PEPTIDOMIMETIC MACROCYCLES AND USES THEREOF

Information

  • Patent Application
  • 20170114098
  • Publication Number
    20170114098
  • Date Filed
    September 02, 2016
    8 years ago
  • Date Published
    April 27, 2017
    7 years ago
Abstract
Provided herein are peptidomimetic macrocycles and methods of using such macrocycles for the treatment of disease. Also provided here in are methods of using such macrocycles in combination with at least one additional pharmaceutically active agent for treatment of disorders, for example for treatment of cancer.
Description
BACKGROUND OF THE INVENTION

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, leads to export from the nucleus of p53 protein, and 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. In this context, inhibition of the activities of MDM2 has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. MDMX (MDM4) has more recently been identified as a similar negative regulator of p53, and studies have revealed 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. There remains a considerable need for compounds capable of binding to and modulating the activity of p53, MDM2 and/or MDMX. Provided herein are p53-based peptidomimetic macrocycles that modulate an activity of p53. Also provided herein are p53-based peptidomimetic macrocycles that inhibit the interactions between p53, MDM2 and/or MDMX proteins. Further, provided herein are p53-based peptidomimetic macrocycles that can be used for treating diseases including but not limited to cancer and other hyperproliferative diseases.


SUMMARY OF THE INVENTION

In one embodiment, the disclosure provides 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 peptidomimetic macrocycle has a Formula:




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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




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[—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 —H, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with R5;
    • each L or 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, w>2 and each of the first two amino acid 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 large hydrophobic side chain.


In some embodiments, w is between 3 and 1000. For example, the third amino acid represented by E comprises a large hydrophobic side chain.


In other embodiments, the peptidomimetic macrocycle excludes the sequence of:


Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2, Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2,


Ac-$r8SQQTFS$LWRLLAibQN—NH2, Ac-QSQ$r8TFSNLW$LLAibQN—NH2,


Ac-QS$r5QTFStNLW$LLAibQN—NH2, or Ac-QSQQ$r8FSNLWR$LAibQN—NH2.


In other embodiments, the peptidomimetic macrocycle excludes the sequence of:


Ac-Q$r8QQTFSN$WRLLAibQN—NH2.


In another embodiment, the disclosure provides 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 peptidomimetic macrocycle has a formula:




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    • 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, 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, 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 another embodiment, the disclosure provides 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 peptidomimetic macrocycle has a Formula:




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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, Xaa5, 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, wherein each X is an amino acid;
    • each D is independently an amino acid;
    • each E is independently an amino acid, 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 are 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 another embodiment, the disclosure provides 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 peptidomimetic macrocycle has a Formula:




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In some embodiments of any of the Formulas described herein, [D]v is -Leu1-Thr2. In other embodiments of the Formulas described herein, 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).


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, for example 2-5. In some embodiments, v is 2.


In some embodiments, peptides disclosed herein bind a binding site defined at least in part by the MDMX amino acid side chains of L17, V46, M50, Y96 (forming the rim of the pocket) and L99. Without being bound by theory, binding to such a binding site improves one or more properties such as binding affinity, induction of apoptosis, in vitro or in vivo anti-tumor efficacy, or reduced ratio of binding affinities to MDMX versus MDM2.


In some embodiments, the peptidomimetic macrocycle has improved binding affinity to MDM2 or MDMX relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1 or 2. In other instances, 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. In still other instances, 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. In some embodiments, 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. In other instances, the peptidomimetic macrocycle of claim 1, 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. In some instances the improved efficacy ratio in vitro, is 1-29, ≧30-49, or ≧50. In still other instances, 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. In some instances the improved efficacy ratio in vivo is −29, ≧30-49, or ≧50. In yet other instances, the peptidomimetic macrocycle has improved in vivo induction of apoptosis in p53 positive tumors relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1 or 2. In some embodiments, the peptidomimetic macrocycle has improved cell permeability relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1 or 2. In other cases, the peptidomimetic macrocycle has improved solubility relative to a corresponding peptidomimetic macrocycle wherein w is 0, 1 or 2. Exemplary cell lines include of MCF-7, HCT-116, MV4-11, DOHH2, MEL-HO, MEL-JUSO, SK-MEL-5, HT1080, MES-SA, SR, MDA-MB-134-VI, ZR-75-1, A427, A549, MOLM-13, SJSA-1, U2OS, RKO, A498, Caki-2, 22RV1, MSTO-211H, C3A, AGS, SNU-1, RMG-1, HEC-151, HEC-265, MOLT-3 and A375 cell lines.


In some embodiments, Xaa5 is Glu or an amino acid analog thereof. In some embodiments, Xaa5 is Glu or an amino acid analog thereof and wherein the peptidomimetic macrocycle has an improved property, such as improved binding affinity, improved solubility, improved cellular efficacy, 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.


In some embodiments, the peptidomimetic macrocycle has improved binding affinity to MDM2 or MDMX relative to a corresponding peptidomimetic macrocycle wherein Xaa5 is Ala. In other embodiments, the peptidomimetic macrocycle has a reduced ratio of binding affinities to MDMX vs MDM2 relative to a corresponding peptidomimetic macrocycle wherein Xaa5 is Ala. In some embodiments, the peptidomimetic macrocycle has improved solubility relative to a corresponding peptidomimetic macrocycle wherein Xaa5 is Ala, or the peptidomimetic macrocycle has improved cellular efficacy relative to a corresponding peptidomimetic macrocycle wherein Xaa5 is Ala.


In some embodiments, Xaa5 is Glu or an amino acid analog thereof and wherein the peptidomimetic macrocycle has improved biological activity, such as 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.


In some embodiments, the peptidomimetic macrocycle has an activity against a p53+/+ cell line which is at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 70-fold, or 100-fold greater than its binding affinity against a p53−/− cell line. In some embodiments, the peptidomimetic macrocycle has an activity against a p53+/+ cell line which is between 1 and 29-fold, between 30 and 49-fold, or ≧50-fold greater than its binding affinity against a p53−/− cell line. Activity can be measured, for example, as an IC50 value. For example, the p53+/+ cell line is SJSA-1, RKO, HCT-116, or MCF-7 and the p53−/− cell line is RKO-E6 or SW-480. In some embodiments, the peptide has an IC50 against the p53+/+ cell line of less than 1 μM.


In some embodiments, Xaa5 is Glu or an amino acid analog thereof and the peptidomimetic macrocycle has an activity against a p53+/+ cell line which is at least 10-fold greater than its binding affinity against a p53−/− cell line.


In another aspect, the disclosure provides 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 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:




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or pharmaceutically acceptable salt thereof,


wherein:

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




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[—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 and 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.


In another aspect, the disclosure provides 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 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:




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or pharmaceutically acceptable salt thereof,


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,




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[—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 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.


In some embodiments, 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.


In some embodiments, the at least one additional pharmaceutically active agent is a nucleoside metabolic inhibitor, a microtubule inhibitor, a platinum-based drug, a hypomethylating agent, a protein kinase inhibitor, a bruton's tyrosine kinase inhibitor, a CDK4 and/or CDK6 inhibitor, a B-raf inhibitor, a K-ras inhibitor, a MEK-1 and/or MEK-2 inhibitor, an estrogen receptor antagonist, an HDAC inhibitor, an anti-CD20 monoclonal antibody, an anti-PD-1 monoclonal antibody, a hormonal antagonist, an agent the alleviates CDK2NA deletion, an agent that alleviates CDK9 abnormality, an AMT regulator, an agent that alleviates AKT activation, an agent that alleviates PTEN deletion, an agent that alleviates Wip-1Alpha overexpression, an agent that upregulates BIM, or an aromatase inhibitor.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates B-raf. In some embodiments, the at least one additional pharmaceutically active agent is a B-raf inhibitor. In some embodiments, the B-raf inhibitor is vemurafenib, dabrafenib, trametinib, sorafenib, C-1, or NVP-LGX818. In some embodiments, the B-raf inhibitor is vemurafenib or dabrafenib and the cancer is melanoma.


In some embodiments, the at least one additional pharmaceutically active agent is a nucleoside metabolic regulator or modulator. In some embodiments, the at least one additional pharmaceutically active agent is a nucleoside metabolic inhibitor. In some embodiments, the nucleoside metabolic inhibitor is capecitabine, gemcitabine or cytarabine. In some embodiments, the nucleoside metabolic inhibitor is capecitabine and the cancer is colon or breast cancer. In some embodiments, the nucleoside metabolic inhibitor is gemcitabine and the cancer is ovarian, NSCLC, or breast cancer. In some embodiments, the nucleoside metabolic inhibitor is cytarabine and the cancer is Leukemia or Lymphoma.


In some embodiments, the at least one additional pharmaceutically active agent is an estrogen receptor antagonist. In some embodiments, the estrogen receptor antagonist is fulvestrant. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is an estrogen receptor positive breast cancer. In some embodiments, the cancer is a Her2 negative positive breast cancer.


In some embodiments, the at least one additional pharmaceutically active agent is a microtubule regulator or modulator. In some embodiments, the at least one additional pharmaceutically active agent is a microtubule inhibitor. In some embodiments, the microtubule inhibitor is paclitaxel, abraxane or docetaxel. In some embodiments, the microtubule inhibitor is paclitaxel and the cancer is ovarian cancer. In some embodiments, the microtubule inhibitor is abraxane and the cancer is ovarian cancer. In some embodiments, the microtubule inhibitor is docetaxel and the cancer is NSCLC, breast cancer, prostate cancer or stomach cancer.


In some embodiments, the at least one additional pharmaceutically active agent is a platinum-based drug. In some embodiments, the platinum-based drug is carboplatin or cisplatin. In some embodiments, the platinum-based drug is carboplatin and the cancer is NSCLC or ovarian cancer. In some embodiments, the platinum-based drug is cisplatin and the cancer is NSCLC, mesothelioma or ovarian cancer.


In some embodiments, the at least one additional pharmaceutically active agent is a hypomethylating agent. In some embodiments, the hypomethylating agent is azacitidine or dacogen. In some embodiments, the hypomethylating agent is azacitidine or dacogen and the cancer is myelodysplastic syndrome.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates a protein kinase. In some embodiments, the additional pharmaceutically active is a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor is sorafenib, midostaurin (PKC412), or quizartinib. In some embodiments, the protein kinase inhibitor is sorafenib and the cancer is kidney or liver cancer.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates a bruton's tyrosine kinase. In some embodiments, the at least one additional pharmaceutically active agent is a bruton's tyrosine kinase inhibitor. In some embodiments, the bruton's tyrosine kinase inhibitor is ibrutinib. In some embodiments, the bruton's tyrosine kinase inhibitor is ibrutinib and the cancer is non-Hodgkin lymphoma (NHL). In some embodiments, the bruton's tyrosine kinase inhibitor is ibrutinib and the cancer is non-Hodgkin lymphoma (NHL).


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates CDK4 and/or CDK6. In some embodiments, the at least one additional pharmaceutically active agent is a CDK4 and/or CDK6 inhibitor. In some embodiments, the CDK4 and/or CDK6 inhibitor is palbociclib. In some embodiments, the CDK4 and/or CDK6 inhibitor is palbociclib and the cancer is breast cancer In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is an estrogen receptor positive breast cancer. In some embodiments, the cancer is a Her2 negative positive breast cancer.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates MEK-1 and/or MEK-2. In some embodiments, the at least one additional pharmaceutically active agent is a MEK-1 and/or MEK-2 inhibitor. In some embodiments, the MEK-1 and/or MEK-2 inhibitor is trametinib, pimasertib, or PD0325901. In some embodiments, the MEK-1 and/or MEK-2 inhibitor is trametinib and the cancer is melanoma. In some embodiments, the MEK-1 and/or MEK-2 inhibitor is pimasertib. In some embodiments, the MEK-1 and/or MEK-2 inhibitor is pimasertib and the cancer is NSCLC. In some embodiments, the MEK-1 and/or MEK-2 inhibitor is PD0325901.


In some embodiments, the at least one additional pharmaceutically active agent is an anti-CD20 monoclonal antibody. In some embodiments, the anti-CD20 monoclonal antibody is rituximab or obinutuzumab. In some embodiments, the cancer is NHL or a B-cell lymphoma.


In some embodiments, the at least one additional pharmaceutically active agent is an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is pembrolizumab or nivolumab. In some embodiments, the anti-PD-1 monoclonal antibody is pembrolizumab or nivolumab and the cancer is melanoma or NSCLC.


In some embodiments, the at least one additional pharmaceutically active agent is an aromatase inhibitor. In some embodiments, the aromatase inhibitor is letrozole or exemestane. In some embodiments, the aromatase inhibitor is letrozole or exemestane and the cancer is breast cancer.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates topoisomerase I or II. In some embodiments, the at least one additional pharmaceutically active agent is an inhibitor of topoisomerase I or II. In some embodiments, the at least one additional pharmaceutically active agent is topotecan, rinotecan, idarubicin, teniposide or epirubicin. In some embodiments, the at least one additional pharmaceutically active agent is topotecan, rinotecan or epirubicin.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates BCR-ABL kinase or BCR-ABL and Src family tyrosine kinase. In some embodiments, the at least one additional pharmaceutically active agent is an inhibitor of BCR-ABL kinase or BCR-ABL and Src family tyrosine kinase. In some embodiments, the at least one additional pharmaceutically active agent is nilotinib, bosutinib, dasatinib or imatinib.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates PI3K. In some embodiments, the at least one additional pharmaceutically active agent is a PI3K inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is GDC-0941 or AMG511.


In some embodiments, the at least one additional pharmaceutically active agent is a hormone antagonist. In some embodiments, the at least one additional pharmaceutically active agent is letrozole or casodex. In some embodiments, the at least one additional pharmaceutically active agent is fluoroucil. In some embodiments, the at least one additional pharmaceutically active agent is a purine analog.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates mTOR. In some embodiments, the at least one additional pharmaceutically active agent is an mTOR inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is AD8005. In some embodiments, the at least one additional pharmaceutically active agent is everolimus. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is an estrogen receptor positive breast cancer. In some embodiments, the cancer is a Her2 negative positive breast cancer. In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates both PI3K/mTOR kinase. In some embodiments, the at least one additional pharmaceutically active agent is a dual PI3K/mTOR kinase inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is BEZ235.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates both BCL-2 and/or BCL-XL. In some embodiments, the at least one additional pharmaceutically active agent is BCL-2 and/or BCL-XL inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is venetoclax (ABT-199) or ABT-263. In some embodiments, the at least one additional pharmaceutically active agent is a purine analog. In some embodiments, the at least one additional pharmaceutically active agent is fludarabine.


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates wild type or mutant K-ras.


In some embodiments, the at least one additional pharmaceutically active agent is radiation.


In some embodiments, the at least one additional pharmaceutically active agent is a multi-targeted tyrosine kinase modulator or binder. In some embodiments, the at least one additional pharmaceutically active agent is multi-targeted tyrosine kinase inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is ponatinib.


In some embodiments, the at least one additional pharmaceutically active agent is a pan-histone deacetylase (HDAC) modulator or binder. In some embodiments, the at least one additional pharmaceutically active agent is a pan-histone deacetylase (HDAC) inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is romidepsin. In some embodiments, the at least one additional pharmaceutically active agent is panobinostat. In some embodiments, the cancer is adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), or periphieral T-cell lymphoma (PTCL).


In some embodiments, the at least one additional pharmaceutically active agent binds to or modulates AKT kinase. In some embodiments, the at least one additional pharmaceutically active agent is an AKT kinase inhibitor. In some embodiments, the at least one additional pharmaceutically active agent is a MK-2206.


In some embodiments, the at least one additional pharmaceutically active agent alleviates CDKN2A (cyclin-dependent kinase inhibitor 2A) deletion. In some embodiments, the at least one additional pharmaceutically active agent alleviates CDK9 (cyclin-dependent kinase 9) abnormality. In some embodiments, the at least one additional pharmaceutically active agent alleviates ATM deficiency. In some embodiments, the at least one additional pharmaceutically active agent alleviates AKT activation. In some embodiments, the at least one additional pharmaceutically active agent alleviates PTEN deletion. In some embodiments, the at least one additional pharmaceutically active agent alleviates Wip-1Alpha over expression.


In some embodiments, the at least one additional pharmaceutically active agent upregulates BIM or is a BIM mimetic. In some embodiments, the at least one additional pharmaceutically active agent is pegylated IFN2a, vinblastine, dexamethasone, or asparaginase. In some embodiments, the at least one additional pharmaceutically active agent is dexamethasone. In some embodiments, the cancer is a B-cell lymphoma.


In some embodiments, the peptidomimetic macrocycle and the additional pharmaceutically active agent are present in a single formulation. In some embodiments, the peptidomimetic macrocycle and the additional pharmaceutically active agent are present in two different formulations. In some embodiments, the two different formulations are administered simultaneously. In some embodiments, the two different formulations are administered sequentially. In some embodiments, a sub-therapeutic amount of the additional therapeutic agent is administered. In some embodiments, a therapeutically effective amount of the additional therapeutic agent is administered.


In some embodiments, the subject comprises cancer cells that overexpress PD-L1. In some embodiments, the subject comprises cancer cells that overexpress PD-1. In some embodiments, the subject comprises cancer cells that overexpress miR-34. In some embodiments, the at least one additional pharmaceutically active agent is a PD-1 antagonist. In some embodiments, the at least one additional pharmaceutically active agent is a PD-L1 antagonist. In some embodiments, the at least one additional pharmaceutically active agent is an agent that blocks the binding of PD-L1 to PD-1. In some embodiments, the at least one additional pharmaceutically active agent specifically binds to PD-1. In some embodiments, the at least one additional pharmaceutically active agent specifically binds to PD-L1. In some embodiments, PD-L1 expression is downregulated. In some embodiments, PD-1 expression is downregulated.


In some embodiments, the at least one additional pharmaceutically active agent is selected from the group consisting of venetoclax (ABT-199), clofarabine, cyclophosphamide, cytarabine, doxorubicin, imatinib mesylate, methotrexate, prednisone, vincristine, azacitadine, cyclophosphamide, cytarabine, dabrafenib, decitabine, doxorubicin, etoposide, vincristine, doxorubicin, methotrexate, capecitabine, cyclophosphamide, docetaxel, doxorubicin, eribulin mesylate, everolimus, exemestane, fluorouracil, fluorouracil, fulvestrant, gemcitabine, goserelin acetate, letrozole, megestrol acetate, methotrexate, paclitaxel, palbociclib, pertuzumab, tamoxifen citrate, trastuzumab, capecitabine, cetuximab, fluorouracil, irinotecan, ramucirumab, carboplatin, cisplatin, doxorubicin, megestrol acetate, paclitaxel, docetaxel, doxorubicin, fluorouracil, ramucirumab, trastuzumab, axitinib, everolimus, pazopanib, sorafenib tosylate, sorafenib tosylate, dacarbazine, paclitaxel, trametinib, vemurafenib, cisplatin, pemetrexed, bendamustine, bortezomib, brentuximab vedotin, chlorambucil, cyclophosphamide, dexamethasone, doxorubicin, ibrutinib, lenalidomide, methotrexate, prednisone, rituximab, vincristine, afatinib dimaleate, carboplatin, cisplatin, crizotinib, docetaxel, erlotinib, gemcitabine, methotrexate, paclitaxel, pemetrexed, ramucirumab, carboplatin, cisplatin, cyclophosphamide, gemcitabine, olaparib, paclitaxel, topotecan, abiraterone, cabazitaxel, docetaxel, enzalutamide, goserelin acetate, prednisone, doxorubicin, imatinib mesylate, romidepsin, obinutuzumab, pazopanib, and combinations thereof.


In some embodiments, the at least one additional pharmaceutically active agent inhibits S-phase. In some embodiments, the at least one additional pharmaceutically active agent inhibits M-phase.


In some embodiments, the peptidomimetic macrocycle antagonizes an interaction between p53 and MDM2 proteins. In some embodiments, the peptidomimetic macrocycle antagonizes an interaction between p53 and MDMX proteins. In some embodiments, the peptidomimetic macrocycle antagonizes an interaction between p53 and MDM2 proteins and p53 and MDMX proteins. In some embodiments, the peptidomimetic macrocycle antagonizes an interaction between p53 and MDM2 proteins and p53 and MDMX proteins.


In one aspect, provided herein is a method of selecting a peptidomimetic macrocycle that reduces PD-L1 expression, comprising: contacting a cancer cell line expressing a first level of PD-L1 with a peptidomimetic macrocycle comprising a polypeptide with a crosslinker connecting a first amino acid and a second amino acid; incubating the cancer cell line for an incubation period; measuring a second level of PD-L1 expression after the incubation period; selecting the peptidomimetic macrocycle as a peptidomimetic macrocycle that reduces PD-L1 expression when the second level of PD-L1 expression is at least 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, or 100 fold lower than the first level of PD-L1 expression.


In some embodiments, the measuring comprises flow cytometry. In some embodiments, the cancer cell line is selected from the group consisting of MCF-7, HCT-116, MV4-11, DOHH2, and A375. In some embodiments, the method further comprises measuring a level of p53 expression before (a), after (b), or both. In some embodiments, the method further comprises measuring a level of p21 expression before (a), after (b), or both. In some embodiments, the method further comprises measuring a level of miR-34 expression before (a), after (b), or both. In some embodiments, the miR-34 is miR-34a, miR-34b, miR-34c, or a combination thereof. In some embodiments, the first level of PD-L1 expression in the cancer cell line is high. In some embodiments, the first level of PD-L1 expression in the cancer cell line is low. In some embodiments, the cancer cell line is p53 wild-type. In some embodiments, the incubation period is about 24, 48, or 72 hours after the contacting. In some embodiments, the incubation period is at least 6, 12, 24, 36, 48, 60, or 72 hours after the contacting. In some embodiments, the method further comprises measuring a level apoptosis after the incubation period.


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.





BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:



FIG. 1A depicts western blots demonstrating Aileron peptide 1 activates the p53-pathway in AML cell lines treated with increasing amounts of Aileron peptide 1.



FIG. 1B depicts a western blot demonstrating Aileron peptide 1 activates the p53-pathway in AML cell lines treated with the indicated amounts of Aileron peptide 1.



FIG. 1C depicts a western blot demonstrating Aileron peptide 1 activates the p53-pathway in primary AML cells lines treated with the indicated amounts of Aileron peptide 1.



FIG. 2 depicts graphs of relative mRNA expression normalized to GAPDH in AML cell lines treated with increasing amounts of Aileron peptide 1.



FIG. 3A depicts a western blot demonstrating that p53 is stabilized in response to Aileron peptide 1 treatment in a dose-dependent manner.



FIG. 3B depicts a western blot demonstrating that p53 is stabilized in response to Aileron peptide 1 treatment in a time-dependent manner.



FIG. 4A depicts a western blot of immunoprecipitations demonstrating Aileron peptide 1 is an inhibitor of the p53-MDMX interaction.



FIG. 4B depicts a western blot of immunoprecipitations demonstrating Aileron peptide 1 is a dual inhibitor of the p53-MDM2 and p53-MDMX interaction.



FIG. 4C depicts a western blot of immunoprecipitations demonstrating Aileron peptide 1 is an inhibitor of the p53-MDM2 interaction.



FIG. 5A depicts a graph demonstrating inhibition of cellular proliferation of AML cell lines treated with the indicated amount of Aileron peptide 1 (AP1).



FIG. 5B depicts a graph demonstrating inhibition of cellular proliferation of AML cell lines treated with the indicated amount of AP1.



FIG. 5C depicts a graph demonstrating inhibition of cellular proliferation of AML cell lines treated with the indicated amount of AP1.



FIG. 5D depicts a graph demonstrating inhibition of cellular proliferation of AML cell lines treated with the indicated amount of AP1.



FIG. 6 depicts a graph demonstrating inhibition of clonogenic capacity of AML cell lines treated with the indicated amount of AP1.



FIG. 7A depicts a graph demonstrating inhibition of cellular proliferation of AML cell lines treated with the indicated amount of AP1.



FIG. 7B depicts a graph demonstrating inhibition of cellular proliferation of AML cell lines treated with the indicated amount of AP1.



FIG. 8A depicts a graph (left) and corresponding FACS data (right) demonstrating Aileron peptide 1 induces apoptotic cell death in a p53 wild type AML cell line.



FIG. 8B depicts a graph (left) and corresponding FACS data (right) demonstrating Aileron peptide 1 induces apoptotic cell death in a p53 wild type AML cell line.



FIG. 8C depicts a graph (left) and corresponding FACS data (right) demonstrating Aileron peptide 1 does not induce apoptotic cell death in a p53 null AML cell line.



FIG. 8D depicts a graph (left) and corresponding FACS data (right) demonstrating Aileron peptide 1 induces apoptotic cell death in a p53 wild type AML cell line.



FIG. 8E depicts a graph (left) and corresponding FACS data (right) demonstrating Aileron peptide 1 induces apoptotic cell death in a p53 wild type AML cell line.



FIG. 9A depicts a graph demonstrating cytarabine (Ara-C) treatment inhibits proliferation of AML cell lines.



FIG. 9B depicts a graph demonstrating Ara-C synergizes with AP1 to inhibit proliferation of AML cell lines.



FIG. 9C depicts a graph demonstrating Ara-C synergizes with AP1 to inhibit proliferation of AML cell lines.



FIG. 10A depicts a graph demonstrating inhibition of cellular proliferation of primary AML cells treated with the indicated amount of AP1.



FIG. 10B depicts a graph demonstrating inhibition of cellular proliferation of primary AML cells treated with the indicated amount of AP1.



FIG. 10C depicts a graph demonstrating inhibition of cellular proliferation of primary AML cells treated with the indicated amount of AP1.



FIG. 10D depicts a graph demonstrating inhibition of cellular proliferation of primary AML cells treated with the indicated amount of AP1.



FIG. 11A depicts a graph demonstrating inhibition of clonogenic capacity of primary AML cells treated with the indicated amount of AP1.



FIG. 11B depicts a graph demonstrating inhibition of clonogenic capacity of primary AML cells treated with the indicated amount of AP1.



FIG. 12 depicts a graph (top) and corresponding FACS data (bottom) demonstrating AP1 induces apoptotic cell death in primary AML cells.



FIG. 13 shows a structure of peptidomimetic macrocycle 46 (Table 2b), a p53 peptidomimetic macrocycle, complexed with MDMX (Primary SwissProt accession number Q7ZUW7; Entry MDM4_DANRE).



FIG. 14 shows overlaid structures of p53 peptidomimetic macrocycles 142 (Table 2b) and SP43 bound to MDMX (Primary SwissProt accession number Q7ZUW7; Entry MDM4_DANRE).



FIG. 15 shows the effect of SP154, a peptidomimetic macrocycle, on tumor growth in a mouse MCF-7 xenograft model.



FIG. 16 shows the effect of SP249, a peptidomimetic macrocycle, on tumor growth in a mouse MCF-7 xenograft model.



FIG. 17 shows the effect of SP315, a peptidomimetic macrocycle, on tumor growth in a mouse MCF-7 xenograft model.



FIG. 18 shows the effect of SP252, a point mutation of SP154, on tumor growth in a mouse MCF-7 xenograft model.



FIG. 19 shows a plot of solubility for peptidomimetic macrocycles with varying C-terminal extensions.



FIG. 20 shows that the peptidomimetic macrocycles of the disclosure show synergy with Zelboraf (Vemurafenib, a.k.a. PLX4032) in B-Raf-mutant Melanoma Cell Line A375.



FIG. 21 shows that the peptidomimetic macrocycles of the disclosure show synergy with Zelboraf in B-Raf-mutant melanoma cell line Mel-Ho but not in B-Raf-WT Mel-Juso.



FIG. 22 shows that the peptidomimetic macrocycles of the disclosure can reduce expression levels of PD-L1 in HCT116 p53+/+ cells.



FIG. 23 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. 24A shows a bar graph of MCF-7 breast cancer cell proliferation when treated with the indicated concentrations of Aileron peptide 1. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Treatment with Aileron peptide 1 supresses MCF-7 breast cancer cell growth.



FIG. 24B shows a bar graph of MOLT-3 cell proliferation when treated with the indicated concentrations of Aileron peptide 1. Cells were evaluated for viability by a WST-1 assay 72 hours after beginning treatment. Treatment with Aileron peptide 1 supresses MOLT-3 cell growth.



FIG. 25A shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated amounts of Aileron petide 1 (log μM), Aileron peptide 1+400 nM everolimus, or Aileron peptide 1+10 nM fulvestrant. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Aileron peptide 1 in combination with fulvestrant and everolimus yields enhanced inhibition of cancer cell proliferation.



FIG. 25B shows a graph of MCF-7 breast cancer cell proliferation inhibition (fraction of control) when treated with the indicated amounts of AP1 (μM), AP1+400 nM everolimus, or AP1+10 nM fulvestrant. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. AP1 in combination with fulvestrant and everolimus yields enhanced inhibition of cancer cell proliferation.



FIG. 26 shows a bar graph of MCF-7 cell proliferation when treated with the indicated concentrations of fulvestrant. Cells were evaluated for viability by a WST-1 assay 5 days after beginning treatment. Fulvestrant treatment inhibited MCF-7 breast cancer cell proliferation with limited cell killing as a single agent.



FIG. 27A shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated amounts of Aileron petide 1, Aileron peptide 1+3 nM fulvestrant, Aileron peptide 1+10 nM fulvestrant, or Aileron peptide 1+30 nM fulvestrant. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with fulvestrant enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 27B shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated amounts of fulvestrant, fulvestrant+0.13 μM Aileron peptide 1, fulvestrant+0.4 μM Aileron peptide 1, or fulvestrant+1.2 μM Aileron peptide 1. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances fulvestrant inhibition of cancer cell proliferation and cell killing.



FIG. 28A shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated fixed amounts of Aileron petide 1 (AP1) in combination with the indicated fixed amounts of fulvestrant (FU). Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances fulvestrant inhibition of cancer cell proliferation and cell killing.



FIG. 28B shows a graph of MCF-7 breast cancer cell proliferation when treated with 0.1 μM Aileron petide 1, 3 nM fulvestrant, or 0.1 μM Aileron petide 1 and 3 nM fulvestrant. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances fulvestrant inhibition of cancer cell proliferation and cell killing.



FIG. 29 shows a bar graph of MCF-7 cell proliferation when treated with the indicated concentrations of everolimus. Cells were evaluated for viability by a WST-1 assay 5 days after beginning treatment. Everolimus treatment inhibited MCF-7 breast cancer cell proliferation with limited cell killing as a single agent.



FIG. 30A shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated amounts of Aileron petide 1, Aileron peptide 1+1 nM everolimus, Aileron peptide 1+3 nM everolimus, Aileron peptide 1+10 nM everolimus, or Aileron peptide 1+100 nM everolimus. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with everolimus enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 30B shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated amounts of everolimus, everolimus+0.13 μM Aileron peptide 1, everolimus+0.4 μM Aileron peptide 1, or everolimus+1.2 μM Aileron peptide 1. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances everolimus inhibition of cancer cell proliferation and cell killing.



FIG. 31A shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated fixed amounts of Aileron petide 1 (AP1) in combination with the indicated fixed amounts of everolimus (EV). Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances everolimus inhibition of cancer cell proliferation and cell killing.



FIG. 31B shows a graph of MCF-7 breast cancer cell proliferation when treated with 0.1 μM Aileron petide 1, 3 nM everolimus, or 0.1 μM Aileron petide 1 and 3 nM everolimus. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances everolimus inhibition of cancer cell proliferation and cell killing.



FIG. 32 shows a bar graph of MOLT-3 cell proliferation when treated with the indicated concentrations of romidepsin. Cells were evaluated for viability by a WST-1 assay 72 hours after beginning treatment. Romidepsin treatment inhibited MOLT-3 cell proliferation.



FIG. 33A shows a graph of MOLT-3 cell proliferation when treated with the indicated amounts of Aileron petide 1, Aileron peptide 1+0.5 nM romidepsin, Aileron peptide 1+1.5 nM romidepsin, or Aileron peptide 1+3 nM romidepsin. Cells were evaluated for viability by MTT assay 72 hours after beginning treatment. Combination with romidepsin enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 33B shows a graph of MOLT-3 cell proliferation when treated with the indicated amounts of romidepsin, romidepsin+0.05 μM Aileron peptide 1, romidepsin+0.2 μM Aileron peptide 1, or romidepsin+0.8 μM Aileron peptide 1. Cells were evaluated for viability by a WST-1 assay 72 hours after beginning treatment. MOLT-3 cells were pretreated with Aileron peptide 1 for 2 hours before adding romedepsin. Combination with Aileron peptide 1 enhances romidepsin inhibition of cancer cell proliferation and cell killing.



FIG. 34A shows a graph of MOLT-3 cell proliferation when treated with the indicated fixed amounts of Aileron petide 1 (AP1) in combination with the indicated fixed amounts of romidepsin (RO). Cells were evaluated for viability by a WST-1 assay 72 hours after beginning treatment. MOLT-3 cells were pretreated with Aileron peptide 1 for 2 hours before adding romedepsin. Aileron peptide 1 and romidepsin suppressed MOLT-3 cell growth. Combination with romidepsin enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 34B shows a graph of MOLT-3 cell proliferation when treated with the indicated fixed amounts of Aileron petide 1 (AP1) in combination with the indicated fixed amounts of romidepsin (RO). Cells were evaluated for viability by MTT assay 72 hours after beginning treatment. MOLT-3 cells were pretreated with Aileron peptide 1 for 2 hours before adding romedepsin. Aileron peptide 1 and romidepsin suppressed MOLT-3 cell growth. Combination with romidepsin enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 34C shows a graph of MOLT-3 cell proliferation when treated with 0.1 μM Aileron petide 1, 1.5 nM romidepsin, or 0.1 μM Aileron petide 1 and 1.5 nM romidepsin. Cells were evaluated for viability by MTT assay 72 hours after beginning treatment. MOLT-3 cells were pretreated with Aileron peptide 1 for 2 hours before adding romedepsin. Aileron peptide 1 and romidepsin suppressed MOLT-3 cell growth. Combination with romidepsin enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 35 shows a bar graph of MCF-7 cell proliferation when treated with the indicated concentrations of palbociclib. Cells were evaluated for viability by a WST-1 assay 5 days after beginning treatment. Palbociclib treatment inhibited MCF-7 breast cancer cell proliferation with limited cell killing as a single agent.



FIG. 36A shows a graph of MCF-7 breast cancer cell viability when treated with the indicated amounts of Aileron petide 1, Aileron peptide 1+0.3 μM palbociclib, Aileron peptide 1+1 μM palbociclib, Aileron peptide 1+3 μM palbociclib, or Aileron peptide 1+10 μM palbociclib. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Palbociclib has anti-proliferative effects when dosed with Aileron peptide 1. Aileron peptide 1 and palbociclib combination studies show complementary in vitro anticancer activity. Combination with palbociclib enhances Aileron peptide 1 inhibition of cancer cell proliferation and cell killing.



FIG. 36B shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated amounts of palbociclib, palbociclib+0.13 μM Aileron peptide 1, palbociclib+0.4 μM Aileron peptide 1, or palbociclib+1.2 μM Aileron peptide 1. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Combination with Aileron peptide 1 enhances palbociclib inhibition of cancer cell proliferation and cell killing.



FIG. 37A shows a graph of MCF-7 breast cancer cell proliferation when treated with the indicated fixed amounts of Aileron petide 1 (AP1) in combination with the indicated fixed amounts of palbociclib (PO). Cells were evaluated for viability by MTT assay 5 days after beginning treatment.



FIG. 37B shows a graph of MCF-7 breast cancer cell viability when treated with 0.3 μM palbociclib or 0.3 μM Aileron petide 1 and 0.3 μM palbociclib. Cells were evaluated for viability by MTT assay 5 days after beginning treatment. Aileron peptide 1 kills cancer cells when dosed with palbociclib.



FIG. 38A shows MV4-11 cell proliferation when treated with the indicated concentrations of Ara-C.



FIG. 38B shows MV4-11 cell viability when treated with varying concentrations of AP1 and Ara-C. Combination with Ara-C enhanced AP1 inhibition of cancer cell proliferation and cell killing.



FIG. 38C shows a combination index profile of treatment with AP1 and Ara-C.



FIG. 39A shows MV4-11 cell proliferation when treated with the indicated concentrations of azacitidine.



FIG. 39B shows MV4-11 cell viability when treated with varying concentrations of AP1 and azacitidine. Combination with azacitidine enhanced AP1 inhibition of cancer cell proliferation and cell killing.



FIG. 39C shows a combination index profile of treatment with AP land azacitidine.



FIG. 40A shows MV4-11 cell proliferation when treated with the indicated concentrations of decitabine.



FIG. 40B shows MV4-11 cell viability when treated with varying concentrations of AP1 and decitabine. Combination with decitabine enhanced AP1 inhibition of cancer cell proliferation and cell killing.



FIG. 40C shows a combination index profile of treatment with AP land decitabine.



FIG. 41A shows MV4-11 cell proliferation when treated with the indicated concentrations of midostaurin.



FIG. 41B shows MV4-11 cell viability when treated with varying concentrations of AP1 and midostaurin. Combination with midostaurin enhanced AP1 inhibition of cancer cell proliferation and cell killing.



FIG. 41C shows a combination index profile of treatment with AP land midostaurin.



