibitor efficacy test in the A549 xenograft model.

	TABLE 13
	Median
	tumor	% TGI		Median time	Median time
	volume	(±SEM) on	Animals	to tumor	to tumor
	(mm3)	d221 (end	with disease	volume > 500	volume > 1000
Treatment	D1	D22	of dosing)	progression2	mm3 (days)	mm3 (days)
Vehicle	126	500	—	9/10	18	ND
					9/10 animals	1/10 animals
					have reached	have reached
					endpoint	endpoint
AP1 20 mg/kg	126	405	42 (7)	10/10	25	ND
qwkx4					8/10 animals
					have reached
					endpoint
Palbociclib 75	126	288	62 (7)	9/10	5/10 animals	ND
mg/kg qdx22					have reached
					endpoint
AP1 +	126	343	48 (7)	10/10	6/10 animals	ND
Palbociclib					have reached
(dose 6 h post					endpoint
AP1)
Palbociclib +	126	256	77 (9)	7/9	4/9 animals	ND
AP1 (6 h post					have reached
palbociclib					endpoint
1Calculated using vehicle median volumes on d0 and d22
2Defined as three consecutive measurements > 150% of d1 volume

Example 28: Combination Therapy with AP1 and MEK Inhibitors

a. Combination Therapy with AP1 and Trametinib

The combination of AP1 and trametinib was tested on human melanoma tumor C32 cells. FIG. 38 shows C32 cell proliferation when the cells were treated with trametinib alone or trametinib in combination with varying concentrations of AP1. FIG. 39 shows the combination index plot of the treatment of C32 cells with AP1 and trametinib. FIG. 40 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 with varying concentrations of trametinib. FIG. 41 shows C32 cell proliferation when the cells were treated with varying concentrations of AP1 and varying concentrations of trametinib.

The combination of AP1 and trametinib was tested on MEL-JUSO cells. FIG. 42 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 and varying concentrations of trametinib. FIG. 43 shows MEL-JUSO cell proliferation when the cells were treated with no agent, AP1 alone, trametinib alone, or 0.03 μM AP1 and 1.0 nM trametinib. FIG. 44 shows MEL-JUSO cell proliferation when the cells were treated with trametinib alone or trametinib with varying concentrations of AP1. FIG. 45 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and trametinib.

The combination of AP1 and trametinib was tested on A375 human melanoma cells. Various A375 cell numbers were plated and evaluated 3-7 later to determine the optimal number of cells to be treated and to determine the optimal treatment duration. The optimal number of cells were plated and treated with various concentrations of AP1 alone or trametinib alone. The cells were evaluated for viability using a WST-1 assay or MTT assay 3-7 days after treatment. A number of concentrations around the IC₅₀ of AP1 and a number of concentrations around the IC₅₀ of trametinib were determined. The EC₅₀ of AP1 on A375 cells was 70 nM.

The cell viability of A375 cells were tested against treatment with AP1 at the selected concentrations in combination with trametinib. The optimal number of A375 cells was plated, and the cells were treated with AP1 and trametinib. The cells were evaluated for viability using a WST-1 assay or MTT assay 3-7 days after beginning simultaneous or sequential treatments with AP1 and trametinib. FIG. 46 shows A375 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of trametinib. FIG. 47 shows A375 cell proliferation when the cells were treated with trametinib alone or trametinib in combination with varying concentrations of AP1. FIG. 48 shows the combination index plot of the treatment of A375 melanoma cells with AP1 and trametinib.

b. Combination Therapy with AP1 and Binimetinib

The combination of AP1 and binimetinib was tested on human melanoma tumor C32 cells. The C32 cells were grown in EMEM medium supplemented with 10% (v/v) fetal calf serum, 100 units of penicillin, and 100 μg/mL of streptomycin at 37° C. and 5% CO₂. One day prior to performing the assay, the C32 cells were trypsinized, counted, and seeded at 3000 cells/well/200 μL in 96-well plates. The cells were dosed with AP1 alone, binimetinib alone, or AP1 and binimetinib. The cells were incubated for 72 h, and cell viability was measured using a WST-1 variant of the MTT assay. FIG. 49 shows C32 cell proliferation when the cells were treated with varying concentrations of binimetinib and AP1. FIG. 50 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of binimetinib. FIG. 51 shows C32 cell proliferation when the cells were treated with binimetinib alone or binimetinib in combination with varying concentrations of AP1. FIG. 52 shows the combination index plot of the treatment of C32 cells with AP1 and binimetinib. The combination index plot showed additive or increased complimentarily for treatment with AP1 and binimetinib in C32 cells.

The combination of AP1 and binimetinib was tested on MEL-JUSO cells. MEL-JUSO cells were grown in EMEM medium supplemented with 10% (v/v) fetal calf serum, 100 units of penicillin, and 100 μg/mL of streptomycin at 37° C. and 5% CO₂. One day prior to performing the assay, the MEL-JUSO cells were trypsinized, counted, and seeded at 3000 cells/well/200 μL in 96-well plates. The cells were dosed with AP1 alone, binimetinib alone, or AP1 and binimetinib. The cells were incubated for 72 h, and cell viability was measured using a WST-1 variant of the MTT assay.

FIG. 53 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of binimetinib. FIG. 54 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of binimetinib. FIG. 55 shows MEL-JUSO cell proliferation when the cells were treated with binimetinib alone or binimetinib in combination with varying concentrations of AP1. FIG. 56 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and binimetinib. The combination index plot showed additive or increased complimentarily for treatment with AP1 and binimetinib in MEL-JUSO cells.

c. Combination Therapy with AP1 and Pimasertib

The combination of AP1 and pimasertib was tested on human melanoma tumor C32 cells. FIG. 57 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying combinations of pimasertib. FIG. 58 shows C32 cell proliferation when the cells were treated with varying concentrations of AP1 and pimasertib. FIG. 59 shows C32 cell proliferation when the cells were treated with pimasertib alone or pimasertib in combination with varying concentrations of AP1. FIG. 60 shows the combination index plot of the treatment of C32 cells with AP1 and pimasertib.

The combination of AP1 and pimasertib was tested on MEL-JUSO cells. FIG. 61 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of pimasertib. FIG. 62 shows MEL-JUSO cell proliferation when the cells were treated with AP1 and pimasertib. FIG. 63 shows MEL-JUSO cell proliferation when the cells were treated with pimasertib alone or pimasertib in combination with varying concentrations of AP1. FIG. 64 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and pimasertib.

d. Combination Therapy with AP1 and Selumetinib

The combination of AP1 and selumetinib was tested on human melanoma tumor C32 cells. FIG. 65 shows C32 cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying combinations of selumetinib. FIG. 66 shows C32 cell proliferation when the cells were treated with varying concentrations of AP1 and selumetinib. FIG. 67 shows C32 cell proliferation when the cells were treated with selumetinib alone or selumetinib in combination with varying concentrations of AP1. FIG. 68 shows the combination index plot of the treatment of C32 cells with AP1 and selumetinib.

The combination of AP1 and pimasertib was tested on MEL-JUSO cells. FIG. 69 shows MEL-JUSO cell proliferation when the cells were treated with AP1 alone or AP1 in combination with varying concentrations of pimasertib. FIG. 70 shows MEL-JUSO cell proliferation when the cells were treated with AP1 and pimasertib. FIG. 71 shows MEL-JUSO cell proliferation when the cells were treated with pimasertib alone or pimasertib in combination with varying concentrations of AP1. FIG. 72 shows the combination index plot of the treatment of MEL-JUSO cells with AP1 and pimasertib.