FIG. 42A shows DOHH-2 cell proliferation when treated with the indicated concentrations of vincristine (VCR).



FIG. 42B shows DOHH-2 cell viability when treated with varying concentrations of AP1 and VCR. Combination with vincristine enhanced AP1 inhibition of cancer cell proliferation and cell killing.



FIG. 42C shows a combination index profile of treatment with AP1 and vincristine.



FIG. 43 shows DOHH-2 cell viability when treated with AP1 alone, vincristine alone, and AP1 in combination with vincristine.



FIG. 44A shows DOHH-2 cell proliferation when treated with the indicated concentrations of AP1.



FIG. 44B shows DOHH-2 cell viability when treated with varying concentrations of cyclophosphamide (CTX) and AP1. Combination with vincristine enhanced CTX inhibition of cancer cell proliferation and cell killing.



FIG. 44C shows a combination index profile of treatment with AP land CTX.



FIG. 45 shows DOHH-2 cell viability when treated with AP1 alone, CTX alone, and AP1 in combination with CTX.



FIG. 46 shows the order of addition effects on DOHH-2 cell viability using various concentrations of AP1 in combination with VCR.



FIG. 47 shows DOHH-2 cell viability based on the order of addition when treated with varying concentrations of AP1 and VCR after pretreatment with AP1 for 24 hrs.



FIG. 48 shows DOHH-2 cell viability based on the order of addition when treated with varying concentrations of AP1 and VCR after pretreatment with VCR for 24 hrs.



FIG. 49 shows the order of addition effects on DOHH-2 cell viability using various concentrations of AP1 in combination with CTX.



FIG. 50 shows DOHH-2 cell viability based on the order of addition when treated with varying concentrations of AP1 and CTX after pretreatment with AP1 for 24 hrs.



FIG. 51 shows DOHH-2 cell viability based on the order of addition when treated with varying concentrations of AP1 and CTX after pretreatment with CTX for 24 hrs.



FIG. 52 shows the order of addition effects on MV4-11 cell viability using various concentrations of AP1 and midostaurin.



FIG. 53 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and midostaurin after pretreatment with midostaurin for 24 hrs.



FIG. 54 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and midostaurin after pretreatment with AP1 for 24 hrs.



FIG. 55 shows the order of addition effects on MV4-11 cell viability using various concentrations of AP1 in combination with decitabine.



FIG. 56 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and decitabine after pretreatment with decitabine for 24 hrs.



FIG. 57 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and decitabine after pretreatment with AP1 for 24 hrs.



FIG. 58 shows the order of addition effects on MV4-11 cell viability using various concentrations of AP1 in combination with Ara-C.



FIG. 59 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and Ara-C after pretreatment with AP1 for 24 hrs.



FIG. 60 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and Ara-C after pretreatment with Ara-C for 24 hrs.



FIG. 61 shows the order of addition effects on MV4-11 cell viability using various concentrations of AP1 in combination with azacitidine.



FIG. 62 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and azacitidine after 24 hrs pretreatment with AP1.



FIG. 63 shows MV4-11 cell viability based on the order of addition when treated with varying concentrations of AP1 and azacitidine after pretreatment with azacitidine for 24 hrs.



FIG. 64A shows MCF-7 cell proliferation when treated with the indicated concentrations of fulvestrant.



FIG. 64B shows MCF-7 cell viability when treated with varying concentrations of AP1 and fulvestrant.



FIG. 65A shows MCF-7 cell proliferation when treated with varying concentrations of AP1.



FIG. 65B shows MCF-7 cell viability when treated with varying concentrations of AP1 and fulvestrant.



FIG. 65C shows the IC50 values of AP1 alone and AP1 with varying concentrations of fulvestrant (FUL).



FIG. 66A shows MCF-7 cell proliferation when treated with varying concentrations of everolimus.



FIG. 66B shows MCF-7 cell viability when treated with varying concentrations of AP1 and everolimus.



FIG. 67A shows MCF-7 cell proliferation when treated with varying concentrations of AP1. AP1 treatment suppressed MCF-7 breast cancer cell proliferation.



FIG. 67B shows a graph of MCF-7 cell viability when treated with varying concentrations of AP1 and everolimus.



FIG. 68A shows the effects of rituximab alone on DOHH-2 cell growth.



FIG. 68B shows DOHH-2 cell viability when treated with varying concentrations of AP1 and rituximab.



FIG. 69A shows the effects of AP1 alone on DOHH-2 cell growth.



FIG. 69B shows DOHH-2 cell viability when treated with varying concentrations of AP1 and rituximab.



FIG. 70 shows DOHH-2 cell viability when treated with AP1 alone, rituximab alone, and varying concentrations of AP1 in combination with rituximab.



FIG. 71A shows the effects of AP1 alone on MOLT-3 cell growth.



FIG. 71B shows MOLT-3 cell viability when treated with varying concentrations of AP1 and romidepsin.



FIG. 72A shows the effects of romidepsin alone on MOLT-3 cell growth.



FIG. 72B shows MOLT-3 cell viability when treated with varying concentrations of AP1 and romidepsin.



FIG. 72C shows the IC50 values of AP1 alone and AP1 with varying concentrations of romidepsin.



FIG. 73 shows MOLT-3 cell viability when treated with AP1 alone, romidepsin alone, and AP1 in combination with romidepsin.



FIG. 74A shows MCF-7 cell proliferation when treated with varying concentrations of ribociclib.



FIG. 74B shows MCF-7 cell viability when treated with varying concentrations of AP1 and ribociclib.



FIG. 75A shows MCF-7 cell proliferation when treated with varying concentrations of AP1.



FIG. 75B shows MCF-7 cell viability when treated with varying concentrations of AP1 and ribociclib.



FIG. 76A shows MCF-7 cell proliferation when treated with varying concentrations of abemaciclib.



FIG. 76B shows MCF-7 cell viability when treated with varying concentrations of AP1 and abemaciclib.



FIG. 77A shows MCF-7 cell proliferation when treated with varying concentrations of AP1.



FIG. 77B shows MCF-7 cell viability when treated with varying concentrations of AP1 and abemaciclib.



FIG. 78A shows MCF-7 cell proliferation when treated with varying concentrations of palbociclib.



FIG. 78B shows MCF-7 cell viability when treated with varying concentrations of AP1 and palbociclib.



FIG. 79A shows MCF-7 cell proliferation when treated with varying concentrations of AP1.



FIG. 79B shows MCF-7 cell viability when treated with varying concentrations of AP1 and palbociclib.



FIG. 80 shows the order of addition effects of AP1 and palbociclib on MCF-7 cell growth.



FIG. 81 shows MCF-7 cell viability based on the order of addition when treated with varying concentrations of AP1 and palbociclib after 24 hrs pretreatment with AP1.



FIG. 82 shows MCF-7 cell viability when treated with varying concentrations of AP1 and palbociclib determined using CyQUANT.



FIG. 83 shows MCF-7 cell viability based on the order of addition when treated with varying concentrations of AP1 and dexamethasone (Dex).



FIG. 84A shows A375 cell viability when treated with varying concentrations of zelboraf.



FIG. 84B shows A375 cell viability with treatment with varying concentrations of AP1 and zelboraf.



FIG. 85A shows A375 cell viability when treated with varying concentrations of AP1.



FIG. 85B shows A375 cell viability with treatment with varying concentrations of zelboraf and AP1.



FIG. 86A shows A375 cell viability when treated with varying concentrations of tafinlar.



FIG. 86B shows A375 cell viability with treatment with varying concentrations of AP1 and tafinlar.



FIG. 87A shows A375 cell viability when treated with varying concentrations of AP1.



FIG. 87B shows A375 cell viability with treatment with varying concentrations of tafinlar and AP1.



FIG. 88A shows A375 cell viability when treated with varying concentrations of mekinist.



FIG. 88B shows cancer cell viability with treatment with varying concentrations of AP1 and mekinist.



FIG. 89A shows A375 cell viability when treated with varying concentrations of AP1.



FIG. 89B shows A375 cell viability with treatment with varying concentrations of mekinist and AP1.



FIG. 90A shows a combination index plot of fulvestrant in MCF-7 cells.



FIG. 90B shows a combination index plot of everolimus in MCF-7 cells.



FIG. 90C shows a combination index plot of palbociclib (WST-1) in MCF-7 cells



FIG. 90D shows a combination index plot of palbociclib (CyQUANT) in MCF-7 cells.



FIG. 90E shows a combination index plot of romidepsin in MCF-7 cells.



FIG. 91A shows a combination index plot of Ara-C in MV4-11 cells.



FIG. 91B shows a combination index plot of decitabine in MV4-11 cells.



FIG. 91C shows a combination index plot of azacitidine in MV4-11 cells.



FIG. 91D shows a combination index plot of midostuarin in MV4-11 cells.



FIG. 92A shows a combination index plot of vincristine in DOHH-2 cells.



FIG. 92B shows a combination index plot of cyclophosphamide in DOHH-2 cells.



FIG. 92C shows a combination index plot ofrituximab in DOHH-2 cells.



FIG. 93 shows a combination index plot of romidepsin in MOLT-3 cells.



FIG. 94A shows a combination index plot of mekinist in A375 cells



FIG. 94B shows a combination index plot of zelboraf in A375 cells.



FIG. 94C shows a combination index plot of tafinlar in A375 cells.





DETAILED DESCRIPTION OF THE INVENTION

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 analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule. Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the α-carbon of the first amino acid residue (or analog) to the α-carbon of the second amino acid residue (or analog). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog 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.


Aileron peptide 1 is an alpha helical hydrocarbon cross-linked 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 and that 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. Aileron peptide 1 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. Aileron peptide 1 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 α helical structure by a peptidomimetic macrocycle as measured by circular dichroism or NMR. For example, in some embodiments, a peptidomimetic macrocycle exhibits 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 analogs.


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 analogs thereof. “Large hydrophobic amino acids” are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. “Polar amino acids” are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. “Charged amino acids” are lysine, arginine, histidine, aspartate, glutamate, and analogs thereof.


The term “amino acid analog” 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 analogs 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 analogs include, without limitation, structures according to the following:




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Amino acid analogs include β-amino acid analogs. Examples of β-amino acid analogs include, but are not limited to, the following: cyclic β-amino acid analogs; 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 analogs include analogs of alanine, valine, glycine or leucine. Examples of amino acid analogs 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-c, β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-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-β-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 analogs include analogs of arginine or lysine. Examples of amino acid analogs 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-ornithine; (Nδ-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine; 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 analogs include analogs of aspartic or glutamic acids. Examples of amino acid analogs 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 analogs include analogs of cysteine and methionine. Examples of amino acid analogs 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 analogs include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine include β-methyl-phenylalanine, -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 analogs include analogs of proline. Examples of amino acid analogs 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 analogs include analogs of serine and threonine. Examples of amino acid analogs 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 analogs include analogs of tryptophan. Examples of amino acid analogs of tryptophan include, but are not limited to, the following: α-methyl-tryptophan; 3-(3-benzothienyl)-D-alanine; 3-(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 analogs are racemic. In some embodiments, the D isomer of the amino acid analog is used. In some embodiments, the L isomer of the amino acid analog is used. In other embodiments, the amino acid analog comprises chiral centers that are in the R or S configuration. In still other embodiments, the amino group(s) of a β-amino acid analog 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 analog is protected, e.g., as its ester derivative. In some embodiments the salt of the amino acid analog 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:




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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:




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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 “custom-character” 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 α-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, Cul 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. Various catalysts are disclosed in Grubbs et al., Acc. Chem. Res. 1995, 28, 446-452, U.S. Pat. No. 5,811,515; U.S. Pat. No. 7,932,397; U.S. Application No. 2011/0065915; U.S. Application No. 2011/0245477; Yu et al., Nature 2011, 479, 88; and Peryshkov et al., J. Am. Chem. Soc. 2011, 133, 20754. In yet other cases, the reactive groups are thiol groups. In such 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 in it.


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 in it. 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 in it.


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 in it. 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 in it.


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, —CH2CH2CH2CH2CH2OH, —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 “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 an equilibrium dissociation constant (“KD”), which is expressed in units which are a measure of concentration (e.g. M, mM, μM, nM etc). Numerically, binding affinity and KD values vary inversely, such that a lower binding affinity corresponds to a higher KD value, while a higher binding affinity corresponds to a lower KD value. Where high binding affinity is desirable, “improved” binding affinity refers to higher binding affinity and therefore lower KD values.


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.


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 oesophagus, 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 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 waldenström 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) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); 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 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), periphieral 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.


The details of one or more particular embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.


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.


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
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, intraarterially, subcutaneously or by infusion. In some examples, the peptidomimetic macrocycle is administered intravenously. In some examples, the peptidomimetic macrocycle is administered intraarterially.


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 nonaqueous 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 (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC) can be used.


In some embodiments, the subject is a human subject and the amount of the peptidomimetic macrocycle administered is 0.01-100 mg per kilogram body weight of the human subject. For example, in various examples, the amount of the peptidomimetic macrocycle administered is about 0.01-50 mg/kg, about 0.01-20 mg/kg, about 0.01-10 mg/kg, about 0.1-100 mg/kg, about 0.1-50 mg/kg, about 0.1-20 mg/kg, about 0.1-10 mg/kg, about 0.5-100 mg/kg, about 0.5-50 mg/kg, about 0.5-20 mg/kg, about 0.5-10 mg/kg, about 1-100 mg/kg, about 1-50 mg/kg, about 1-20 mg/kg, about 1-10 mg/kg body weight of the human subject. In one embodiment, about 0.5 mg-10 mg of the peptidomimetic macrocycle per kilogram body weight of the human subject is administered. In some examples 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 human subject. In some examples 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, or about 14.24 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 0.16 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 0.32 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 0.64 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 1.28 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 3.56 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 7.12 mg per kilogram body weight of the human subject. In some examples the amount of the peptidomimetic macrocycle administered is about 14.24 mg per kilogram body weight of the human subject.


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 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 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, 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, the peptidomimetic macrocycle is administered gradually over a period of time. A desired amount of peptidomimetic macrocycle can, for example can be administered gradually over a period of from about 0.1 h-24 h. In some cases a desired amount of peptidomimetic macrocycle is administered gradually over a period of 0.1 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h. In some examples, a desired amount of peptidomimetic macrocycle is administered gradually over a period of 0.25-12 h, for example over a period of 0.25-1 h, 0.25-2 h, 0.25-3 h, 0.25-4 h, 0.25-6 h, 0.25-8 h, 0.25-10 h. In some examples, a desired amount of peptidomimetic macrocycle is administered gradually over a period of 0.25-2 h. In some examples, a desired amount of peptidomimetic macrocycle is administered gradually over a period of 0.25-1 h. In some examples, a desired amount of peptidomimetic macrocycle is administered gradually over a period of 0.25 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, or 2.0 h. In some examples, a desired amount of peptidomimetic macrocycle is administered gradually over a period of 1 h. In some examples, a desired amount of peptidomimetic macrocycle is administered gradually over a period of 2 h.


Administration of the peptidomimetic macrocycles can continue as long as necessary. In some embodiments, one or more peptidomimetic macrocycle of the disclosure is 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, the 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 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 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.


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.


Various methods and software programs can be used to determine the homology between two or more peptides, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm.


Peptidomimetic Macrocycles

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




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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,




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[—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 intrahelical 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




embedded image


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:




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wherein:

    • each of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, 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, 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. In other embodiments, at least four of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, 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-Leu11-X11-Ser2. 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. In other embodiments, at least six of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, 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. In other embodiments, at least seven of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, 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.


In some embodiments, a peptidomimetic macrocycle has the Formula:




embedded image


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-X1-Ala12, 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-Alas-Gln9-Leu10/Cba10-X11-Ala12. In other embodiments of the above Formula, at least four of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, 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. 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-Alas-Gln9-Leu10/Cba10-X11-Ala12. In other embodiments of the above Formula, at least six of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, 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. In other embodiments of the above Formula, at least seven of Xaa3, Xaa5, Xaa6, Xaa7, Xaa5, Xaa9, and Xaa10 are the same amino acid as the amino acid at the corresponding position of the sequence Phe3-X4-Glu5-Tyr6-Trp7-Alas-Gln9-Leu10/Cba10-X11-Ala2.


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, for example 2-5. 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 intrahelical 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




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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):




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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,




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[—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; and—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 may encompass peptidomimetic macrocycles which are the same or different. For example, a compound may 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 intrahelical 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




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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:




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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:




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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:




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wherein “AA” represents any natural or non-natural amino acid side chain and “custom-character” 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.




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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):




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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,




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[—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




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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 ia 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 intrahelical 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




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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




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




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or a tautomer thereof.


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




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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):




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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,




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[—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 intrahelical 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




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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.




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




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wherein:

    • each Aa, Ca, Da, Ea, Ab, Cb, and Db is independently a natural or non-natural amino acid or an amino acid analog;
    • each Ba and Bb is independently a natural or non-natural amino acid, amino acid analog,




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

    • each Ra1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or Ra1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the Da or Ea amino acids; or together with La forms a ring that is unsubstituted or substituted;
    • each Ra2 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or Ra2 forms a macrocycle-forming linker L′ connected to the alpha position of one of the Da or Ea amino acids; or together with La forms a ring that is unsubstituted or substituted;
    • each Rb1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or Rb1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the Db amino acids; or together with Lb 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 La is independently a macrocycle-forming linker, and optionally forms a ring with Ra1 or Ra2 that is unsubstituted or substituted;
    • each Lb is independently a macrocycle-forming linker, and optionally forms a ring with Rb1 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, NR3q, CONR3q, OCONR3q, or OSO2NR3q, wherein each R3q is independently a point of attachment to Ra1, Ra2, or Rb1;
    • Ra7 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 Da amino acid;
    • Rb7 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 Db amino acid;
    • Ra8 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 Ea amino acid;
    • Rb8 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):




embedded image


wherein:

    • each Aa, Ca, Da, Ea, Ab, Cb, and Db is independently a natural or non-natural amino acid or an amino acid analog;
    • each Ba and Bb is independently a natural or non-natural amino acid, amino acid analog,




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

    • each Ra1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or Ra1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the Da or Ea amino acids; or together with La forms a ring that is unsubstituted or substituted;
    • each Ra2 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or Ra2 forms a macrocycle-forming linker L′ connected to the alpha position of one of the Da or Ea amino acids; or together with La forms a ring that is unsubstituted or substituted;
    • each Rb1 is independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or substituted; or H; or Rb1 forms a macrocycle-forming linker L′ connected to the alpha position of one of the Db amino acids; or together with Lb 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 La is independently a macrocycle-forming linker, and optionally forms a ring with Ra1 or Ra2 that is unsubstituted or substituted;
    • each Lb is independently a macrocycle-forming linker, and optionally forms a ring with Rb1 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, NR3q, CONR3q, OCONR3q, or OSO2NR3q, wherein each R3q is independently a point of attachment to Ra1, Ra2, or Rb1;
    • 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 Ra7 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 Da amino acid;
    • Rb7 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 Db amino acid;
    • each Ra8 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 Ea amino acid;
    • Rb8 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 La is independently a macrocycle-forming linker of the formula -L1-L2-, and optionally forms a ring with Ra1 or Ra2 that is unsubstituted or substituted;
    • each Lb is independently a macrocycle-forming linker of the formula -L1-L2-, and optionally forms a ring with Rb1 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, NR3q, CONR3q, OCONR3q, or OSO2NR3q, wherein each R3q is independently a point of attachment to Ra1, Ra2, or Rb1;
    • 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 La and Lb is a bis-thioether-containing macrocycle-forming linker. In some embodiments, one of La and Lb 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 La and Lb is a bis-sulfone-containing macrocycle-forming linker. In some embodiments, one of La and Lb 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 La and Lb is a bis-sulfoxide-containing macrocycle-forming linker. In some embodiments, one of La and Lb 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, ua is 0. In some embodiments, ua is 0, and Lb is a macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, ua is 0, and Lb is a macrocycle-forming linker that crosslinks a α-hairpin secondary structure. In some embodiments, ua is 0, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, ua is 0, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure.


In some embodiments, ub is 0. In some embodiments, Ub is 0, and La is a macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, ub is 0, and La is a macrocycle-forming linker that crosslinks a β-hairpin secondary structure. In some embodiments, ub is 0, and L. is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, Ub is 0, and La is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-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, La and Lb are a combination of hydrocarbon-, triazole, or sulfur-containing macrocycle-forming linkers. In some embodiments, the peptidomimetic macrocycle comprises La and Lb, wherein La is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and Lb is a triazole-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, the peptidomimetic macrocycle comprises La and Lb, wherein La is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure, and Lb is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, the peptidomimetic macrocycle comprises La and Lb, wherein La is a triazole-containing macrocycle-forming linker that crosslinks an α-helical structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, the peptidomimetic macrocycle comprises La and Lb, wherein La is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure.


In some embodiments, ua+ub is at least 1. In some embodiments, ua+ub=2.


In some embodiments, ua is 1, ub is 1, La is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, ua is 1, ub is 1, La is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, ua is 1, ub is 1, La is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, ua is 1, ub is 1, La is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure.


In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and Lb is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical secondary structure, and Lb is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure. In some embodiments, ua is 1, Ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and Lb is a triazole-containing macrocycle-forming linker that crosslinks an α-helical secondary structure. In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure, and Lb is a triazole-containing macrocycle-forming linker that crosslinks a β-hairpin secondary structure.


In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker with an α-helical secondary structure, and Lb is a sulfur-containing macrocycle-forming linker. In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker with a β-hairpin secondary structure, and Lb is a sulfur-containing macrocycle-forming linker.


In some embodiments, ua is 1, ub is 1, La is a sulfur-containing macrocycle-forming linker, and Lb is a hydrocarbon-containing macrocycle-forming linker with an α-helical secondary structure. In some embodiments, ua is 1, ub is 1, La is a sulfur-containing macrocycle-forming linker, and Lb is a hydrocarbon-containing macrocycle-forming linker with a β-hairpin secondary structure.


In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure. In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks an α-helical structure. In some embodiments, ua is 1, ub is 1, La is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure, and Lb is a hydrocarbon-containing macrocycle-forming linker that crosslinks a β-hairpin structure.


In some embodiments, Rb1 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.


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 by a deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this invention.


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.


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, or Table 1c 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.


Various methods to effect formation of peptidomimetic macrocycles are known in the art. For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. The α,α-disubstituted amino acids and amino acid precursors disclosed in the cited references 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:




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In other embodiments, the peptidomimetic macrocycles are of Formula IV or IVa. Methods for the preparation of such macrocycles are described, for example, in U.S. Pat. No. 7,202,332.


Additional methods of forming peptidomimetic macrocycles which are envisioned as suitable include those disclosed by Mustapa et al., J. Org. Chem. (2003), 68, pp. 8193-8198; Yang et al. Bioorg. Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat. No. 5,446,128; U.S. Pat. No. 5,824,483; U.S. Pat. No. 6,713,280; and U.S. Pat. No. 7,202,332. 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.


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.


Biological Samples

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.


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, H&E staining, microscopic assessment of tumors, next-generation DNA sequencing (NGS) (e.g. extraction, purification, quantification, and amplification of DNA, 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. See e.g., Saiki et al., Science, Vol. 239, p. 487, 1988; U.S. Pat. No. 4,683,202; and U.S. Pat. No. 4,683,195. 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. See, e.g., Cotton et al., Proc. Natl. Acad. Sci. USA, vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad. Sci. USA, vol. 72, p. 989, 1975. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, Human Genetics, vol. 42, p. 726, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) 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 (Lips et al., 2005; Lai et al., 2007).


Examples of p53 gene sequence and single nucleotide polymorphism arrays include p53 Gene Chip (Affymetrix, Santa Clara, Calif.), Roche p53 Ampli-Chip (Roche Molecular Systems, Pleasanton, Calif.), GeneChip Mapping arrays (Affymetrix, Santa Clara, Calif.), SNP Array 6.0 (Affymetrix, Santa Clara, Calif.), BeadArrays (Illumina, San Diego, Calif.), etc.


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.


Assay 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 (e.g., Jasco J-710) 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 (Yang et al. (1986), Methods Enzymol. 130:208)).


Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle comprising a secondary structure such as an α-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Typically peptidomimetic macrocycles exhibit Tm 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 Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710) 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).


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, typically 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 (Pierce) (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).


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.


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 (e.g. Perkin-Elmer LS50B). Kd values can be determined by nonlinear regression analysis using, for example, GraphPad Prism software (GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic macrocycle shows, In some embodiments, similar or lower Kd than a corresponding uncrosslinked polypeptide.


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 (e.g. Perkin-Elmer LS50B). Kd values can be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.).


Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.


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 (Phosphate-buffered saline: 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.


Assay for Protein-Ligand Kd 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 Kd 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 Kd as described in Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hifner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.


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. Additional details on these and other methods are provided in “A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions” D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hifner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.


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.


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, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.


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.


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.


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 (e.g., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, Mass.). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, Mass.). 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.


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.


Pharmaceutical Compositions and Routes of Administration

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.


Pharmaceutically acceptable salts of the compounds disclosed herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases. 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.


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. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.


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 cross-linked 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 packeted 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.


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.


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.


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) (reviewed in Vaickus (1991), Crit Rev. Oncol./Hemotol. 11:267-97); 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

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.


In some embodiments, the combination therapy can be particularly advantageous, since not only the therapeutic (for e.g. anti-cancerous) effect may be enhanced compared to the effect of each compound alone, the dosage of each agent in a combination therapy may also be reduced as compared to monotherapy with each agent, while still achieving an overall therapeutic (e.g. anti-tumor) effect. In addition, in some embodiments, the peptidomimetic macrocycles of the disclosure can exhibit synergistic effect with the additional pharmaceutical agents. In such cases, due to the synergistic effect, the total amount of drugs administered to a patient can advantageously be reduced, which may result in decreased side effects.


The present disclosure also provides methods for combination therapies in which the peptidomimetic macrocycles of the disclosure are used in combination with at least one additional pharmaceutically active agent. In various embodiments, the at least one additional pharmaceutically active agent may be capable of modulating the same or a different target as the peptidomimetic macrocycles of the disclosure. In some embodiments, the at least one additional pharmaceutically active agent may modulate the same target as the peptidomimetic macrocycles of the disclosure, or other components of the same pathway, or even overlapping sets of target enzymes. In some embodiments, the at least one additional pharmaceutically active agent may modulate a different target as the peptidomimetic macrocycles of the disclosure.


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.


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 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.


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, e.g., stem cells, or any combination thereof.


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an estrogen receptor antagonist. In one example, the peptidomimetic macrocycles of the disclosure are used in combination with the estrogen receptor antagonist fulvestrant (FASLODEX). 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 typically administered via intramuscular injection. Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin beta1 (see, e.g., Hutcheson et al., Breast cancer Research (2011) 13:R29).


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with an aromatase inhibitor. In one example, 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 a mTOR inhibitor. In one example, the peptidomimetic macrocycles of the disclosure are used in combination with everolimus (AFINITOR). Everolimus affects the mTORC 1 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 FKBP 12, a protein receptor which directly interacts with mTORC 1 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 macrocyclyes can be used in combination with everolimus and exemestane. Everolimus shows clinical efficacy in combination with tamoxifen, letrozole, or exemestane for the treatment of estrogen receptor-positive breast cancer (see, e.g., Chen et al., Mol. Cancer Res. 11(10); 1269-78 (2013).


In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with one or more antimetabolites, for example in combination with Capccitabine (XELODA), Gemcitabine (GEMZAR) and Cytarabine (cytosine arabinoside also known as Ara-C(arabinofuranosyl cytidine; Cytosar-U)).


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with taxanes. Exemplary non-limiting taxanes that may be used in combination with the instant peptidomimetic macrocycles include 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.


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with therapeutic antibodies. Examples of therapeutic antibodies that can be combined with compounds of this disclosure include but are not limited to anti CD20 antibodies, for example rituximab (MABTHERA/RITUXAN) or obinutuzumab (GAZYVA). 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).


PD-1 antagonists useful in the any of the treatment method, medicaments and uses of the present invention include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1. A PD-1 antagonist can be any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and may also block binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the treatment method, medicaments and uses of the present invention in which a human individual is being treated, the PD-1 antagonist can block binding of human PD-L1 to human PD-1, and may block binding of both human PD-L1 and PD-L2 to human PD-1.


Examples of mAbs that bind to human PD-1, and useful in the treatment method, medicaments and uses of the present invention, are described in U.S. Pat. No. 7,521,051, U.S. Pat. No. 8,008,449, and U.S. Pat. No. 8,354,509. Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention include: MK-3475, a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013), nivolumab (BMS-936558), a human IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013); the humanized antibodies h409A11, h409A16 and h409A17, which are described in WO2008/156712, and AMP-514.


Other PD-1 antagonists useful in the any of the treatment method, medicaments and uses of the present invention include an immunoadhesin that specifically binds to PD-1 or PD-L1. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in WO2010/027827 and WO2011/066342. Specific fusion proteins useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1.


Other antibodies that can be used in combination with the peptidomimetic macrocycles of the disclosure include antibodies against human PD-L1. Examples of antibodies that bind to human PD-L1 and useful in the treatment method, medicaments and uses of the present invention, are described in WO2013/019906, WO2010/077634 A1 and U.S. Pat. No. 8,383,796. Specific anti-human PD-L1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C and an antibody which comprises the heavy chain and light chain variable regions of SEQ ID NO:24 and SEQ ID NO:21, respectively, of WO2013/019906. Exemplary useful antibodies targeting PD-1 receptors include Pidilizumab, BMS 936559, and MPDL328OA. An exemplary anti-PD-L1 antibody is human monoclonal antibody MDX-1105 which binds PD-L1 and blocks its binding to and activation of its receptor PD-1, which may enhance the T cell-mediated immune response to neoplasms and reverse T-cell inactivation in chronic infections disease. An exemplary anti-PD-1 antibody is human monoclonal antibody MDX-1106 which binds and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2, resulting in the activation of T cells and cell-mediated immune responses against tumor cell


Several approaches have been described for quantifying PD-L1 protein expression in IHC assays of tumor tissue sections. See, e.g., Thompson, R. H., et al, PNAS 101 (49); 17174-17179 (2004); Thompson, R. H. et al, Cancer Res. 66:3381-3385 (2006); Gadiot, J., et al, Cancer 117:2192-2201 (2011); Taube, J. M. et al, Sci Transl Med 4, 127ra37 (2012); and Toplian, S. L. et al, New Eng. J Med. 366 (26): 2443-2454 (2012). One approach employs a simple binary end-point of positive or negative for PD-LI expression, with a positive result defined in terms of the percentage of tumor cells that exhibit histologic evidence of cell-surface membrane staining. A tumor tissue section is counted as positive for PD-L1 expression is at least 1%, and preferably 5% of total tumor cells. In another approach, PD-L1 expression in the tumor tissue section is quantified in the tumor cells as well as in infiltrating immune cells, which predominantly comprise lymphocytes. The percentage of tumor cells and infiltrating immune cells that exhibit membrane staining are separately quantified as <5%, 5 to 9%, and then in 10% increments up to 100%. For tumor cells, PD-L1 expression is counted as negative if the score is <5% score and positive if the score is >5%. PD-L1 expression in the immune infiltrate is reported as a semi-quantitative measurement called the adjusted inflammation score (AIS), which is determined by multiplying the percent of membrane staining cells by the intensity of the infiltrate, which is graded as none (0), mild (score of 1, rare lymphocytes), moderate (score of 2, focal infiltration of tumor by lymphohistiocytic aggregates), or severe (score of 3, diffuse infiltration). A tumor tissue section is counted as positive for PD-L1 expression by immune infiltrates if the AIS is >5. A tissue section from a tumor that has been stained by IHC with a diagnostic PD-LI antibody may also be scored for PD-L1 protein expression by assessing PD-L1 expression in both the tumor cells and infiltrating immune cells in the tissue section. This PD-L1 scoring process can comprise examining each tumor nest in the tissue section for staining, and assigning to the tissue section one or both of a modified H score (MHS) and a modified proportion score (MPS). To assign the MHS, four separate percentages are estimated across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests: (a) cells that have no staining (intensity=0), (b) weak staining (intensity=1+), (c) moderate staining (intensity=2+) and (d) strong staining (intensity=3+). A cell must have at least partial membrane staining to be included in the weak, moderate or strong staining percentages. The estimated percentages, the sum of which is 100%, are then input into the formula of 1×(percent of weak staining cells)+2×(percent of moderate staining cells)+3×(percent of strong staining cells), and the result is assigned to the tissue section as the MHS. The MPS is assigned by estimating, across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests, the percentage of cells that have at least partial membrane staining of any intensity, and the resulting percentage is assigned to the tissue section as the MPS. In some embodiments, the tumor is designated as positive for PD-L1 expression if the MHS or the MPS is positive. The level of PD-L mRNA expression may be compared to the mRNA expression levels of one or more reference genes that are frequently used in quantitative RT-PCR, such as ubiquitin C. In some embodiments, a level of PD-L1 expression (protein and/or mRNA) by malignant cells and/or by infiltrating immune cells within a tumor is determined to be “overexpressed” or “elevated” based on comparison with the level of PD-L1 expression (protein and/or mRNA) by an appropriate control. For example, a control PD-L1 protein or mRNA expression level may be the level quantified in nonmalignant cells of the same type or in a section from a matched normal tissue. In some preferred embodiments, PD-L1 expression in a tumor sample is determined to be elevated if PD-L1 protein (and/or PD-L1 mRNA) in the sample is at least 10%, 20%, or 30% greater than in the control.


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in combination with antihormone therapy. Exemplary hormone antagonists that may be used in combination with the peptidomimetic macrocycles of the instant disclosure include letrozole (FEMARA) and casodex.


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with hypomethylating agents or demethylating agents. Examples of such agents that may be used in combination with the peptidomimetic macrocycles of the disclosure include azacitidine (VIDAZA, AZADINE) and decitabine (Dacogen).


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with an anti-inflammatory agent. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with a corticosteroid. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with a glucocorticosteroid. In one example, the peptidomimetic macrocycles of the disclosure are used in combination with dexamethasone.


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with a histone deacetylase (HDAC) inhibitor. In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with a depsipeptide. In one example, the peptidomimetic macrocycles of the disclosure are used in combination with romidepsin (ISTODAX). Exemplary cancers for treatment with the peptidomimetic macrocycles of the disclosure and HDAC inhibitors, such as romidepsin, include T-cell lymphomas, for example, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), or periphieral T-cell lymphoma (PTCL). HDAC inhibitors may interact synergistically with MDM2 inhibitors by mediating hyperacetylation of p53. Acetylation may be required for p53 activation. HDAC inhibitors may enhance the antitumor action of MDM2 inhibitors by diminishing MDM2 inhibitor-induced MDM2 expression. MDM2 is upregulated by p53 activation in a feedback loop that negatively controls p53 activity. MDM2 inhibitors may elicit cancer cell death by downregulating MDM4 expression. MDM4 is the second main negative regulator of p53, which is structurally homologues, but functionally not redundant to MDM2. Nutlin-3 and vorinostat cooperate in affecting cell viability and in inducing cell death and Δψm loss in A549 cells and cooperate in inducing cell death, Δψm loss and caspase-3 activity in A2780 cells (see, e.g., J. Sonnemann et al., Invest New Drugs (2012) 30:25-36).


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with platinum-based antineoplastic drugs (platinum drugs or platins). Examples of the platins that may be used in combination with the peptidomimetic macrocycles of the disclosure include cisplatin (also known as cisplatinum, platamin, neoplatin, cismaplat, cis-diamminedichloroplatinum(II), or CDDP; tradename PLATINOL) and carboplatin (also known as cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II); tradenames PARAPLATIN and PARAPLATIN-AQ).


In some embodiments, the peptidomimetic macrocycles of the disclosure are used in in combination with a kinase inhibitor drug. The compounds described herein can be used in combination with MEK inhibitors. The compounds described herein can be used in combination with MEK1 inhibitors. The compounds described herein can be used in combination with MEK2 inhibitors. The compounds described herein can be used in combination with inhibitors of MEK1 and MEK2. In one example, he peptidomimetic macrocycles of the disclosure are used in in combination with trametinib (MEKINIST). The compounds described herein can be used in combination with BRAF inhibitors. The BRAF inhibitors used in combination with the peptidomimetic macrocycles of the disclosure may be inhibitor of either wild type or mutated BRAF. In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with at least one additional pharmaceutically active agent that is an inhibitor of wild type BRAF. In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with at least one additional pharmaceutically active agent that is an inhibitor of mutated BRAF. In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with at least one additional pharmaceutically active agent that is an inhibitor of a V600E mutated BRAF. In some embodiments the compounds described herein can be used in combination with one or more BRAF inhibitors selected from vemurafenib (ZELBORAF a.k.a. PLX4032), dabrafenib (TAFINLAR), C-1, NVP-LGX818 and sorafenib (NEXAVAR). In some embodiments the compounds described herein can synergize with one or more BRAF inhibitors. In some embodiments one or more of the compounds described herein can synergize with all BRAF inhibitors.


The compounds described herein can be used in combination with KRAS inhibitors. The KRAS inhibitors used in combination with the peptidomimetic macrocycles of the disclosure may be inhibitor of either wild type or mutated KRAS. In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with at least one additional pharmaceutically active agent that is an inhibitor of wild type KRAS. In some examples, the peptidomimetic macrocycles of the disclosure are used in combination with at least one additional pharmaceutically active agent that is an inhibitor of mutated KRAS. In some embodiments the compounds described herein can synergize with one or more KRAS inhibitors. In some embodiments one or more of the compounds described herein can synergize with all KRAS inhibitors.


The peptidomimetic macrocycles of the disclosure may also be used in combination with Bruton's tyrosine kinase (BTK) inhibitor, for example in combination with ibrutinib (IMBRUVICA). In some embodiments the compounds described herein can synergize with one or more BTK inhibitor. In some embodiments one or more of the compounds described herein can synergize with all BTK inhibitors.


In some examples, the peptidomimetic macrocycles of the disclosure may also be used in combination with inhibitors of the cyclin-dependent kinases, for example with an inhibitor of CDK4 and/or CDK6. An example of such inhibitor that may be used in combination with the instant peptidomimetic macrocycle is palbociclib (IBRANCE) (see, e.g., Clin. Cancer Res.; 2015, 21(13); 2905-10). 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 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. In some examples, the p53 agent antagonizes an interaction between p53 and MDM2 proteins and/or between p53 and MDMX proteins. In some examples, the at least one additional pharmaceutically active agent binds to CDK4 and/or CDK6. 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 any one of claims 1-56; 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 examples, 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 (XI006); JNJ26854165 (serdemetan); RITA (NSC652287); YH239EE; and any combination thereof. In some examples, 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 analog, a peptide derivative; an antibody, an antibody fragment, a peptidomimetic; a peptidomimetic macrocycle of any one of claims 1-56; 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 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.


In some examples, the peptidomimetic macrocycles of the disclosure may also be used in combination with inhibitors of the cyclin-dependent kinases and an estrogen receptor antagonist. An example of such inhibitors that may be used in combination with the instant peptidomimetic macrocycle is palbociclib and fulvestrant. 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.


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.


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.


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 various 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,
Yes: S phase




Linfolizin


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









The peptidomimetic macrocycles or a composition comprising same and the at least one additional pharmaceutically active agent 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 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 are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the drugs are administered simultaneously, the peptidomimetic macrocycle and the at least one additional pharmaceutically active agent 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 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 are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60 or more minutes. Either the peptidomimetic macrocycle or the pharmaceutically active agent may be administered first. The peptidomimetic macrocycle and the additional pharmaceutically active agent 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 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 are non-concurrent. For example, in some embodiments, the administration of the peptidomimetic macrocycles is terminated before the additional pharmaceutically active agent is administered. In some embodiments, the administration of the additional pharmaceutically active agent 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 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 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 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 can be administered more or less frequently. In addition, the peptidomimetic macrocycle and the at least one additional pharmaceutically active agent can be administered using the same route of administration or using different routes of administration.


According to certain embodiments, the peptidomimetic macrocycles and the additional pharmaceutically active agent are administered within a single pharmaceutical composition. 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 are administered within different pharmaceutical composition.


According to certain embodiments, peptidomimetic macrocycles is administered in an amount of from 0 mg/kg body weight to 100 mg/kg body weight. According to other embodiments, the peptidomimetic macrocycle is administered at an amount of from 0.5 mg/kg body weight to 20 mg/kg body weight. According to additional embodiments, the peptidomimetic macrocycle is administered at an amount of from 1.0 mg/kg body weight to 10 mg/kg body weight. The at least one additional pharmaceutical agent is administered at the therapeutic amount known to be used for treating the specific type of cancer. According to other embodiments, the at least one additional pharmaceutical agent 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.


While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments described herein can be employed in practicing the disclosure. It is intended that the following claims define the scope and that methods and structures within the scope of these claims and their equivalents be covered thereby.


Examples
Example 1: Synthesis of 6-chlorotryptophan Fmoc amino acids



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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 the same temperature 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 allowed to warm to room temperature and stirred for an additional 2.5 h. Water (50 mL) was added 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 directly 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 give the 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 solution was stirred an additional 30 min at 40° C. Then NBS (3.38 g, 19 mmol, 1.3 eq.) was added. The resulting mixture was allowed to warm 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 in a silica plug and quickly eluted with 25% EtOAc in Hexanes. The solution was concentrated to give 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.) in solution of DMF (5.0 mL) was added via 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). αMe-6Cl-Trp(Boc)-Ni—S-BPB, 4: M+H calc. 775.21, M+H obs. 775.26; 1H NMR (CDCl3) δ: 1.23 (s, 3H, αMe); 1.56 (m, 11H, Boc+CH2); 1.82-2.20 (m, 4H, 2CH2); 3.03 (m, 1H, CHα); 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).


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 more equivalents of Na2CO3 (1.95 g, 18.4 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (1.68 g, 4.5 mmol, 2 eq.) was then added and the 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. Afterwards, 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). Fmoc-αMe-6Cl-Trp(Boc)-OH, 6: 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).


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.) in solution of DMF (10 mL) was added via 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). 6Cl-Trp(Boc)-Ni—S-BPB, 5: 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, CHα); 3.22 (m, 2H, CH2); 3.45 and 4.25 (AB system, 2H, CH2 (benzyl), J=12.8 Hz); 4.27 (m, 1H, CHα); 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-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 more equivalents of Na2CO3 (5.57 g, 52 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (4.89 g, 13.1 mmol, 2 eq.) and the 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. Afterwards, 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). Fmoc-6Cl-Trp(Boc)-OH, 7: 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 synthesized, purified and analyzed as previously described and as described below (Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No. 7,192,713). 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 either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.


Purification of cross-linked compounds was achieved by high performance liquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18 column (Varian) to yield the pure compounds. Chemical composition of the pure products was confirmed by LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).


The following protocol was used in the 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 preactivated 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 deprotected 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 deprotected 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 deprotected 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.


The following protocol was used in the 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 preactivated 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 deprotected 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 deprotected 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, 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. Molybdenumhexacarbonyl (0.01 eq, Sigma Aldrich 199959) was added. Anhydrous chlorobenzene was added to the reaction vessel. Then 2-fluorophenol (1 eq, Sigma Aldrich F12804) was added. The reaction was then loaded into the microwave and held at 130° C. for 10 minutes. Reaction may need to be pushed a subsequent time for completion. The alkyne metathesized resin-bound peptides were deprotected and cleaved from the solid support by treatment 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 shows a list of peptidomimetic macrocycles prepared.
