Example 29: Combination Therapy with AP1 and Cancer Agents

a. Clinical Development for the Treatment of Acute Myeloid Leukemia

AP1 was tested for the treatment of patients with the hematological cancers, Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS), expressing WT p53. AML and MDS patients received AP1 or AP1 in combination with cytarabine. Cytarabine is an important agent for the treatment of patients with AML or MDS. Combination treatment is a standard treatment practice in oncology used to improve patient outcomes. FIG. 73 shows combination treatment and dosing regimens used to study the effects of AP1 to treat AML.

TABLE 14 shows initial patient analyses of the AML study. Of the evaluable patients where bone marrow biopsies were available before and after dosing with AP1, 3 patients demonstrated stabilization of their disease.

TABLE 14
				Days on	Best overall
Patient #	Disease	Dose level	Status	treatment	response
1	MDS	3.1 alone	Disease	98	SD
			progression
2	MDS	3.1 alone	Ongoing	138	SD
3	AML	3.1 alone	Withdrew	99	SD
			consent
4	AML	3.1 combo	Disease	68	ODP
			progression
5	AML	3.1 combo	Ongoing	41	tbd
6	AML	3.1 combo	Ongoing	19	tbd
7	MDS	4.4 alone	Ongoing	82	tbd
8	MDS	4.4 alone	Ongoing	47	tbd

b. Combination Therapy with AP1, Paclitaxel, and Eribulin

The efficacy of AP1 alone and in combination with paclitaxel or eribulin was tested in the MCF-7.1 human breast carcinoma xenograft model using female athymic nude mice. TABLE 15 shows the dosing group numbers and amounts of paclitaxel and eribulin for the combination treatment.

	TABLE 15
	AP1
	Drug	Amount	0 mg/kg	5 mg/kg	10 mg/kg
	Paclitaxel	0 mg/kg	1	3	2
		10 mg/kg	5	10	9
		15 mg/kg	4	8	7
	Eribulin	0.1 mg/kg	6	12	11

Animals were provided with drinking water with 10 μg/mL of 17 beta-estradiol supplementation, 3 days prior to cell implementation and for the duration of the study. Charles River NCr nu/nu mice were treated with subcutaneous injections of 1×10⁷ MCF-7.1 tumor cells in 0% Matrigel® in the flank. The cell injection volume was 0.1 mL/mouse. The mice were 8-12 weeks of age at the start of the study. Body weight and caliper measurements were made biweekly to the end of the study. Any individual animal with a single observation of >30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized. Any group with a mean body weight loss of >20% or >10% mortality was not given further dosages. The groups were not euthanized and recovery was allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint was euthanized. If the group treatment-related body weight loss was recovered within 10% of the original weight, dosing was resumed at a loser dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1000 mm³ or 60 days, whichever came first. Responders were followed for a longer period of time. When the endpoint was reached, the animals were euthanized.

AP1 was prepared as a phosphate-buffered aqueous solution. Paclitaxel was prepared in 5% ethanol and 5% cremaphor EL® in D5W. The vehicle was a phosphate-buffered aqueous solution. The dosing volume was 10 mL/kg (0.2 mL/20 g mouse). The volume was adjusted accordingly for the body weight of each mouse.

TABLE 16 shows results from the paclitaxel combination therapy efficacy test in MCF-7 subjects using AP1, paclitaxel, and eribulin. After 28 days, the surviving animals were followed to the tumor size endpoint or death.

TABLE 16
Arm	Treatment A	Treatment B	N	Dosing schedule
1	Vehicle	Vehicle	10	Days 1, 4, 8, 11, 15, 18, 22, 25
2	AP1 10 mg/kg	Vehicle	10	Days 1, 4, 8, 11, 15, 18, 22, 25
3	AP1 5 mg/kg	Vehicle	10	Days 1, 4, 8, 11, 15, 18, 22, 25
4	Paclitaxel 15 mg/kg	Vehicle	10	Days 1, 8, 15, 22
5	Paclitaxel 10 mg/kg	Vehicle	10	Days 1, 8, 15, 22
6	Eribulin 0.1 mg/kg	Vehicle	10	Days 1, 8, 15, 22
7	AP1 10 mg/kg	Paclitaxel 15 mg/kg	10	Days 1, 8, 15, 22: Paclitaxel followed by
				AP1 6 h later
				Days 4, 11, 18, 25: AP1 only
8	AP1 5 mg/kg	Paclitaxel 15 mg/kg	10	Days 1, 8, 15, 22: Paclitaxel followed by
				AP1 6 h later
				Days 4, 11, 18, 25: AP1 only
9	AP1 10 mg/kg	Paclitaxel 10 mg/kg	10	Days 1, 8, 15, 22: Paclitaxel followed by
				AP1 6 h later
				Days 4, 11, 18, 25: AP1 only
10	AP1 5 mg/kg	Paclitaxel 10 mg/kg	10	Days 1, 8, 15, 22: Paclitaxel followed by
				AP1 6 h later
				Days 4, 11, 18, 25: AP1 only
11	AP1 10 mg/kg	Eribulin 0.1 mg/kg	10	Days 1, 8, 15, 22: Eribulin followed by
				AP1 6 h later
				Days 4, 11, 18, 25: AP1 only
12	AP1 5 mg/kg	Eribulin 0.1 mg/kg	10	Days 1, 8, 15, 22: Eribulin followed by
				AP1 6 h later
				Days 4, 11, 18, 25: AP1 only

FIG. 74 shows the results of treatment with AP1 or Paclitaxel on individual mouse tumor volume by day. FIG. 75 shows the results of combination treatment with AP1+paclitaxel on individual mouse tumor volume by day. FIG. 76 shows the results of treatment with AP1 or Paclitaxel on individual mouse tumor volume by day on a Log₁₀ axis to show growth. FIG. 77 shows the results of combination treatment with AP1+paclitaxel on individual mouse tumor volume by day on a Log₁₀ axis to show growth. FIG. 78 shows the results of treatment with AP1 or Paclitaxel on individual mouse tumor volume % change from baseline by day. FIG. 79 shows the results of combination treatment with AP1+paclitaxel on individual mouse tumor volume % change from baseline by day.

FIG. 80 shows the results of treatment with AP1 or Paclitaxel on median tumor volume % change from baseline by day. FIG. 81 shows the results of combination treatment with AP1+paclitaxel on median tumor volume % change from baseline by day. FIG. 82 shows the results of treatment with AP1 or Paclitaxel on average (±1 StDev) tumor volume % change from baseline by day. FIG. 83 shows the results of combination treatment with AP1+paclitaxel on average (±1 StDev) tumor volume % change from baseline by day.

FIG. 84 compares the results of treatment with AP1, paclitaxel, or combination treatment with AP1+paclitaxel on the average % change in tumor volume from baseline per day. The data show that combination therapy with 5 mg/kg AP1 and 10 mg/kg paclitaxel; or 5 mg/kg AP1 and 15 mg/kg paclitaxel minimized the average % change in tumor volume from baseline for the duration of the study. FIG. 85 compares the results of treatment with AP1, paclitaxel, or combination treatment with AP1+paclitaxel on the average % change in tumor volume from baseline per day. The data show that combination therapy with 10 mg/kg AP1 and 10 mg/kg paclitaxel; or 5 mg/kg AP1 and 15 mg/kg paclitaxel minimized the average % change in tumor volume from baseline for the duration of the study.