TABLE 1








Exact
Found
Calc
Calc
Calc


SP
Sequence
Isomer
Mass
Mass
(M + 1)/1
(M + 2)/2
(M + 3)/3






















SP1
Ac-F$r8AYWEAc3cL$AAA-NH2

1456.78
729.44
1457.79
729.4
486.6


SP2
Ac-F$r8AYWEAc3cL$AAibA-NH2

1470.79
736.4
1471.8
736.4
491.27


SP3
Ac-LTF$r8AYWAQL$SANle-NH2

1715.97
859.02
1716.98
858.99
573


SP4
Ac-LTF$r8AYWAQL$SAL-NH2

1715.97
859.02
1716.98
858.99
573


SP5
Ac-LTF$r8AYWAQL$SAM-NH2

1733.92
868.48
1734.93
867.97
578.98


SP6
Ac-LTF$r8AYWAQL$SAhL-NH2

1729.98
865.98
1730.99
866
577.67


SP7
Ac-LTF$r8AYWAQL$SAF-NH2

1749.95
876.36
1750.96
875.98
584.32


SP8
Ac-LTF$r8AYWAQL$SAI-NH2

1715.97
859.02
1716.98
858.99
573


SP9
Ac-LTF$r8AYWAQL$SAChg-NH2

1741.98
871.98
1742.99
872
581.67


SP10
Ac-LTF$r8AYWAQL$SAAib-NH2

1687.93
845.36
1688.94
844.97
563.65


SP11
Ac-LTF$r8AYWAQL$SAA-NH2

1673.92
838.01
1674.93
837.97
558.98


SP12
Ac-LTF$r8AYWA$L$S$Nle-NH2

1767.04
884.77
1768.05
884.53
590.02


SP13
Ac-LTF$r8AYWA$L$S$A-NH2

1724.99
864.23
1726
863.5
576


SP14
Ac-F$r8AYWEAc3cL$AANle-NH2

1498.82
750.46
1499.83
750.42
500.61


SP15
Ac-F$r8AYWEAc3cL$AAL-NH2

1498.82
750.46
1499.83
750.42
500.61


SP16
Ac-F$r8AYWEAc3cL$AAM-NH2

1516.78
759.41
1517.79
759.4
506.6


SP17
Ac-F$r8AYWEAc3cL$AAhL-NH2

1512.84
757.49
1513.85
757.43
505.29


SP18
Ac-F$r8AYWEAc3cL$AAF-NH2

1532.81
767.48
1533.82
767.41
511.94


SP19
Ac-F$r8AYWEAc3cL$AAI-NH2

1498.82
750.39
1499.83
750.42
500.61


SP20
Ac-F$r8AYWEAc3cL$AAChg-NH2

1524.84
763.48
1525.85
763.43
509.29


SP21
Ac-F$r8AYWEAc3cL$AACha-NH2

1538.85
770.44
1539.86
770.43
513.96


SP22
Ac-F$r8AYWEAc3cL$AAAib-NH2

1470.79
736.84
1471.8
736.4
491.27


SP23
Ac-LTF$r8AYWAQL$AAAibV-NH2

1771.01
885.81
1772.02
886.51
591.34


SP24
Ac-LTF$r8AYWAQL$AAAibV-NH2
iso2
1771.01
886.26
1772.02
886.51
591.34


SP25
Ac-LTF$r8AYWAQL$SAibAA-NH2

1758.97
879.89
1759.98
880.49
587.33


SP26
Ac-LTF$r8AYWAQL$SAibAA-NH2
iso2
1758.97
880.34
1759.98
880.49
587.33


SP27
Ac-HLTF$r8HHWHQL$AANleNle-NH2

2056.15
1028.86
2057.16
1029.08
686.39


SP28
Ac-DLTF$r8HHWHQL$RRLV-NH2

2190.23
731.15
2191.24
1096.12
731.08


SP29
Ac-HHTF$r8HHWHQL$AAML-NH2

2098.08
700.43
2099.09
1050.05
700.37


SP30
Ac-F$r8HHWHQL$RRDCha-NH2

1917.06
959.96
1918.07
959.54
640.03


SP31
Ac-F$r8HHWHQL$HRFV-NH2

1876.02
938.65
1877.03
939.02
626.35


SP32
Ac-HLTF$r8HHWHQL$AAhLA-NH2

2028.12
677.2
2029.13
1015.07
677.05


SP33
Ac-DLTF$r8HHWHQL$RRChgl-NH2

2230.26
1115.89
2231.27
1116.14
744.43


SP34
Ac-DLTF$r8HHWHQL$RRChgl-NH2
iso2
2230.26
1115.96
2231.27
1116.14
744.43


SP35
Ac-HHTF$r8HHWHQL$AAChav-NH2

2106.14
1053.95
2107.15
1054.08
703.05


SP36
Ac-F$r8HHWHQL$RRDa-NH2

1834.99
918.3
1836
918.5
612.67


SP37
Ac-F$r8HHWHQL$HRAibG-NH2

1771.95
886.77
1772.96
886.98
591.66


SP38
Ac-F$r8AYWAQL$HHNleL-NH2

1730.97
866.57
1731.98
866.49
578


SP39
Ac-F$r8AYWSAL$HQANle-NH2

1638.89
820.54
1639.9
820.45
547.3


SP40
Ac-F$r8AYWVQL$QHChgl-NH2

1776.01
889.44
1777.02
889.01
593.01


SP41
Ac-F$r8AYWTAL$QQNlev-NH2

1671.94
836.97
1672.95
836.98
558.32


SP42
Ac-F$r8AYWYQL$HAibAa-NH2

1686.89
844.52
1687.9
844.45
563.3


SP43
Ac-LTF$r8AYWAQL$HHLa-NH2

1903.05
952.27
1904.06
952.53
635.36


SP44
Ac-LTF$r8AYWAQL$HHLa-NH2
iso2
1903.05
952.27
1904.06
952.53
635.36


SP45
Ac-LTF$r8AYWAQL$HQNlev-NH2

1922.08
962.48
1923.09
962.05
641.7


SP46
Ac-LTF$r8AYWAQL$HQNlev-NH2
iso2
1922.08
962.4
1923.09
962.05
641.7


SP47
Ac-LTF$r8AYWAQL$QQMl-NH2

1945.05
973.95
1946.06
973.53
649.36


SP48
Ac-LTF$r8AYWAQL$QQMl-NH2
iso2
1945.05
973.88
1946.06
973.53
649.36


SP49
Ac-LTF$r8AYWAQL$HAibhLV-NH2

1893.09
948.31
1894.1
947.55
632.04


SP50
Ac-LTF$r8AYWAQL$AHFA-NH2

1871.01
937.4
1872.02
936.51
624.68


SP51
Ac-HLTF$r8HHWHQL$AANlel-NH2

2056.15
1028.79
2057.16
1029.08
686.39


SP52
Ac-DLTF$r8HHWHQL$RRLa-NH2

2162.2
721.82
2163.21
1082.11
721.74


SP53
Ac-HHTF$r8HHWHQL$AAMv-NH2

2084.07
1042.92
2085.08
1043.04
695.7


SP54
Ac-F$r8HHWHQL$RRDA-NH2

1834.99
612.74
1836
918.5
612.67


SP55
Ac-F$r8HHWHQL$HRFCha-NH2

1930.06
966.47
1931.07
966.04
644.36


SP56
Ac-F$r8AYWEAL$AA-NHAm

1443.82
1445.71
1444.83
722.92
482.28


SP57
Ac-F$r8AYWEAL$AA-NHiAm

1443.82
723.13
1444.83
722.92
482.28


SP58
Ac-F$r8AYWEAL$AA-NHnPr3Ph

1491.82
747.3
1492.83
746.92
498.28


SP59
Ac-F$r8AYWEAL$AA-NHnBu33Me

1457.83
1458.94
1458.84
729.92
486.95


SP60
Ac-F$r8AYWEAL$AA-NHnPr

1415.79
709.28
1416.8
708.9
472.94


SP61
Ac-F$r8AYWEAL$AA-NHnEt2Ch

1483.85
1485.77
1484.86
742.93
495.62


SP62
Ac-F$r8AYWEAL$AA-NHnEt2Cp

1469.83
1470.78
1470.84
735.92
490.95


SP63
Ac-F$r8AYWEAL$AA-NHHex

1457.83
730.19
1458.84
729.92
486.95


SP64
Ac-LTF$r8AYWAQL$AAIA-NH2

1771.01
885.81
1772.02
886.51
591.34


SP65
Ac-LTF$r8AYWAQL$AAIA-NH2
iso2
1771.01
866.8
1772.02
886.51
591.34


SP66
Ac-LTF$r8AYWAAL$AAMA-NH2

1731.94
867.08
1732.95
866.98
578.32


SP67
Ac-LTF$r8AYWAAL$AAMA-NH2
iso2
1731.94
867.28
1732.95
866.98
578.32


SP68
Ac-LTF$r8AYWAQL$AANleA-NH2

1771.01
867.1
1772.02
886.51
591.34


SP69
Ac-LTF$r8AYWAQL$AANleA-NH2
iso2
1771.01
886.89
1772.02
886.51
591.34


SP70
Ac-LTF$r8AYWAQL$AAIa-NH2

1771.01
886.8
1772.02
886.51
591.34


SP71
Ac-LTF$r8AYWAQL$AAIa-NH2
iso2
1771.01
887.09
1772.02
886.51
591.34


SP72
Ac-LTF$r8AYWAAL$AAMa-NH2

1731.94
867.17
1732.95
866.98
578.32


SP73
Ac-LTF$r8AYWAAL$AAMa-NH2
iso2
1731.94
867.37
1732.95
866.98
578.32


SP74
Ac-LTF$r8AYWAQL$AANlea-NH2

1771.01
887.08
1772.02
886.51
591.34


SP75
Ac-LTF$r8AYWAQL$AANlea-NH2
iso2
1771.01
887.08
1772.02
886.51
591.34


SP76
Ac-LTF$r8AYWAAL$AAIv-NH2

1742.02
872.37
1743.03
872.02
581.68


SP77
Ac-LTF$r8AYWAAL$AAIv-NH2
iso2
1742.02
872.74
1743.03
872.02
581.68


SP78
Ac-LTF$r8AYWAQL$AAMv-NH2

1817
910.02
1818.01
909.51
606.67


SP79
Ac-LTF$r8AYWAAL$AANlev-NH2

1742.02
872.37
1743.03
872.02
581.68


SP80
Ac-LTF$r8AYWAAL$AANlev-NH2
iso2
1742.02
872.28
1743.03
872.02
581.68


SP81
Ac-LTF$r8AYWAQL$AAIl-NH2

1813.05
907.81
1814.06
907.53
605.36


SP82
Ac-LTF$r8AYWAQL$AAIl-NH2
iso2
1813.05
907.81
1814.06
907.53
605.36


SP83
Ac-LTF$r8AYWAAL$AAMl-NH2

1773.99
887.37
1775
888
592.34


SP84
Ac-LTF$r8AYWAQL$AANlel-NH2

1813.05
907.61
1814.06
907.53
605.36


SP85
Ac-LTF$r8AYWAQL$AANlel-NH2
iso2
1813.05
907.71
1814.06
907.53
605.36


SP86
Ac-F$r8AYWEAL$AAMA-NH2

1575.82
789.02
1576.83
788.92
526.28


SP87
Ac-F$r8AYWEAL$AANleA-NH2

1557.86
780.14
1558.87
779.94
520.29


SP88
Ac-F$r8AYWEAL$AAIa-NH2

1557.86
780.33
1558.87
779.94
520.29


SP89
Ac-F$r8AYWEAL$AAMa-NH2

1575.82
789.3
1576.83
788.92
526.28


SP90
Ac-F$r8AYWEAL$AANlea-NH2

1557.86
779.4
1558.87
779.94
520.29


SP91
Ac-F$r8AYWEAL$AAIv-NH2

1585.89
794.29
1586.9
793.95
529.64


SP92
Ac-F$r8AYWEAL$AAMv-NH2

1603.85
803.08
1604.86
802.93
535.62


SP93
Ac-F$r8AYWEAL$AANlev-NH2

1585.89
793.46
1586.9
793.95
529.64


SP94
Ac-F$r8AYWEAL$AAIl-NH2

1599.91
800.49
1600.92
800.96
534.31


SP95
Ac-F$r8AYWEAL$AAMl-NH2

1617.86
809.44
1618.87
809.94
540.29


SP96
Ac-F$r8AYWEAL$AANlel-NH2

1599.91
801.7
1600.92
800.96
534.31


SP97
Ac-F$r8AYWEAL$AANlel-NH2
iso2
1599.91
801.42
1600.92
800.96
534.31


SP98
Ac-LTF$r8AY6clWAQL$SAA-NH2

1707.88
855.72
1708.89
854.95
570.3


SP99
Ac-LTF$r8AY6clWAQL$SAA-NH2
iso2
1707.88
855.35
1708.89
854.95
570.3


SP100
Ac-WTF$r8FYWSQL$AVAa-NH2

1922.01
962.21
1923.02
962.01
641.68


SP101
Ac-WTF$r8FYWSQL$AVAa-NH2
iso2
1922.01
962.49
1923.02
962.01
641.68


SP102
Ac-WTF$r8VYWSQL$AVA-NH2

1802.98
902.72
1803.99
902.5
602


SP103
Ac-WTF$r8VYWSQL$AVA-NH2
iso2
1802.98
903
1803.99
902.5
602


SP104
Ac-WTF$r8FYWSQL$SAAa-NH2

1909.98
956.47
1910.99
956
637.67


SP105
Ac-WTF$r8FYWSQL$SAAa-NH2
iso2
1909.98
956.47
1910.99
956
637.67


SP106
Ac-WTF$r8VYWSQL$AVAaa-NH2

1945.05
974.15
1946.06
973.53
649.36


SP107
Ac-WTF$r8VYWSQL$AVAaa-NH2
iso2
1945.05
973.78
1946.06
973.53
649.36


SP108
Ac-LTF$r8AYWAQL$AVG-NH2

1671.94
837.52
1672.95
836.98
558.32


SP109
Ac-LTF$r8AYWAQL$AVG-NH2
iso2
1671.94
837.21
1672.95
836.98
558.32


SP110
Ac-LTF$r8AYWAQL$AVQ-NH2

1742.98
872.74
1743.99
872.5
582


SP111
Ac-LTF$r8AYWAQL$AVQ-NH2
iso2
1742.98
872.74
1743.99
872.5
582


SP112
Ac-LTF$r8AYWAQL$SAa-NH2

1673.92
838.23
1674.93
837.97
558.98


SP113
Ac-LTF$r8AYWAQL$SAa-NH2
iso2
1673.92
838.32
1674.93
837.97
558.98


SP114
Ac-LTF$r8AYWAQhL$SAA-NH2

1687.93
844.37
1688.94
844.97
563.65


SP115
Ac-LTF$r8AYWAQhL$SAA-NH2
iso2
1687.93
844.81
1688.94
844.97
563.65


SP116
Ac-LTF$r8AYWEQLStSA$-NH2

1826
905.27
1827.01
914.01
609.67


SP117
Ac-LTF$r8AYWAQL$SLA-NH2

1715.97
858.48
1716.98
858.99
573


SP118
Ac-LTF$r8AYWAQL$SLA-NH2
iso2
1715.97
858.87
1716.98
858.99
573


SP119
Ac-LTF$r8AYWAQL$SWA-NH2

1788.96
895.21
1789.97
895.49
597.33


SP120
Ac-LTF$r8AYWAQL$SWA-NH2
iso2
1788.96
895.28
1789.97
895.49
597.33


SP121
Ac-LTF$r8AYWAQL$SVS-NH2

1717.94
859.84
1718.95
859.98
573.65


SP122
Ac-LTF$r8AYWAQL$SAS-NH2

1689.91
845.85
1690.92
845.96
564.31


SP123
Ac-LTF$r8AYWAQL$SVG-NH2

1687.93
844.81
1688.94
844.97
563.65


SP124
Ac-ETF$r8VYWAQL$SAa-NH2

1717.91
859.76
1718.92
859.96
573.64


SP125
Ac-ETF$r8VYWAQL$SAA-NH2

1717.91
859.84
1718.92
859.96
573.64


SP126
Ac-ETF$r8VYWAQL$SVA-NH2

1745.94
873.82
1746.95
873.98
582.99


SP127
Ac-ETF$r8VYWAQL$SLA-NH2

1759.96
880.85
1760.97
880.99
587.66


SP128
Ac-ETF$r8VYWAQL$SWA-NH2

1832.95
917.34
1833.96
917.48
611.99


SP129
Ac-ETF$r8KYWAQL$SWA-NH2

1861.98
931.92
1862.99
932
621.67


SP130
Ac-ETF$r8VYWAQL$SVS-NH2

1761.93
881.89
1762.94
881.97
588.32


SP131
Ac-ETF$r8VYWAQL$SAS-NH2

1733.9
867.83
1734.91
867.96
578.97


SP132
Ac-ETF$r8VYWAQL$SVG-NH2

1731.92
866.87
1732.93
866.97
578.31


SP133
Ac-LTF$r8VYWAQL$SSa-NH2

1717.94
859.47
1718.95
859.98
573.65


SP134
Ac-ETF$r8VYWAQL$SSa-NH2

1733.9
867.83
1734.91
867.96
578.97


SP135
Ac-LTF$r8VYWAQL$SNa-NH2

1744.96
873.38
1745.97
873.49
582.66


SP136
Ac-ETF$r8VYWAQL$SNa-NH2

1760.91
881.3
1761.92
881.46
587.98


SP137
Ac-LTF$r8VYWAQL$SAa-NH2

1701.95
851.84
1702.96
851.98
568.32


SP138
Ac-LTF$r8VYWAQL$SVA-NH2

1729.98
865.53
1730.99
866
577.67


SP139
Ac-LTF$r8VYWAQL$SVA-NH2
iso2
1729.98
865.9
1730.99
866
577.67


SP140
Ac-LTF$r8VYWAQL$SWA-NH2

1816.99
909.42
1818
909.5
606.67


SP141
Ac-LTF$r8VYWAQL$SVS-NH2

1745.98
873.9
1746.99
874
583


SP142
Ac-LTF$r8VYWAQL$SVS-NH2
iso2
1745.98
873.9
1746.99
874
583


SP143
Ac-LTF$r8VYWAQL$SAS-NH2

1717.94
859.84
1718.95
859.98
573.65


SP144
Ac-LTF$r8VYWAQL$SAS-NH2
iso2
1717.94
859.91
1718.95
859.98
573.65


SP145
Ac-LTF$r8VYWAQL$SVG-NH2

1715.97
858.87
1716.98
858.99
573


SP146
Ac-LTF$r8VYWAQL$SVG-NH2
iso2
1715.97
858.87
1716.98
858.99
573


SP147
Ac-LTF$r8EYWAQCha$SAA-NH2

1771.96
886.85
1772.97
886.99
591.66


SP148
Ac-LTF$r8EYWAQCha$SAA-NH2
iso2
1771.96
886.85
1772.97
886.99
591.66


SP149
Ac-LTF$r8EYWAQCpg$SAA-NH2

1743.92
872.86
1744.93
872.97
582.31


SP150
Ac-LTF$r8EYWAQCpg$SAA-NH2
iso2
1743.92
872.86
1744.93
872.97
582.31


SP151
Ac-LTF$r8EYWAQF$SAA-NH2

1765.91
883.44
1766.92
883.96
589.64


SP152
Ac-LTF$r8EYWAQF$SAA-NH2
iso2
1765.91
883.89
1766.92
883.96
589.64


SP153
Ac-LTF$r8EYWAQCba$SAA-NH2

1743.92
872.42
1744.93
872.97
582.31


SP154
Ac-LTF$r8EYWAQCba$SAA-NH2
iso2
1743.92
873.39
1744.93
872.97
582.31


SP155
Ac-LTF3Cl$r8EYWAQL$SAA-NH2

1765.89
883.89
1766.9
883.95
589.64


SP156
Ac-LTF3Cl$r8EYWAQL$SAA-NH2
iso2
1765.89
883.96
1766.9
883.95
589.64


SP157
Ac-LTF34F2$r8EYWAQL$SAA-NH2

1767.91
884.48
1768.92
884.96
590.31


SP158
Ac-LTF34F2$r8EYWAQL$SAA-NH2
iso2
1767.91
884.48
1768.92
884.96
590.31


SP159
Ac-LTF34F2$r8EYWAQhL$SAA-NH2

1781.92
891.44
1782.93
891.97
594.98


SP160
Ac-LTF34F2$r8EYWAQhL$SAA-NH2
iso2
1781.92
891.88
1782.93
891.97
594.98


SP161
Ac-ETF$r8EYWAQL$SAA-NH2

1747.88
874.34
1748.89
874.95
583.63


SP162
Ac-LTF$r8AYWVQL$SAA-NH2

1701.95
851.4
1702.96
851.98
568.32


SP163
Ac-LTF$r8AHWAQL$SAA-NH2

1647.91
824.83
1648.92
824.96
550.31


SP164
Ac-LTF$r8AEWAQL$SAA-NH2

1639.9
820.39
1640.91
820.96
547.64


SP165
Ac-LTF$r8ASWAQL$SAA-NH2

1597.89
799.38
1598.9
799.95
533.64


SP166
Ac-LTF$r8AEWAQL$SAA-NH2
iso2
1639.9
820.39
1640.91
820.96
547.64


SP167
Ac-LTF$r8ASWAQL$SAA-NH2
iso2
1597.89
800.31
1598.9
799.95
533.64


SP168
Ac-LTF$r8AF4coohWAQL$SAA-NH2

1701.91
851.4
1702.92
851.96
568.31


SP169
Ac-LTF$r8AF4coohWAQL$SAA-NH2
iso2
1701.91
851.4
1702.92
851.96
568.31


SP170
Ac-LTF$r8AHWAQL$AAIa-NH2

1745
874.13
1746.01
873.51
582.67


SP171
Ac-ITF$r8FYWAQL$AAIa-NH2

1847.04
923.92
1848.05
924.53
616.69


SP172
Ac-ITF$r8EHWAQL$AAIa-NH2

1803.01
903.17
1804.02
902.51
602.01


SP173
Ac-ITF$r8EHWAQL$AAIa-NH2
iso2
1803.01
903.17
1804.02
902.51
602.01


SP174
Ac-ETF$r8EHWAQL$AAIa-NH2

1818.97
910.76
1819.98
910.49
607.33


SP175
Ac-ETF$r8EHWAQL$AAIa-NH2
iso2
1818.97
910.85
1819.98
910.49
607.33


SP176
Ac-LTF$r8AHWVQL$AAIa-NH2

1773.03
888.09
1774.04
887.52
592.02


SP177
Ac-ITF$r8FYWVQL$AAIa-NH2

1875.07
939.16
1876.08
938.54
626.03


SP178
Ac-ITF$r8EYWVQL$AAIa-NH2

1857.04
929.83
1858.05
929.53
620.02


SP179
Ac-ITF$r8EHWVQL$AAIa-NH2

1831.04
916.86
1832.05
916.53
611.35


SP180
Ac-LTF$r8AEWAQL$AAIa-NH2

1736.99
869.87
1738
869.5
580


SP181
Ac-LTF$r8AF4coohWAQL$AAIa-NH2

1799
900.17
1800.01
900.51
600.67


SP182
Ac-LTF$r8AF4coohWAQL$AAIa-NH2
iso2
1799
900.24
1800.01
900.51
600.67


SP183
Ac-LTF$r8AHWAQL$AHFA-NH2

1845.01
923.89
1846.02
923.51
616.01


SP184
Ac-ITF$r8FYWAQL$AHFA-NH2

1947.05
975.05
1948.06
974.53
650.02


SP185
Ac-ITF$r8FYWAQL$AHFA-NH2
iso2
1947.05
976.07
1948.06
974.53
650.02


SP186
Ac-ITF$r8FHWAQL$AEFA-NH2

1913.02
958.12
1914.03
957.52
638.68


SP187
Ac-ITF$r8FHWAQL$AEFA-NH2
iso2
1913.02
957.86
1914.03
957.52
638.68


SP188
Ac-ITF$r8EHWAQL$AHFA-NH2

1903.01
952.94
1904.02
952.51
635.34


SP189
Ac-ITF$r8EHWAQL$AHFA-NH2
iso2
1903.01
953.87
1904.02
952.51
635.34


SP190
Ac-LTF$r8AHWVQL$AHFA-NH2

1873.04
937.86
1874.05
937.53
625.35


SP191
Ac-ITF$r8FYWVQL$AHFA-NH2

1975.08
988.83
1976.09
988.55
659.37


SP192
Ac-ITF$r8EYWVQL$AHFA-NH2

1957.05
979.35
1958.06
979.53
653.36


SP193
Ac-ITF$r8EHWVQL$AHFA-NH2

1931.05
967
1932.06
966.53
644.69


SP194
Ac-ITF$r8EHWVQL$AHFA-NH2
iso2
1931.05
967.93
1932.06
966.53
644.69


SP195
Ac-ETF$r8EYWAAL$SAA-NH2

1690.86
845.85
1691.87
846.44
564.63


SP196
Ac-LTF$r8AYWVAL$SAA-NH2

1644.93
824.08
1645.94
823.47
549.32


SP197
Ac-LTF$r8AHWAAL$SAA-NH2

1590.89
796.88
1591.9
796.45
531.3


SP198
Ac-LTF$r8AEWAAL$SAA-NH2

1582.88
791.9
1583.89
792.45
528.63


SP199
Ac-LTF$r8AEWAAL$SAA-NH2
iso2
1582.88
791.9
1583.89
792.45
528.63


SP200
Ac-LTF$r8ASWAAL$SAA-NH2

1540.87
770.74
1541.88
771.44
514.63


SP201
Ac-LTF$r8ASWAAL$SAA-NH2
iso2
1540.87
770.88
1541.88
771.44
514.63


SP202
Ac-LTF$r8AYWAAL$AAIa-NH2

1713.99
857.39
1715
858
572.34


SP203
Ac-LTF$r8AYWAAL$AAIa-NH2
iso2
1713.99
857.84
1715
858
572.34


SP204
Ac-LTF$r8AYWAAL$AHFA-NH2

1813.99
907.86
1815
908
605.67


SP205
Ac-LTF$r8EHWAQL$AHIa-NH2

1869.03
936.1
1870.04
935.52
624.02


SP206
Ac-LTF$r8EHWAQL$AHIa-NH2
iso2
1869.03
937.03
1870.04
935.52
624.02


SP207
Ac-LTF$r8AHWAQL$AHIa-NH2

1811.03
906.87
1812.04
906.52
604.68


SP208
Ac-LTF$r8EYWAQL$AHIa-NH2

1895.04
949.15
1896.05
948.53
632.69


SP209
Ac-LTF$r8AYWAQL$AAFa-NH2

1804.99
903.2
1806
903.5
602.67


SP210
Ac-LTF$r8AYWAQL$AAFa-NH2
iso2
1804.99
903.28
1806
903.5
602.67


SP211
Ac-LTF$r8AYWAQL$AAWa-NH2

1844
922.81
1845.01
923.01
615.67


SP212
Ac-LTF$r8AYWAQL$AAVa-NH2

1756.99
878.86
1758
879.5
586.67


SP213
Ac-LTF$r8AYWAQL$AAVa-NH2
iso2
1756.99
879.3
1758
879.5
586.67


SP214
Ac-LTF$r8AYWAQL$AALa-NH2

1771.01
886.26
1772.02
886.51
591.34


SP215
Ac-LTF$r8AYWAQL$AALa-NH2
iso2
1771.01
886.33
1772.02
886.51
591.34


SP216
Ac-LTF$r8EYWAQL$AAIa-NH2

1829.01
914.89
1830.02
915.51
610.68


SP217
Ac-LTF$r8EYWAQL$AAIa-NH2
iso2
1829.01
915.34
1830.02
915.51
610.68


SP218
Ac-LTF$r8EYWAQL$AAFa-NH2

1863
932.87
1864.01
932.51
622.01


SP219
Ac-LTF$r8EYWAQL$AAFa-NH2
iso2
1863
932.87
1864.01
932.51
622.01


SP220
Ac-LTF$r8EYWAQL$AAVa-NH2

1815
908.23
1816.01
908.51
606.01


SP221
Ac-LTF$r8EYWAQL$AAVa-NH2
iso2
1815
908.31
1816.01
908.51
606.01


SP222
Ac-LTF$r8EHWAQL$AAIa-NH2

1803.01
903.17
1804.02
902.51
602.01


SP223
Ac-LTF$r8EHWAQL$AAIa-NH2
iso2
1803.01
902.8
1804.02
902.51
602.01


SP224
Ac-LTF$r8EHWAQL$AAWa-NH2

1876
939.34
1877.01
939.01
626.34


SP225
Ac-LTF$r8EHWAQL$AAWa-NH2
iso2
1876
939.62
1877.01
939.01
626.34


SP226
Ac-LTF$r8EHWAQL$AALa-NH2

1803.01
902.8
1804.02
902.51
602.01


SP227
Ac-LTF$r8EHWAQL$AALa-NH2
iso2
1803.01
902.9
1804.02
902.51
602.01


SP228
Ac-ETF$r8EHWVQL$AALa-NH2

1847
924.82
1848.01
924.51
616.67


SP229
Ac-LTF$r8AYWAQL$AAAa-NH2

1728.96
865.89
1729.97
865.49
577.33


SP230
Ac-LTF$r8AYWAQL$AAAa-NH2
iso2
1728.96
865.89
1729.97
865.49
577.33


SP231
Ac-LTF$r8AYWAQL$AAAibA-NH2

1742.98
872.83
1743.99
872.5
582


SP232
Ac-LTF$r8AYWAQL$AAAibA-NH2
iso2
1742.98
872.92
1743.99
872.5
582


SP233
Ac-LTF$r8AYWAQL$AAAAa-NH2

1800
901.42
1801.01
901.01
601.01


SP234
Ac-LTF$r5AYWAQL$s8AAIa-NH2

1771.01
887.17
1772.02
886.51
591.34


SP235
Ac-LTF$r5AYWAQL$s8SAA-NH2

1673.92
838.33
1674.93
837.97
558.98


SP236
Ac-LTF$r8AYWAQCba$AANleA-NH2

1783.01
892.64
1784.02
892.51
595.34


SP237
Ac-ETF$r8AYWAQCba$AANleA-NH2

1798.97
900.59
1799.98
900.49
600.66


SP238
Ac-LTF$r8EYWAQCba$AANleA-NH2

1841.01
922.05
1842.02
921.51
614.68


SP239
Ac-LTF$r8AYWAQCba$AWNleA-NH2

1898.05
950.46
1899.06
950.03
633.69


SP240
Ac-ETF$r8AYWAQCba$AWNleA-NH2

1914.01
958.11
1915.02
958.01
639.01


SP241
Ac-LTF$r8EYWAQCba$AWNleA-NH2

1956.06
950.62
1957.07
979.04
653.03


SP242
Ac-LTF$r8EYWAQCba$SAFA-NH2

1890.99
946.55
1892
946.5
631.34


SP243
Ac-LTF34F2$r8EYWAQCba$SANleA-

1892.99
947.57
1894
947.5
632



NH2


SP244
Ac-LTF$r8EF4coohWAQCba$SANleA-

1885
943.59
1886.01
943.51
629.34



NH2


SP245
Ac-LTF$r8EYWSQCba$SANleA-NH2

1873
937.58
1874.01
937.51
625.34


SP246
Ac-LTF$r8EYWWQCba$SANleA-NH2

1972.05
987.61
1973.06
987.03
658.36


SP247
Ac-LTF$r8EYWAQCba$AAIa-NH2

1841.01
922.05
1842.02
921.51
614.68


SP248
Ac-LTF34F2$r8EYWAQCba$AAIa-NH2

1876.99
939.99
1878
939.5
626.67


SP249
Ac-LTF$r8EF4coohWAQCba$AAIa-NH2

1869.01
935.64
1870.02
935.51
624.01


SP250
Pam-ETF$r8EYWAQCba$SAA-NH2

1956.1
979.57
1957.11
979.06
653.04


SP251
Ac-LThF$r8EFWAQCba$SAA-NH2

1741.94
872.11
1742.95
871.98
581.65


SP252
Ac-LTA$r8EYWAQCba$SAA-NH2

1667.89
835.4
1668.9
834.95
556.97


SP253
Ac-LTF$r8EYAAQCba$SAA-NH2

1628.88
815.61
1629.89
815.45
543.97


SP254
Ac-LTF$r8EY2NalAQCba$SAA-NH2

1754.93
879.04
1755.94
878.47
585.98


SP255
Ac-LTF$r8AYWAQCba$SAA-NH2

1685.92
844.71
1686.93
843.97
562.98


SP256
Ac-LTF$r8EYWAQCba$SAF-NH2

1819.96
911.41
1820.97
910.99
607.66


SP257
Ac-LTF$r8EYWAQCba$SAFa-NH2

1890.99
947.41
1892
946.5
631.34


SP258
Ac-LTF$r8AYWAQCba$SAF-NH2

1761.95
882.73
1762.96
881.98
588.32


SP259
Ac-LTF34F2$r8AYWAQCba$SAF-NH2

1797.93
900.87
1798.94
899.97
600.32


SP260
Ac-LTF$r8AF4coohWAQCba$SAF-NH2

1789.94
896.43
1790.95
895.98
597.65


SP261
Ac-LTF$r8EY6clWAQCba$SAF-NH2

1853.92
929.27
1854.93
927.97
618.98


SP262
Ac-LTF$r8AYWSQCba$SAF-NH2

1777.94
890.87
1778.95
889.98
593.65


SP263
Ac-LTF$r8AYWWQCba$SAF-NH2

1876.99
939.91
1878
939.5
626.67


SP264
Ac-LTF$r8AYWAQCba$AAIa-NH2

1783.01
893.19
1784.02
892.51
595.34


SP265
Ac-LTF34F2$r8AYWAQCba$AAIa-NH2

1818.99
911.23
1820
910.5
607.34


SP266
Ac-LTF$r8AY6clWAQCba$AAIa-NH2

1816.97
909.84
1817.98
909.49
606.66


SP267
Ac-LTF$r8AF4coohWAQCba$AAIa-NH2

1811
906.88
1812.01
906.51
604.67


SP268
Ac-LTF$r8EYWAQCba$AAFa-NH2

1875
938.6
1876.01
938.51
626.01


SP269
Ac-LTF$r8EYWAQCba$AAFa-NH2
iso2
1875
938.6
1876.01
938.51
626.01


SP270
Ac-ETF$r8AYWAQCba$AWNlea-NH2

1914.01
958.42
1915.02
958.01
639.01


SP271
Ac-LTF$r8EYWAQCba$AWNlea-NH2

1956.06
979.42
1957.07
979.04
653.03


SP272
Ac-ETF$r8EYWAQCba$AWNlea-NH2

1972.01
987.06
1973.02
987.01
658.34


SP273
Ac-ETF$r8EYWAQCba$AWNlea-NH2
iso2
1972.01
987.06
1973.02
987.01
658.34


SP274
Ac-LTF$r8AYWAQCba$SAFa-NH2

1832.99
917.89
1834
917.5
612


SP275
Ac-LTF$r8AYWAQCba$SAFa-NH2
iso2
1832.99
918.07
1834
917.5
612


SP276
Ac-ETF$r8AYWAQL$AWNlea-NH2

1902.01
952.22
1903.02
952.01
635.01


SP277
Ac-LTF$r8EYWAQL$AWNlea-NH2

1944.06
973.5
1945.07
973.04
649.03


SP278
Ac-ETF$r8EYWAQL$AWNlea-NH2

1960.01
981.46
1961.02
981.01
654.34


SP279
Dmaac-LTF$r8EYWAQhL$SAA-NH2

1788.98
896.06
1789.99
895.5
597.33


SP280
Hexac-LTF$r8EYWAQhL$SAA-NH2

1802
902.9
1803.01
902.01
601.67


SP281
Napac-LTF$r8EYWAQhL$SAA-NH2

1871.99
937.58
1873
937
625


SP282
Decac-LTF$r8EYWAQhL$SAA-NH2

1858.06
930.55
1859.07
930.04
620.36


SP283
Admac-LTF$r8EYWAQhL$SAA-NH2

1866.03
934.07
1867.04
934.02
623.02


SP284
Tmac-LTF$r8EYWAQhL$SAA-NH2

1787.99
895.41
1789
895
597


SP285
Pam-LTF$r8EYWAQhL$SAA-NH2

1942.16
972.08
1943.17
972.09
648.39


SP286
Ac-LTF$r8AYWAQCba$AANleA-NH2
iso2
1783.01
892.64
1784.02
892.51
595.34


SP287
Ac-LTF34F2$r8EYWAQCba$AAIa-NH2
iso2
1876.99
939.62
1878
939.5
626.67


SP288
Ac-LTF34F2$r8EYWAQCba$SAA-NH2

1779.91
892.07
1780.92
890.96
594.31


SP289
Ac-LTF34F2$r8EYWAQCba$SAA-NH2
iso2
1779.91
891.61
1780.92
890.96
594.31


SP290
Ac-LTF$r8EF4coohWAQCba$SAA-NH2

1771.92
887.54
1772.93
886.97
591.65


SP291
Ac-LTF$r8EF4coohWAQCba$SAA-NH2
iso2
1771.92
887.63
1772.93
886.97
591.65


SP292
Ac-LTF$r8EYWSQCba$SAA-NH2

1759.92
881.9
1760.93
880.97
587.65


SP293
Ac-LTF$r8EYWSQCba$SAA-NH2
iso2
1759.92
881.9
1760.93
880.97
587.65


SP294
Ac-LTF$r8EYWAQhL$SAA-NH2

1745.94
875.05
1746.95
873.98
582.99


SP295
Ac-LTF$r8AYWAQhL$SAF-NH2

1763.97
884.02
1764.98
882.99
589


SP296
Ac-LTF$r8AYWAQhL$SAF-NH2
iso2
1763.97
883.56
1764.98
882.99
589


SP297
Ac-LTF34F2$r8AYWAQhL$SAA-NH2

1723.92
863.67
1724.93
862.97
575.65


SP298
Ac-LTF34F2$r8AYWAQhL$SAA-NH2
iso2
1723.92
864.04
1724.93
862.97
575.65


SP299
Ac-LTF$r8AF4coohWAQhL$SAA-NH2

1715.93
859.44
1716.94
858.97
572.98


SP300
Ac-LTF$r8AF4coohWAQhL$SAA-NH2
iso2
1715.93
859.6
1716.94
858.97
572.98


SP301
Ac-LTF$r8AYWSQhL$SAA-NH2

1703.93
853.96
1704.94
852.97
568.98


SP302
Ac-LTF$r8AYWSQhL$SAA-NH2
iso2
1703.93
853.59
1704.94
852.97
568.98


SP303
Ac-LTF$r8EYWAQL$AANleA-NH2

1829.01
915.45
1830.02
915.51
610.68


SP304
Ac-LTF34F2$r8AYWAQL$AANleA-NH2

1806.99
904.58
1808
904.5
603.34


SP305
Ac-LTF$r8AF4coohWAQL$AANleA-NH2

1799
901.6
1800.01
900.51
600.67


SP306
Ac-LTF$r8AYWSQL$AANleA-NH2

1787
894.75
1788.01
894.51
596.67


SP307
Ac-LTF34F2$r8AYWAQhL$AANleA-NH2

1821
911.79
1822.01
911.51
608.01


SP308
Ac-LTF34F2$r8AYWAQhL$AANleA-NH2
iso2
1821
912.61
1822.01
911.51
608.01


SP309
Ac-LTF$r8AF4coohWAQhL$AANleA-

1813.02
907.95
1814.03
907.52
605.35



NH2


SP310
Ac-LTF$r8AF4coohWAQhL$AANleA-
iso2
1813.02
908.54
1814.03
907.52
605.35



NH2


SP311
Ac-LTF$r8AYWSQhL$AANleA-NH2

1801.02
901.84
1802.03
901.52
601.35


SP312
Ac-LTF$r8AYWSQhL$AANleA-NH2
iso2
1801.02
902.62
1802.03
901.52
601.35


SP313
Ac-LTF$r8AYWAQhL$AAAAa-NH2

1814.01
908.63
1815.02
908.01
605.68


SP314
Ac-LTF$r8AYWAQhL$AAAAa-NH2
iso2
1814.01
908.34
1815.02
908.01
605.68


SP315
Ac-LTF$r8AYWAQL$AAAAAa-NH2

1871.04
936.94
1872.05
936.53
624.69


SP316
Ac-LTF$r8AYWAQL$AAAAAAa-NH2
iso2
1942.07
972.5
1943.08
972.04
648.37


SP317
Ac-LTF$r8AYWAQL$AAAAAAa-NH2
iso1
1942.07
972.5
1943.08
972.04
648.37


SP318
Ac-LTF$r8EYWAQhL$AANleA-NH2

1843.03
922.54
1844.04
922.52
615.35


SP319
Ac-AATF$r8AYWAQL$AANleA-NH2

1800
901.39
1801.01
901.01
601.01


SP320
Ac-LTF$r8AYWAQL$AANleAA-NH2

1842.04
922.45
1843.05
922.03
615.02


SP321
Ac-ALTF$r8AYWAQL$AANleAA-NH2

1913.08
957.94
1914.09
957.55
638.7


SP322
Ac-LTF$r8AYWAQCba$AANleAA-NH2

1854.04
928.43
1855.05
928.03
619.02


SP323
Ac-LTF$r8AYWAQhL$AANleAA-NH2

1856.06
929.4
1857.07
929.04
619.69


SP324
Ac-LTF$r8EYWAQCba$SAAA-NH2

1814.96
909.37
1815.97
908.49
605.99


SP325
Ac-LTF$r8EYWAQCba$SAAA-NH2
iso2
1814.96
909.37
1815.97
908.49
605.99


SP326
Ac-LTF$r8EYWAQCba$SAAAA-NH2

1886
944.61
1887.01
944.01
629.67


SP327
Ac-LTF$r8EYWAQCba$SAAAA-NH2
iso2
1886
944.61
1887.01
944.01
629.67


SP328
Ac-ALTF$r8EYWAQCba$SAA-NH2

1814.96
909.09
1815.97
908.49
605.99


SP329
Ac-ALTF$r8EYWAQCba$SAAA-NH2

1886
944.61
1887.01
944.01
629.67


SP330
Ac-ALTF$r8EYWAQCba$SAA-NH2
iso2
1814.96
909.09
1815.97
908.49
605.99


SP331
Ac-LTF$r8EYWAQL$AAAAAa-NH2
iso2
1929.04
966.08
1930.05
965.53
644.02


SP332
Ac-LTF$r8EY6clWAQCba$SAA-NH2

1777.89
890.78
1778.9
889.95
593.64


SP333
Ac-

1918.96
961.27
1919.97
960.49
640.66



LTF$r8EF4cooh6clWAQCba$SANleA-



NH2


SP334
Ac-
iso2
1918.96
961.27
1919.97
960.49
640.66



LTF$r8EF4cooh6clWAQCba$SANleA-



NH2


SP335
Ac-LTF$r8EF4cooh6clWAQCba$AAIa-

1902.97
953.03
1903.98
952.49
635.33



NH2


SP336
Ac-LTF$r8EF4cooh6clWAQCba$AAIa-
iso2
1902.97
953.13
1903.98
952.49
635.33



NH2


SP337
Ac-LTF$r8AY6clWAQL$AAAAAa-NH2

1905
954.61
1906.01
953.51
636.01


SP338
Ac-LTF$r8AY6clWAQL$AAAAAa-NH2
iso2
1905
954.9
1906.01
953.51
636.01


SP339
Ac-F$r8AY6clWEAL$AAAAAAa-NH2

1762.89
883.01
1763.9
882.45
588.64


SP340
Ac-ETF$r8EYWAQL$AAAAAa-NH2

1945
974.31
1946.01
973.51
649.34


SP341
Ac-ETF$r8EYWAQL$AAAAAa-NH2
iso2
1945
974.49
1946.01
973.51
649.34


SP342
Ac-LTF$r8EYWAQL$AAAAAAa-NH2

2000.08
1001.6
2001.09
1001.05
667.7


SP343
Ac-LTF$r8EYWAQL$AAAAAAa-NH2
iso2
2000.08
1001.6
2001.09
1001.05
667.7


SP344
Ac-LTF$r8AYWAQL$AANleAAa-NH2

1913.08
958.58
1914.09
957.55
638.7


SP345
Ac-LTF$r8AYWAQL$AANleAAa-NH2
iso2
1913.08
958.58
1914.09
957.55
638.7


SP346
Ac-LTF$r8EYWAQCba$AAAAAa-NH2

1941.04
972.55
1942.05
971.53
648.02


SP347
Ac-LTF$r8EYWAQCba$AAAAAa-NH2
iso2
1941.04
972.55
1942.05
971.53
648.02


SP348
Ac-LTF$r8EF4coohWAQCba$AAAAAa-

1969.04
986.33
1970.05
985.53
657.35



NH2


SP349
Ac-LTF$r8EF4coohWAQCba$AAAAAa-
iso2
1969.04
986.06
1970.05
985.53
657.35



NH2


SP350
Ac-LTF$r8EYWSQCba$AAAAAa-NH2

1957.04
980.04
1958.05
979.53
653.35


SP351
Ac-LTF$r8EYWSQCba$AAAAAa-NH2
iso2
1957.04
980.04
1958.05
979.53
653.35


SP352
Ac-LTF$r8EYWAQCba$SAAa-NH2

1814.96
909
1815.97
908.49
605.99


SP353
Ac-LTF$r8EYWAQCba$SAAa-NH2
iso2
1814.96
909
1815.97
908.49
605.99


SP354
Ac-ALTF$r8EYWAQCba$SAAa-NH2

1886
944.52
1887.01
944.01
629.67


SP355
Ac-ALTF$r8EYWAQCba$SAAa-NH2
iso2
1886
944.98
1887.01
944.01
629.67


SP356
Ac-ALTF$r8EYWAQCba$SAAAa-NH2

1957.04
980.04
1958.05
979.53
653.35


SP357
Ac-ALTF$r8EYWAQCba$SAAAa-NH2
iso2
1957.04
980.04
1958.05
979.53
653.35


SP358
Ac-AALTF$r8EYWAQCba$SAAAa-NH2

2028.07
1016.1
2029.08
1015.04
677.03


SP359
Ac-AALTF$r8EYWAQCba$SAAAa-NH2
iso2
2028.07
1015.57
2029.08
1015.04
677.03


SP360
Ac-RTF$r8EYWAQCba$SAA-NH2

1786.94
895.03
1787.95
894.48
596.65


SP361
Ac-LRF$r8EYWAQCba$SAA-NH2

1798.98
901.51
1799.99
900.5
600.67


SP362
Ac-LTF$r8EYWRQCba$SAA-NH2

1828.99
916.4
1830
915.5
610.67


SP363
Ac-LTF$r8EYWARCba$SAA-NH2

1771.97
887.63
1772.98
886.99
591.66


SP364
Ac-LTF$r8EYWAQCba$RAA-NH2

1812.99
908.08
1814
907.5
605.34


SP365
Ac-LTF$r8EYWAQCba$SRA-NH2

1828.99
916.12
1830
915.5
610.67


SP366
Ac-LTF$r8EYWAQCba$SAR-NH2

1828.99
916.12
1830
915.5
610.67


SP367
5-FAM-BaLTF$r8EYWAQCba$SAA-NH2

2131
1067.09
2132.01
1066.51
711.34


SP368
5-FAM-BaLTF$r8AYWAQL$AANleA-NH2

2158.08
1080.6
2159.09
1080.05
720.37


SP369
Ac-LAF$r8EYWAQL$AANleA-NH2

1799
901.05
1800.01
900.51
600.67


SP370
Ac-ATF$r8EYWAQL$AANleA-NH2

1786.97
895.03
1787.98
894.49
596.66


SP371
Ac-AAF$r8EYWAQL$AANleA-NH2

1756.96
880.05
1757.97
879.49
586.66


SP372
Ac-AAAF$r8EYWAQL$AANleA-NH2

1827.99
915.57
1829
915
610.34


SP373
Ac-AAAAF$r8EYWAQL$AANleA-NH2

1899.03
951.09
1900.04
950.52
634.02


SP374
Ac-AATF$r8EYWAQL$AANleA-NH2

1858
930.92
1859.01
930.01
620.34


SP375
Ac-AALTF$r8EYWAQL$AANleA-NH2

1971.09
987.17
1972.1
986.55
658.04


SP376
Ac-AAALTF$r8EYWAQL$AANleA-NH2

2042.12
1023.15
2043.13
1022.07
681.71


SP377
Ac-LTF$r8EYWAQL$AANleAA-NH2

1900.05
952.02
1901.06
951.03
634.36


SP378
Ac-ALTF$r8EYWAQL$AANleAA-NH2

1971.09
987.63
1972.1
986.55
658.04


SP379
Ac-AALTF$r8EYWAQL$AANleAA-NH2

2042.12
1022.69
2043.13
1022.07
681.71


SP380
Ac-LTF$r8EYWAQCba$AANleAA-NH2

1912.05
958.03
1913.06
957.03
638.36


SP381
Ac-LTF$r8EYWAQhL$AANleAA-NH2

1914.07
958.68
1915.08
958.04
639.03


SP382
Ac-ALTF$r8EYWAQhL$AANleAA-NH2

1985.1
994.1
1986.11
993.56
662.71


SP383
Ac-LTF$r8ANmYWAQL$AANleA-NH2

1785.02
894.11
1786.03
893.52
596.01


SP384
Ac-LTF$r8ANmYWAQL$AANleA-NH2
iso2
1785.02
894.11
1786.03
893.52
596.01


SP385
Ac-LTF$r8AYNmWAQL$AANleA-NH2

1785.02
894.11
1786.03
893.52
596.01


SP386
Ac-LTF$r8AYNmWAQL$AANleA-NH2
iso2
1785.02
894.11
1786.03
893.52
596.01


SP387
Ac-LTF$r8AYAmwAQL$AANleA-NH2

1785.02
894.01
1786.03
893.52
596.01


SP388
Ac-LTF$r8AYAmwAQL$AANleA-NH2
iso2
1785.02
894.01
1786.03
893.52
596.01


SP389
Ac-LTF$r8AYWAibQL$AANleA-NH2

1785.02
894.01
1786.03
893.52
596.01


SP390
Ac-LTF$r8AYWAibQL$AANleA-NH2
iso2
1785.02
894.01
1786.03
893.52
596.01


SP391
Ac-LTF$r8AYWAQL$AAibNleA-NH2

1785.02
894.38
1786.03
893.52
596.01


SP392
Ac-LTF$r8AYWAQL$AAibNleA-NH2
iso2
1785.02
894.38
1786.03
893.52
596.01


SP393
Ac-LTF$r8AYWAQL$AaNleA-NH2

1771.01
887.54
1772.02
886.51
591.34


SP394
Ac-LTF$r8AYWAQL$AaNleA-NH2
iso2
1771.01
887.54
1772.02
886.51
591.34


SP395
Ac-LTF$r8AYWAQL$ASarNleA-NH2

1771.01
887.35
1772.02
886.51
591.34


SP396
Ac-LTF$r8AYWAQL$ASarNleA-NH2
iso2
1771.01
887.35
1772.02
886.51
591.34


SP397
Ac-LTF$r8AYWAQL$AANleAib-NH2

1785.02
894.75
1786.03
893.52
596.01


SP398
Ac-LTF$r8AYWAQL$AANleAib-NH2
iso2
1785.02
894.75
1786.03
893.52
596.01


SP399
Ac-LTF$r8AYWAQL$AANleNmA-NH2

1785.02
894.6
1786.03
893.52
596.01


SP400
Ac-LTF$r8AYWAQL$AANleNmA-NH2
iso2
1785.02
894.6
1786.03
893.52
596.01


SP401
Ac-LTF$r8AYWAQL$AANleSar-NH2

1771.01
886.98
1772.02
886.51
591.34


SP402
Ac-LTF$r8AYWAQL$AANleSar-NH2
iso2
1771.01
886.98
1772.02
886.51
591.34


SP403
Ac-LTF$r8AYWAQL$AANleAAib-NH2

1856.06

1857.07
929.04
619.69


SP404
Ac-LTF$r8AYWAQL$AANleAAib-NH2
iso2
1856.06

1857.07
929.04
619.69


SP405
Ac-LTF$r8AYWAQL$AANleANmA-NH2

1856.06
930.37
1857.07
929.04
619.69


SP406
Ac-LTF$r8AYWAQL$AANleANmA-NH2
iso2
1856.06
930.37
1857.07
929.04
619.69


SP407
Ac-LTF$r8AYWAQL$AANleAa-NH2

1842.04
922.69
1843.05
922.03
615.02


SP408
Ac-LTF$r8AYWAQL$AANleAa-NH2
iso2
1842.04
922.69
1843.05
922.03
615.02


SP409
Ac-LTF$r8AYWAQL$AANleASar-NH2

1842.04
922.6
1843.05
922.03
615.02


SP410
Ac-LTF$r8AYWAQL$AANleASar-NH2
iso2
1842.04
922.6
1843.05
922.03
615.02


SP411
Ac-LTF$r8AYWAQL$AANleA-NH2

1799.04
901.14
1800.05
900.53
600.69


SP412
Ac-LTFAibAYWAQLAibAANleA-NH2

1648.9
826.02
1649.91
825.46
550.64


SP413
Ac-LTF$r8Cou4YWAQL$AANleA-NH2

1975.05
989.11
1976.06
988.53
659.36


SP414
Ac-LTF$r8Cou4YWAQL$AANleA-NH2
iso2
1975.05
989.11
1976.06
988.53
659.36


SP415
Ac-LTF$r8AYWCou4QL$AANleA-NH2

1975.05
989.11
1976.06
988.53
659.36


SP416
Ac-LTF$r8AYWAQL$Cou4ANleA-NH2

1975.05
989.57
1976.06
988.53
659.36


SP417
Ac-LTF$r8AYWAQL$Cou4ANleA-NH2
iso2
1975.05
989.57
1976.06
988.53
659.36


SP418
Ac-LTF$r8AYWAQL$ACou4NleA-NH2

1975.05
989.57
1976.06
988.53
659.36


SP419
Ac-LTF$r8AYWAQL$ACou4NleA-NH2
iso2
1975.05
989.57
1976.06
988.53
659.36


SP420
Ac-LTF$r8AYWAQL$AANleA-OH

1771.99
887.63
1773
887
591.67


SP421
Ac-LTF$r8AYWAQL$AANleA-OH
iso2
1771.99
887.63
1773
887
591.67


SP422
Ac-LTF$r8AYWAQL$AANleA-NHnPr

1813.05
908.08
1814.06
907.53
605.36


SP423
Ac-LTF$r8AYWAQL$AANleA-NHnPr
iso2
1813.05
908.08
1814.06
907.53
605.36


SP424
Ac-LTF$r8AYWAQL$AANleA-

1855.1
929.17