FIG. 86 shows the effect of treatment with AP1, paclitaxel, or combination treatment with AP1+paclitaxel on individual tumor volume % change from baseline on Day 28 per study group. Group 1: control; Group 2: AP110 mg/kg; Group 3: AP1 5 mg/kg; Group 4: paclitaxel 15 mg/kg; Group 5: paclitaxel 10 mg/kg; Group 7: combination treatment AP1 10 mg/kg+paclitaxel 15 mg/kg; Group 8: combination treatment AP1 15 mg/kg+paclitaxel 15 mg/kg; Group 9: combination treatment AP1 10 mg/kg+paclitaxel 10 mg/kg; Group 10: AP1 5 mg/kg+paclitaxel 10 mg/kg. FIG. 87 shows the effect of treatment with AP1, eribulin, or combination treatment with AP1+eribulin on the average % change of tumor volume. Line 1: control; Line 2: combination treatment with AP1 10 mg/kg+eribulin 0.1 mg/kg; Line 3: combination treatment with AP1 5 mg/kg+eribulin 0.1 mg/kg; Line 4: AP1 10 mg/kg; Line 5: AP1 5 mg/kg; Line 6: eribulin 0.1 mg/kg. FIG. 88 shows the effect of treatment with AP1, eribulin, or combination treatment with AP1+eribulin on individual tumor volume? % change from baseline on Day 28. Group 1: control; Group 2: AP1 10 mg/kg; Group 3: AP1 5 mg/kg; Group 6: eribulin 0.1 mg/kg; Group 11: combination treatment with AP1 10 mg/kg+eribulin 0.1 mg/kg; Group 12: combination treatment with AP1 5 mg/kg+eribulin 0.1 mg/kg.

c. Combination Therapy with AP1 and Abraxane®

Abraxane®, also known as protein-bound paclitaxel or nanoparticle albumin-bound paclitaxel, is an injectable formulation of paclitaxel used to treat breast cancer, lung cancer, and pancreatic cancer. The efficacy of AP1 alone and in combination with Abraxane® was tested in the MCF-7.1 human breast carcinoma xenograft model using female athymic nude mice, following the method used to test the efficacy of AP1 in combination with paclitaxel.

FIG. 89 shows changes in the normalized body weights of mice treated under various dosing regimens of AP1, Abraxane®, or combination treatment with AP1+Abraxane® over a period of 12 days in the MCF-7.1 human breast carcinoma xenograft model. FIG. 90 shows changes in tumor volumes (mm³) of mice treated under various dosing regimens of AP1, Abraxane®, or combination treatment with AP1+Abraxane® over a period of 12 days in the MCF-7.1 human breast carcinoma xenograft model.

TABLE 17 shows the dosing regimens used to obtain data on the efficacy of combination treatment using AP1 and Abraxane®.

TABLE 17
Group # Dosing
Group 1 vehicle (i.v., days 2, 5, 9, 12, 16, 19, 23, 26)
Group 2 AP1 5 mg/kg (i.v., days 2, 5, 9, 12, 16, 19, 23, 26)
Group 3 Abraxane ® 15 mg/kg (i.v., qwk × 4 starting on day 2)
Group 4 combination treatment with AP1 5 mg/kg (i.v., days 2, 5, 9,
        12, 16, 19, 23, 26) + Abraxane ® 15 mg/kg
        (i.v., qwk × 4 starting on day 4)
Group 5 combination treatment with AP1 5 mg/kg (i.v., days 2, 5, 9, 12,
        16, 19, 23, 26; dose 6 hours prior to Abraxane ®) +
        Abraxane ® 15 mg/kg (i.v., qwk × 4 starting on day 2)
Group 6 combination treatment with Abraxane ® 15 mg/kg
        (i.v., qwk × 4 starting on day 2) + AP1 5 mg/kg (i.v., days 2, 5,
        9, 12, 16, 19, 23, 26; dose 6 hours post-Abraxane ®)
Group 7 Combination treatment with Abraxane ® 15 mg/kg
        (i.v., qwk × 4) + AP1 5 mg/kg (i.v., days 2, 5, 9, 12, 16, 19,
        23, 26; dose 24 hours post-Abraxane ®)
Group 8 Combination treatment with AP1 5 mg/kg (i.v., days 2, 5, 9, 12,
        16, 19, 23, 26; dose 24 hours prior to Abraxane ®) +
        Abraxane ® 15 mg/kg (i.v., qwk × 4 starting on day 3)

The data show that Group 7, Group 6, Group 5, and Group 4 resulted in an overall reduction in tumor volume upon treatment. Group 7 had the highest reduction in tumor volume 5 days after treatment.

Example 30: Combination Therapy with AP1 and PD-1 or PD-L1 Antagonists

a. Mice Treated with CloudmanS91 Malignant Melanoma Tumors

The efficacy of AP1 in combination with murine anti-PD-1, anti-PD-L1, or anti-CTLA-4 was tested in syngeneic mouse models. The murine syngeneic models used for the studies were CT-26 for CTLA-4; CloudmanS91, Colon26, EMT-6, A20, and MC-38 for PD-1; and CloudmanS91, A20, MC-38, and B16F10 for PD-L1.

AP1 was administered intravenously starting on D1 at dosages of 5 mg/kg, 10 mg/kg, or 20 mg/kg per body weight of each mouse. AP1 was administered 2 times per week for 2 weeks. Anti-PD-1 was administered I.P. on day 3 at a dose of 5 mg/kg, twice a week for two weeks. Anti-PD-L1 was administered I.P. on day 3 at a dose of 5 mg/kg, twice a week for two weeks. Anti-CTLA-4 was administered I.P. on day 3 at a dose of 5 mg/kg, and then at a dose of 2.5 mg/kg on day 6 and day 10. End points were based on tumor volume, body weight, and clinical observations.

FIG. 91 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 91 PANEL B shows the results of treatment with anti-PD-1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 91 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 91 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. The dotted line indicates the median tumor volume for the vehicle control.

FIG. 92 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 92 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 92 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. FIG. 92 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using a CloudmanS91 malignant melanoma model. The dotted line indicates the median tumor volume for the vehicle control.

b. Mice Treated with A20 Lymphoma

The efficacy of treatment with AP1 alone and in combination with anti-PD-1 was tested in the A20 murine lymphoma model using female BALB/c mice. Charles River female BALB/c mice were treated subcutaneously in the flank with 1×10⁶ A20 cells in 0% Matrigel®. The cell injection volume was 0.1 mL/mouse. The mice were 8 to 12 weeks of age at the start of the experiment. A pair match was performed when tumors reached an average size of 90-120 mm³, and treatment began. Body weight and caliper measurements were made biweekly throughout the experiment. Dosing volume was 10 mL/kg, and the volume was adjusted accordingly for the body weight of each mouse.

Any individual animal with a single observation of >30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized. Any group with a mean body weight loss of >20% or >10% mortality was not given further dosages. The groups were not euthanized and recovery was allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint was euthanized. If the group treatment-related body weight loss was recovered within 10% of the original weight, dosing was resumed at a loser dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Animals were monitored individually. The endpoint of the experiment was a tumor volume of 2000 mm³ or 45 days, whichever came first. Responders were followed for a longer period of time. When the endpoint was reached, the animals were euthanized.

Anti-PD-1 RMP1-14 (ratIgG) was used to test the efficacy of combination treatment using AP1 and anti-PD-1. TABLE 18 shows the treatment regimens used to test the efficacy of combination treatment using AP1 and anti-PD-1.

	TABLE 18
	Regimen 1	Regimen 2
Gr.	N	Agent	mg/kg	Route	Schedule	Agent	mg/kg	Route	Schedule
1#	10	vehicle	—	iv	biwk x 2 (start	PBS	—	ip	biwk x 2 (start
					on day 1)				on day 3)
2	10	anti-PD-1	5	ip	biwk x 2 (start	—	—	—	—
		RMP1-14			on day 3)
3	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 1)
4	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 3)
5	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 5)
6	10	AR16	5	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 1)	RMP1-14			on day 3)
7	10	AR16	10	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 1)	RMP1-14			on day 3)
8	10	AR16	20	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 1)	RMP1-14			on day 3)
9	10	AR16	5	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 3)	RMP1-14			on day 3)
10	10	AR16	10	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 3)	RMP1-14			on day 3)
11	10	AR16	20	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 3)	RMP1-14			on day 3)
12	10	AR16	5	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 5)	RMP1-14			on day 3)
13	10	AR16	10	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 5)	RMP1-14			on day 3)
14	10	AR16	20	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 5)	RMP1-14			on day 3)
#control

FIG. 93 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 93 PANEL B shows the results of treatment with anti-PD-1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 93 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 93 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model. The dotted line indicates the median tumor volume for the vehicle control.

Anti-PD-L1 10F.9G2 in PBS was used to test the efficacy of combination treatment using AP1 and anti-PD-L1. The dosing volume for the vehicle and AP1 was 10 mL/kg, and was adjusted accordingly for the body weight of each mouse. The dosing volume for PBS and anti-PD-L1 was 0.2 mL/mouse, and was not adjusted for body weight. TABLE 19 shows the treatment regimens used to test the efficacy of combination treatment using AP1 and anti-PD-L1.

	TABLE 19
	Regimen 1	Regimen 2
Gr.	N	Agent	mg/kg	Route	Schedule	Agent	mg/kg	Route	Schedule
1#	10	vehicle	—	iv	biwk x 2 (start	PBS	—	ip	biwk x 2 (start
					on day 1)				on day 3)
2	10	anti-PD-L1	100*	ip	biwk x 2 (start	—	—	—	—
					on day 3)
3	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 1)
4	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 3)	—	—	—	—
5	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 5)
6	10	AR16	5	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 1)				on day 3)
7	10	AR16	10	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 1)				on day 3)
8	10	AR16	20	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 1)				on day 3)
9	10	AR16	5	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 3)				on day 3)
10	10	AR16	10	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 3)				on day 3)
11	10	AR16	20	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 3)				on day 3)
12	10	AR16	5	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 5)				on day 3)
13	10	AR16	10	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 5)				on day 3)
14	10	AR16	20	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 5)				on day 3)
#control
*μg/animal

FIG. 94 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 94 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 94 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the A20 murine lymphoma model. FIG. 94 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using the A20 murine lymphoma model. The dotted line indicates the median tumor volume for the vehicle control.

c. Mice Treated with M38 Syngeneic Colon Carcinoma

The efficacy of AP1 alone and in combination with anti-PD-1 and anti-PD-L1 was tested in the M38 syngeneic colon carcinoma model using C57BL/6 female mice.

Mice were anesthetized with isoflurane for the implantation of cells to reduce ulcerations. Charles River female C57BL/6 mice were treated subcutaneously in the flank with 5×10⁵ MC38 tumor cells in 0% Matrigel®. The cell injection volume was 0.1 mL/mouse. The mice were 8-12 weeks of age at the beginning of the experiments. A pair match was performed when tumors reached an average size of 80-120 mm³. Body weight and caliper measurements were made biweekly throughout the duration of the experiment.

Any individual animal with a single observation of >30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized. Any group with a mean body weight loss of >20% or >10% mortality was not given further dosages. The groups were not euthanized and recovery was allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint was euthanized. If the group treatment-related body weight loss was recovered within 10% of the original weight, dosing was resumed at a loser dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1000 mm³ or 45 days, whichever came first. Responders were followed for a longer period of time. When the endpoint was reached, the animals were euthanized.

Anti-PD-1 RMP1-14 (ratIgG) was used to test the efficacy of combination treatment using AP1 and anti-PD-1. TABLE 20 shows the treatment regimens used to test the efficacy of combination treatment using AP1 and anti-PD-1.

	TABLE 20
	Regimen 1	Regimen 2
Gr.	N	Agent	mg/kg	Route	Schedule	Agent	mg/kg	Route	Schedule
1#	10	vehicle	—	iv	biwk x 2 (start	PBS	—	ip	biwk x 2 (start
					on day 1)				on day 3)
2	10	anti-PD-1	5	ip	biwk x 2 (start	—	—	—	—
		RMP1-14			on day 3)
3	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 1)
4	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 3)
5	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 5)
6	10	AR16	5	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 1)	RMP1-14			on day 3)
7	10	AR16	10	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 1)	RMP1-14			on day 3)
8	10	AR16	20	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 1)	RMP1-14			on day 3)
9	10	AR16	5	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 3)	RMP1-14			on day 3)
10	10	AR16	10	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 3)	RMP1-14			on day 3)
11	10	AR16	20	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 3)	RMP1-14			on day 3)
12	10	AR16	5	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 5)	RMP1-14			on day 3)
13	10	AR16	10	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 5)	RMP1-14			on day 3)
14	10	AR16	20	iv	biwk x 2 (start	anti-PD-1	5	ip	biwk x 2 (start
					on day 5)	RMP1-14			on day 3)
#Control group

FIG. 95 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 95 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 95 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 95 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. The dotted line indicates the median tumor volume for the vehicle control.

Anti-PD-L1 10F.9G2 in PBS was used to test the efficacy of combination treatment using AP1 and anti-PD-L1. The dosing volume for the vehicle and AP1 was 10 mL/kg, and was adjusted accordingly for the body weight of each mouse. The dosing volume for PBS and anti-PD-L1 was 0.2 mL/mouse, and was not adjusted for body weight. TABLE 21 shows the treatment regimens used to test the efficacy of combination treatment using AP1 and anti-PD-L1.

	TABLE 21
	Regimen 1	Regimen 2
Gr.	N	Agent	mg/kg	Route	Schedule	Agent	mg/kg	Route	Schedule
1#	10	vehicle	—	iv	biwk x 2 (start	PBS	—	ip	biwk x 2 (start
					on day 1)				on day 3)
2	10	anti-PDL-1	100*	ip	biwk x 2 (start	—	—	—	—
					on day 3)
3	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 1)
4	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 3)
5	10	AR16	20	iv	biwk x 2 (start	—	—	—	—
					on day 5)
6	10	AR16	5	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 1)				on day 3)
7	10	AR16	10	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 1)				on day 3)
8	10	AR16	20	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 1)				on day 3)
9	10	AR16	5	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 3)				on day 3)
10	10	AR16	10	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 3)				on day 3)
11	10	AR16	20	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 3)				on day 3)
12	10	AR16	5	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 5)				on day 3)
13	10	AR16	10	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 5)				on day 3)
14	10	AR16	20	iv	biwk x 2 (start	anti-PDL-1	100*	ip	biwk x 2 (start
					on day 5)				on day 3)
#Control group
*μg/animal

FIG. 96 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 96 PANEL B shows the results of treatment with anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 96 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. FIG. 96 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-PD-L1 on tumor volumes (mm³) of mice using the M38 syngeneic colon carcinoma model. The dotted line indicates the median tumor volume for the vehicle control.

d. Mice Treated with CT26 Undifferentiated Colon Carcinoma Cell Line

The efficacy of AP1 alone and in combination with anti-CTLA-4 was tested in the CT26 undifferentiated colon carcinoma cell line in mice.