1856.11
928.56
619.37



NHnBu33Me


SP425
Ac-LTF$r8AYWAQL$AANleA-
iso2
1855.1
929.17
1856.11
928.56
619.37



NHnBu33Me


SP426
Ac-LTF$r8AYWAQL$AANleA-NHHex

1855.1
929.17
1856.11
928.56
619.37


SP427
Ac-LTF$r8AYWAQL$AANleA-NHHex
iso2
1855.1
929.17
1856.11
928.56
619.37


SP428
Ac-LTA$r8AYWAQL$AANleA-NH2

1694.98
849.33
1695.99
848.5
566


SP429
Ac-LThL$r8AYWAQL$AANleA-NH2

1751.04
877.09
1752.05
876.53
584.69


SP430
Ac-LTF$r8AYAAQL$AANleA-NH2

1655.97
829.54
1656.98
828.99
553


SP431
Ac-LTF$r8AY2NalAQL$AANleA-NH2

1782.01
892.63
1783.02
892.01
595.01


SP432
Ac-LTF$r8EYWCou4QCba$SAA-NH2

1947.97
975.8
1948.98
974.99
650.33


SP433
Ac-LTF$r8EYWCou7QCba$SAA-NH2

16.03
974.9
17.04
9.02
6.35


SP434
Ac-LTF%r8EYWAQCba%SAA-NH2

1745.94
874.8
1746.95
873.98
582.99


SP435
Dmaac-LTF$r8EYWAQCba$SAA-NH2

1786.97
894.8
1787.98
894.49
596.66


SP436
Dmaac-LTF$r8AYWAQL$AAAAAa-NH2

1914.08
958.2
1915.09
958.05
639.03


SP437
Dmaac-LTF$r8AYWAQL$AAAAAa-NH2
iso2
1914.08
958.2
1915.09
958.05
639.03


SP438
Dmaac-LTF$r8EYWAQL$AAAAAa-NH2

1972.08
987.3
1973.09
987.05
658.37


SP439
Dmaac-LTF$r8EYWAQL$AAAAAa-NH2
iso2
1972.08
987.3
1973.09
987.05
658.37


SP440
Dmaac-LTF$r8EF4coohWAQCba$AAIa-

1912.05
957.4
1913.06
957.03
638.36



NH2


SP441
Dmaac-LTF$r8EF4coohWAQCba$AAIa-
iso2
1912.05
957.4
1913.06
957.03
638.36



NH2


SP442
Dmaac-LTF$r8AYWAQL$AANleA-NH2

1814.05
908.3
1815.06
908.03
605.69


SP443
Dmaac-LTF$r8AYWAQL$AANleA-NH2
iso2
1814.05
908.3
1815.06
908.03
605.69


SP444
Ac-LTF%r8AYWAQL%AANleA-NH2

1773.02
888.37
1774.03
887.52
592.01


SP445
Ac-LTF%r8EYWAQL%AAAAAa-NH2

1931.06
966.4
1932.07
966.54
644.69


SP446
Cou6BaLTF$r8EYWAQhL$SAA-NH2

2018.05
1009.9
2019.06
1010.03
673.69


SP447
Cou8BaLTF$r8EYWAQhL$SAA-NH2

1962.96
982.34
1963.97
982.49
655.32


SP448
Ac-LTF4I$r8EYWAQL$AAAAAa-NH2

2054.93
1028.68
2055.94
1028.47
685.98


SP449
Ac-LTF$r8EYWAQL$AAAAAa-NH2

1929.04
966.17
1930.05
965.53
644.02


SP550
Ac-LTF$r8EYWAQL$AAAAAa-OH

1930.02
966.54
1931.03
966.02
644.35


SP551
Ac-LTF$r8EYWAQL$AAAAAa-OH
iso2
1930.02
965.89
1931.03
966.02
644.35


SP552
Ac-LTF$r8EYWAEL$AAAAAa-NH2

1930.02
966.82
1931.03
966.02
644.35


SP553
Ac-LTF$r8EYWAEL$AAAAAa-NH2
iso2
1930.02
966.91
1931.03
966.02
644.35


SP554
Ac-LTF$r8EYWAEL$AAAAAa-OH

1931.01
967.28
1932.02
966.51
644.68


SP555
Ac-LTF$r8EY6clWAQL$AAAAAa-NH2

1963
983.28
1964.01
982.51
655.34


SP556
Ac-LTF$r8EF4bOH2WAQL$AAAAAa-NH2

1957.05
980.04
1958.06
979.53
653.36


SP557
Ac-AAALTF$r8EYWAQL$AAAAAa-NH2

2142.15
1072.83
2143.16
1072.08
715.06


SP558
Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2

1965.02
984.3
1966.03
983.52
656.01


SP559
Ac-RTF$r8EYWAQL$AAAAAa-NH2

1972.06
987.81
1973.07
987.04
658.36


SP560
Ac-LTA$r8EYWAQL$AAAAAa-NH2

1853.01
928.33
1854.02
927.51
618.68


SP561
Ac-LTF$r8EYWAibQL$AAAAAa-NH2

1943.06
973.48
1944.07
972.54
648.69


SP562
Ac-LTF$r8EYWAQL$AAibAAAa-NH2

1943.06
973.11
1944.07
972.54
648.69


SP563
Ac-LTF$r8EYWAQL$AAAibAAa-NH2

1943.06
973.48
1944.07
972.54
648.69


SP564
Ac-LTF$r8EYWAQL$AAAAibAa-NH2

1943.06
973.48
1944.07
972.54
648.69


SP565
Ac-LTF$r8EYWAQL$AAAAAiba-NH2

1943.06
973.38
1944.07
972.54
648.69


SP566
Ac-LTF$r8EYWAQL$AAAAAiba-NH2
iso2
1943.06
973.38
1944.07
972.54
648.69


SP567
Ac-LTF$r8EYWAQL$AAAAAAib-NH2

1943.06
973.01
1944.07
972.54
648.69


SP568
Ac-LTF$r8EYWAQL$AaAAAa-NH2

1929.04
966.54
1930.05
965.53
644.02


SP569
Ac-LTF$r8EYWAQL$AAaAAa-NH2

1929.04
966.35
1930.05
965.53
644.02


SP570
Ac-LTF$r8EYWAQL$AAAaAa-NH2

1929.04
966.54
1930.05
965.53
644.02


SP571
Ac-LTF$r8EYWAQL$AAAaAa-NH2
iso2
1929.04
966.35
1930.05
965.53
644.02


SP572
Ac-LTF$r8EYWAQL$AAAAaa-NH2

1929.04
966.35
1930.05
965.53
644.02


SP573
Ac-LTF$r8EYWAQL$AAAAAA-NH2

1929.04
966.35
1930.05
965.53
644.02


SP574
Ac-LTF$r8EYWAQL$ASarAAAa-NH2

1929.04
966.54
1930.05
965.53
644.02


SP575
Ac-LTF$r8EYWAQL$AASarAAa-NH2

1929.04
966.35
1930.05
965.53
644.02


SP576
Ac-LTF$r8EYWAQL$AAASarAa-NH2

1929.04
966.35
1930.05
965.53
644.02


SP577
Ac-LTF$r8EYWAQL$AAAASara-NH2

1929.04
966.35
1930.05
965.53
644.02


SP578
Ac-LTF$r8EYWAQL$AAAAASar-NH2

1929.04
966.08
1930.05
965.53
644.02


SP579
Ac-7LTF$r8EYWAQL$AAAAAa-NH2

1918.07
951.99
1919.08
960.04
640.37


SP581
Ac-TF$r8EYWAQL$AAAAAa-NH2

1815.96
929.85
1816.97
908.99
606.33


SP582
Ac-F$r8EYWAQL$AAAAAa-NH2

1714.91
930.92
1715.92
858.46
572.64


SP583
Ac-LVF$r8EYWAQL$AAAAAa-NH2

1927.06
895.12
1928.07
964.54
643.36


SP584
Ac-AAF$r8EYWAQL$AAAAAa-NH2

1856.98
859.51
1857.99
929.5
620


SP585
Ac-LTF$r8EYWAQL$AAAAa-NH2

1858
824.08
1859.01
930.01
620.34


SP586
Ac-LTF$r8EYWAQL$AAAa-NH2

1786.97
788.56
1787.98
894.49
596.66


SP587
Ac-LTF$r8EYWAQL$AAa-NH2

1715.93
1138.57
1716.94
858.97
572.98


SP588
Ac-LTF$r8EYWAQL$Aa-NH2

1644.89
1144.98
1645.9
823.45
549.3


SP589
Ac-LTF$r8EYWAQL$a-NH2

1573.85
1113.71
1574.86
787.93
525.62


SP590
Ac-LTF$r8EYWAQL$AAA-OH

1716.91
859.55
1717.92
859.46
573.31


SP591
Ac-LTF$r8EYWAQL$A-OH

1574.84
975.14
1575.85
788.43
525.95


SP592
Ac-LTF$r8EYWAQL$AAA-NH2

1715.93
904.75
1716.94
858.97
572.98


SP593
Ac-LTF$r8EYWAQCba$SAA-OH

1744.91
802.49
1745.92
873.46
582.64


SP594
Ac-LTF$r8EYWAQCba$S-OH

1602.83
913.53
1603.84
802.42
535.28


SP595
Ac-LTF$r8EYWAQCba$S-NH2

1601.85
979.58
1602.86
801.93
534.96


SP596
4-FBz1-LTF$r8EYWAQL$AAAAAa-NH2

2009.05
970.52
2010.06
1005.53
670.69


SP597
4-FBz1-LTF$r8EYWAQCba$SAA-NH2

1823.93
965.8
1824.94
912.97
608.98


SP598
Ac-LTF$r8RYWAQL$AAAAAa-NH2

1956.1
988.28
1957.11
979.06
653.04


SP599
Ac-LTF$r8HYWAQL$AAAAAa-NH2

1937.06
1003.54
1938.07
969.54
646.69


SP600
Ac-LTF$r8QYWAQL$AAAAAa-NH2

1928.06
993.92
1929.07
965.04
643.69


SP601
Ac-LTF$r8CitYWAQL$AAAAAa-NH2

1957.08
987
1958.09
979.55
653.37


SP602
Ac-LTF$r8GlaYWAQL$AAAAAa-NH2

1973.03
983
1974.04
987.52
658.68


SP603
Ac-LTF$r8F4gYWAQL$AAAAAa-NH2

2004.1
937.86
2005.11
1003.06
669.04


SP604
Ac-LTF$r82mRYWAQL$AAAAAa-NH2

1984.13
958.58
1985.14
993.07
662.38


SP605
Ac-LTF$r8ipKYWAQL$AAAAAa-NH2

1970.14
944.52
1971.15
986.08
657.72


SP606
Ac-LTF$r8F4NH2YWAQL$AAAAAa-NH2

1962.08
946
1963.09
982.05
655.03


SP607
Ac-LTF$r8EYWAAL$AAAAAa-NH2

1872.02
959.32
1873.03
937.02
625.01


SP608
Ac-LTF$r8EYWALL$AAAAAa-NH2

1914.07
980.88
1915.08
958.04
639.03


SP609
Ac-LTF$r8EYWAAibL$AAAAAa-NH2

1886.03
970.61
1887.04
944.02
629.68


SP610
Ac-LTF$r8EYWASL$AAAAAa-NH2

1888.01
980.51
1889.02
945.01
630.34


SP611
Ac-LTF$r8EYWANL$AAAAAa-NH2

1915.02
1006.41
1916.03
958.52
639.35


SP612
Ac-LTF$r8EYWACitL$AAAAAa-NH2

1958.07

1959.08
980.04
653.7


SP613
Ac-LTF$r8EYWAHL$AAAAAa-NH2

1938.04
966.24
1939.05
970.03
647.02


SP614
Ac-LTF$r8EYWARL$AAAAAa-NH2

1957.08

1958.09
979.55
653.37


SP615
Ac-LTF$r8EpYWAQL$AAAAAa-NH2

2009.01

2010.02
1005.51
670.68


SP616
Cbm-LTF$r8EYWAQCba$SAA-NH2

1590.85

1591.86
796.43
531.29


SP617
Cbm-LTF$r8EYWAQL$AAAAAa-NH2

1930.04

1931.05
966.03
644.35


SP618
Ac-LTF$r8EYWAQL$SAAAAa-NH2

1945.04
1005.11
1946.05
973.53
649.35


SP619
Ac-LTF$r8EYWAQL$AAAASa-NH2

1945.04
986.52
1946.05
973.53
649.35


SP620
Ac-LTF$r8EYWAQL$SAAASa-NH2

1961.03
993.27
1962.04
981.52
654.68


SP621
Ac-LTF$r8EYWAQTba$AAAAAa-NH2

1943.06
983.1
1944.07
972.54
648.69


SP622
Ac-LTF$r8EYWAQAdm$AAAAAa-NH2

2007.09
990.31
2008.1
1004.55
670.04


SP623
Ac-LTF$r8EYWAQCha$AAAAAa-NH2

1969.07
987.17
1970.08
985.54
657.36


SP624
Ac-LTF$r8EYWAQhCha$AAAAAa-NH2

1983.09
1026.11
1984.1
992.55
662.04


SP625
Ac-LTF$r8EYWAQF$AAAAAa-NH2

1963.02
957.01
1964.03
982.52
655.35


SP626
Ac-LTF$r8EYWAQhF$AAAAAa-NH2

1977.04
1087.81
1978.05
989.53
660.02


SP627
Ac-LTF$r8EYWAQL$AANleAAa-NH2

1971.09
933.45
1972.1
986.55
658.04


SP628
Ac-LTF$r8EYWAQAdm$AANleAAa-NH2

2049.13
1017.97
2050.14
1025.57
684.05


SP629
4-FBz-BaLTF$r8EYWAQL$AAAAAa-NH2

2080.08

2081.09
1041.05
694.37


SP630
4-FBz-BaLTF$r8EYWAQCba$SAA-NH2

1894.97

1895.98
948.49
632.66


SP631
Ac-LTF$r5EYWAQL$s8AAAAAa-NH2

1929.04
1072.68
1930.05
965.53
644.02


SP632
Ac-LTF$r5EYWAQCba$s8SAA-NH2

1743.92
1107.79
1744.93
872.97
582.31


SP633
Ac-LTF$r8EYWAQL$AAhhLAAa-NH2

1999.12

2000.13
1000.57
667.38


SP634
Ac-LTF$r8EYWAQL$AAAAAAAa-NH2

2071.11

2072.12
1036.56
691.38


SP635
Ac-LTF$r8EYWAQL$AAAAAAAAa-NH2

2142.15
778.1
2143.16
1072.08
715.06


SP636
Ac-LTF$r8EYWAQL$AAAAAAAAAa-NH2

2213.19
870.53
2214.2
1107.6
738.74


SP637
Ac-LTA$r8EYAAQCba$SAA-NH2

1552.85

1553.86
777.43
518.62


SP638
Ac-LTA$r8EYAAQL$AAAAAa-NH2

1737.97
779.45
1738.98
869.99
580.33


SP639
Ac-LTF$r8EPmpWAQL$AAAAAa-NH2

2007.03
779.54
2008.04
1004.52
670.02


SP640
Ac-LTF$r8EPmpWAQCba$SAA-NH2

1821.91
838.04
1822.92
911.96
608.31


SP641
Ac-ATF$r8HYWAQL$S-NH2

1555.82
867.83
1556.83
778.92
519.61


SP642
Ac-LTF$r8HAWAQL$S-NH2

1505.84
877.91
1506.85
753.93
502.95


SP643
Ac-LTF$r8HYWAQA$S-NH2

1555.82
852.52
1556.83
778.92
519.61


SP644
Ac-LTF$r8EYWAQCba$SA-NH2

1672.89
887.18
1673.9
837.45
558.64


SP645
Ac-LTF$r8EYWAQL$SAA-NH2

1731.92
873.32
1732.93
866.97
578.31


SP646
Ac-LTF$r8HYWAQCba$SAA-NH2

1751.94
873.05
1752.95
876.98
584.99


SP647
Ac-LTF$r8SYWAQCba$SAA-NH2

1701.91
844.88
1702.92
851.96
568.31


SP648
Ac-LTF$r8RYWAQCba$SAA-NH2

1770.98
865.58
1771.99
886.5
591.33


SP649
Ac-LTF$r8KYWAQCba$SAA-NH2

1742.98
936.57
1743.99
872.5
582


SP650
Ac-LTF$r8QYWAQCba$SAA-NH2

1742.94
930.93
1743.95
872.48
581.99


SP651
Ac-LTF$r8EYWAACba$SAA-NH2

1686.9
1032.45
1687.91
844.46
563.31


SP652
Ac-LTF$r8EYWAQCba$AAA-NH2

1727.93
895.46
1728.94
864.97
576.98


SP653
Ac-LTF$r8EYWAQL$AAAAA-OH

1858.99
824.54
1860
930.5
620.67


SP654
Ac-LTF$r8EYWAQL$AAAA-OH

1787.95
894.48
1788.96
894.98
596.99


SP655
Ac-LTF$r8EYWAQL$AA-OH

1645.88
856
1646.89
823.95
549.63


SP656
Ac-LTF$r8AF4bOH2WAQL$AAAAAa-NH2


SP657
Ac-LTF$r8AF4bOH2WAAL$AAAAAa-NH2


SP658
Ac-LTF$r8EF4bOH2WAQCba$SAA-NH2


SP659
Ac-LTF$r8ApYWAQL$AAAAAa-NH2


SP660
Ac-LTF$r8ApYWAAL$AAAAAa-NH2


SP661
Ac-LTF$r8EpYWAQCba$SAA-NH2


SP662
Ac-LTF$rda6AYWAQL$da5AAAAAa-NH2

1974.06
934.44


SP663
Ac-LTF$rda6EYWAQCba$da5SAA-NH2

1846.95
870.52

869.94


SP664
Ac-LTF$rda6EYWAQL$da5AAAAAa-NH2


SP665
Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2


936.57

935.51


SP666
Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2


SP667
Ac-LTF$ra9EYWAQCba$a6SAA-NH2


SP668
Ac-LTA$ra9EYWAQCba$a6SAA-NH2


SP669
5-FAM-BaLTF$ra9EYWAQCba$a6SAA-



NH2


SP670
5-FAM-BaLTF$r8EYWAQL$AAAAAa-NH2

2316.11


SP671
5-FAM-BaLTF$/r8EYWAQL$/AAAAAa-

2344.15



NH2


SP672
5-FAM-BaLTA$r8EYWAQL$AAAAAa-NH2

2240.08


SP673
5-FAM-BaLTF$r8AYWAQL$AAAAAa-NH2

2258.11


SP674
5-FAM-BaATF$r8EYWAQL$AAAAAa-NH2

2274.07


SP675
5-FAM-BaLAF$r8EYWAQL$AAAAAa-NH2

2286.1


SP676
5-FAM-BaLTF$r8EAWAQL$AAAAAa-NH2

2224.09


SP677
5-FAM-BaLTF$r8EYAAQL$AAAAAa-NH2

2201.07


SP678
5-FAM-BaLTA$r8EYAAQL$AAAAAa-NH2

2125.04


SP679
5-FAM-BaLTF$r8EYWAAL$AAAAAa-NH2

2259.09


SP680
5-FAM-BaLTF$r8EYWAQA$AAAAAa-NH2

2274.07


SP681
5-FAM-BaLTF$/r8EYWAQCba$/SAA-

2159.03



NH2


SP682
5-FAM-BaLTA$r8EYWAQCba$SAA-NH2

2054.97


SP683
5-FAM-BaLTF$r8EYAAQCba$SAA-NH2

2015.96


SP684
5-FAM-BaLTA$r8EYAAQCba$SAA-NH2

1939.92


SP685
5-FAM-BaQSQQTF$r8NLWRLL$QN-NH2

2495.23


SP686
5-TAMRA-BaLTF$r8EYWAQCba$SAA-

2186.1



NH2


SP687
5-TAMRA-BaLTA$r8EYWAQCba$SAA-

2110.07



NH2


SP688
5-TAMRA-BaLTF$r8EYAAQCba$SAA-

2071.06



NH2


SP689
5-TAMRA-BaLTA$r8EYAAQCba$SAA-

1995.03



NH2


SP690
5-TAMRA-BaLTF$/r8EYWAQCba$/SAA-

2214.13



NH2


SP691
5-TAMRA-BaLTF$r8EYWAQL$AAAAAa-

2371.22



NH2


SP692
5-TAMRA-BaLTA$r8EYWAQL$AAAAAa-

2295.19



NH2


SP693
5-TAMRA-

2399.25



BaLTF$/r8EYWAQL$/AAAAAa-NH2


SP694
Ac-LTF$r8EYWCou7QCba$SAA-OH

1947.93


SP695
Ac-LTF$r8EYWCou7QCba$S-OH

1805.86


SP696
Ac-LTA$r8EYWCou7QCba$SAA-NH2

1870.91


SP697
Ac-LTF$r8EYACou7QCba$SAA-NH2

1831.9


SP698
Ac-LTA$r8EYACou7QCba$SAA-NH2

1755.87


SP699
Ac-LTF$/r8EYWCou7QCba$/SAA-NH2

1974.98


SP700
Ac-LTF$r8EYWCou7QL$AAAAAa-NH2

2132.06


SP701
Ac-LTF$/r8EYWCou7QL$/AAAAAa-NH2

2160.09


SP702
Ac-LTF$r8EYWCou7QL$AAAAA-OH

2062.01


SP703
Ac-LTF$r8EYWCou7QL$AAAA-OH

1990.97


SP704
Ac-LTF$r8EYWCou7QL$AAA-OH

1919.94


SP705
Ac-LTF$r8EYWCou7QL$AA-OH

1848.9


SP706
Ac-LTF$r8EYWCou7QL$A-OH

1777.86


SP707
Ac-LTF$r8EYWAQL$AAAASa-NH2
iso2

974.4

973.53


SP708
Ac-LTF$r8AYWAAL$AAAAAa-NH2
iso2
1814.01
908.82
1815.02
908.01
605.68


SP709
Biotin-BaLTF$r8EYWAQL$AAAAAa-

2184.14
1093.64
2185.15
1093.08
729.05



NH2


SP710
Ac-LTF$r8HAWAQL$S-NH2
iso2
1505.84
754.43
1506.85
753.93
502.95


SP711
Ac-LTF$r8EYWAQCba$SA-NH2
iso2
1672.89
838.05
1673.9
837.45
558.64


SP712
Ac-LTF$r8HYWAQCba$SAA-NH2
iso2
1751.94
877.55
1752.95
876.98
584.99


SP713
Ac-LTF$r8SYWAQCba$SAA-NH2
iso2
1701.91
852.48
1702.92
851.96
568.31


SP714
Ac-LTF$r8RYWAQCba$SAA-NH2
iso2
1770.98
887.45
1771.99
886.5
591.33


SP715
Ac-LTF$r8KYWAQCba$SAA-NH2
iso2
1742.98
872.92
1743.99
872.5
582


SP716
Ac-LTF$r8EYWAQCba$AAA-NH2
iso2
1727.93
865.71
1728.94
864.97
576.98


SP717
Ac-LTF$r8EYWAQL$AAAAAaBaC-NH2

2103.09
1053.12
2104.1
1052.55
702.04


SP718
Ac-LTF$r8EYWAQL$AAAAAadPeg4C-

2279.19
1141.46
2280.2
1140.6
760.74



NH2


SP719
Ac-LTA$r8AYWAAL$AAAAAa-NH2

1737.98
870.43
1738.99
870
580.33


SP720
Ac-LTF$r8AYAAAL$AAAAAa-NH2

1698.97
851
1699.98
850.49
567.33


SP721
5-FAM-BaLTF$r8AYWAAL$AAAAAa-NH2

2201.09
1101.87
2202.1
1101.55
734.7


SP722
Ac-LTA$r8AYWAQL$AAAAAa-NH2

1795
898.92
1796.01
898.51
599.34


SP723
Ac-LTF$r8AYAAQL$AAAAAa-NH2

1755.99
879.49
1757
879
586.34


SP724
Ac-LTF$rda6AYWAAL$da5AAAAAa-NH2

1807.97

1808.98
904.99
603.66


SP725
FITC-BaLTF$r8EYWAQL$AAAAAa-NH2

2347.1
1174.49
2348.11
1174.56
783.37


SP726
FITC-BaLTF$r8EYWAQCba$SAA-NH2

2161.99
1082.35
2163
1082
721.67


SP733
Ac-LTF$r8EYWAQL$EAAAAa-NH2

1987.05
995.03
1988.06
994.53
663.36


SP734
Ac-LTF$r8AYWAQL$EAAAAa-NH2

1929.04
966.35
1930.05
965.53
644.02


SP735
Ac-LTF$r8EYWAQL$AAAAAaBaKbio-

2354.25
1178.47
2355.26
1178.13
785.76



NH2


SP736
Ac-LTF$r8AYWAAL$AAAAAa-NH2

1814.01
908.45
1815.02
908.01
605.68


SP737
Ac-LTF$r8AYAAAL$AAAAAa-NH2
iso2
1698.97
850.91
1699.98
850.49
567.33


SP738
Ac-LTF$r8AYAAQL$AAAAAa-NH2
iso2
1755.99
879.4
1757
879
586.34


SP739
Ac-LTF$r8EYWAQL$EAAAAa-NH2
iso2
1987.05
995.21
1988.06
994.53
663.36


SP740
Ac-LTF$r8AYWAQL$EAAAAa-NH2
iso2
1929.04
966.08
1930.05
965.53
644.02


SP741
Ac-LTF$r8EYWAQCba$SAAAAa-NH2

1957.04
980.04
1958.05
979.53
653.35


SP742
Ac-LTF$r8EYWAQLStAAA$r5AA-NH2

2023.12
1012.83
2024.13
1012.57
675.38


SP743
Ac-LTF$r8EYWAQL$A$AAA$A-NH2

2108.17
1055.44
2109.18
1055.09
703.73


SP744
Ac-LTF$r8EYWAQL$AA$AAA$A-NH2

2179.21
1090.77
2180.22
1090.61
727.41


SP745
Ac-LTF$r8EYWAQL$AAA$AAA$A-NH2

2250.25
1126.69
2251.26
1126.13
751.09


SP746
Ac-AAALTF$r8EYWAQL$AAA-OH

1930.02

1931.03
966.02
644.35


SP747
Ac-AAALTF$r8EYWAQL$AAA-NH2

1929.04
965.85
1930.05
965.53
644.02


SP748
Ac-AAAALTF$r8EYWAQL$AAA-NH2

2000.08
1001.4
2001.09
1001.05
667.7


SP749
Ac-AAAAALTF$r8EYWAQL$AAA-NH2

2071.11
1037.13
2072.12
1036.56
691.38


SP750
Ac-AAAAAALTF$r8EYWAQL$AAA-NH2

2142.15

2143.16
1072.08
715.06


SP751
Ac-LTF$rda6EYWAQCba$da6SAA-NH2
iso2
1751.89
877.36
1752.9
876.95
584.97


SP752
Ac-t$r5wya$r5f4CF3ekllr-NH2


844.25


SP753
Ac-tawy$r5nf4CF3e$r5llr-NH2


837.03


SP754
Ac-tawya$r5f4CF3ek$r5lr-NH2


822.97


SP755
Ac-tawyanf4CF3e$r5llr$r5a-NH2


908.35


SP756
Ac-t$s8wyanf4CF3e$r5llr-NH2


858.03


SP757
Ac-tawy$s8nf4CF3ekll$r5a-NH2


879.86


SP758
Ac-tawya$s8f4CF3ekllr$r5a-NH2


936.38


SP759
Ac-tawy$s8naekll$r5a-NH2


844.25


SP760
5-FAM-Batawy$s8nf4CF3ekll$r5a-



NH2


SP761
5-FAM-Batawy$s8naekll$r5a-NH2


SP762
Ac-tawy$s8nf4CF3eall$r5a-NH2


SP763
Ac-tawy$s8nf4CF3ekll$r5aaaaa-



NH2


SP764
Ac-tawy$s8nf4CF3eall$r5aaaaa-



NH2























TABLE 1a








Exact
Found
Calc
Calc
Calc


SP
Sequence
Isomer
Mass
Mass
(M + 1)/1
(M + 2)/2
(M + 3)/3






















SP244
Ac-LTF$r8EF4coohWAQCba$SANleA-NH2

1885
943.59
1886.01
943.51
629.34


SP331
Ac-LTF$r8EYWAQL$AAAAAa-NH2
iso2
1929.04
966.08
1930.05
965.53
644.02


SP555
Ac-LTF$r8EY6clWAQL$AAAAAa-NH2

1963
983.28
1964.01
982.51
655.34


SP557
Ac-AAALTF$r8EYWAQL$AAAAAa-NH2

2142.15
1072.83
2143.16
1072.08
715.06


SP558
Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2

1965.02
984.3
1966.03
983.52
656.01


SP562
Ac-LTF$r8EYWAQL$AAibAAAa-NH2

1943.06
973.11
1944.07
972.54
648.69


SP564
Ac-LTF$r8EYWAQL$AAAAibAa-NH2

1943.06
973.48
1944.07
972.54
648.69


SP566
Ac-LTF$r8EYWAQL$AAAAAiba-NH2
iso2
1943.06
973.38
1944.07
972.54
648.69


SP567
Ac-LTF$r8EYWAQL$AAAAAAib-NH2

1943.06
973.01
1944.07
972.54
648.69


SP572
Ac-LTF$r8EYWAQL$AAAAaa-NH2

1929.04
966.35
1930.05
965.53
644.02


SP573
Ac-LTF$r8EYWAQL$AAAAAA-NH2

1929.04
966.35
1930.05
965.53
644.02


SP578
Ac-LTF$r8EYWAQL$AAAAASar-NH2

1929.04
966.08
1930.05
965.53
644.02


SP551
Ac-LTF$r8EYWAQL$AAAAAa-OH
iso2
1930.02
965.89
1931.03
966.02
644.35


SP662
Ac-LTF$rda6AYWAQL$da5AAAAAa-NH2

1974.06
934.44

933.49



SP367
5-FAM-BaLTF$r8EYWAQCba$SAA-NH2

2131
1067.09
2132.01
1066.51
711.34


SP349
Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH2
iso2
1969.04
986.06
1970.05
985.53
657.35


SP347
Ac-LTF$r8EYWAQCba$AAAAAa-NH2
iso2
1941.04
972.55
1942.05
971.53
648.02









Table 1b shows a further selection of peptidomimetic macrocycles.
















TABLE 1b








Exact
Found
Calc
Calc
Calc


SP
Sequence
Isomer
Mass
Mass
(M + 1)/1
(M + 2)/2
(M + 3)/3






















SP581
Ac-TF$r8EYWAQL$AAAAAa-NH2

1815.96
929.85
1816.97
908.99
606.33


SP582
Ac-F$r8EYWAQL$AAAAAa-NH2

1714.91
930.92
1715.92
858.46
572.64


SP583
Ac-LVF$r8EYWAQL$AAAAAa-NH2

1927.06
895.12
1928.07
964.54
643.36


SP584
Ac-AAF$r8EYWAQL$AAAAAa-NH2

1856.98
859.51
1857.99
929.5
620


SP585
Ac-LTF$r8EYWAQL$AAAAa-NH2

1858
824.08
1859.01
930.01
620.34


SP586
Ac-LTF$r8EYWAQL$AAAa-NH2

1786.97
788.56
1787.98
894.49
596.66


SP587
Ac-LTF$r8EYWAQL$AAa-NH2

1715.93
1138.57
1716.94
858.97
572.98


SP588
Ac-LTF$r8EYWAQL$Aa-NH2

1644.89
1144.98
1645.9
823.45
549.3


SP589
Ac-LTF$r8EYWAQL$a-NH2

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:




embedded image


embedded image


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





SP #
Structure







SP154


embedded image








Chemical Formula: C87H125N17O21



Exact Mass: 1743.92



Molecular Weight: 1745.02





SP115


embedded image








Chemical Formula: C85H125N17O19



Exact Mass: 1687.93



Molecular Weight: 1689.00





SP114


embedded image








Chemical Formula: C85H125N17O19



Exact Mass: 1687.93



Molecular Weight: 1689.00





SP99 


embedded image








Chemical Formula: C84H122ClN17O19



Exact Mass: 1707.88



Molecular Weight: 1709.42





SP388


embedded image








Chemical Formula: C91H136N18O19



Exact Mass: 1785.02



Molecular Weight: 1786.16





SP331


embedded image








Chemical Formula: C95H140N20O23



Exact Mass: 1929.04



Molecular Weight: 1930.25





SP445


embedded image








Chemical Formula: C95H142N20O23



Exact Mass: 1931.06



Molecular Weight: 1932.26





SP351


embedded image








Chemical Formula: C96H140N20O24



Exact Mass: 1957.03



Molecular Weight: 1958.26





SP71 


embedded image








Chemical Formula: C90H134N18O19



Exact Mass: 1771.01



Molecular Weight: 1772.14





SP69 


embedded image








Chemical Formula: C90H134N18O19



Exact Mass: 1771.01



Molecular Weight: 1772.14





SP7 


embedded image








Chemical Formula: C90H127N17O19



Exact Mass: 1749.95



Molecular Weight: 1751.07





SP160


embedded image








Chemical Formula: C87H125F2N17O21



Exact Mass: 1781.92



Molecular Weight: 1783.02





SP315


embedded image








Chemical Formula: C93H138N20O21



Exact Mass: 1871.03



Molecular Weight: 1872.21





SP249


embedded image








Chemical Formula: C94H136N18O22



Exact Mass: 1869.01



Molecular Weight: 1870.19





SP437


embedded image








Chemical Formula: C95H143N21O21



Exact Mass: 1914.08



Molecular Weight: 1915.28





SP349


embedded image








Chemical Formula: C97H140N20O24



Exact Mass: 1969.03



Molecular Weight: 1970.27





SP555


embedded image








Chemical Formula: C95H139ClN20O23



Exact Mass: 1963.69



Molecular Weight: 1964.69





SP557


embedded image








Chemical Formula: C104H155N23O26



Exact Mass: 2142.15



Molecular Weight: 2143.48





SP558


embedded image








Chemical Formula: C95H138F2N20O23



Exact Mass: 1965.02



Molecular Weight: 1966.23





SP367


embedded image







SP562


embedded image








Chemical Formula: C96H142N20O23



Exact Mass: 1943.06



Molecular Weight: 1944.27





SP564


embedded image








Chemical Formula: C96H142N20O23



Exact Mass: 1943.06



Molecular Weight: 1944.27





SP566


embedded image







SP567


embedded image








Chemical Formula: C96H142N20O23



Exact Mass: 1943.06



Molecular Weight: 1944.27





SP572


embedded image








Chemical Formula: C95H140N20O23



Exact Mass: 1929.04



Molecular Weight: 1930.25





SP573


embedded image








Chemical Formula: C95H140N20O23



Exact Mass: 1929.04



Molecular Weight: 1930.25





SP578


embedded image








Chemical Formula: C95H140N20O23



Exact Mass: 1929.04



Molecular Weight: 1930.25





SP664


embedded image








Chemical Formula: C95H134N20O23



Exact Mass: 1922.99



Molecular Weight: 1924.20





SP662


embedded image








Chemical Formula: C95H134N20O23



Exact Mass: 1922.99



Molecular Weight: 1924.20








embedded image








Chemical Formula: C96H136N20O23



Exact Mass: 1937.01



Molecular Weight: 1938.23









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









TABLE 2a





Sequence

















L$r5QETFSD$s8WKLLPEN



LSQ$r5TFSDLW$s8LLPEN



LSQE$r5FSDLWK$s8LPEN



LSQET$r5SDLWKL$s8PEN



LSQETF$r5DLWKLL$s8EN



LXQETFS$r5LWKLLP$s8N



LSQETFSD$r5WKLLPE$s8



LSQQTF$r5DLWKLL$s8EN



LSQETF$r5DLWKLL$s8QN



LSQQTF$r5DLWKLL$s8QN



LSQETF$r5NLWKLL$s8QN



LSQQTF$r5NLWKLL$s8QN



LSQQTF$r5NLWRLL$s8QN



QSQQTF$r5NLWKLL$s8QN



QSQQTF$r5NLWRLL$s8QN



QSQQTA$r5NLWRLL$s8QN



L$r8QETFSD$WKLLPEN



LSQ$r8TFSDLW$LLPEN



LSQE$r8FSDLWK$LPEN



LSQET$r8SDLWKL$PEN



LSQETF$r8DLWKLL$EN



LXQETFS$r8LWKLLP$N



LSQETFSD$r8WKLLPE$



LSQQTF$r8DLWKLL$EN



LSQETF$r8DLWKLL$QN



LSQQTF$r8DLWKLL$QN



LSQETF$r8NLWKLL$QN



LSQQTF$r8NLWKLL$QN



LSQQTF$r8NLWRLL$QN



QSQQTF$r8NLWKLL$QN



QSQQTF$r8NLWRLL$QN



QSQQTA$r8NLWRLL$QN



QSQQTF$r8NLWRKK$QN



QQTF$r8DLWRLL$EN



QQTF$r8DLWRLL$



LSQQTF$DLW$LL



QQTF$DLW$LL



QQTA$r8DLWRLL$EN



QSQQTF$r5NLWRLL$s8QN



(dihydroxylated olefin)



QSQQTA$r5NLWRLL$s8QN



(dihydroxylated olefin)



QSQQTF$r8DLWRLL$QN



QTF$r8NLWRLL$



QSQQTF$NLW$LLPQN



QS$QTF$NLWRLLPQN



$TFS$LWKLL



ETF$DLW$LL



QTF$NLW$LL



$SQE$FSNLWKLL










In Table 2a, X represents S or any amino acid. Peptides shown 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









Observed




Exact

mass


Number
Sequence
Mass
M + 2
(m/e)



















1
Ac-LSQETF$r8DLWKLL$EN-NH2
2068.13
1035.07
1035.36


2
Ac-LSQETF$r8NLWKLL$QN-NH2
2066.16
1034.08
1034.31


3
Ac-LSQQTF$r8NLWRLL$QN-NH2
2093.18
1047.59
1047.73


4
Ac-QSQQTF$r8NLWKLL$QN-NH2
2080.15
1041.08
1041.31


5
Ac-QSQQTF$r8NLWRLL$QN-NH2
2108.15
1055.08
1055.32


6
Ac-QSQQTA$r8NLWRLL$QN-NH2
2032.12
1017.06
1017.24


7
Ac-QAibQQTF$r8NLWRLL$QN-NH2
2106.17
1054.09
1054.34


8
Ac-QSQQTFSNLWRLLPQN-NH2
2000.02
1001.01
1001.26


9
Ac-QSQQTF$/r8NLWRLL$/QN-NH2
2136.18
1069.09
1069.37


10
Ac-QSQAibTF$r8NLWRLL$QN-NH2
2065.15
1033.58
1033.71


11
Ac-QSQQTF$r8NLWRLL$AN-NH2
2051.13
1026.57
1026.70


12
Ac-ASQQTF$r8NLWRLL$QN-NH2
2051.13
1026.57
1026.90


13
Ac-QSQQTF$r8ALWRLL$QN-NH2
2065.15
1033.58
1033.41


14
Ac-QSQETF$r8NLWRLL$QN-NH2
2109.14
1055.57
1055.70


15
Ac-RSQQTF$r8NLWRLL$QN-NH2
2136.20
1069.10
1069.17


16
Ac-RSQQTF$r8NLWRLL$EN-NH2
2137.18
1069.59
1069.75


17
Ac-LSQETFSDLWKLLPEN-NH2
1959.99
981.00
981.24


18
Ac-QSQ$TFS$LWRLLPQN-NH2
2008.09
1005.05
1004.97


19
Ac-QSQQ$FSN$WRLLPQN-NH2
2036.06
1019.03
1018.86


20
Ac-QSQQT$SNL$RLLPQN-NH2
1917.04
959.52
959.32


21
Ac-QSQQTF$NLW$LLPQN-NH2
2007.06
1004.53
1004.97


22
Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2
2310.26
1156.13
1156.52


23
Ac-QSQQTF$r8NLWRLL$RN-NH2
2136.20
1069.10
1068.94


24
Ac-QSQRTF$r8NLWRLL$QN-NH2
2136.20
1069.10
1068.94


25
Ac-QSQQTF$r8NNleWRLL$QN-NH2
2108.15
1055.08
1055.44


26
Ac-QSQQTF$r8NLWRNleL$QN-NH2
2108.15
1055.08
1055.84


27
Ac-QSQQTF$r8NLWRLNle$QN-NH2
2108.15
1055.08
1055.12


28
Ac-QSQQTY$r8NLWRLL$QN-NH2
2124.15
1063.08
1062.92


29
Ac-RAibQQTF$r8NLWRLL$QN-NH2
2134.22
1068.11
1068.65


30
Ac-MPRFMDYWEGLN-NH2
1598.70
800.35
800.45


31
Ac-RSQQRF$r8NLWRLL$QN-NH2
2191.25
1096.63
1096.83


32
Ac-QSQQRF$r8NLWRLL$QN-NH2
2163.21
1082.61
1082.87


33
Ac-RAibQQRF$r8NLWRLL$QN-NH2
2189.27
1095.64
1096.37


34
Ac-RSQQRF$r8NFWRLL$QN-NH2
2225.23
1113.62
1114.37


35
Ac-RSQQRF$r8NYWRLL$QN-NH2
2241.23
1121.62
1122.37


36
Ac-RSQQTF$r8NLWQLL$QN-NH2
2108.15
1055.08
1055.29


37
Ac-QSQQTF$r8NLWQAmlL$QN-NH2
2094.13
1048.07
1048.32


38
Ac-QSQQTF$r8NAmlWRLL$QN-NH2
2122.17
1062.09
1062.35


39
Ac-NlePRF$r8DYWEGL$QN-NH2
1869.98
935.99
936.20


40
Ac-NlePRF$r8NYWRLL$QN-NH2
1952.12
977.06
977.35


41
Ac-RF$r8NLWRLL$Q-NH2
1577.96
789.98
790.18


42
Ac-QSQQTF$r8N2ffWRLL$QN-NH2
2160.13
1081.07
1081.40


43
Ac-QSQQTF$r8N3ffWRLL$QN-NH2
2160.13
1081.07
1081.34


44
Ac-QSQQTF#r8NLWRLL#QN-NH2
2080.12
1041.06
1041.34


45
Ac-RSQQTA$r8NLWRLL$QN-NH2
2060.16
1031.08
1031.38


46
Ac-QSQQTF%r8NLWRLL%QN-NH2
2110.17
1056.09
1056.55


47
HepQSQ$TFSNLWRLLPQN-NH2
2051.10
1026.55
1026.82


48
HepQSQ$TF$r8NLWRLL$QN-NH2
2159.23
1080.62
1080.89


49
Ac-QSQQTF$r8NL6clWRLL$QN-NH2
2142.11
1072.06
1072.35


50
Ac-QSQQTF$r8NLMe6clwRLL$QN-NH2
2156.13
1079.07
1079.27


51
Ac-LTFEHYWAQLTS-NH2
1535.74
768.87
768.91


52
Ac-LTF$HYW$QLTS-NH2
1585.83
793.92
794.17


53
Ac-LTFE$YWA$LTS-NH2
1520.79
761.40
761.67


54
Ac-LTF$zr8HYWAQL$zS-NH2
1597.87
799.94
800.06


55
Ac-LTF$r8HYWRQL$S-NH2
1682.93
842.47
842.72


56
Ac-QS$QTFStNLWRLL$s8QN-NH2
2145.21
1073.61
1073.90


57
Ac-QSQQTASNLWRLLPQN-NH2
1923.99
963.00
963.26


58
Ac-QSQQTA$/r8NLWRLL$/QN-NH2
2060.15
1031.08
1031.24


59
Ac-ASQQTF$/r8NLWRLL$/QN-NH2
2079.16
1040.58
1040.89


60
Ac-$SQQ$FSNLWRLLAibQN-NH2
2009.09
1005.55
1005.86


61
Ac-QS$QTF$NLWRLLAibQN-NH2
2023.10
1012.55
1012.79


62
Ac-QSQQ$FSN$WRLLAibQN-NH2
2024.06
1013.03
1013.31


63
Ac-QSQQTF$NLW$LLAibQN-NH2
1995.06
998.53
998.87


64
Ac-QSQQTFS$LWR$LAibQN-NH2
2011.06
1006.53
1006.83


65
Ac-QSQQTFSNLW$LLA$N-NH2
1940.02
971.01
971.29


66
Ac-$/SQQ$/FSNLWRLLAibQN-NH2
2037.12
1019.56
1019.78


67
Ac-QS$/QTF$/NLWRLLAibQN-NH2
2051.13
1026.57
1026.90


68
Ac-QSQQ$/FSN$/WRLLAibQN-NH2
2052.09
1027.05
1027.36


69
Ac-QSQQTF$/NLW$/LLAibQN-NH2
2023.09
1012.55
1013.82


70
Ac-QSQ$TFS$LWRLLAibQN-NH2
1996.09
999.05
999.39


71
Ac-QSQ$/TFS$/LWRLLAibQN-NH2
2024.12
1013.06
1013.37


72
Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2
2201.27
1101.64
1102.00


73
Ac-$r8SQQTFS$LWRLLAibQN-NH2
2038.14
1020.07
1020.23


74
Ac-QSQ$r8TFSNLW$LLAibQN-NH2
1996.08
999.04
999.32


75
Ac-QSQQTFS$r8LWRLLA$N-NH2
2024.12
1013.06
1013.37


76
Ac-QS$r5QTFStNLW$LLAibQN-NH2
2032.12
1017.06
1017.39


77
Ac-$/r8SQQTFS$/LWRLLAibQN-NH2
2066.17
1034.09
1034.80


78
Ac-QSQ$/r8TFSNLW$/LLAibQN-NH2
2024.11
1013.06
1014.34


79
Ac-QSQQTFS$/r8LWRLLA$/N-NH2
2052.15
1027.08
1027.16


80
Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2
2088.18
1045.09
1047.10


81
Ac-QSQQTFSNLWRLLAibQN-NH2
1988.02
995.01
995.31


82
Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2
2215.29
1108.65
1108.93


83
Ac-ASQQTF$r8NLRWLL$QN-NH2
2051.13
1026.57
1026.90


84
Ac-QSQQTF$/r8NLWRLL$/Q-NH2
2022.14
1012.07
1012.66


85
Ac-QSQQTF$r8NLWRLL$Q-NH2
1994.11
998.06
998.42


86
Ac-AAARAA$r8AAARAA$AA-NH2
1515.90
758.95
759.21


87
Ac-LTFEHYWAQLTSA-NH2
1606.78
804.39
804.59


88
Ac-LTF$r8HYWAQL$SA-NH2
1668.90
835.45
835.67


89
Ac-ASQQTFSNLWRLLPQN-NH2
1943.00
972.50
973.27


90
Ac-QS$QTFStNLW$r5LLAibQN-NH2
2032.12
1017.06
1017.30


91
Ac-QSQQTFAibNLWRLLAibQN-NH2
1986.04
994.02
994.19


92
Ac-QSQQTFNleNLWRLLNleQN-NH2
2042.11
1022.06
1022.23


93
Ac-QSQQTF$/r8NLWRLLAibQN-NH2
2082.14
1042.07
1042.23


94
Ac-QSQQTF$/r8NLWRLLNleQN-NH2
2110.17
1056.09
1056.29


95
Ac-QSQQTFAibNLWRLL$/QN-NH2
2040.09
1021.05
1021.25


96
Ac-QSQQTFNleNLWRLL$/QN-NH2
2068.12
1035.06
1035.31


97
Ac-QSQQTF%r8NL6clWRNleL%QN-NH2
2144.13
1073.07
1073.32


98
Ac-QSQQTF%r8NLMe6clWRLL%QN-NH2
2158.15
1080.08
1080.31


101
Ac-FNle$YWE$L-NH2
1160.63

1161.70


102
Ac-F$r8AYWELL$A-NH2
1344.75

1345.90


103
Ac-F$r8AYWQLL$A-NH2
1343.76

1344.83


104
Ac-NlePRF$r8NYWELL$QN-NH2
1925.06
963.53
963.69


105
Ac-NlePRF$r8DYWRLL$QN-NH2
1953.10
977.55
977.68


106
Ac-NlePRF$r8NYWRLL$Q-NH2
1838.07
920.04
920.18


107
Ac-NlePRF$r8NYWRLL$-NH2
1710.01
856.01
856.13


108
Ac-QSQQTF$r8DLWRLL$QN-NH2
2109.14
1055.57
1055.64


109
Ac-QSQQTF$r8NLWRLL$EN-NH2
2109.14
1055.57
1055.70


110
Ac-QSQQTF$r8NLWRLL$QD-NH2
2109.14
1055.57
1055.64


111
Ac-QSQQTF$r8NLWRLL$S-NH2
1953.08
977.54
977.60


112
Ac-ESQQTF$r8NLWRLL$QN-NH2
2109.14
1055.57
1055.70


113
Ac-LTF$r8NLWRNleL$Q-NH2
1635.99
819.00
819.10


114
Ac-LRF$r8NLWRNleL$Q-NH2
1691.04
846.52
846.68


115
Ac-QSQQTF$r8NWWRNleL$QN-NH2
2181.15
1091.58
1091.64


116
Ac-QSQQTF$r8NLWRNleL$Q-NH2
1994.11
998.06
998.07


117
Ac-QTF$r8NLWRNleL$QN-NH2
1765.00
883.50
883.59


118
Ac-NlePRF$r8NWWRLL$QN-NH2
1975.13
988.57
988.75


119
Ac-NlePRF$r8NWWRLL$A-NH2
1804.07
903.04
903.08


120
Ac-TSFAEYWNLLNH2
1467.70
734.85
734.90


121
Ac-QTF$r8HWWSQL$S-NH2
1651.85
826.93
827.12


122
Ac-FM$YWE$L-NH2
1178.58

1179.64


123
Ac-QTFEHWWSQLLS-NH2
1601.76
801.88
801.94


124
Ac-QSQQTF$r8NLAmwRLNle$QN-NH2
2122.17
1062.09
1062.24


125
Ac-FMAibY6clWEAc3cL-NH2
1130.47

1131.53


126
Ac-FNle$Y6clWE$L-NH2
1194.59

1195.64


127
Ac-F$zr8AY6clWEAc3cL$z-NH2
1277.63
639.82
1278.71


128
Ac-F$r8AY6clWEAc3cL$A-NH2
1348.66

1350.72


129
Ac-NlePRF$r8NY6clWRLL$QN-NH2
1986.08
994.04
994.64


130
Ac-AF$r8AAWALA$A-NH2
1223.71

1224.71


131
Ac-TF$r8AAWRLA$Q-NH2
1395.80
698.90
399.04


132
Pr-TF$r8AAWRLA$Q-NH2
1409.82
705.91
706.04


133
Ac-QSQQTF%r8NLWRNleL%QN-NH2
2110.17
1056.09
1056.22


134
Ac-LTF%r8HYWAQL%SA-NH2
1670.92
836.46
836.58


135
Ac-NlePRF%r8NYWRLL%QN-NH2
1954.13
978.07
978.19


136
Ac-NlePRF%r8NY6clWRLL%QN-NH2
1988.09
995.05
995.68


137
Ac-LTF%r8HY6clWAQL%S-NH2
1633.84
817.92
817.93


138
Ac-QS%QTF%StNLWRLL%s8QN-NH2
2149.24
1075.62
1075.65


139
Ac-LTF%r8HY6clWRQL%S-NH2
1718.91
860.46
860.54


140
Ac-QSQQTF%r8NL6clWRLL%QN-NH2
2144.13
1073.07
1073.64


141
Ac-%r8SQQTFS%LWRLLAibQN-NH2
2040.15
1021.08
1021.13


142
Ac-LTF%r8HYWAQL%S-NH2
1599.88
800.94
801.09


143
Ac-TSF%r8QYWNLL%P-NH2
1602.88
802.44
802.58


147
Ac-LTFEHYWAQLTS-NH2
1535.74
768.87
769.5


152
Ac-F$er8AY6clWEAc3cL$e-NH2
1277.63
639.82
1278.71


153
Ac-AF$r8AAWALA$A-NH2
1277.63
639.82
1277.84


154
Ac-TF$r8AAWRLA$Q-NH2
1395.80
698.90
699.04


155
Pr-TF$r8AAWRLA$Q-NH2
1409.82
705.91
706.04


156
Ac-LTF$er8HYWAQL$eS-NH2
1597.87
799.94
800.44


159
Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2
2745.30
1373.65
1372.99