FIG. 97 PANEL A shows the results of vehicle treatment on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. FIG. 97 PANEL B shows the results of treatment with anti-CTLA-4 9H10 on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. FIG. 97 PANEL C shows the effect of treatment with twice a week treatment of AP1 at 20 mg/kg on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. FIG. 97 PANEL D shows the effect of combination treatment with twice a week treatment of AP1 at 20 mg/kg and anti-CTLA-4 on tumor volumes (mm³) of mice using the CT26 undifferentiated colon carcinoma cell line. The dotted line indicates the median tumor volume for the vehicle control.

EMBODIMENTS

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;wherein the peptidomimetic macrocycle has a Formula:

wherein:

• • each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 8), wherein each X is an amino acid;
  • each D and E is independently an amino acid;
  • R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • v is an integer from 1-1000;
  • w is an integer from 3-1000; and
  • n is an integer from 1-5.

Embodiment 2

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;wherein the peptidomimetic macrocycle has a Formula:

wherein:

• • each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually an amino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at the corresponding position of the sequence Phe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 9), wherein each X is an amino acid;
  • each D and E is independently an amino acid;
  • R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • v is an integer from 1-1000;
  • w is an integer from 3-1000; and
  • n is an integer from 1-5.

Embodiment 3

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle has improved binding affinity to MDM2 or MDMX relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1 or 2.

Embodiment 4

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle has a reduced ratio of binding affinities to MDMX versus MDM2 relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 5

The method of any one of embodiment 1-4, wherein the peptidomimetic macrocycle has improved in vitro anti-tumor efficacy against p53 positive tumor cell lines relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 6

The method of any one of embodiments 1-5, wherein the peptidomimetic macrocycle shows improved in vitro induction of apoptosis in p53 positive tumor cell lines relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 7

The method of any one of embodiments 1-6, wherein the peptidomimetic macrocycle has an improved in vitro anti-tumor efficacy ratio for p53 positive versus p53 negative or mutant tumor cell lines relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 8

The method of any one of embodiments 1-6, wherein the peptidomimetic macrocycle has improved in vivo anti-tumor efficacy against p53 positive tumors relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 9

The method of any one of embodiments 1-8, wherein the peptidomimetic macrocycle has improved cell permeability relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 10

The method of any one of embodiments 1-9, wherein the peptidomimetic macrocycle has improved solubility relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1, or 2.

Embodiment 11

The method of any one of embodiments 1-10, wherein Xaa₅ is Glu or an amino acid analogue thereof.

Embodiment 12

The method of any one of embodiments 1-11, wherein Xaa₅ is Glu or an amino acid analogue thereof and wherein the peptidomimetic macrocycle has an improved binding affinity, improved solubility, improved cellular efficacy, improved helicity, improved cell permeability, improved in vivo or in vitro anti-tumor efficacy, or improved induction of apoptosis relative to a corresponding peptidomimetic macrocycle wherein Xaa₅ is Ala.

Embodiment 13 The method of any one of embodiments 1-12, wherein each E is independently an amino acid selected from Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine).

Embodiment 14

The method of any one of embodiments 1-13, wherein [D]_v is -Leu₁-Thr₂.

Embodiment 15

The method of any one of embodiments 1-14, wherein w is 3-10.

Embodiment 16

The method of any one of embodiments 1-15, wherein w is 3-6.

Embodiment 17

The method of any one of embodiments 1-15, wherein w is 6-10.

Embodiment 18

The method of any one of embodiments 1-17, wherein w is 6.

Embodiment 19

The method of any one of any one of embodiments 1-18, wherein v is 1-10.

Embodiment 20

The method of any one of embodiments 1-19, wherein v is 2-10.

Embodiment 21

The method of any one of embodiments 1-20, wherein v is 2-5.

Embodiment 22

The method of any one of embodiments 1-21, wherein v is 2.

Embodiment 23

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;

wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1, Table 1a, Table 1b, or Table 1c and wherein the peptidomimetic macrocycle has the formula:

wherein:

• • each A, C, D, and E is independently an amino acid;
  • each B is independently an amino acid, amino acid analogue,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v and w is independently an integer from 1-1000;
  • u is an integer from 1-10;
  • each x, y, and z is independently an integer from 0-10;
  • n is an integer from 1-5; andwherein the peptidomimetic macrocycle is not a peptidomimetic macrocycle of Tables 2a or 2b.

Embodiment 24

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;

wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1, Table 1a, Table 1b, or Table 1c, wherein the peptidomimetic macrocycle has the formula:

wherein:

• • each A, C, D, and E is independently an amino acid;
  • each B is independently an amino acid, amino acid analogue,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v and w is independently an integer from 1-1000;
  • u is an integer from 1-10;
  • each x, y, and z is independently an integer from 0-10;
  • n is an integer from 1-5;
• wherein w>2 and each of the first two amino acid represented by E comprises an uncharged side chain or a negatively charged side chain,
• with the proviso that the peptidomimetic macrocycle is not a peptidomimetic macrocycle of Table 2a and does not have the sequence:

(SEQ ID NO: 762)
Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2,
(SEQ ID NO: 813)
Ac-Sr8SQQTFS$LWRLLAibQN-NH2,
(SEQ ID NO: 814)
Ac-QSQ$r8TFSNLW$LLAibQN-NH2,
(SEQ ID NO: 816)
Ac-QS$r5QTFStNLW$LLAibQN-NH2,
or
(SEQ ID NO: 896)
Ac-QSQQ$r8FSNLWR$LAibQN-NH2,

wherein Aib represents 2-aminoisobutyric acid, $ represents an alpha-Me S5-pentenyl-alanine olefin amino acid connected to another amino acid side chain by an all-carbon crosslinker comprising one double bond, $r5 represents an alpha-Me R₅-pentenyl-alanine olefin amino acid connected to another amino acid side chain by an all-carbon comprising one double bond, and $r8 represents an alpha-Me R₈-octenyl-alanine olefin amino acid connected to another amino acid side chain by an all-carbon crosslinker comprising one double bond.

Embodiment 25

The method of embodiments 24 or 25, wherein each E is independently an amino acid selected from Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine).

Embodiment 26

The method of any one of embodiments 24-25, wherein the first C-terminal amino acid and/or the second C-terminal amino acid represented by E comprise a hydrophobic side chain.

Embodiment 27

The method of embodiment 27, wherein the hydrophobic chain is a large hydrophobic side chain.

Embodiment 28

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;

wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1, Table 1a, Table 1b, or Table 1c, wherein the peptidomimetic macrocycle has the formula:

wherein:

• • each A, C, D, and E is independently an amino acid;
  • each B is independently an amino acid, amino acid analogue,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or at least one of R₁ and R₂ forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₅ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v and w is independently an integer from 1-1000;
  • u is an integer from 1-10;
  • each x, y, and z is independently an integer from 0-10;
  • n is an integer from 1-5; and
  • w>2,

wherein the third amino acid represented by E comprises a large hydrophobic side chain, with the proviso that the peptidomimetic macrocycle is not a peptidomimetic macrocycle of Table 2a and does not have the sequence of: Ac-Q$r8QQTFSN$WRLLAibQN-NH₂ (SEQ ID NO: 895).

Embodiment 29

The method of embodiment 28, wherein each E other than the third amino acid represented by E is an amino acid selected from Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine).

Embodiment 30

The method of any one of embodiments 23-29, wherein w is 3-10.

Embodiment 31

The method of any one of embodiments 23-30, wherein w is 3-6.

Embodiment 32

The method of any one of embodiments 23-29, wherein w is 6-10.

Embodiment 33

The method of any one of embodiments 23-32, wherein w is 6.

Embodiment 34

The method of any one of embodiments 24-33, wherein v is 1-10.

Embodiment 35

The method of any one of embodiments 23-34, wherein v is 3-10.

Embodiment 36

The method of any one of embodiments 23-35, wherein v is 3-5.

Embodiment 37

The method of any one of embodiments 23-36, wherein v is 3.

Embodiment 38

The method of any one of embodiments 34-37, wherein [D]_v is -Leu₁-Thr₂-Phe₃.

Embodiment 39

The method of any one of embodiments 28-38, wherein each of the first two amino acid represented by E comprises an uncharged side chain or a negatively charged side chain.

Embodiment 40

The method of any one of embodiments 28-38, wherein the third amino acid represented by E is an amino acid selected from the group consisting of: isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), and tyrosine (Y).

Embodiment 41

The method of any one of embodiments 1-40, wherein L₁ and L₂ are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each being optionally substituted with R₅.

Embodiment 42

The method of any one of embodiments 1-40, wherein L₁ and L₂ are independently alkylene or alkenylene.

Embodiment 43

The method of any one of embodiments 1-40, wherein L is alkylene, alkenylene, or alkynylene.

Embodiment 44

The method of any one of embodiments 1-43, wherein L is alkylene.

Embodiment 45

The method of any one of embodiments 1-44, wherein L is C₃-C₁₆ alkylene.

Embodiment 46

The method of any one of embodiments 1-44, wherein L is C₁₀-C₁₄ alkylene.

Embodiment 47

The method of any one of embodiments 1-46, wherein R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo.

Embodiment 48

The method of any one of embodiments 1-47, wherein R₁ and R₂ are H.

Embodiment 49

The method of any one of embodiments 1-48, wherein R₁ and R₂ are independently alkyl.

Embodiment 50

The method of any one of embodiments 1-49, wherein R₁ and R₂ are methyl.

Embodiment 51

The method of any one of embodiments 1-50, wherein x+y+z=6.

Embodiment 52

The method of any one of embodiments 1-51, wherein u is 1.

Embodiment 53

The method of any one of embodiments 1-52, wherein the peptidomimetic macrocycle is not a macrocycle of Table 2a or Table 2b.

Embodiment 54

The method of any one of embodiments 1-53, wherein each E is Ser or Ala or an analogue thereof.

Embodiment 55

The method of any one of embodiments 1-54, wherein the peptidomimetic macrocycle comprises at least one amino acid which is an amino acid analogue.

Embodiment 56

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject

(a) a therapeutically effective amount of a p53 agent that

• • (i) inhibits the interaction between p53 and MDM2 and/or p53 and MDMX, and/or
  • (ii) modulates the activity of p53 and/or MDM2 and/or MDMX; and

(b) at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent

• • (i) modulates the activity of CDK4 and/or CDK6, and/or
  • (ii) inhibits CDK4 and/or CDK6;wherein the at least one additional pharmaceutically active agent and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes.

Embodiment 57

The method of embodiment 56, wherein the p53 agent antagonizes an interaction between p53 and MDM2 proteins and/or between p53 and MDMX proteins.

Embodiment 58

The method of embodiments 56 or 57, wherein the at least one additional pharmaceutically active agent binds to CDK4 and/or CDK6.

Embodiment 59

The method of any one of embodiments 56-58, wherein the p53 agent is selected from the group consisting of a small organic or inorganic molecule; a saccharine; an oligosaccharide; a polysaccharide; a peptide, a protein, a peptide analogue, a peptide derivative; an antibody, an antibody fragment, a peptidomimetic; a peptidomimetic macrocycle of any one of embodiments 1-55 a nucleic acid; a nucleic acid analogue, a nucleic acid derivative; an extract made from biological materials; a naturally occurring or synthetic composition; and any combination thereof.

Embodiment 60

The method of any one of embodiments 56-59, wherein the p53 agent is selected from the group consisting of RG7388 (RO5503781, idasanutlin); RG7112 (RO5045337); nutlin3a; nutlin3b; nutlin3; nutlin2; spirooxindole containing small molecules; 1,4-diazepines; 1,4-benzodiazepine-2,5-dione compounds; WK23; WK298; SJ172550; RO2443; RO5963; RO5353; RO2468; MK8242 (SCH900242); MI888; MI773 (SAR405838); NVPCGM097; DS3032b; AM8553; AMG232; NSC207895 (XI006); JNJ26854165 (serdemetan); RITA (NSC652287); YH239EE; and any combination thereof.

Embodiment 61

The method of any one of embodiments 56-60, wherein the at least one additional pharmaceutically active agent is selected from the group consisting of a small organic or inorganic molecule; a saccharine; an oligosaccharide; a polysaccharide; a peptide, a protein, a peptide analogue, a peptide derivative; an antibody, an antibody fragment, a peptidomimetic; a peptidomimetic macrocycle of any one of embodiments 1-55; a nucleic acid; a nucleic acid analogue, a nucleic acid derivative; an extract made from biological materials; a naturally occurring or synthetic composition; and any combination thereof.

Embodiment 62

The method of any one of embodiments 1-61, wherein the at least one additional pharmaceutically active agent is selected from the group consisting of palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; arcyriaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-amino thioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (ryuvidine); and flavopiridol (alvocidib); seliciclib; dinaciclib; milciclib; roniciclib; atuveciclib; briciclib; riviciclib; trilaciclib (G1T28); and any combination thereof.

Embodiment 63

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 163)

or a pharmaceutically acceptable salt thereof.

Embodiment 64

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 124)

or a pharmaceutically acceptable salt thereof.

Embodiment 65

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 123):

or a pharmaceutically acceptable salt thereof.

Embodiment 66

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 108)

or a pharmaceutically acceptable salt thereof.

Embodiment 67

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 397)

or a pharmaceutically acceptable salt thereof.

Embodiment 68

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 340)

or a pharmaceutically acceptable salt thereof.

Embodiment 69

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 454)

or a pharmaceutically acceptable salt thereof.

Embodiment 70

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 360)

or a pharmaceutically acceptable salt thereof.

Embodiment 71

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 80)

or a pharmaceutically acceptable salt thereof.

Embodiment 72

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 78)

or a pharmaceutically acceptable salt thereof.

Embodiment 73

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 16)

or a pharmaceutically acceptable salt thereof.

Embodiment 74

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 169)

or a pharmaceutically acceptable salt thereof.

Embodiment 75

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 324)

or a pharmaceutically acceptable salt thereof.

Embodiment 76

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 258)

or a pharmaceutically acceptable salt thereof.

Embodiment 77

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 446)

or a pharmaceutically acceptable salt thereof.

Embodiment 78

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 358)

or a pharmaceutically acceptable salt thereof.

Embodiment 79

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 464)

or a pharmaceutically acceptable salt thereof.

Embodiment 80

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 466)

or a pharmaceutically acceptable salt thereof.

Embodiment 81

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 467)

or a pharmaceutically acceptable salt thereof.