160
Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-NH2
2669.27
1335.64
1336.09


161
Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2
2589.26
1295.63
1296.2


162
Ac-LTF$/r8HYWAQLS/S-NH2
1625.90
813.95
814.18


163
Ac-F%r8HY6clWRAc3cL%-NH2
1372.72
687.36
687.59


164
Ac-QTF%r8HWWSQL%S-NH2
1653.87
827.94
827.94


165
Ac-LTA$r8HYWRQL$S-NH2
1606.90
804.45
804.66


166
Ac-Q$r8QQTFSN$WRLLAibQN-NH2
2080.12
1041.06
1041.61


167
Ac-QSQQ$r8FSNLWR$LAibQN-NH2
2066.11
1034.06
1034.58


168
Ac-F$r8AYWEAc3cL$A-NH2
1314.70
658.35
1315.88


169
Ac-F$r8AYWEAc3cL$S-NH2
1330.70
666.35
1331.87


170
Ac-F$r8AYWEAc3cL$Q-NH2
1371.72
686.86
1372.72


171
Ac-F$r8AYWEAibL$S-NH2
1332.71
667.36
1334.83


172
Ac-F$r8AYWEAL$S-NH2
1318.70
660.35
1319.73


173
Ac-F$r8AYWEQL$S-NH2
1375.72
688.86
1377.53


174
Ac-F$r8HYWEQL$S-NH2
1441.74
721.87
1443.48


175
Ac-F$r8HYWAQL$S-NH2
1383.73
692.87
1385.38


176
Ac-F$r8HYWAAc3cL$S-NH2
1338.71
670.36
1340.82


177
Ac-F$r8HYWRAc3cL$S-NH2
1423.78
712.89
713.04


178
Ac-F$r8AYWEAc3cL#A-NH2
1300.69
651.35
1302.78


179
Ac-NlePTF%r8NYWRLL%QN-NH2
1899.08
950.54
950.56


180
Ac-TF$r8AAWRAL$Q-NH2
1395.80
698.90
699.13


181
Ac-TSF%r8HYWAQL%S-NH2
1573.83
787.92
787.98


184
Ac-F%r8AY6clWEAc3cL%A-NH2
1350.68
676.34
676.91


185
Ac-LTF$r8HYWAQI$S-NH2
1597.87
799.94
800.07


186
Ac-LTF$r8HYWAQNle$S-NH2
1597.87
799.94
800.07


187
Ac-LTF$r8HYWAQL$A-NH2
1581.87
791.94
792.45


188
Ac-LTF$r8HYWAQL$Abu-NH2
1595.89
798.95
799.03


189
Ac-LTF$r8HYWAbuQL$S-NH2
1611.88
806.94
807.47


190
Ac-LTF$er8AYWAQL$eS-NH2
1531.84
766.92
766.96


191
Ac-LAF$r8HYWAQL$S-NH2
1567.86
784.93
785.49


192
Ac-LAF$r8AYWAQL$S-NH2
1501.83
751.92
752.01


193
Ac-LTF$er8AYWAQL$eA-NH2
1515.85
758.93
758.97


194
Ac-LAF$r8AYWAQL$A-NH2
1485.84
743.92
744.05


195
Ac-LTF$r8NLWANleL$Q-NH2
1550.92
776.46
776.61


196
Ac-LTF$r8NLWANleL$A-NH2
1493.90
747.95
1495.6


197
Ac-LTF$r8ALWANleL$Q-NH2
1507.92
754.96
755


198
Ac-LAF$r8NLWANleL$Q-NH2
1520.91
761.46
761.96


199
Ac-LAF$r8ALWANleL$A-NH2
1420.89
711.45
1421.74


200
Ac-A$r8AYWEAc3cL$A-NH2
1238.67
620.34
1239.65


201
Ac-F$r8AYWEAc3cL$AA-NH2
1385.74
693.87
1386.64


202
Ac-F$r8AYWEAc3cL$Abu-NH2
1328.72
665.36
1330.17


203
Ac-F$r8AYWEAc3cL$Nle-NH2
1356.75
679.38
1358.22


204
Ac-F$r5AYWEAc3cL$s8A-NH2
1314.70
658.35
1315.51


205
Ac-F$AYWEAc3cL$r8A-NH2
1314.70
658.35
1315.66


206
Ac-F$r8AYWEAc3cl$A-NH2
1314.70
658.35
1316.18


207
Ac-F$r8AYWEAc3cNle$A-NH2
1314.70
658.35
1315.66


208
Ac-F$r8AYWEAmlL$A-NH2
1358.76
680.38
1360.21


209
Ac-F$r8AYWENleL$A-NH2
1344.75
673.38
1345.71


210
Ac-F$r8AYWQAc3cL$A-NH2
1313.72
657.86
1314.7


211
Ac-F$r8AYWAAc3cL$A-NH2
1256.70
629.35
1257.56


212
Ac-F$r8AYWAbuAc3cL$A-NH2
1270.71
636.36
1272.14


213
Ac-F$r8AYWNleAc3cL$A-NH2
1298.74
650.37
1299.67


214
Ac-F$r8AbuYWEAc3cL$A-NH2
1328.72
665.36
1329.65


215
Ac-F$r8NleYWEAc3cL$A-NH2
1356.75
679.38
1358.66


216
5-FAM-BaLTFEHYWAQLTS-NH2
1922.82
962.41
962.87


217
5-FAM-BaLTF%r8HYWAQL%S-NH2
1986.96
994.48
994.97


218
Ac-LTF$r8HYWAQhL$S-NH2
1611.88
806.94
807


219
Ac-LTF$r8HYWAQTle$S-NH2
1597.87
799.94
799.97


220
Ac-LTF$r8HYWAQAdm$S-NH2
1675.91
838.96
839.09


221
Ac-LTF$r8HYWAQhCha$S-NH2
1651.91
826.96
826.98


222
Ac-LTF$r8HYWAQCha$S-NH2
1637.90
819.95
820.02


223
Ac-LTF$r8HYWAc6cQL$S-NH2
1651.91
826.96
826.98


224
Ac-LTF$r8HYWAc5cQL$S-NH2
1637.90
819.95
820.02


225
Ac-LThF$r8HYWAQL$S-NH2
1611.88
806.94
807


226
Ac-LTIgl$r8HYWAQL$S-NH2
1625.90
813.95
812.99


227
Ac-LTF$r8HYWAQChg$S-NH2
1623.88
812.94
812.99


228
Ac-LTF$r8HYWAQF$S-NH2
1631.85
816.93
816.99


229
Ac-LTF$r8HYWAQIgl$S-NH2
1659.88
830.94
829.94


230
Ac-LTF$r8HYWAQCba$S-NH2
1609.87
805.94
805.96


231
Ac-LTF$r8HYWAQCpg$S-NH2
1609.87
805.94
805.96


232
Ac-LTF$r8HhYWAQL$S-NH2
1611.88
806.94
807


233
Ac-F$r8AYWEAc3chL$A-NH2
1328.72
665.36
665.43


234
Ac-F$r8AYWEAc3cTle$A-NH2
1314.70
658.35
1315.62


235
Ac-F$r8AYWEAc3cAdm$A-NH2
1392.75
697.38
697.47


236
Ac-F$r8AYWEAc3chCha$A-NH2
1368.75
685.38
685.34


237
Ac-F$r8AYWEAc3cCha$A-NH2
1354.73
678.37
678.38


238
Ac-F$r8AYWEAc6cL$A-NH2
1356.75
679.38
679.42


239
Ac-F$r8AYWEAc5cL$A-NH2
1342.73
672.37
672.46


240
Ac-hF$r8AYWEAc3cL$A-NH2
1328.72
665.36
665.43


241
Ac-Igl$r8AYWEAc3cL$A-NH2
1342.73
672.37
671.5


243
Ac-F$r8AYWEAc3cF$A-NH2
1348.69
675.35
675.35


244
Ac-F$r8AYWEAc3cIgl$A-NH2
1376.72
689.36
688.37


245
Ac-F$r8AYWEAc3cCba$A-NH2
1326.70
664.35
664.47


246
Ac-F$r8AYWEAc3cCpg$A-NH2
1326.70
664.35
664.39


247
Ac-F$r8AhYWEAc3cL$A-NH2
1328.72
665.36
665.43


248
Ac-F$r8AYWEAc3cL$Q-NH2
1371.72
686.86
1372.87


249
Ac-F$r8AYWEAibL$A-NH2
1316.72
659.36
1318.18


250
Ac-F$r8AYWEAL$A-NH2
1302.70
652.35
1303.75


251
Ac-LAF$r8AYWAAL$A-NH2
1428.82
715.41
715.49


252
Ac-LTF$r8HYWAAc3cL$S-NH2
1552.84
777.42
777.5


253
Ac-NleTF$r8HYWAQL$S-NH2
1597.87
799.94
800.04


254
Ac-VTF$r8HYWAQL$S-NH2
1583.85
792.93
793.04


255
Ac-FTF$r8HYWAQL$S-NH2
1631.85
816.93
817.02


256
Ac-WTF$r8HYWAQL$S-NH2
1670.86
836.43
836.85


257
Ac-RTF$r8HYWAQL$S-NH2
1640.88
821.44
821.9


258
Ac-KTF$r8HYWAQL$S-NH2
1612.88
807.44
807.91


259
Ac-LNleF$r8HYWAQL$S-NH2
1609.90
805.95
806.43


260
Ac-LVF$r8HYWAQL$S-NH2
1595.89
798.95
798.93


261
Ac-LFF$r8HYWAQL$S-NH2
1643.89
822.95
823.38


262
Ac-LWF$r8HYWAQL$S-NH2
1682.90
842.45
842.55


263
Ac-LRF$r8HYWAQL$S-NH2
1652.92
827.46
827.52


264
Ac-LKF$r8HYWAQL$S-NH2
1624.91
813.46
813.51


265
Ac-LTF$r8NleYWAQL$S-NH2
1573.89
787.95
788.05


266
Ac-LTF$r8VYWAQL$S-NH2
1559.88
780.94
780.98


267
Ac-LTF$r8FYWAQL$S-NH2
1607.88
804.94
805.32


268
Ac-LTF$r8WYWAQL$S-NH2
1646.89
824.45
824.86


269
Ac-LTF$r8RYWAQL$S-NH2
1616.91
809.46
809.51


270
Ac-LTF$r8KYWAQL$S-NH2
1588.90
795.45
795.48


271
Ac-LTF$r8HNleWAQL$S-NH2
1547.89
774.95
774.98


272
Ac-LTF$r8HVWAQL$S-NH2
1533.87
767.94
767.95


273
Ac-LTF$r8HFWAQL$S-NH2
1581.87
791.94
792.3


274
Ac-LTF$r8HWWAQL$S-NH2
1620.88
811.44
811.54


275
Ac-LTF$r8HRWAQL$S-NH2
1590.90
796.45
796.52


276
Ac-LTF$r8HKWAQL$S-NH2
1562.90
782.45
782.53


277
Ac-LTF$r8HYWNleQL$S-NH2
1639.91
820.96
820.98


278
Ac-LTF$r8HYWVQL$S-NH2
1625.90
813.92
814.03


279
Ac-LTF$r8HYWFQL$S-NH2
1673.90
837.95
838.03


280
Ac-LTF$r8HYWWQL$S-NH2
1712.91
857.46
857.5


281
Ac-LTF$r8HYWKQL$S-NH2
1654.92
828.46
828.49


282
Ac-LTF$r8HYWANleL$S-NH2
1582.89
792.45
792.52


283
Ac-LTF$r8HYWAVL$S-NH2
1568.88
785.44
785.49


284
Ac-LTF$r8HYWAFL$S-NH2
1616.88
809.44
809.47


285
Ac-LTF$r8HYWAWL$S-NH2
1655.89
828.95
829


286
Ac-LTF$r8HYWARL$S-NH2
1625.91
813.96
813.98


287
Ac-LTF$r8HYWAQL$Nle-NH2
1623.92
812.96
813.39


288
Ac-LTF$r8HYWAQL$V-NH2
1609.90
805.95
805.99


289
Ac-LTF$r8HYWAQL$F-NH2
1657.90
829.95
830.26


290
Ac-LTF$r8HYWAQL$W-NH2
1696.91
849.46
849.5


291
Ac-LTF$r8HYWAQL$R-NH2
1666.94
834.47
834.56


292
Ac-LTF$r8HYWAQL$K-NH2
1638.93
820.47
820.49


293
Ac-Q$r8QQTFSN$WRLLAibQN-NH2
2080.12
1041.06
1041.54


294
Ac-QSQQ$r8FSNLWR$LAibQN-NH2
2066.11
1034.06
1034.58


295
Ac-LT2Pal$r8HYWAQL$S-NH2
1598.86
800.43
800.49


296
Ac-LT3Pal$r8HYWAQL$S-NH2
1598.86
800.43
800.49


297
Ac-LT4Pal$r8HYWAQL$S-NH2
1598.86
800.43
800.49


298
Ac-LTF2CF3$r8HYWAQL$S-NH2
1665.85
833.93
834.01


299
Ac-LTF2CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


300
Ac-LTF2Me$r8HYWAQL$S-NH2
1611.88
806.94
807


301
Ac-LTF3Cl$r8HYWAQL$S-NH2
1631.83
816.92
816.99


302
Ac-LTF4CF3$r8HYWAQL$S-NH2
1665.85
833.93
833.94


303
Ac-LTF4tBu$r8HYWAQL$S-NH2
1653.93
827.97
828.02


304
Ac-LTF5F$r8HYWAQL$S-NH2
1687.82
844.91
844.96


305
Ac-LTF$r8HY3BthAAQL$S-NH2
1614.83
808.42
808.48


306
Ac-LTF2Br$r8HYWAQL$S-NH2
1675.78
838.89
838.97


307
Ac-LTF4Br$r8HYWAQL$S-NH2
1675.78
838.89
839.86


308
Ac-LTF2Cl$r8HYWAQL$S-NH2
1631.83
816.92
816.99


309
Ac-LTF4Cl$r8HYWAQL$S-NH2
1631.83
816.92
817.36


310
Ac-LTF3CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


311
Ac-LTF4CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


312
Ac-LTF34Cl2$r8HYWAQL$S-NH2
1665.79
833.90
833.94


313
Ac-LTF34F2$r8HYWAQL$S-NH2
1633.85
817.93
817.95


314
Ac-LTF35F2$r8HYWAQL$S-NH2
1633.85
817.93
817.95


315
Ac-LTDip$r8HYWAQL$S-NH2
1673.90
837.95
838.01


316
Ac-LTF2F$r8HYWAQL$S-NH2
1615.86
808.93
809


317
Ac-LTF3F$r8HYWAQL$S-NH2
1615.86
808.93
809


318
Ac-LTF4F$r8HYWAQL$S-NH2
1615.86
808.93
809


319
Ac-LTF4I$r8HYWAQL$S-NH2
1723.76
862.88
862.94


320
Ac-LTF3Me$r8HYWAQL$S-NH2
1611.88
806.94
807.07


321
Ac-LTF4Me$r8HYWAQL$S-NH2
1611.88
806.94
807


322
Ac-LT1Nal$r8HYWAQL$S-NH2
1647.88
824.94
824.98


323
Ac-LT2Nal$r8HYWAQL$S-NH2
1647.88
824.94
825.06


324
Ac-LTF3CF3$r8HYWAQL$S-NH2
1665.85
833.93
834.01


325
Ac-LTF4NO2$r8HYWAQL$S-NH2
1642.85
822.43
822.46


326
Ac-LTF3NO2$r8HYWAQL$S-NH2
1642.85
822.43
822.46


327
Ac-LTF$r82ThiYWAQL$S-NH2
1613.83
807.92
807.96


328
Ac-LTF$r8HBipWAQL$S-NH2
1657.90
829.95
830.01


329
Ac-LTF$r8HF4tBuWAQL$S-NH2
1637.93
819.97
820.02


330
Ac-LTF$r8HF4CF3WAQL$S-NH2
1649.86
825.93
826.02


331
Ac-LTF$r8HF4ClWAQL$S-NH2
1615.83
808.92
809.37


332
Ac-LTF$r8HF4MeWAQL$S-NH2
1595.89
798.95
799.01


333
Ac-LTF$r8HF4BrWAQL$S-NH2
1659.78
830.89
830.98


334
Ac-LTF$r8HF4CNWAQL$S-NH2
1606.87
804.44
804.56


335
Ac-LTF$r8HF4NO2WAQL$S-NH2
1626.86
814.43
814.55


336
Ac-LTF$r8H1NalWAQL$S-NH2
1631.89
816.95
817.06


337
Ac-LTF$r8H2NalWAQL$S-NH2
1631.89
816.95
816.99


338
Ac-LTF$r8HWAQL$S-NH2
1434.80
718.40
718.49


339
Ac-LTF$r8HY1NalAQL$S-NH2
1608.87
805.44
805.52


340
Ac-LTF$r8HY2NalAQL$S-NH2
1608.87
805.44
805.52


341
Ac-LTF$r8HYWAQI$S-NH2
1597.87
799.94
800.07


342
Ac-LTF$r8HYWAQNle$S-NH2
1597.87
799.94
800.44


343
Ac-LTF$er8HYWAQL$eA-NH2
1581.87
791.94
791.98


344
Ac-LTF$r8HYWAQL$Abu-NH2
1595.89
798.95
799.03


345
Ac-LTF$r8HYWAbuQL$S-NH2
1611.88
806.94
804.47


346
Ac-LAF$r8HYWAQL$S-NH2
1567.86
784.93
785.49


347
Ac-LTF$r8NLWANleL$Q-NH2
1550.92
776.46
777.5


348
Ac-LTF$r8ALWANleL$Q-NH2
1507.92
754.96
755.52


349
Ac-LAF$r8NLWANleL$Q-NH2
1520.91
761.46
762.48


350
Ac-F$r8AYWAAc3cL$A-NH2
1256.70
629.35
1257.56


351
Ac-LTF$r8AYWAAL$S-NH2
1474.82
738.41
738.55


352
Ac-LVF$r8AYWAQL$S-NH2
1529.87
765.94
766


353
Ac-LTF$r8AYWAbuQL$S-NH2
1545.86
773.93
773.92


354
Ac-LTF$r8AYWNleQL$S-NH2
1573.89
787.95
788.17


355
Ac-LTF$r8AbuYWAQL$S-NH2
1545.86
773.93
773.99


356
Ac-LTF$r8AYWHQL$S-NH2
1597.87
799.94
799.97


357
Ac-LTF$r8AYWKQL$S-NH2
1588.90
795.45
795.53


358
Ac-LTF$r8AYWOQL$S-NH2
1574.89
788.45
788.5


359
Ac-LTF$r8AYWRQL$S-NH2
1616.91
809.46
809.51


360
Ac-LTF$r8AYWSQL$S-NH2
1547.84
774.92
774.96


361
Ac-LTF$r8AYWRAL$S-NH2
1559.89
780.95
780.95


362
Ac-LTF$r8AYWRQL$A-NH2
1600.91
801.46
801.52


363
Ac-LTF$r8AYWRAL$A-NH2
1543.89
772.95
773.03


364
Ac-LTF$r5HYWAQL$s8S-NH2
1597.87
799.94
799.97


365
Ac-LTF$HYWAQL$r8S-NH2
1597.87
799.94
799.97


366
Ac-LTF$r8HYWAAL$S-NH2
1540.84
771.42
771.48


367
Ac-LTF$r8HYWAAbuL$S-NH2
1554.86
778.43
778.51


368
Ac-LTF$r8HYWALL$S-NH2
1582.89
792.45
792.49


369
Ac-F$r8AYWHAL$A-NH2
1310.72
656.36
656.4


370
Ac-F$r8AYWAAL$A-NH2
1244.70
623.35
1245.61


371
Ac-F$r8AYWSAL$A-NH2
1260.69
631.35
1261.6


372
Ac-F$r8AYWRAL$A-NH2
1329.76
665.88
1330.72


373
Ac-F$r8AYWKAL$A-NH2
1301.75
651.88
1302.67


374
Ac-F$r8AYWOAL$A-NH2
1287.74
644.87
1289.13


375
Ac-F$r8VYWEAc3cL$A-NH2
1342.73
672.37
1343.67


376
Ac-F$r8FYWEAc3cL$A-NH2
1390.73
696.37
1392.14


377
Ac-F$r8WYWEAc3cL$A-NH2
1429.74
715.87
1431.44


378
Ac-F$r8RYWEAc3cL$A-NH2
1399.77
700.89
700.95


379
Ac-F$r8KYWEAc3cL$A-NH2
1371.76
686.88
686.97


380
Ac-F$r8ANleWEAc3cL$A-NH2
1264.72
633.36
1265.59


381
Ac-F$r8AVWEAc3cL$A-NH2
1250.71
626.36
1252.2


382
Ac-F$r8AFWEAc3cL$A-NH2
1298.71
650.36
1299.64


383
Ac-F$r8AWWEAc3cL$A-NH2
1337.72
669.86
1338.64


384
Ac-F$r8ARWEAc3cL$A-NH2
1307.74
654.87
655


385
Ac-F$r8AKWEAc3cL$A-NH2
1279.73
640.87
641.01


386
Ac-F$r8AYWVAc3cL$A-NH2
1284.73
643.37
643.38


387
Ac-F$r8AYWFAc3cL$A-NH2
1332.73
667.37
667.43


388
Ac-F$r8AYWWAc3cL$A-NH2
1371.74
686.87
686.97


389
Ac-F$r8AYWRAc3cL$A-NH2
1341.76
671.88
671.94


390
Ac-F$r8AYWKAc3cL$A-NH2
1313.75
657.88
657.88


391
Ac-F$r8AYWEVL$A-NH2
1330.73
666.37
666.47


392
Ac-F$r8AYWEFL$A-NH2
1378.73
690.37
690.44


393
Ac-F$r8AYWEWL$A-NH2
1417.74
709.87
709.91


394
Ac-F$r8AYWERL$A-NH2
1387.77
694.89
1388.66


395
Ac-F$r8AYWEKL$A-NH2
1359.76
680.88
1361.21


396
Ac-F$r8AYWEAc3cL$V-NH2
1342.73
672.37
1343.59


397
Ac-F$r8AYWEAc3cL$F-NH2
1390.73
696.37
1392.58


398
Ac-F$r8AYWEAc3cL$W-NH2
1429.74
715.87
1431.29


399
Ac-F$r8AYWEAc3cL$R-NH2
1399.77
700.89
700.95


400
Ac-F$r8AYWEAc3cL$K-NH2
1371.76
686.88
686.97


401
Ac-F$r8AYWEAc3cL$AV-NH2
1413.77
707.89
707.91


402
Ac-F$r8AYWEAc3cL$AF-NH2
1461.77
731.89
731.96


403
Ac-F$r8AYWEAc3cL$AW-NH2
1500.78
751.39
751.5


404
Ac-F$r8AYWEAc3cL$AR-NH2
1470.80
736.40
736.47


405
Ac-F$r8AYWEAc3cL$AK-NH2
1442.80
722.40
722.41


406
Ac-F$r8AYWEAc3cL$AH-NH2
1451.76
726.88
726.93


407
Ac-LTF2NO2$r8HYWAQL$S-NH2
1642.85
822.43
822.54


408
Ac-LTA$r8HYAAQL$S-NH2
1406.79
704.40
704.5


409
Ac-LTF$r8HYAAQL$S-NH2
1482.82
742.41
742.47


410
Ac-QSQQTF$r8NLWALL$AN-NH2
1966.07
984.04
984.38


411
Ac-QAibQQTF$r8NLWALL$AN-NH2
1964.09
983.05
983.42


412
Ac-QAibQQTF$r8ALWALL$AN-NH2
1921.08
961.54
961.59


413
Ac-AAAATF$r8AAWAAL$AA-NH2
1608.90
805.45
805.52


414
Ac-F$r8AAWRAL$Q-NH2
1294.76
648.38
648.48


415
Ac-TF$r8AAWAAL$Q-NH2
1310.74
656.37
1311.62


416
Ac-TF$r8AAWRAL$A-NH2
1338.78
670.39
670.46


417
Ac-VF$r8AAWRAL$Q-NH2
1393.82
697.91
697.99


418
Ac-AF$r8AAWAAL$A-NH2
1223.71
612.86
1224.67


420
Ac-TF$r8AAWKAL$Q-NH2
1367.80
684.90
684.97


421
Ac-TF$r8AAWOAL$Q-NH2
1353.78
677.89
678.01


422
Ac-TF$r8AAWSAL$Q-NH2
1326.73
664.37
664.47


423
Ac-LTF$r8AAWRAL$Q-NH2
1508.89
755.45
755.49


424
Ac-F$r8AYWAQL$A-NH2
1301.72
651.86
651.96


425
Ac-F$r8AWWAAL$A-NH2
1267.71
634.86
634.87


426
Ac-F$r8AWWAQL$A-NH2
1324.73
663.37
663.43


427
Ac-F$r8AYWEAL$-NH2
1231.66
616.83
1232.93


428
Ac-F$r8AYWAAL$-NH2
1173.66
587.83
1175.09


429
Ac-F$r8AYWKAL$-NH2
1230.72
616.36
616.44


430
Ac-F$r8AYWOAL$-NH2
1216.70
609.35
609.48


431
Ac-F$r8AYWQAL$-NH2
1230.68
616.34
616.44


432
Ac-F$r8AYWAQL$-NH2
1230.68
616.34
616.37


433
Ac-F$r8HYWDQL$S-NH2
1427.72
714.86
714.86


434
Ac-F$r8HFWEQL$S-NH2
1425.74
713.87
713.98


435
Ac-F$r8AYWHQL$S-NH2
1383.73
692.87
692.96


436
Ac-F$r8AYWKQL$S-NH2
1374.77
688.39
688.45


437
Ac-F$r8AYWOQL$S-NH2
1360.75
681.38
681.49


438
Ac-F$r8HYWSQL$S-NH2
1399.73
700.87
700.95


439
Ac-F$r8HWWEQL$S-NH2
1464.76
733.38
733.44


440
Ac-F$r8HWWAQL$S-NH2
1406.75
704.38
704.43


441
Ac-F$r8AWWHQL$S-NH2
1406.75
704.38
704.43


442
Ac-F$r8AWWKQL$S-NH2
1397.79
699.90
699.92


443
Ac-F$r8AWWOQL$S-NH2
1383.77
692.89
692.96


444
Ac-F$r8HWWSQL$S-NH2
1422.75
712.38
712.42


445
Ac-LTF$r8NYWANleL$Q-NH2
1600.90
801.45
801.52


446
Ac-LTF$r8NLWAQL$Q-NH2
1565.90
783.95
784.06


447
Ac-LTF$r8NYWANleL$A-NH2
1543.88
772.94
773.03


448
Ac-LTF$r8NLWAQL$A-NH2
1508.88
755.44
755.49


449
Ac-LTF$r8AYWANleL$Q-NH2
1557.90
779.95
780.06


450
Ac-LTF$r8ALWAQL$Q-NH2
1522.89
762.45
762.45


451
Ac-LAF$r8NYWANleL$Q-NH2
1570.89
786.45
786.5


452
Ac-LAF$r8NLWAQL$Q-NH2
1535.89
768.95
769.03


453
Ac-LAF$r8AYWANleL$A-NH2
1470.86
736.43
736.47


454
Ac-LAF$r8ALWAQL$A-NH2
1435.86
718.93
719.01


455
Ac-LAF$r8AYWAAL$A-NH2
1428.82
715.41
715.41


456
Ac-F$r8AYWEAc3cL$AAib-NH2
1399.75
700.88
700.95


457
Ac-F$r8AYWAQL$AA-NH2
1372.75
687.38
687.78


458
Ac-F$r8AYWAAc3cL$AA-NH2
1327.73
664.87
664.84


459
Ac-F$r8AYWSAc3cL$AA-NH2
1343.73
672.87
672.9


460
Ac-F$r8AYWEAc3cL$AS-NH2
1401.73
701.87
701.84


461
Ac-F$r8AYWEAc3cL$AT-NH2
1415.75
708.88
708.87


462
Ac-F$r8AYWEAc3cL$AL-NH2
1427.79
714.90
714.94


463
Ac-F$r8AYWEAc3cL$AQ-NH2
1442.76
722.38
722.41


464
Ac-F$r8AFWEAc3cL$AA-NH2
1369.74
685.87
685.93


465
Ac-F$r8AWWEAc3cL$AA-NH2
1408.75
705.38
705.39


466
Ac-F$r8AYWEAc3cL$SA-NH2
1401.73
701.87
701.99


467
Ac-F$r8AYWEAL$AA-NH2
1373.74
687.87
687.93


468
Ac-F$r8AYWENleL$AA-NH2
1415.79
708.90
708.94


469
Ac-F$r8AYWEAc3cL$AbuA-NH2
1399.75
700.88
700.95


470
Ac-F$r8AYWEAc3cL$NleA-NH2
1427.79
714.90
714.86


471
Ac-F$r8AYWEAibL$NleA-NH2
1429.80
715.90
715.97


472
Ac-F$r8AYWEAL$NleA-NH2
1415.79
708.90
708.94


473
Ac-F$r8AYWENleL$NleA-NH2
1457.83
729.92
729.96


474
Ac-F$r8AYWEAibL$Abu-NH2
1330.73
666.37
666.39


475
Ac-F$r8AYWENleL$Abu-NH2
1358.76
680.38
680.39


476
Ac-F$r8AYWEAL$Abu-NH2
1316.72
659.36
659.36


477
Ac-LTF$r8AFWAQL$S-NH2
1515.85
758.93
759.12


478
Ac-LTF$r8AWWAQL$S-NH2
1554.86
778.43
778.51


479
Ac-LTF$r8AYWAQI$S-NH2
1531.84
766.92
766.96


480
Ac-LTF$r8AYWAQNle$S-NH2
1531.84
766.92
766.96


481
Ac-LTF$r8AYWAQL$SA-NH2
1602.88
802.44
802.48


482
Ac-LTF$r8AWWAQL$A-NH2
1538.87
770.44
770.89


483
Ac-LTF$r8AYWAQI$A-NH2
1515.85
758.93
759.42


484
Ac-LTF$r8AYWAQNle$A-NH2
1515.85
758.93
759.42


485
Ac-LTF$r8AYWAQL$AA-NH2
1586.89
794.45
794.94


486
Ac-LTF$r8HWWAQL$S-NH2
1620.88
811.44
811.47


487
Ac-LTF$r8HRWAQL$S-NH2
1590.90
796.45
796.52


488
Ac-LTF$r8HKWAQL$S-NH2
1562.90
782.45
782.53


489
Ac-LTF$r8HYWAQL$W-NH2
1696.91
849.46
849.5


491
Ac-F$r8AYWAbuAL$A-NH2
1258.71
630.36
630.5


492
Ac-F$r8AbuYWEAL$A-NH2
1316.72
659.36
659.51


493
Ac-NlePRF%r8NYWRLL%QN-NH2
1954.13
978.07
978.54


494
Ac-TSF%r8HYWAQL%S-NH2
1573.83
787.92
787.98


495
Ac-LTF%r8AYWAQL%S-NH2
1533.86
767.93
768


496
Ac-HTF$r8HYWAQL$S-NH2
1621.84
811.92
811.96


497
Ac-LHF$r8HYWAQL$S-NH2
1633.88
817.94
818.02


498
Ac-LTF$r8HHWAQL$S-NH2
1571.86
786.93
786.94


499
Ac-LTF$r8HYWHQL$S-NH2
1663.89
832.95
832.38


500
Ac-LTF$r8HYWAHL$S-NH2
1606.87
804.44
804.48


501
Ac-LTF$r8HYWAQL$H-NH2
1647.89
824.95
824.98


502
Ac-LTF$r8HYWAQL$S-NHPr
1639.91
820.96
820.98


503
Ac-LTF$r8HYWAQL$S-NHsBu
1653.93
827.97
828.02


504
Ac-LTF$r8HYWAQL$S-NHiBu
1653.93
827.97
828.02


505
Ac-LTF$r8HYWAQL$S-NHBn
1687.91
844.96
844.44


506
Ac-LTF$r8HYWAQL$S-NHPe
1700.92
851.46
851.99


507
Ac-LTF$r8HYWAQL$S-NHChx
1679.94
840.97
841.04


508
Ac-ETF$r8AYWAQL$S-NH2
1547.80
774.90
774.96


509
Ac-STF$r8AYWAQL$S-NH2
1505.79
753.90
753.94


510
Ac-LEF$r8AYWAQL$S-NH2
1559.84
780.92
781.25


511
Ac-LSF$r8AYWAQL$S-NH2
1517.83
759.92
759.93


512
Ac-LTF$r8EYWAQL$S-NH2
1589.85
795.93
795.97


513
Ac-LTF$r8SYWAQL$S-NH2
1547.84
774.92
774.96


514
Ac-LTF$r8AYWEQL$S-NH2
1589.85
795.93
795.9


515
Ac-LTF$r8AYWAEL$S-NH2
1532.83
767.42
766.96


516
Ac-LTF$r8AYWASL$S-NH2
1490.82
746.41
746.46


517
Ac-LTF$r8AYWAQL$E-NH2
1573.85
787.93
787.98


518
Ac-LTF2CN$r8HYWAQL$S-NH2
1622.86
812.43
812.47


519
Ac-LTF3Cl$r8HYWAQL$S-NH2
1631.83
816.92
816.99


520
Ac-LTDip$r8HYWAQL$S-NH2
1673.90
837.95
838.01


521
Ac-LTF$r8HYWAQTle$S-NH2
1597.87
799.94
800.04


522
Ac-F$r8AY6clWEAL$A-NH2
1336.66
669.33
1338.56


523
Ac-F$r8AYdl6brWEAL$A-NH2
1380.61
691.31
692.2


524
Ac-F$r8AYdl6fWEAL$A-NH2
1320.69
661.35
1321.61


525
Ac-F$r8AYdl4mWEAL$A-NH2
1316.72
659.36
659.36


526
Ac-F$r8AYdl5clWEAL$A-NH2
1336.66
669.33
669.35


527
Ac-F$r8AYdl7mWEAL$A-NH2
1316.72
659.36
659.36


528
Ac-LTF%r8HYWAQL%A-NH2
1583.89
792.95
793.01


529
Ac-LTF$r8HCouWAQL$S-NH2
1679.87
840.94
841.38


530
Ac-LTFEHCouWAQLTS-NH2
1617.75
809.88
809.96


531
Ac-LTA$r8HCouWAQL$S-NH2
1603.84
802.92
803.36


532
Ac-F$r8AYWEAL$AbuA-NH2
1387.75
694.88
694.88


533
Ac-F$r8AYWEAI$AA-NH2
1373.74
687.87
687.93


534
Ac-F$r8AYWEANle$AA-NH2
1373.74
687.87
687.93


535
Ac-F$r8AYWEAmlL$AA-NH2
1429.80
715.90
715.97


536
Ac-F$r8AYWQAL$AA-NH2
1372.75
687.38
687.48


537
Ac-F$r8AYWAAL$AA-NH2
1315.73
658.87
658.92


538
Ac-F$r8AYWAbuAL$AA-NH2
1329.75
665.88
665.95


539
Ac-F$r8AYWNleAL$AA-NH2
1357.78
679.89
679.94


540
Ac-F$r8AbuYWEAL$AA-NH2
1387.75
694.88
694.96


541
Ac-F$r8NleYWEAL$AA-NH2
1415.79
708.90
708.94


542
Ac-F$r8FYWEAL$AA-NH2
1449.77
725.89
725.97


543
Ac-LTF$r8HYWAQhL$S-NH2
1611.88
806.94
807


544
Ac-LTF$r8HYWAQAdm$S-NH2
1675.91
838.96
839.04


545
Ac-LTF$r8HYWAQIgl$S-NH2
1659.88
830.94
829.94


546
Ac-F$r8AYWAQL$AA-NH2
1372.75
687.38
687.48


547
Ac-LTF$r8ALWAQL$Q-NH2
1522.89
762.45
762.52


548
Ac-F$r8AYWEAL$AA-NH2
1373.74
687.87
687.93


549
Ac-F$r8AYWENleL$AA-NH2
1415.79
708.90
708.94


550
Ac-F$r8AYWEAibL$Abu-NH2
1330.73
666.37
666.39


551
Ac-F$r8AYWENleL$Abu-NH2
1358.76
680.38
680.38


552
Ac-F$r8AYWEAL$Abu-NH2
1316.72
659.36
659.36


553
Ac-F$r8AYWEAc3cL$AbuA-NH2
1399.75
700.88
700.95


554
Ac-F$r8AYWEAc3cL$NleA-NH2
1427.79
714.90
715.01


555
H-LTF$r8AYWAQL$S-NH2
1489.83
745.92
745.95


556
mdPEG3-LTF$r8AYWAQL$S-NH2
1679.92
840.96
840.97


557
mdPEG7-LTF$r8AYWAQL$S-NH2
1856.02
929.01
929.03


558
Ac-F$r8ApmpEt6clWEAL$A-NH2
1470.71
736.36
788.17


559
Ac-LTF3Cl$r8AYWAQL$S-NH2
1565.81
783.91
809.18


560
Ac-LTF3Cl$r8HYWAQL$A-NH2
1615.83
808.92
875.24


561
Ac-LTF3Cl$r8HYWWQL$S-NH2
1746.87
874.44
841.65


562
Ac-LTF3Cl$r8AYWWQL$S-NH2
1680.85
841.43
824.63


563
Ac-LTF$r8AYWWQL$S-NH2
1646.89
824.45
849.98


564
Ac-LTF$r8HYWWQL$A-NH2
1696.91
849.46
816.67


565
Ac-LTF$r8AYWWQL$A-NH2
1630.89
816.45
776.15


566
Ac-LTF4F$r8AYWAQL$S-NH2
1549.83
775.92
776.15


567
Ac-LTF2F$r8AYWAQL$S-NH2
1549.83
775.92
776.15


568
Ac-LTF3F$r8AYWAQL$S-NH2
1549.83
775.92
785.12


569
Ac-LTF34F2$r8AYWAQL$S-NH2
1567.83
784.92
785.12


570
Ac-LTF35F2$r8AYWAQL$S-NH2
1567.83
784.92
1338.74


571
Ac-F3Cl$r8AYWEAL$A-NH2
1336.66
669.33
705.28


572
Ac-F3Cl$r8AYWEAL$AA-NH2
1407.70
704.85
680.11


573
Ac-F$r8AY6clWEAL$AA-NH2
1407.70
704.85
736.83


574
Ac-F$r8AY6clWEAL$-NH2
1265.63
633.82
784.1


575
Ac-LTF$r8HYWAQLSt/S-NH2
16.03
9.02
826.98


576
Ac-LTF$r8HYWAQL$S-NHsBu
1653.93
827.97
828.02


577
Ac-STF$r8AYWAQL$S-NH2
1505.79
753.90
753.94


578
Ac-LTF$r8AYWAEL$S-NH2
1532.83
767.42
767.41


579
Ac-LTF$r8AYWAQL$E-NH2
1573.85
787.93
787.98


580
mdPEG3-LTF$r8AYWAQL$S-NH2
1679.92
840.96
840.97


581
Ac-LTF$r8AYWAQhL$S-NH2
1545.86
773.93
774.31


583
Ac-LTF$r8AYWAQCha$S-NH2
1571.88
786.94
787.3


584
Ac-LTF$r8AYWAQChg$S-NH2
1557.86
779.93
780.4


585
Ac-LTF$r8AYWAQCba$S-NH2
1543.84
772.92
780.13


586
Ac-LTF$r8AYWAQF$S-NH2
1565.83
783.92
784.2


587
Ac-LTF4F$r8HYWAQhL$S-NH2
1629.87
815.94
815.36


588
Ac-LTF4F$r8HYWAQCha$S-NH2
1655.89
828.95
828.39


589
Ac-LTF4F$r8HYWAQChg$S-NH2
1641.87
821.94
821.35


590
Ac-LTF4F$r8HYWAQCba$S-NH2
1627.86
814.93
814.32


591
Ac-LTF4F$r8AYWAQhL$S-NH2
1563.85
782.93
782.36


592
Ac-LTF4F$r8AYWAQCha$S-NH2
1589.87
795.94
795.38


593
Ac-LTF4F$r8AYWAQChg$S-NH2
1575.85
788.93
788.35


594
Ac-LTF4F$r8AYWAQCba$S-NH2
1561.83
781.92
781.39


595
Ac-LTF3Cl$r8AYWAQhL$S-NH2
1579.82
790.91
790.35


596
Ac-LTF3Cl$r8AYWAQCha$S-NH2
1605.84
803.92
803.67


597
Ac-LTF3Cl$r8AYWAQChg$S-NH2
1591.82
796.91
796.34


598
Ac-LTF3Cl$r8AYWAQCba$S-NH2
1577.81
789.91
789.39


599
Ac-LTF$r8AYWAQhF$S-NH2
1579.84
790.92
791.14


600
Ac-LTF$r8AYWAQF3CF3$S-NH2
1633.82
817.91
818.15


601
Ac-LTF$r8AYWAQF3Me$S-NH2
1581.86
791.93
791.32


602
Ac-LTF$r8AYWAQ1Nal$S-NH2
1615.84
808.92
809.18


603
Ac-LTF$r8AYWAQBip$S-NH2
1641.86
821.93
822.13


604
Ac-LTF$r8FYWAQL$A-NH2
1591.88
796.94
797.33


605
Ac-LTF$r8HYWAQL$S-NHAm
1667.94
834.97
835.92


606
Ac-LTF$r8HYWAQL$S-NHiAm
1667.94
834.97
835.55


607
Ac-LTF$r8HYWAQL$S-NHnPr3Ph
1715.94
858.97
859.79


608
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me
1681.96
841.98
842.49


610
Ac-LTF$r8HYWAQL$S-NHnPr
1639.91
820.96
821.58


611
Ac-LTF$r8HYWAQL$S-NHnEt2Ch
1707.98
854.99
855.35


612
Ac-LTF$r8HYWAQL$S-NHHex
1681.96
841.98
842.4


613
Ac-LTF$r8AYWAQL$S-NHmdPeg2
1633.91
817.96
818.35


614
Ac-LTF$r8AYWAQL$A-NHmdPeg2
1617.92
809.96
810.3


615
Ac-LTF$r8AYWAQL$A-NHmdPeg4
1705.97
853.99
854.33


616
Ac-F$r8AYdl4mWEAL$A-NH2
1316.72
659.36
659.44


617
Ac-F$r8AYdl5clWEAL$A-NH2
1336.66
669.33
669.43


618
Ac-LThF$r8AYWAQL$S-NH2
1545.86
773.93
774.11


619
Ac-LT2Nal$r8AYWAQL$S-NH2
1581.86
791.93
792.43


620
Ac-LTA$r8AYWAQL$S-NH2
1455.81
728.91
729.15


621
Ac-LTF$r8AYWVQL$S-NH2
1559.88
780.94
781.24


622
Ac-LTF$r8HYWAAL$A-NH2
1524.85
763.43
763.86


623
Ac-LTF$r8VYWAQL$A-NH2
1543.88
772.94
773.37


624
Ac-LTF$r8IYWAQL$S-NH2
1573.89
787.95
788.17


625
Ac-FTF$r8VYWSQL$S-NH2
1609.85
805.93
806.22


626
Ac-ITF$r8FYWAQL$S-NH2
1607.88
804.94
805.2


627
Ac-2NalTF$r8VYWSQL$S-NH2
1659.87
830.94
831.2


628
Ac-ITF$r8LYWSQL$S-NH2
1589.89
795.95
796.13


629
Ac-FTF$r8FYWAQL$S-NH2
1641.86
821.93
822.13


630
Ac-WTF$r8VYWAQL$S-NH2
1632.87
817.44
817.69


631
Ac-WTF$r8WYWAQL$S-NH2
1719.88
860.94
861.36


632
Ac-VTF$r8AYWSQL$S-NH2
1533.82
767.91
768.19


633
Ac-WTF$r8FYWSQL$S-NH2
1696.87
849.44
849.7


634
Ac-FTF$r8IYWAQL$S-NH2
1607.88
804.94
805.2


635
Ac-WTF$r8VYWSQL$S-NH2
1648.87
825.44
824.8


636
Ac-FTF$r8LYWSQL$S-NH2
1623.87
812.94
812.8


637
Ac-YTF$r8FYWSQL$S-NH2
1673.85
837.93
837.8


638
Ac-LTF$r8AY6clWEAL$A-NH2
1550.79
776.40
776.14


639
Ac-LTF$r8AY6clWSQL$S-NH2
1581.80
791.90
791.68


640
Ac-F$r8AY6clWSAL$A-NH2
1294.65
648.33
647.67


641
Ac-F$r8AY6clWQAL$AA-NH2
1406.72
704.36
703.84


642
Ac-LHF$r8AYWAQL$S-NH2
1567.86
784.93
785.21


643
Ac-LTF$r8AYWAQL$S-NH2
1531.84
766.92
767.17


644
Ac-LTF$r8AHWAQL$S-NH2
1505.84
753.92
754.13


645
Ac-LTF$r8AYWAHL$S-NH2
1540.84
771.42
771.61


646
Ac-LTF$r8AYWAQL$H-NH2
1581.87
791.94
792.15


647
H-LTF$r8AYWAQL$A-NH2
1473.84
737.92
737.29


648
Ac-HHF$r8AYWAQL$S-NH2
1591.83
796.92
797.35


649
Ac-aAibWTF$r8VYWSQL$S-NH2
1804.96
903.48
903.64


650
Ac-AibWTF$r8HYWAQL$S-NH2
1755.91
878.96
879.4


651
Ac-AibAWTF$r8HYWAQL$S-NH2
1826.95
914.48
914.7


652
Ac-fWTF$r8HYWAQL$S-NH2
1817.93
909.97
910.1


653
Ac-AibWWTF$r8HYWAQL$S-NH2
1941.99
972.00
972.2


654
Ac-WTF$r8LYWSQL$S-NH2
1662.88
832.44
832.8


655
Ac-WTF$r8NleYWSQL$S-NH2
1662.88
832.44
832.6


656
Ac-LTF$r8AYWSQL$a-NH2
1531.84
766.92
767.2


657
Ac-LTF$r8EYWARL$A-NH2
1601.90
801.95
802.1


658
Ac-LTF$r8EYWAHL$A-NH2
1582.86
792.43
792.6


659
Ac-aTF$r8AYWAQL$S-NH2
1489.80
745.90
746.08


660
Ac-AibTF$r8AYWAQL$S-NH2
1503.81
752.91
753.11


661
Ac-AmfTF$r8AYWAQL$S-NH2
1579.84
790.92
791.14


662
Ac-AmwTF$r8AYWAQL$S-NH2
1618.86
810.43
810.66


663
Ac-NmLTF$r8AYWAQL$S-NH2
1545.86
773.93
774.11


664
Ac-LNmTF$r8AYWAQL$S-NH2
1545.86
773.93
774.11


665
Ac-LSarF$r8AYWAQL$S-NH2
1501.83
751.92
752.18


667
Ac-LGF$r8AYWAQL$S-NH2
1487.82
744.91
745.15


668
Ac-LTNmF$r8AYWAQL$S-NH2
1545.86
773.93
774.2


669
Ac-TF$r8AYWAQL$S-NH2
1418.76
710.38
710.64


670
Ac-ETF$r8AYWAQL$A-NH2
1531.81
766.91
767.2


671
Ac-LTF$r8EYWAQL$A-NH2
1573.85
787.93
788.1


672
Ac-LT2Nal$r8AYWSQL$S-NH2
1597.85
799.93
800.4


673
Ac-LTF$r8AYWAAL$S-NH2
1474.82
738.41
738.68


674
Ac-LTF$r8AYWAQhCha$S-NH2
1585.89
793.95
794.19


675
Ac-LTF$r8AYWAQChg$S-NH2
1557.86
779.93
780.97


676
Ac-LTF$r8AYWAQCba$S-NH2
1543.84
772.92
773.19


677
Ac-LTF$r8AYWAQF3CF3$S-NH2
1633.82
817.91
818.15


678
Ac-LTF$r8AYWAQ1Nal$S-NH2
1615.84
808.92
809.18


679
Ac-LTF$r8AYWAQBip$S-NH2
1641.86
821.93
822.32


680
Ac-LT2Nal$r8AYWAQL$S-NH2
1581.86
791.93
792.15


681
Ac-LTF$r8AYWVQL$S-NH2
1559.88
780.94
781.62


682
Ac-LTF$r8AWWAQL$S-NH2
1554.86
778.43
778.65


683
Ac-FTF$r8VYWSQL$S-NH2
1609.85
805.93
806.12


684
Ac-ITF$r8FYWAQL$S-NH2
1607.88
804.94
805.2


685
Ac-ITF$r8LYWSQL$S-NH2
1589.89
795.95
796.22


686
Ac-FTF$r8FYWAQL$S-NH2
1641.86
821.93
822.41


687
Ac-VTF$r8AYWSQL$S-NH2
1533.82
767.91
768.19


688
Ac-LTF$r8AHWAQL$S-NH2
1505.84
753.92
754.31


689
Ac-LTF$r8AYWAQL$H-NH2
1581.87
791.94
791.94


690
Ac-LTF$r8AYWAHL$S-NH2
1540.84
771.42
771.61


691
Ac-aAibWTF$r8VYWSQL$S-NH2
1804.96
903.48
903.9


692
Ac-AibWTF$r8HYWAQL$S-NH2
1755.91
878.96
879.5


693
Ac-AibAWTF$r8HYWAQL$S-NH2
1826.95
914.48
914.7


694
Ac-fWTF$r8HYWAQL$S-NH2
1817.93
909.97
910.2


695
Ac-AibWWTF$r8HYWAQL$S-NH2
1941.99
972.00
972.7


696
Ac-WTF$r8LYWSQL$S-NH2
1662.88
832.44
832.7


697
Ac-WTF$r8NleYWSQL$S-NH2
1662.88
832.44
832.7


698
Ac-LTF$r8AYWSQL$a-NH2
1531.84
766.92
767.2


699
Ac-LTF$r8EYWARL$A-NH2
1601.90
801.95
802.2


700
Ac-LTF$r8EYWAHL$A-NH2
1582.86
792.43
792.6


701
Ac-aTF$r8AYWAQL$S-NH2
1489.80
745.90
746.1


702
Ac-AibTF$r8AYWAQL$S-NH2
1503.81
752.91
753.2


703
Ac-AmfTF$r8AYWAQL$S-NH2
1579.84
790.92
791.2


704
Ac-AmwTF$r8AYWAQL$S-NH2
1618.86
810.43
810.7


705
Ac-NmLTF$r8AYWAQL$S-NH2
1545.86
773.93
774.1


706
Ac-LNmTF$r8AYWAQL$S-NH2
1545.86
773.93
774.4


707
Ac-LSarF$r8AYWAQL$S-NH2
1501.83
751.92
752.1


708
Ac-TF$r8AYWAQL$S-NH2
1418.76
710.38
710.8


709
Ac-ETF$r8AYWAQL$A-NH2
1531.81
766.91
767.4


710
Ac-LTF$r8EYWAQL$A-NH2
1573.85
787.93
788.2


711
Ac-WTF$r8VYWSQL$S-NH2
1648.87
825.44
825.2


713
Ac-YTF$r8FYWSQL$S-NH2
1673.85
837.93
837.3


714
Ac-F$r8AY6clWSAL$A-NH2
1294.65
648.33
647.74


715
Ac-ETF$r8EYWVQL$S-NH2
1633.84
817.92
817.36


716
Ac-ETF$r8EHWAQL$A-NH2
1563.81
782.91
782.36


717
Ac-ITF$r8EYWAQL$S-NH2
1589.85
795.93
795.38


718
Ac-ITF$r8EHWVQL$A-NH2
1575.88
788.94
788.42


719
Ac-ITF$r8EHWAQL$S-NH2
1563.85
782.93
782.43


720
Ac-LTF4F$r8AYWAQCba$S-NH2
1561.83
781.92
781.32


721
Ac-LTF3Cl$r8AYWAQhL$S-NH2
1579.82
790.91
790.64


722
Ac-LTF3Cl$r8AYWAQCha$S-NH2
1605.84
803.92
803.37


723
Ac-LTF3Cl$r8AYWAQChg$S-NH2
1591.82
796.91
796.27


724
Ac-LTF3Cl$r8AYWAQCba$S-NH2
1577.81
789.91
789.83


725
Ac-LTF$r8AY6clWSQL$S-NH2
1581.80
791.90
791.75


726
Ac-LTF4F$r8HYWAQhL$S-NH2
1629.87
815.94
815.36


727
Ac-LTF4F$r8HYWAQCba$S-NH2
1627.86
814.93
814.32


728
Ac-LTF4F$r8AYWAQhL$S-NH2
1563.85
782.93
782.36


729
Ac-LTF4F$r8AYWAQChg$S-NH2
1575.85
788.93
788.35


730
Ac-ETF$r8EYWVAL$S-NH2
1576.82
789.41
788.79


731
Ac-ETF$r8EHWAAL$A-NH2
1506.79
754.40
754.8


732
Ac-ITF$r8EYWAAL$S-NH2
1532.83
767.42
767.75


733
Ac-ITF$r8EHWVAL$A-NH2
1518.86
760.43
760.81


734
Ac-ITF$r8EHWAAL$S-NH2
1506.82
754.41
754.8


735
Pam-LTF$r8EYWAQL$S-NH2
1786.07
894.04
894.48


736
Pam-ETF$r8EYWAQL$S-NH2
1802.03
902.02
902.34


737
Ac-LTF$r8AYWLQL$S-NH2
1573.89
787.95
787.39


738
Ac-LTF$r8EYWLQL$S-NH2
1631.90
816.95
817.33


739
Ac-LTF$r8EHWLQL$S-NH2
1605.89
803.95
804.29


740
Ac-LTF$r8VYWAQL$S-NH2
1559.88
780.94
781.34


741
Ac-LTF$r8AYWSQL$S-NH2
1547.84
774.92
775.33


742
Ac-ETF$r8AYWAQL$S-NH2
1547.80
774.90
775.7


743
Ac-LTF$r8EYWAQL$S-NH2
1589.85
795.93
796.33


744
Ac-LTF$r8HYWAQL$S-NHAm
1667.94
834.97
835.37


745
Ac-LTF$r8HYWAQL$S-NHiAm
1667.94
834.97
835.27


746
Ac-LTF$r8HYWAQL$S-NHnPr3Ph
1715.94
858.97
859.42


747
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me
1681.96
841.98
842.67


748
Ac-LTF$r8HYWAQL$S-NHnBu
1653.93
827.97
828.24


749
Ac-LTF$r8HYWAQL$S-NHnPr
1639.91
820.96
821.31


750
Ac-LTF$r8HYWAQL$S-NHnEt2Ch
1707.98
854.99
855.35


751
Ac-LTF$r8HYWAQL$S-NHHex
1681.96
841.98
842.4


752
Ac-LTF$r8AYWAQL$S-NHmdPeg2
1633.91
817.96
855.35


753
Ac-LTF$r8AYWAQL$A-NHmdPeg2
1617.92
809.96
810.58


754
Ac-LTF$r5AYWAAL$s8S-NH2
1474.82
738.41
738.79


755
Ac-LTF$r8AYWCouQL$S-NH2
1705.88
853.94
854.61


756
Ac-LTF$r8CouYWAQL$S-NH2
1705.88
853.94
854.7


757
Ac-CouTF$r8AYWAQL$S-NH2
1663.83
832.92
833.33


758
H-LTF$r8AYWAQL$A-NH2
1473.84
737.92
737.29


759
Ac-HHF$r8AYWAQL$S-NH2
1591.83
796.92
797.72


760
Ac-LT2Nal$r8AYWSQL$S-NH2
1597.85
799.93
800.68


761
Ac-LTF$r8HCouWAQL$S-NH2
1679.87
840.94
841.38


762
Ac-LTF$r8AYWCou2QL$S-NH2
1789.94
895.97
896.51


763
Ac-LTF$r8Cou2YWAQL$S-NH2
1789.94
895.97
896.5


764
Ac-Cou2TF$r8AYWAQL$S-NH2
1747.90
874.95
875.42


765
Ac-LTF$r8ACou2WAQL$S-NH2
1697.92
849.96
850.82


766
Dmaac-LTF$r8AYWAQL$S-NH2
1574.89
788.45
788.82


767
Hexac-LTF$r8AYWAQL$S-NH2
1587.91
794.96
795.11


768
Napac-LTF$r8AYWAQL$S-NH2
1657.89
829.95
830.36


769
Pam-LTF$r8AYWAQL$S-NH2
1728.06
865.03
865.45


770
Ac-LT2Nal$r8HYAAQL$S-NH2
1532.84
767.42
767.61


771
Ac-LT2Nal$/r8HYWAQL$/S-NH2
1675.91
838.96
839.1


772
Ac-LT2Nal$r8HYFAQL$S-NH2
1608.87
805.44
805.9


773
Ac-LT2Nal$r8HWAAQL$S-NH2
1555.86
778.93
779.08


774
Ac-LT2Nal$r8HYAWQL$S-NH2
1647.88
824.94
825.04


775
Ac-LT2Nal$r8HYAAQW$S-NH2
1605.83
803.92
804.05


776
Ac-LTW$r8HYWAQL$S-NH2
1636.88
819.44
819.95


777
Ac-LT1Nal$r8HYWAQL$S-NH2
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








Exact
Found
Calc
Calc
Calc


SP
Sequence
Isomer
Mass
Mass
(M + 1)/1
(M + 2)/2
(M + 3)/3







SP765
Ac-tawyanfekllr-NH2


777.46





SP766
Ac-tawyanf4CF3ekllr-NH2


811.41









Example 3: 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 the zebrafish MDMX protein solution and allowed to sit overnight at 4° C. before setting up crystallization experiments. Procedures were similar to those described by Popowicz et al. with some variations, as noted below. 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. Protein was purified using Ni-NT Agarose followed by Superdex 75 buffered with 50 mM NaPO4, pH 8.0, 150 mM NaCl, 2 mM TCEP and then concentrated to 24 mg/ml. The buffer was exchanged to 20 mM Tris, pH 8.0, 50 mM NaCl, 2 mM DTT for crystallization experiments. Initial crystals were obtained with the Nextal (Qiagen) 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 them 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; see The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D50, 760-763 (1994); P. R. Evans. Joint CCP4 and ESF-EACBM Newsletter 33, 22-24 (1997)) in space group C2 (unit cell: a=109.2786, b=81.0836, c=30.9058 Å, α=90, β=89.8577, γ=900). Molecular replacement with program Molrep (CCP4; see A. Vagin & A. Teplyakov. J. Appl. Cryst. 30, 1022-1025 (1997)) was performed with the MDMX component of the structure determined by Popowicz et al. (2Z5S; see G. M. Popowicz, A. Czarna, U. Rothweiler, A. Szwagierczak, M. Krajewski, L. Weber & T. A. Holak. Cell Cycle 6, 2386-2392 (2007)) and identified two molecules in the asymmetric unit. Initial refinement of just the two molecules of the zebrafish MDMX with program Refmac (CCP4; see G. N. Murshudov, A. A. Vagin & E. J. Dodson. Acta Crystallogr. D53, 240-255 (1997)) resulted in an R-factor of 0.3424 (Rfree=0.3712) and rmsd values for bonds (0.018 Å) and angles (1.698°). The electron density for the stapled peptide components, starting with Gln19 and including all of the aliphatic staple, was very clear. Further refinement with CNX (Accelrys) 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 is well refined (Rf=0.2601, Rfree=0.3162, rmsd bonds=0.007 Å and rmsd angles=0.916°).


Results from this Example are shown in FIGS. 13 and 14.


Example 4: Circular Dichroism (CD) Analysis of Alpha-Helicity

Peptide solutions were analyzed by CD spectroscopy using a Jasco J-815 spectropolarimeter (Jasco Inc., Easton, Md.) with the Jasco Spectra Manager Ver.2 system software. A Peltier temperature controller was used to maintain temperature control of the optical cell. Results are expressed as mean molar ellipticity [θ] (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), 1 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 stocks 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 pathlength 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 was reported.


Table 3 shows circular dichroism data for selected peptidomimetic macrocycles:














TABLE 3






Molar
Molar
Molar
% Helix
% Helix



Ellipticity
Ellipticity
Ellipticity
50% TFE
benign



Benign
50% TFE
TFE - Molar
compared to
compared to



(222 in
(222 in
Ellipticity
50% TFE
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 5: 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. KD with 5-FAM-BaLTFEHYWAQLTS-NH2 is ˜13.38 nM.


Example 6: Competitive Fluorescence Polarization Assay for MDM2

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 84 nM (2×) working stock solution.


2. Add 20 μl of 84 nM (2×) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices)


3. 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.


4. Make unlabeled peptide dose plate with FP buffer starting with 1 μM (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 μM with H2O (dilution 1:10) and then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25). Making 5 fold serial dilutions from 4 μM (4×) for 6 points.


5. Transfer 10 μl of serial diluted unlabeled peptides to each well which is filled with 20 μl of 84 nM of protein.


6. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well and incubate for 3 hr to read.


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

The assay was performed according to the following general protocol:


1. Dilute MDMX (In-house, 40 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. KD with 5-FAM-BaLTFEHYWAQLTS-NH2 is −51 nM.


Example 8: Competitive Fluorescence Polarization Assay for MDMX

The assay was performed according to the following general protocol:


1. Dilute MDMX (In-house, 40 kD) into FP buffer (High salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5.) to make 300 nM (2×) working stock solution.


2. Add 20 μl of 300 nM (2×) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices)


3. 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.


4. Make unlabeled peptide dose plate with FP buffer starting with 5 μM (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme.


5. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 μM with H2O (dilution 1:10) and then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25). Making 5 fold serial dilutions from 20 μM (4×) for 6 points.


6. Transfer 10 μl of serial diluted unlabeled peptides to each well which is filled with 20 μl of 300 nM of protein.


7. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well and incubate for 3 hr to read. Results from Examples 5-8 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 9: Competition Binding ELISA (MDM2 & MDMX)

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


Example 10: Cell Viability Assay

The assay was performed according to the following general protocol:


Cell Plating: Trypsinize, count and seed cells at the pre-determined densities in 96-well plates a day prior to assay. Following cell densities are used for each cell line in use:

    • SJSA-1: 7500 cells/well
    • RKO: 5000 cells/well
    • RKO-E6: 5000 cells/well
    • HCT-116: 5000 cells/well
    • SW-480: 2000 cells/well
    • MCF-7: 5000 cells/well


On the day of study, replace media with fresh media with 11% FBS (assay media) at room temperature. Add 180 μL of the assay media per well. Control wells with no cells, receive 200 μL media.


Peptide dilution: all dilutions are made at room temperature and added to cells at room temperature.

    • Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water. This gives range of 10× working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells.
    • Thus the working stocks concentration range μM will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using multichannel.
    • Row H has controls. H1-H3 will receive 20 μL of assay media. H4-H9 will receive 20 μL of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells.
    • Positive control: MDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.


Addition of working stocks to cells:

    • Add 20 μL of 10× desired concentration to appropriate well to achieve the final concentrations in total 200 μL volume in well. (20 μL of 300 μM peptide+180 μL of cells in media=30 μM final concentration in 200 μL volume in wells). Mix gently a few times using pipette. Thus final concentration range used will be 30, 10, 3, 1, 0.3, 0.1, 0.03 & 0 μM (for potent peptides further dilutions are included).
    • Controls include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS.
    • Incubate for 72 hours at 37° C. in humidified 5% CO2 atmosphere.
    • The viability of cells is determined using MTT reagent from Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116 cells is determined on day 3, MCF-7 cells on day 5 and SW-480 cells on day 6. At the end of designated incubation time, allow the plates to come to room temperature. Remove 80 μL of assay media from each well. Add 15 μL of thawed MTT reagent to each well.
    • Allow plate to incubate for 2 h at 37° C. in humidified 5% CO2 atmosphere and add 100 μL solubilization reagent as per manufacturer's protocol. Incubate with agitation for 1 h at room temperature and read on Synergy Biotek multiplate reader for absorbance at 570 nM.
    • Analyze the cell viability against the DMSO controls using GraphPad PRISM analysis tools.


Reagents:

    • Invitrogen cell culture Media
    • Falcon 96-well clear cell culture treated plates (Nunc 353072)
    • DMSO (Sigma D 2650)
    • RPMI 1640 (Invitrogen 72400)
    • MTT (Promega G4000)


Instruments:


Multiplate Reader for Absorbance readout (Synergy 2).


Results from cell viability assays are shown in Tables 5 and 6. The following scale is used: “+” represents a value greater than 30 μM, “++” represents a value greater than 15 μM and less than or equal to 30 μM, “+++” 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



SP#
EC50 (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
RKO
RKO-E6
EC50
IC50


SP#
EC50 (72 h)
EC50 (72 h)
EC50 (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 11: p21 ELISA Assay

The assay was performed according to the following general protocol:


Cell Plating:

    • Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100 μL/well in 96-well plates a day prior to assay.
    • On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 90 μL of the assay media per well. Control wells with no cells, receive 100 μL media.


Peptide dilution:

    • Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10× working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells.
    • Thus the working stocks concentration range μM will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using multichannel.
    • Row H has controls. H1-H3 will receive 10 μL of assay media. H4-H9 will receive 10 μL of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells.
    • Positive control: MDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.


Addition of working stocks to cells:

    • Add 10 μL of 10× desired concentration to appropriate well to achieve the final concentrations in total 100 μL volume in well. (10 μL of 300 μM peptide+90 μL of cells in media=30 μM final concentration in 100 μL volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.
    • Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS.
    • 20 h-post incubation, aspirate the media; wash cells with 1×PBS (without Ca++/Mg++) and lyse in 60 μL of 1× Cell lysis buffer (Cell Signaling technologies 10× buffer diluted to 1× and supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min.
    • Centrifuge plates in at 5000 rpm speed in at 4° C. for 8 min; collect clear supernatants and freeze at −80° C. till further use.


Protein Estimation:

    • Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. Typically about 6-7 μg protein is expected per well.
    • Use 50 μL of the lysate per well to set up p21 ELISA.


Human Total p21 ELISA:


The ELISA assay protocol is followed as per the manufacturer's instructions. 50 μL lysate is used for each well, and each well is set up in triplicate.


Reagents:

    • Cell-Based Assay (−)-Nutlin-3 (10 mM): Cayman Chemicals, catalog #600034
    • OptiMEM, Invitrogen catalog #51985
    • Cell Signaling Lysis Buffer (10×), Cell signaling technology, Catalog #9803
    • Protease inhibitor Cocktail tablets(mini), Roche Chemicals, catalog #04693124001
    • Phosphatase inhibitor Cocktail tablet, Roche Chemicals, catalog #04906837001
    • Human total p21 ELISA kit, R&D Systems, DYC1047-5
    • STOP Solution (1M HCL), Cell Signaling Technologies, Catalog #7002


Instruments: Micro centrifuge-Eppendorf 5415D and Multiplate Reader for Absorbance readout (Synergy 2).


Example 12: Caspase 3 Detection Assay

The assay was performed according to the following general protocol: Cell Plating: Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100 μL/well in 96-well plates a day prior to assay. On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 180 μL of the assay media per well. Control wells with no cells, receive 200 μL media.


Peptide dilution:

    • Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10× working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells.
    • Thus the working stocks concentration range μM will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using multichannel. Add 20 μL of 10× working stocks to appropriate wells.
    • Row H has controls. H1-H3 will receive 20 μL of assay media. H4-H9 will receive 20 μL of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells.
    • Positive control: MDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.


Addition of working stocks to cells:

    • Add 10 μL of 10× desired concentration to appropriate well to achieve the final concentrations in total 100 μL volume in well. (10 μL of 300 μM peptide+90 μL of cells in media=30 μM final concentration in 100 μL volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.
    • Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS.
    • 48 h-post incubation, aspirate 80 μL media from each well; add 100 μL Caspase3/7Glo assay reagent (Promega Caspase 3/7 glo assay system, G8092) per well, incubate with gentle shaking for 1 h at room temperature.
    • read on Synergy Biotek multiplate reader for luminescence.
    • Data is analyzed as Caspase 3 activation over DMSO-treated cells.


Results from Examples 11 and 12 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 13. Cell Lysis by Peptidomimetic Macrocycles

SJSA-1 cells were plated out one day in advance in clear flat-bottom plates (Costar, catalog number 353072) at 7500 cells/well with 100 ul/well of growth media, leaving row H columns 10-12 empty for media alone. On the day of the assay, media was exchanged with RPMI 1% FBS media, 90 uL of media per well.


10 mM stock solutions of the peptidomimetic macrocycles were prepared in 100% DMSO. Peptidomimetic macrocycles were then diluted serially in 100% DMSO, and then further diluted 20-fold in sterile water to prepare working stock solutions in 5% DMSO/water of each peptidomimetic macrocycle at concentrations ranging from 500 μM to 62.5 μM.


10 μL of each compound was added to the 90 uL of SJSA-1 cells to yield final concentrations of 50 μM to 6.25 μM in 0.5% DMSO-containing media. The negative control (non-lytic) sample was 0.5% DMSO alone and positive control (lytic) samples include 10 μM Melittin and 1% Triton X-100.


Cell plates were incubated for 1 hour at 37° C. After the 1 hour incubation, the morphology of the cells is examined by microscope and then the plates were centrifuged at 1200 rpm for 5 minutes at room temperature. 40 μL of supernatant for each peptidomimetic macrocycle and control sample is transferred to clear assay plates. LDH release is measured using the LDH cytotoxicity assay kit from Caymen, catalog#1000882. Results 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 14: p53 GRIP Assay

Thermo Scientific* BioImage p53-MDM2 Redistribution Assay monitors the protein interaction with MDM2 and 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). They provide a ready-to-use assay system for measuring the effects of experimental conditions on the interaction of p53 and MDM2. Imaging and analysis is performed with a HCS platform.


CHO-hIR cells are regularly maintained in Ham's F12 media supplemented with 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin and 10% FBS. Cells seeded into 96-well plates at the density of 7000 cells/100 μL per well 18-24 hours prior to running the assay using culture media. The next day, media is refreshed and PD-177 is added to cells to the final concentration of 3 μM to activate foci formation. Control wells are kept without PD-177 solution. 24 h post stimulation with PD-177, cells are washed once with Opti-MEM Media and 50 μL of the Opti-MEM Media supplemented with PD-177 (6 μM) is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides. 50 μL of 2× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 6 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cells are fixed by gently aspirating out the media and adding 150 μL of fixing solution per well for 20 minutes at room temperature. Fixed cells are washed 4 times with 200 μL PBS per well each time. At the end of last wash, 100 μL of 1 μM Hoechst staining solution is added. Sealed plates incubated for at least 30 min in dark, washed with PBS to remove excess stain and PBS is added to each well. Plates can be stored at 4° C. in dark up to 3 days. The translocation of p53/MDM2 is imaged using Molecular translocation module on Cellomics Arrayscan instrument using 10× objective, XF-100 filter sets for Hoechst and GFP. The output parameters were Mean-CircRINGAveIntenRatio (the ratio of average fluorescence intensities of nucleus and cytoplasm (well average)). The minimally acceptable number of cells per well used for image analysis was set to 500 cells.


Example 15: 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, a point mutation of SP154 (F to A at position 19) was also tested in one group; this peptide had shown no activity in the SJSA-1 in vitro viability assay. Slow release 90 day 0.72 mg 17β-estradiol pellets (Innovative Research, Sarasota, Fla.) 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 (Crl:NU-Foxnlnu) 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 at pH 7. This formulation was prepared once for the duration of the study. This 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 (iv) three times per week from Days 18-39. Groups 2 and 3 received SP154 as an iv injection at 30 mg/kg three times per week or 40 mg/kg twice a week, respectively. Group 4 received 6.7 mg/kg SP249 as an iv injection three times per week. Groups 5, 6, 7 and 8 received SP315 as an iv injection of 26.7 mg/kg three times per week, 20 mg/kg twice per week, 30 mg/kg twice per week, or 40 mg/kg twice per week, respectively. Group 9 received 30 mg/kg SP252 as an iv injection three times per week.


During the dosing period the mice were weighed and tumors measured 1-2 times per week. Results in terms of tumor volume are shown in FIGS. 15-18 and tumor growth inhibition compared with the vehicle group, body weight change 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−[(TuVolTreated-day x−TuVoTreated-day18)/(TuVolVehicle negative control-day x−TuVolVehicle negative control−day18)*100, where x=day that effect of treatment is being assessed. Group 1, the vehicle negative control group, showed good tumor growth rate for this tumor model.