Embodiment 82

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 376)

or a pharmaceutically acceptable salt thereof.

Embodiment 83

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 471)

or a pharmaceutically acceptable salt thereof.

Embodiment 84

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 473)

or a pharmaceutically acceptable salt thereof.

Embodiment 85

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 475)

or a pharmaceutically acceptable salt thereof.

Embodiment 86

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 476)

or a pharmaceutically acceptable salt thereof.

Embodiment 87

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 481)

or a pharmaceutically acceptable salt thereof.

Embodiment 88

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 482)

or a pharmaceutically acceptable salt thereof.

Embodiment 89

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 487)

or a pharmaceutically acceptable salt thereof.

Embodiment 90

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 572)

or a pharmaceutically acceptable salt thereof.

Embodiment 91

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 572)

or a pharmaceutically acceptable salt thereof.

Embodiment 92

The method of embodiments 1 or 2, wherein the peptidomimetic macrocycle is (SEQ ID NO: 1500)

or a pharmaceutically acceptable salt thereof.

Embodiment 94

A method of modulating the activity of p53 and/or MDM2 and/or MDMX in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;

wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60% identical to an amino acid sequence in any of Table 1, Table 1a, Table 1b, and Table 1c, wherein the peptidomimetic macrocycle has the formula:

or pharmaceutically acceptable salt thereof, wherein:

• • each A, C, D, and E is independently an amino acid;
  • each B is independently an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v is independently an integer from 1-1000;
  • each w is independently an integer from 1-1000;
  • u is an integer from 1-10;
  • each x, y and z is independently an integer from 0-10; and
  • each n is independently an integer from 1-5.

Embodiment 95

A method of antagonizing an interaction between p53 and MDM2 proteins and/or between p53 and MDMX proteins in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a peptidomimetic macrocycle and at least one additional pharmaceutically active agent, wherein the at least one additional pharmaceutically active agent:

• (a) is selected from the group consisting of cobimetinib and binimetinib, or
• (b) is a cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes;

wherein the peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60% identical to an amino acid sequence in any of Table 1, Table 1a, Table 1b, and Table 1c and wherein the peptidomimetic macrocycle has the formula:

or pharmaceutically acceptable salt thereof, wherein:

• • each A, C, D, and E is independently an amino acid;
  • each B is independently an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v is independently an integer from 1-1000;
  • each w is independently an integer from 1-1000;
  • u is an integer from 1-10;
  • each x, y and z is independently an integer from 0-10; and
  • each n is independently an integer from 1-5.

Embodiment 96

The method of any one of embodiments 1-95, wherein the cancer is selected from the group consisting of head and neck cancer, melanoma, lung cancer, breast cancer, colon cancer, ovarian cancer, NSCLC, stomach cancer, prostate cancer, leukemia, lymphoma, mesothelioma, renal cancer, non-Hodgkin lymphoma (NHL), and glioma.

Embodiment 97

The method of any one of embodiments 1-96, wherein, a sub-therapeutic amount of the at least one additional pharmaceutically active agent is administered.

Embodiment 98

The method of any one of embodiments 1-97, wherein a therapeutic amount of the at least one additional pharmaceutically active agent is administered.

Embodiment 99

The method of any one of embodiments 1-98, wherein the at least one additional pharmaceutically active agent comprises cobimetinib or binimetinib.

Embodiment 100

The method of any one of embodiments 1-98, wherein the at least one additional pharmaceutically active agent comprises the cyclin dependent kinase inhibitor (CDKI) and the CDKI and the peptidomimetic macrocycle are administered with a time separation of more than about 61 minutes.

Embodiment 101

The method of any one of embodiments 1-98 or 100, wherein the at least one additional pharmaceutically active agent comprises palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; arcyriaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-amino thioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (ryuvidine); and flavopiridol (alvocidib); seliciclib; dinaciclib; milciclib; roniciclib; atuveciclib; briciclib; riviciclib; trilaciclib; and any combination thereof.

Embodiment 102

The method of embodiment 100 or 101, wherein the peptidomimetic macrocycle is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the cyclin dependent kinase inhibitor is administered.

Embodiment 103

The method of embodiment 100 or 101, wherein the peptidomimetic macrocycle is administered at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the cyclin dependent kinase inhibitor is administered.

Embodiment 104

The method of embodiment 100 or 101, wherein the peptidomimetic macrocycle is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, before the cyclin dependent kinase inhibitor is administered.

Embodiment 105

The method of embodiment 100 or 101, wherein the peptidomimetic macrocycle is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, after the cyclin dependent kinase inhibitor is administered.

Embodiment 106

The method of embodiment 100 or 101, wherein the peptidomimetic macrocycle is administered at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, after the cyclin dependent kinase inhibitor is administered.

Embodiment 107

The method of embodiment 100 or 101, wherein the peptidomimetic macrocycle is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 week, 2 weeks, three weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any combination thereof, after the cyclin dependent kinase inhibitor is administered.

Embodiment 108

The method of any one of embodiments 1-107, wherein an additional therapeutic agent is administered.

Embodiment 109

The method of any one of embodiments 1-108, wherein the subject comprises cancer cells that overexpress PD-L1.

Embodiment 110

The method of any one of embodiments 1-109, wherein the subject comprises cancer cells that overexpress PD-1.

Embodiment 111

The method of any one of embodiments 1-110, wherein the subject comprises cancer cells that overexpress miR-34.

Embodiment 112

The method of any one of embodiments 108-111, wherein the additional therapeutic agent is a PD-1 antagonist.

Embodiment 113

The method of any one of embodiments 108-112, wherein the additional therapeutic agent is a PD-L1 antagonist.

Embodiment 114

The method of any one of embodiments 108-113, wherein the additional therapeutic agent is an agent that blocks the binding of PD-L1 to PD-1.

Embodiment 115

The method of any one of embodiments 108-114, wherein the additional therapeutic agent specifically binds to PD-1.

Embodiment 116

The method of any one of embodiments 108-115, wherein the additional therapeutic agent specifically binds to PD-L1.

Embodiment 117

The method of any one of embodiments 1-116, wherein PD-L1 expression is downregulated.

Embodiment 118

The method of any one of embodiments 1-117, wherein PD-1 expression is downregulated.

Embodiment 119

The method of any one of embodiments 1-118, wherein S-phase is inhibited.

Embodiment 120

The method of any one of embodiments 1-119, wherein M-phase is inhibited.

Embodiment 121

The method of any one of embodiments 1-120, wherein the peptidomimetic macrocycle antagonizes an interaction between p53 and MDM2 proteins.

Embodiment 122

The method of any one of embodiments 1-121, wherein the peptidomimetic macrocycle antagonizes an interaction between p53 and MDMX proteins.

Embodiment 123

The method of any one of embodiments 1-122, wherein the peptidomimetic macrocycle antagonizes an interaction between p53 and MDM2 proteins and p53 and MDMX proteins.

Embodiment 124

The method of any one of embodiments 1-123, wherein the peptidomimetic macrocycle antagonizes an interaction between p53 and MDM2 proteins and p53 and MDMX proteins.

Embodiment 201

A method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a peptidomimetic macrocycle and at least one pharmaceutically-active agent, wherein the peptidomimetic macrocycle and the at least one pharmaceutically-active agent are administered with a time separation of more than 61 minutes.

Embodiment 202

The method of embodiment 201, wherein the peptidomimetic macrocycle is of the formula:

or pharmaceutically acceptable salt thereof, wherein:

• • each A, C, D, and E is independently an amino acid;
  • each B is independently an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

• • each R₁ and R₂ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids;
  • each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R₅;
  • each L and L′ is independently a macrocycle-forming linker of the formula -L₁-L₂-;
  • each L₁, L₂, and L₃ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or [—R₄—K—R₄-]_n, each being optionally substituted with R₅;
  • each R₄ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
  • each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;
  • each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₆ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R₇ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with a D residue;
  • each R₈ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R₅, or part of a cyclic structure with an E residue;
  • each v is independently an integer from 1-1000;
  • each w is independently an integer from 1-1000;
  • u is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each x, y and z is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and
  • each n is independently 1, 2, 3, 4, or 5.

Embodiment 203

The method of embodiment 202, wherein v is 3-10.

Embodiment 204

The method of embodiments 202 or 203, wherein v is 3.

Embodiment 205

The method of any one of embodiments 202-204, wherein w is 3-10.

Embodiment 206

The method of any one of embodiments 202-205, wherein w is 6.

Embodiment 207

The method of any one of embodiments 202-206, wherein x+y+z=6.

Embodiment 208

The method of any one of embodiments 202-207, wherein each L₁ and L₂ is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene.

Embodiment 209

The method of any one of embodiments 202-208, wherein each L₁ and L₂ is independently alkylene or alkenylene.

Embodiment 210

The method of any one of embodiments 202-209, wherein each R₁ and R₂ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

Embodiment 211

The method of any one of embodiments 202-210, wherein each R₁ and R₂ is independently hydrogen.

Embodiment 212

The method of any one of embodiments 202-210, wherein each R₁ and R₂ is independently alkyl.

Embodiment 213

The method of any one of embodiments 202-210 or 212, wherein each R₁ and R₂ is independently methyl.

Embodiment 214

The method of any one of embodiments 202-214, wherein u is 1.

Embodiment 215

The method of any one of embodiments 202-214, wherein each E is Ser or Ala, or an analogue thereof.

Embodiment 216

The method of any one of embodiments 201-215, wherein the peptidomimetic macrocycle comprises an amino acid sequence that is at least 60% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

Embodiment 217

The method of any one of embodiments 201-216, wherein the peptidomimetic macrocycle comprises an amino acid sequence that is at least 70% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

Embodiment 218

The method of any one of embodiments 201-217, wherein the peptidomimetic macrocycle comprises an amino acid sequence that is at least 80% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

Embodiment 219

The method of any one of embodiments 201-218, wherein the peptidomimetic macrocycle is at least 60% identical to SP-153, SP-303, SP-331, or SP-671.

Embodiment 220

The method of any one of embodiments 201-219, wherein the condition is cancer.

Embodiment 221

The method of any one of embodiments 201-220, wherein the cancer is lymphoma.

Embodiment 222

The method of any one of embodiments 201-220, wherein the cancer is breast cancer.

Embodiment 223

The method of any one of embodiments 201-220, wherein the cancer is skin cancer.

Embodiment 224

The method of any one of embodiments 201-220, wherein the cancer is leukemia.

Embodiment 225

The method of any one of embodiments 201-220, wherein the cancer is melanoma.

Embodiment 226

The method of any one of embodiments 201-220, wherein the cancer is bone cancer

Embodiment 227

The method of any one of embodiments 201-226, wherein the at least one pharmaceutically-active agent, pharmaceutically-acceptable salt, or conjugate thereof is a cyclin-dependent kinase (CDK) inhibitor.

Embodiment 228

The method of any one of embodiments 201-227, wherein the CDK inhibitor is palbociclib.

Embodiment 229

The method of any one of embodiments 201-227, wherein the CDK inhibitor is abemaciclib.

Embodiment 230

The method of any one of embodiments 201-227, wherein the CDK inhibitor is ribociclib.

Embodiment 231

The method of any one of embodiments 201-226, wherein the at least one pharmaceutically-active agent is a mitogen-activated protein kinase (MEK) inhibitor.

Embodiment 232

The method of any one of embodiments 201-226, wherein the at least one pharmaceutically-active agent is a microtubule inhibitor.

Embodiment 233

The method of any one of embodiments 201-226 or 232, wherein the microtubule inhibitor is eribulin.

Embodiment 234

The method of any one of embodiments 201-226 or 232, wherein the microtubule inhibitor is paclitaxel.

Embodiment 235

The method of any one of embodiments 201-226, 232, or 234, wherein the microtubule inhibitor is nanoparticle albumin-bound paclitaxel.

Claims

1. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a peptidomimetic macrocycle and at least one pharmaceutically-active agent, wherein the peptidomimetic macrocycle and the at least one pharmaceutically-active agent are administered with a time separation of more than 61 minutes.

2. The method of claim 1, wherein the peptidomimetic macrocycle is of the formula:

3. The method of claim 2, wherein v is 3-10.

4. The method of claim 3, wherein v is 3.

5. The method of claim 2, wherein w is 3-10.

6. The method of claim 5, wherein w is 6.

7. The method of claim 2, wherein x+y+z=6.

8. The method of claim 2, wherein each L1 and L2 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene.

9. The method of claim 8, wherein each L1 and L2 is independently alkylene or alkenylene.

10. The method of claim 2, wherein each R1 and R2 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

11. The method of claim 10, wherein each R1 and R2 is independently hydrogen.

12. The method of claim 10, wherein each R1 and R2 is independently alkyl.

13. The method of claim 10, wherein each R1 and R2 is independently methyl.

14. The method of claim 2, wherein u is 1.

15. The method of claim 2, wherein each E is Ser or Ala, or an analogue thereof.

16. The method of claim 1, wherein the peptidomimetic macrocycle comprises an amino acid sequence that is at least 60% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

17. The method of claim 16, wherein the peptidomimetic macrocycle comprises an amino acid sequence that is at least 70% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

18. The method of claim 17, wherein the peptidomimetic macrocycle comprises an amino acid sequence that is at least 80% identical to an amino acid sequence listed in Table 1, Table 1a, Table 1b, Table 1c, Table 2a, or Table 2b.

19. The method of claim 16, wherein the peptidomimetic macrocycle is at least 60% identical to SP-153, SP-303, SP-331, or SP-671.

20. The method of claim 1, wherein the condition is cancer.

21. The method of claim 20, wherein the cancer is lymphoma.

22. The method of claim 20, wherein the cancer is breast cancer.

23. The method of claim 20, wherein the cancer is skin cancer.

24. The method of claim 20, wherein the cancer is leukemia.

25. The method of claim 20, wherein the cancer is melanoma.

26. The method of claim 20, wherein the cancer is bone cancer.

27. The method of claim 1, wherein the at least one pharmaceutically-active agent, pharmaceutically-acceptable salt, or conjugate thereof is a cyclin-dependent kinase (CDK) inhibitor.

28. The method of claim 27, wherein the CDK inhibitor is palbociclib.

29. The method of claim 27, wherein the CDK inhibitor is abemaciclib.

30. The method of claim 27, wherein the CDK inhibitor is ribociclib.

31. The method of claim 1, wherein the at least one pharmaceutically-active agent is a mitogen-activated protein kinase (MEK) inhibitor.

32. The method of claim 1, wherein the at least one pharmaceutically-active agent is a microtubule inhibitor.

33. The method of claim 32, wherein the microtubule inhibitor is eribulin.

34. The method of claim 32, wherein the microtubule inhibitor is paclitaxel.

35. The method of claim 34, wherein the microtubule inhibitor is nanoparticle albumin-bound paclitaxel.