For SP154, in the group dosed with 40 mg/kg twice a week 2 mice died during treatment, indicating that this dosing regimen was not tolerable. The dosing regimen of 30 mg/kg of SP154 three times per week was well-tolerated and yielded a TGI of 84%.


For SP249, the group dosed with 6.7 mg/kg three times per week 4 mice died during treatment, indicating that this dosing regimen was not tolerable.


All dosing regimens used for SP315 showed good tolerability, with no body weight loss or deaths noted. 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.


For SP252, the point mutation of SP154 which shows no appreciable activity in in vitro assays, dosing with 30 mg/kg three times per week was well-tolerated with no body weight loss or deaths noted. While TGI of 88% was noted by Day 32, that TGI was reduced to 41% by Day 39.


Results from this Example are shown in FIGS. 15-18 and are summarized in Table 9.














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 3x/wk iv
+5.7
0/10
0/10
*84


3
SP154 40 mg/kg 2x/wk iv
N/A
0/10
2/10 (2 deaths)
Regimen not







tolerated


4
SP249 6.7 mg/kg 3x/wk iv
N/A
6/10
4/10
Regimen not







tolerated


5
SP315 26.7 mg/kg 3x/wk iv
+3.7
0/10
0/10
*86


6
SP315 20 mg/kg 2x/wk iv
+3.9
0/10
0/10
*82


7
SP315 30 mg/kg 2x/wk iv
+8.0
0/10
0/10
*85


8
SP315 40 mg/kg 2x/wk iv
+2.1
0/10
0/10
*92


9
SP252 30 mg/kg 3x/wk iv
+3.3
0/10
0/10
*41





*p ≦ 0.05 Vs Vehicle Control






Example 16: Solubility Determination for Peptidomimetic Macrocycles

Peptidomimetic macrocycles are first dissolved in neat N, N-dimethylacetamide (DMA, Sigma-Aldrich, 38840-1L-F) to make 20× stock solutions over a concentration range of 20-140 mg/mL. The DMA stock solutions are diluted 20-fold in an aqueous vehicle containing 2% Solutol-HS-15, 25 mM histidine, 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, 45 mg/mL mannitol. The final solutions are mixed gently by repeat pipetting or light vortexing, and then the final solutions are sonicated for 10 min at room temperature in an ultrasonic water bath. Careful visual observation is then performed under hood light using a 7× visual amplifier to determine if precipitate exists on the bottom or as a suspension. Additional concentration ranges are tested as needed to determine the maximum solubility limit for each peptidomimetic macrocycle.


Results from this Example are shown in FIG. 19.


Example 17: Preparation of Peptidomimetic Macrocycles Using a Boc-Protected Amino Acid

Peptidomimetic macrocycle precursors were prepared as described in Example 2 comprising an R8 amino acid at position “i” and an S5 amino acid at position “i+7”. 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 (and commercially available, for example, from Novabiochem) was using during solid phase synthesis:




embedded image


Metathesis was performed using a ruthenium catalyst prior to the cleavage and deprotection steps. The composition obtained following cyclization was determined by HPLC analysis 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 18: Testing of Peptidomimetic Macrocycles for Ability to Reduce Immune Checkpoint Protein Expression or Inhibit Immune Checkpoint Protein Activity

HCT-116 cells that are p53WT (but not p53 null) upregulate p53 and down-regulate PD-L1 in response to dosing with Nutlin3. p53 effects on PD-L1 are mediated by transcription of miR-34a, -b and -c. The peptidomimetic macrocycles described herein can increase p53 levels in cancer cells. p53 expression is inversely correlated with PD-L1 in patients with NSCLC and PD-L1 expression is higher in patients with mutant p53 compared to p53WT. Patients with low PD-L1 expression and high p53 expression have better survival compared to patients with high PD-L1 expression and low p53 expression. p53 regulates PD-L1 and the miR-34 family downregulates PD-L1 expression by directly repressing PD-L1. Furthermore, therapeutic delivery of miR-34a represses PD-L1 in vivo and therapeutic delivery of miR-34a alone or in combination with XRT increases CD8+ T cells. Therapeutic delivery of miR-34a also increases IFN-γ promoting tumor growth delay. miR-34a is directly transactivated by p53 to regulate several pathways in cancer, including tumor immune evasion.


Assays were performed to determine whether the peptidomimetic macrocycles can diminish PD-L1 activity or expression. Briefly, HCT-116 p53+/+ cells and HCT-116 p53−/− cells were treated with DMSO or 10 μM SP or 20 μM SP as indicated in FIG. 22. As shown in FIG. 22, SP treatment led to decreased PD-L1 expression in HCT-116 p53+/+ cells, but not HCT-116 p53−/− cells. Similar assays will be performed in cell lines that express higher levels of PD-L1, such as A549 cells, H460 cells, and syngeneic mouse cell lines.


Assays will be performed to determine whether the peptidomimetic macrocycles can diminish PD-L1 activity or expression via miR-34a to enhance immune response against tumors. Assays will be 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 added benefit of cell cycle arrest and apoptosis). Briefly, cancer cells from different lineages MCF-7 (breast), HCT-116 (large intestine), MV4-11 (leukemia), DOHH2, and A375 (melanoma) will be dosed with peptidomimetic macrocycles. These cell lines and others will be chosen 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 (and p53 and p21 as controls) will be measured, for example, using flow cytometry. RT-PCR assays will be conducted to quantify miR-34a, miR-34b, and/or miR-34c levels in samples taken by FlowMetric in parallel with flow cytometry measurements. Full dose-response curves will be taken 24, 48, and 72 hours after dosing. Additionally, apoptosis measurements will be taken in parallel.


Example 19: WST-1 Cell Proliferation Assays

The human tumor cell lines MCF-7 and MOLT-3 were obtained from American Type Culture Collection (ATCC) and grown in EMEM and RPMI1640, respectively. All media were supplemented with 10% (v/v) fetal calf serum, 100 units penicillin and 100 μg/ml 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, 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. Cells were dosed with Aileron peptide 1, palbociclib, everolimus, fulvestrant, or romidepsin alone or in combination with Aileron peptide 1 and incubated for three to five days. The WST-1 variant of the MTT assay was used to measure cell viability according to the manufacturer's protocol. WST-1 is a cell-impermeable, sulfonated tetrazolium salt that can be used to examine cell viability without killing the cells. Results can be seen in FIG. 23 (MCF-7 cells, no treatment), FIGS. 24A and 24B (MCF-7 or MOLT-3 cells, Aileron peptide 1), FIGS. 25A (fulvestrant) and 25B (everolimus), FIGS. 26, 27A, 27B, 28A, and 28B (fulvestrant), FIGS. 29, 30A, 30B, 31A, and 31B (everolimus), FIGS. 32, 33A, 33B, 34A, 34B, and 34C (romidepsin), and FIGS. 35, 36A, 36B, 37A, and 34B (palbociclib).


Example 20: Synergism Between PLX4032 and the Peptidomimetic Macrocycles of the Disclosure in B-Raf-Mutant Melanoma Cell Line A375 and Mel-Ho (V600E) but not in Mel-Juso (H- & N-Ras Mutations, COSMIC)

The combination of a representative peptidomimetic macrocycle of the disclosure (a p53 hydrocarbon cross-linked polypeptide macrocycle with an observed mass of 950-975 m/e) and commercially available targeted agent PLX4032 BRAF inhibitor was tested at various drug doses. The EC50 of Aileron peptide 1 on A375 cells was determined to be 70 nM. As seen in FIG. 20, the peptidomimetic macrocycle displayed synergy with PLX4032 in B-Raf-mutant melanoma cell line A375. As seen in FIG. 21, the peptidomimetic macrocycle also displayed synergy with PLX4032 in Mel-Ho (V600E) but not in Mel-Juso (H- & N-Ras mutations, COSMIC).


Example 21: Synergism Between Fluvestant and the Peptidomimetic Macrocycles of the Disclosure in the MCF-7 Breast Cancer Cell Line

The combination of a representative peptidomimetic macrocycle of the disclosure (a p53 hydrocarbon cross-linked polypeptide macrocycle with an observed mass of 950-975 m/e) and commercially available targeted agent fluvestrant was tested at various drug doses. Initially, various MCF-7 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration (FIG. 23). Next, the optimal number of cells were plated and treated with various concentrations of Aileron peptide 1 or with various concentrations of fluvestrant alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment (FIGS. 24A and 26). A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of fluvestrant were then determined.















IC50 (nM)



















FUL
0.768



FUL + 0.13 μM Aileron peptide 1
0.4428



FUL + 0.4 μM Aileron peptide 1
0.2609



FUL + 1.2 μM Aileron peptide 1
0.2621










The EC50 of Aileron peptide 1 on MCF-7 cells was determined to be 410 nM. These chosen concentrations were tested on MCF-7 cells for Aileron peptide 1 in combination with fluvestrant. The optimal number of MCF-7 cells was plated and treated with Aileron peptide 1 and fluvestrant in combination. Aileron peptide 1 was added to the cells simultaneously with the fluvestrant. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous treatments (FIGS. 25, 27 and 28). As seen in FIG. 26, fulvestrant inhibited MCF-7 breast cancer cell proliferation with limited cell killing as a single agent. However, as seen in FIGS. 25, 27 and 28, Aileron peptide 1 displayed synergy with fluvestant in the MCF-7 breast cancer cell line. Combination index (CI) values were calculated using the CompuSyn software. The data were 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.


Exemplary cooperativity index calculations are shown in the table below:















Dose Aileron peptide 1
Dose fulvestrant




(μM)
(nM)
Effect
CI


















0.001
3.0
0.323
0.14159


0.003
3.0
0.402
0.09317


0.01
3.0
0.418
0.10712


0.03
3.0
0.482
0.12223


0.1
3.0
0.588
0.17027


0.3
3.0
0.644
0.34356


1.0
3.0
0.709
0.77439


3.0
3.0
0.755
1.74401


10.0
3.0
0.901
1.62697


30.0
3.0
0.92
3.70727


0.001
10.0
0.429
0.23789


0.003
10.0
0.414
0.26661


0.01
10.0
0.466
0.21426


0.03
10.0
0.519
0.19805


0.1
10.0
0.594
0.22753


0.3
10.0
0.701
0.28387


1.0
10.0
0.737
0.68105


3.0
10.0
0.786
1.43567


10.0
10.0
0.911
1.42122


30.0
10.0
0.946
2.26507


0.001
30.0
0.43
0.70343


0.003
30.0
0.418
0.76190


0.01
30.0
0.443
0.67686


0.03
30.0
0.478
0.60025


0.1
30.0
0.586
0.42426


0.3
30.0
0.6
0.66109


1.0
30.0
0.718
0.84264


3.0
30.0
0.758
1.79040


10.0
30.0
0.897
1.73116


30.0
30.0
0.917
3.89829


0.13
0.03
0.269
0.90978


0.13
0.1
0.321
0.68139


0.13
0.3
0.486
0.30536


0.13
1.0
0.552
0.23162


0.13
3.0
0.63
0.17212


0.13
10.0
0.61
0.24727


0.13
30.0
0.611
0.40656


0.13
100.0
0.594
1.07046


0.13
300.0
0.58
3.09815


0.13
900.0
0.627
6.70074


0.4
0.03
0.492
0.89889


0.4
0.1
0.524
0.77451


0.4
0.3
0.58
0.59564


0.4
1.0
0.666
0.39101


0.4
3.0
0.675
0.38341


0.4
10.0
0.693
0.38057


0.4
30.0
0.685
0.49815


0.4
100.0
0.66
0.98770


0.4
300.0
0.679
1.92173


0.4
900.0
0.667
5.45686









Example 22: Synergism Between Everolimus and the Peptidomimetic Macrocycles of the Disclosure in the MCF-7 Breast Cancer Cell Line

The combination of a representative peptidomimetic macrocycle of the disclosure (a p53 hydrocarbon cross-linked polypeptide macrocycle with an observed mass of 950-975 m/e) and commercially available targeted agent everolimus was tested at various drug doses. Initially, various MCF-7 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration (FIG. 23). Next, the optimal number of cells were plated and treated with various concentrations of Aileron peptide 1 or with various concentrations of everolimus alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment (FIGS. 24A and 29). A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of everolimus were then determined. The EC50 of Aileron peptide 1 on MCF-7 cells was determined to be 410 nM. These chosen concentrations were tested on MCF-7 cells for Aileron peptide 1 in combination with everolimus. The optimal number of MCF-7 cells was plated and treated with Aileron peptide 1 and everolimus in combination. Aileron peptide 1 was added to the cells simultaneously with the everolimus. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous treatments (FIGS. 25, 30 and 31). As seen in FIG. 29, everolimus inhibited MCF-7 breast cancer cell proliferation with limited cell killing as a single agent. However, as seen in FIGS. 25, 30 and 31, Aileron peptide 1 displayed synergy with everolimus in the MCF-7 breast cancer cell line.


Exemplary cooperativity index calculations are shown in the table below:















Dose Aileron peptide 1
Dose everolimus




(μM)
(μM)
Effect
CI


















0.001
0.001
0.363
0.46998


0.003
0.001
0.365
0.45978


0.01
0.001
0.406
0.23282


0.03
0.001
0.429
0.21862


0.1
0.001
0.516
0.22558


0.3
0.001
0.703
0.23698


1.0
0.001
0.811
0.39235


3.0
0.001
0.864
0.74302


10.0
0.001
0.952
0.65211


30.0
0.001
0.964
1.37599


0.001
0.003
0.469
0.18255


0.003
0.003
0.495
0.11727


0.01
0.003
0.508
0.10758


0.03
0.003
0.557
0.08415


0.1
0.003
0.609
0.14138


0.3
0.003
0.722
0.21318


1.0
0.003
0.819
0.36874


3.0
0.003
0.871
0.69183


10.0
0.003
0.945
0.77158


30.0
0.003
0.952
1.95633


0.001
0.01
0.524
0.21623


0.003
0.01
0.537
0.17334


0.01
0.01
0.525
0.22955


0.03
0.01
0.554
0.17216


0.1
0.01
0.623
0.15213


0.3
0.01
0.716
0.22398


1.0
0.01
0.799
0.42963


3.0
0.01
0.854
0.81864


10.0
0.01
0.933
0.98709


30.0
0.01
0.953
1.90630


0.001
0.1
0.515
2.53851


0.003
0.1
0.541
1.56244


0.01
0.1
0.522
2.24431


0.03
0.1
0.563
1.07533


0.1
0.1
0.645
0.31323


0.3
0.1
0.735
0.22476


1.0
0.1
0.783
0.48900


3.0
0.1
0.844
0.89820


10.0
0.1
0.909
1.45716


30.0
0.1
0.925
3.41419


0.13
0.0001
0.477
0.31844


0.13
0.0003
0.548
0.22849


0.13
0.001
0.567
0.21454


0.13
0.003
0.626
0.16282


0.13
0.01
0.673
0.13216


0.13
0.03
0.699
0.12434


0.13
0.1
0.717
0.13805


0.13
0.3
0.743
0.15137


0.13
1.0
0.762
0.21739


0.13
3.0
0.789
0.27115


0.4
0.0001
0.633
0.45701


0.4
0.0003
0.664
0.38983


0.4
0.001
0.673
0.37246


0.4
0.003
0.723
0.28218


0.4
0.01
0.74
0.25644


0.4
0.03
0.746
0.25127


0.4
0.1
0.76
0.23938


0.4
0.3
0.8
0.18580


0.4
1.0
0.804
0.21110


0.4
3.0
0.828
0.20196


1.2
0.0001
0.703
0.94557


1.2
0.0003
0.746
0.73428


1.2
0.001
0.768
0.63825


1.2
0.003
0.783
0.57706


1.2
0.01
0.798
0.51924


1.2
0.03
0.798
0.52033


1.2
0.1
0.816
0.45611


1.2
0.3
0.832
0.40364


1.2
1.0
0.814
0.49378


1.2
3.0
0.845
0.39182









Analysis was performed according to Chou et al., Advances in Enzyme Regulation, 22:27-55 (1984) and Zhang et al., Am J Cancer Res., 6:97-104 (2016). Combination index (CI) values were calculated using the CompuSyn software. The data were 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.


Example 23: Treatment with Romidepsin and the Peptidomimetic Macrocycles of the Disclosure in the Human MOLT-3 T-Lymphoid Cell Line

The combination of Aileron peptide 1 and commercially available targeted agent romidepsin was tested at various drug doses. Initially, various MOLT-3 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells were plated and treated with various concentrations of Aileron peptide 1 or with various concentrations of romidepsin alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment (FIGS. 24B and 32). A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of romidepsin were then determined.















IC50 (μM)



















Aileron peptide 1
0.088



Aileron peptide 1 + 0.5 nM Romidepsin
0.1014



Aileron peptide 1 + 1.5 nM Romidepsin
0.038



Aileron peptide 1 + 3 nM Romidepsin
0.028










The EC50 of Aileron peptide 1 on MOLT-3 cells was determined to be 210 nM. These chosen concentrations were tested on MOLT-3 cells for the peptidomimetic macrocycle in combination with romidepsin. The optimal number of MOLT-3 cells was plated and treated with Aileron peptide 1 and romidepsin in combination. Aileron peptide 1 was added to the cells 2 hours prior to addition of the romidepsin. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the sequential treatment (FIGS. 33 and 34).


Example 24: Treatment with Palbociclib and the Peptidomimetic Macrocycles of the Disclosure in the MCF-7 Breast Cancer Cell Line

The combination of Aileron peptide 1 and commercially available targeted agent palbociclib was tested at various drug doses. Initially, various MCF-7 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration (FIG. 23). Next, the optimal number of cells were plated and treated with various concentrations of Aileron peptide 1 or with various concentrations of palbociclib alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days or 120 hrs after beginning treatment (FIGS. 24A and 35). A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of palbociclib were then determined. The EC50 of Aileron peptide 1 on MCF-7 cells was determined to be 410 nM. These chosen concentrations were tested on MCF-7 cells for Aileron peptide 1 in combination with palbociclib. The optimal number of MCF-7 cells was plated and treated with Aileron peptide 1 and palbociclib in combination. Aileron peptide 1 was added to the cells simultaneously with the palbociclib. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous treatments (FIGS. 36 and 37).


Exemplary cooperativity index calculations are shown in the table below:















Dose Aileron peptide 1
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









Cells were also evaluated for viability using the CyQUANT method after beginning treatment (FIGS. 78A and 79A). Cells were evaluated for viability using the CyQUANT method after beginning the simultaneous treatments (FIGS. 78B and 79B). Analysis was performed according to Chou et al., Advances in Enzyme Regulation, 22:27-55 (1984) and Zhang et al., Am J Cancer Res., 6:97-104 (2016). Combination index (CI) values were calculated using the CompuSyn software. The data were 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.


Example 25: Treatment with Dexamethasone and the Peptidomimetic Macrocycles of the Disclosure in the DOHH-2 Human Lymphoma B-Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agent dexamethasone are tested at various drug doses. Initially, various DOHH-2 cell numbers are plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells are plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of dexamethasone alone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of dexamethasone are then determined. The EC50 of Aileron peptide 1 on DOHH-2 cells was determined to be 60 nM. These chosen concentrations are tested on DOHH-2 cells for the peptidomimetic macrocycle in combination with dexamethasone. The optimal number of DOHH-2 cells are plated and treated with the representative peptidomimetic macrocycle of the disclosure and dexamethasone in combination. In some cases, the peptidomimetic macrocycle are added to the cells simultaneously with the dexamethasone. In some cases, the peptidomimetic macrocycle are added to the cells prior to addition of the dexamethasone. In some cases, the peptidomimetic macrocycle are added to the cells after addition of the dexamethasone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 26: Treatment with Trametinib and the Peptidomimetic Macrocycles of the Disclosure in the A375 Human Melanoma Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agent trametinib are tested at various drug doses. Initially, various A375 cell numbers are plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells are plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of trametinib alone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of trametinib are then determined. The EC50 of Aileron peptide 1 on A375 cells was determined to be 70 nM. These chosen concentrations are tested on A375 cells for the peptidomimetic macrocycle in combination with trametinib. The optimal number of A375 cells are plated and treated with the representative peptidomimetic macrocycle of the disclosure and trametinib in combination. In some cases, the peptidomimetic macrocycle are added to the cells simultaneously with the trametinib. In some cases, the peptidomimetic macrocycle are added to the cells prior to addition of the trametinib. In some cases, the peptidomimetic macrocycle are added to the cells after addition of the trametinib. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 27: Treatment with Rituximab and the Peptidomimetic Macrocycles of the Disclosure in the DOHH-2 Human Lymphoma B-Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agent rituximab were tested at various drug doses. Initially, various DOHH-2 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells were plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of rituximab alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of rituximab were then determined. The EC50 of Aileron peptide 1 on DOHH-2 cells was determined to be 60 nM. These chosen concentrations were tested on DOHH-2 cells for the peptidomimetic macrocycle in combination with rituximab. The optimal number of DOHH-2 cells were plated and treated with the representative peptidomimetic macrocycle of the disclosure and rituximab in combination. In some cases, the peptidomimetic macrocycle were added to the cells simultaneously with the rituximab. In some cases, the peptidomimetic macrocycle were added to the cells prior to addition of the rituximab. In some cases, the peptidomimetic macrocycle were added to the cells after addition of the rituximab. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 28: Treatment with Obinutuzumab and the Peptidomimetic Macrocycles of the Disclosure in the DOHH-2 Human Lymphoma B-Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agent obinutuzumab are tested at various drug doses. Initially, various DOHH-2 cell numbers are plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells are plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of obinutuzumab alone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of obinutuzumab are then determined. The EC50 of Aileron peptide 1 on DOHH-2 cells was determined to be 60 nM. These chosen concentrations are tested on DOHH-2 cells for the peptidomimetic macrocycle in combination with obinutuzumab. The optimal number of DOHH-2 cells are plated and treated with the representative peptidomimetic macrocycle of the disclosure and obinutuzumab in combination. In some cases, the peptidomimetic macrocycle are added to the cells simultaneously with the obinutuzumab. In some cases, the peptidomimetic macrocycle are added to the cells prior to addition of the obinutuzumab. In some cases, the peptidomimetic macrocycle are added to the cells after addition of the obinutuzumab. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 29: Treatment with Dabrafenib and the Peptidomimetic Macrocycles of the Disclosure in the A375 Human Melanoma Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agent dabrafenib are tested at various drug doses. Initially, various A375 cell numbers are plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells are plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of dabrafenib alone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of dabrafenib are then determined. The EC50 of Aileron peptide 1 on A375 cells was determined to be 70 nM. These chosen concentrations are tested on A375 cells for the peptidomimetic macrocycle in combination with dabrafenib. The optimal number of A375 cells are plated and treated with the representative peptidomimetic macrocycle of the disclosure and dabrafenib in combination. In some cases, the peptidomimetic macrocycle are added to the cells simultaneously with the dabrafenib. In some cases, the peptidomimetic macrocycle are added to the cells prior to addition of the dabrafenib. In some cases, the peptidomimetic macrocycle are added to the cells after addition of the dabrafenib. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 30: Treatment with Vemurafenib and the Peptidomimetic Macrocycles of the Disclosure in the A375 Human Melanoma Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agent vemurafenib are tested at various drug doses. Initially, various A375 cell numbers are plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration. Next, the optimal number of cells are plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of vemurafenib alone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of vemurafenib are then determined. The EC50 of Aileron peptide 1 on A375 cells was determined to be 70 nM. These chosen concentrations are tested on A375 cells for the peptidomimetic macrocycle in combination with vemurafenib. The optimal number of A375 cells are plated and treated with the representative peptidomimetic macrocycle of the disclosure and vemurafenib in combination. In some cases, the peptidomimetic macrocycle are added to the cells simultaneously with the vemurafenib. In some cases, the peptidomimetic macrocycle are added to the cells prior to addition of the vemurafenib. In some cases, the peptidomimetic macrocycle are added to the cells after addition of the vemurafenib. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 31: Treatment with Dabrafenib, Vemurafenib and the Peptidomimetic Macrocycles of the Disclosure in the A375 Human Melanoma Cell Line

The combination of one or more representative peptidomimetic macrocycles of the disclosure and commercially available targeted agents dabrafenib and vemurafenib are tested at various drug doses. Initially, various A375 cell numbers are plated and evaluated 3-7 days later to determine the optimal number of cells plated and treatment duration. Next, the optimal number of cells are plated and treated with various concentrations of a representative peptidomimetic macrocycle of the disclosure or with various concentrations of vemurafenib alone or with various concentrations of dabrafenib alone. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of dabrafenib and vemurafenib are then determined. The EC50 of Aileron peptide 1 on A375 cells is determined to be 70 nM. These chosen concentrations are tested on A375 cells for the peptidomimetic macrocycle in combination with dabrafenib and vemurafenib. The optimal number of A375 cells are plated and treated with the representative peptidomimetic macrocycle of the disclosure and dabrafenib and vemurafenib in combination. In some cases, the peptidomimetic macrocycle are added to the cells simultaneously with the dabrafenib and vemurafenib. In some cases, the peptidomimetic macrocycle are added to the cells prior to addition of the dabrafenib and vemurafenib. In some cases, the peptidomimetic macrocycle are added to the cells after addition of the dabrafenib and vemurafenib. Cells are evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning the simultaneous or sequential treatments.


Example 32: Treatment with Cytarabine (Ara-C), Azacitidine, Decitabine, and Midostaurin with the Peptidomimetic Macrocycles of the Disclosure in the MV4-11 Leukemia Cancer Cell Line

The combinations of Aileron peptide 1 (AP1) and commercially available Ara-C(FIG. 38A), azacitidine (FIG. 39A), decitabine (FIG. 40A), and midostaurin (FIG. 41A) were tested at various drug doses. Initially, various MV4-11 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration.


Next, the optimal number of cells were plated and treated with various concentrations of AP1 or with various concentrations of Ara-C, azacitidine, decitabine, or midostaurin alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment. AP1 in combination with Ara-C (FIG. 38B), azacitidine (FIG. 39B), decitabine (FIG. 40B), or midostaurin (FIG. 41B). All showed complementary in vitro anticancer activity. Combination with Ara-C, azacitidine, decitabine, or midostaurin enhanced AP1 inhibition of cancer cell proliferation and cell killing.


A drug combination index plot was used to assess the synergistic, additive, or antagonistic properties of each combination treatment. The anti-proliferative effect of the Ara-C and AP1 combination was mostly additive with some degree of synergy (FIG. 38C). The anti-proliferative effect of the azacitidine and AP1 combination was mostly additive with some synergy (FIG. 39C). The anti-proliferative effect of the decitabine and AP1 combination was mostly additive (FIG. 40C). The anti-proliferative effect of the midostaurin and AP1 combination was mostly synergistic (FIG. 41C). Combination index (CI) values were calculated using the CompuSyn software. The data were 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.


Example 33: Treatment with Vincristine (VCR) and Cyclophosphamide (CTX) with the Peptidomimetic Macrocycles of the Disclosure in the DOHH-2 Lymphoma B-Cell Cancer Cell Line

The combinations of AP1 and commercially available VCR (FIG. 42A) and CTX (FIG. 44A) were tested at various drug doses. Initially, various DOHH-2 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration.


Next, the optimal number of cells were plated and treated with various concentrations of AP1 or with various concentrations of VCR or CTX alone. Cells were evaluated for viability by WST-1 assay or MTT assay 72 hours after beginning treatment with VCR (FIG. 42B) or CTX (FIG. 44B). AP1 in combination with VCR showed complementary in vitro anticancer activity (FIG. 43). AP1 in combination with CTX showed complementary in vitro anticancer activity (FIG. 45).


A drug combination index plot was used to assess the synergistic, additive, or antagonistic properties of each combination treatment. The anti-proliferative effect of the VCR and AP1 combination was mostly synergistic (FIG. 42C). The anti-proliferative effect of the CTX and AP1 combination was synergistic (FIG. 44C). Combination index (CI) values were calculated using the CompuSyn software. The data were 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.


A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of VCR were then determined.















IC50 (μM)



















AP1
0.3095



AP1 + 0.3 nM VCR
0.2520



AP1 + 3 nM VCR
0.082










A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of CTX were then determined.















IC50 (mM)



















CTX
1.981



CTX + 0.07 μM AP1
0.4109



CTX + 0.2 μM AP1
0.1718



CTX + 0.6 μM AP1
0.2579










Example 34: The Order of Addition Effects on DOHH-2 Cell Viability Using Various Concentrations of AP1 in Combination with VCR

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. DOHH-2 cells were sequentially treated by varying concentrations of AP1 and VCR for 72 hrs (FIG. 46). AP1 suppressed DOHH-2 cell growth with or without VCR (FIG. 47). Similarly, VCR suppressed DOHH-2 cell growth with or without AP1 (FIG. 48).


Example 35: The Order of Addition Effects on DOHH-2 Cell Viability Using Various Concentrations of AP1 in Combination with CTX

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. DOHH-2 cells were sequentially treated by varying concentrations of AP1 and CTX for 72 hrs (FIG. 49). AP1 suppressed DOHH-2 cell growth with or without CTX (FIG. 50). Similarly, CTX suppressed DOHH-2 cell growth with or without AP1 (FIG. 51).


Example 36: The Order of Addition Effects on MV4-11 Cell Viability Using Various Concentrations of AP1 in Combination with Midostaurin

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. MV4-11 cells were sequentially treated by varying concentrations of AP1 and midostaurin for 72 hrs (FIG. 52). AP1 suppressed MV4-11 cell growth with or without midostaurin (FIG. 53). Similarly, midostaurin suppressed MV4-11 cell growth with or without AP1 (FIG. 54).


Example 37: The Order of Addition Effects on MV4-11 Cell Viability Using Various Concentrations of AP1 in Combination with Decitabine

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. MV4-11 cells were sequentially treated by varying concentrations of AP1 and decitabine for 72 hrs (FIG. 55). AP1 suppressed MV4-11 cell growth with or without decitabine (FIG. 56). Similarly, decitabine suppressed MV4-11 cell growth with or without AP1 (FIG. 57).


Example 38: The Order of Addition Effects on MV4-11 Cell Viability Using Various Concentrations of AP1 in Combination with Ara-C

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. MV4-11 cells were sequentially treated by varying concentrations of AP1 and Ara-C for 72 hrs (FIG. 58). AP1 suppressed MV4-11 cell growth with or without Ara-C(FIG. 59). Similarly, Ara-C suppressed MV4-11 cell growth with or without AP1 (FIG. 60).


Example 39: The Order of Addition Effects on MV4-11 Cell Viability Using Various Concentrations of AP1 in Combination with Azacitidine

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. MV4-11 cells were sequentially treated by varying concentrations of AP1 and azacitidine for 72 hrs (FIG. 61). AP1 suppressed MV4-11 cell growth with or without azacitidine (FIG. 62). Similarly, azacitidine suppressed MV4-11 cell growth with or without AP1 (FIG. 63).


Example 40: Treatment with Fulvestrant (FUL) and Everolimus with the Peptidomimetic Macrocycles of the Disclosure in the MCF-7 Breast Cancer Cell Line

The combinations of AP1 and commercially available fulvestrant (FIGS. 64A and 65A) and everolimus (FIGS. 66A and 67A) were tested at various drug doses. Initially, various MCF-7 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration.


Next, the optimal number of cells were plated and treated with various concentrations of AP1 or with various concentrations of fulvestrant or everolimus alone. Cells were evaluated for viability by WST-1 assay or MTT assay 120 hours after beginning treatment. AP1 suppressed MCF-7 cell growth with or without fulvestrant (FIGS. 64B and 65B). AP1 suppressed MCF-7 cell growth with or without everolimus (FIGS. 66B and 67B).


A number of concentrations around the IC50 of the peptidomimetic macrocycle and a number of concentrations around the IC50 of FUL were then determined.















IC50 (nM)



















FUL
0.768



FUL + 0.13 μM AP1
0.4428



FUL + 0.4 μM AP1
0.2609



FUL + 1.2 μM AP1
0.2621










Example 41: Treatment with Rituximab and Romidepsin with the Peptidomimetic Macrocycles of the Disclosure in the MOLT-3 T-Lymphoid Cancer Cell Line

The combinations of AP1 and commercially available rituximab (FIGS. 68A and 69A) and romidepsin (FIGS. 71A and 72A) were tested at various drug doses. Initially, various MOLT-3 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration.


Next, the optimal number of cells were plated and treated with various concentrations of AP1 or with various concentrations of rituximab or romidepsin alone. Cells were evaluated for viability by WST-1 assay or MTT assay 3-7 days after beginning treatment with rituximab (FIGS. 68B and 69B) or romidepsin (FIGS. 71B and 72B). API in combination with rituximab showed complementary in vitro anticancer activity (FIG. 70). API in combination with romidepsin showed complementary in vitro anticancer activity (FIG. 73). The IC50 values of API alone and API with varying concentrations of romidepsin are shown in FIG. 72C.


Example 42: Treatment with Rituximab and Romidepsin with the Peptidomimetic Macrocycles of the Disclosure in the MCF-7 Breast Cancer Cell Line

The combinations of AP1 and commercially available ribociclib (FIGS. 74A and 75A) and abemaciclib (FIGS. 76A and 77A) were tested at various drug doses. Initially, various MCF-7 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration.


Next, the optimal number of cells were plated and treated with various concentrations of AP1 or with various concentrations of ribociclib or abemaciclib alone. Cells were evaluated for viability by WST-1 assay or MTT assay 72 or 120 hours after beginning treatment with rituximab (FIGS. 74B and 75B) or romidepsin (FIGS. 76B and 77B).


Example 43: The Order of Addition Effects on MCF-7 Cell Viability Using Various Concentrations of AP1 in Combination with Palbociclib Using the CyQUANT Method

The anticancer activity of the combination treatment was assessed based on the order of addition of the drugs. MCF-7 cells were sequentially treated by varying concentrations of AP1 and palbociclib for 72 hrs (FIG. 80). AP1 suppressed MCF-7 cell growth with or without palbociclib (FIG. 81). Similarly, palbociclib suppressed MCF-7 cell growth with or without AP1 (FIG. 82).


Example 44: Treatment with Dexamethasone with the Peptidomimetic Macrocycles of the Disclosure in the MCF-7 Breast Cancer Cell Line

The combination of AP1 and commercially available dexamethasone was tested at various drug doses for 120 hrs. Cells were evaluated for viability by WST-1 assay. AP1 suppressed MCF-7 cell growth with or without dexamethasone (FIG. 83).


Example 45: Treatment with Zelboraf, Tafinlar, and Mekinist with the Peptidomimetic Macrocycles of the Disclosure in the A375 Melanoma Cancer Cell Line

The combinations of AP1 and commercially available zelboraf (FIGS. 84A and 85A), tafinlar (FIGS. 86A and 87A), and mekinist (FIGS. 88A and 89A) were tested at various drug doses. Initially, various A375 cell numbers were plated and evaluated 3-7 days later to determine the optimal number of cells to be plated and treatment duration.


Next, the optimal number of cells were plated and treated with various concentrations of AP1 or with various concentrations of zelboraf or tafinlar alone. Cells were evaluated for viability by WST-1 assay or MTT assay 72 hours after beginning treatment with zelboraf (FIGS. 84B and 85B), tafinlar (FIGS. 86B and 87B), or mekinist (FIGS. 88B and 89B).


Example 46: Combination Index Plots of Fulvestrant, Everolimus, Palbociclib (WST-1), Palbociclib (WST-1), and Romidepsin in MCF-7 Cells

The combination index plots suggest additive or better complimentarily for AP1 in MCF-7 cells using fulvestrant (FIG. 90A), everolimus (FIG. 90B), palbociclib via WST-1 (FIG. 90C), palbociclib via CyQUANT (FIG. 90D), and romidepsin (FIG. 90E). Combination index (CI) values were calculated using the CompuSyn software. The data were 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.


Example 47: Combination Index Plots of Ara-C, Decitabine, Azacitidine, and Midostaurin in MV4-11 Cells

The combination index plots suggest additive or better complimentarity for AP1 in MV4-11 cells using Ara-C(FIG. 91A), decitabine (FIG. 91B), azacitidine (FIG. 91C), and midostaurin (FIG. 91D). Combination index (CI) values were calculated using the CompuSyn software. The data were 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


Example 48: Combination Index Plots of Vincristine, Cyclophosphamide, and Rituximab in DOHH-2 Cells

The combination index plots suggest additive or better complimentarity for AP1 in DOHH-2 cells using vincristine (FIG. 92A), cyclophosphamide (FIG. 92B), and rituximab (FIG. 92C). Combination index (CI) values were calculated using the CompuSyn software. The data were 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


Example 49: Combination Index Plot of Romidepsin in MOLT-3 Cells

The combination index plots suggest mostly additive complimentarity for AP1 in MOLT-3 cells using romidepsin (FIG. 93). Combination index (CI) values were calculated using the CompuSyn software. The data were 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


Example 50: Combination Index Plots of Vincristine, Cyclophosphamide, and Rituximab in A375 Cells

The combination index plots suggest additive or better complimentarity for AP1 in A375 cells using mekinist (FIG. 94A), zelboraf (FIG. 94B), and tafinlar (FIG. 94C). Combination index (CI) values were calculated using the CompuSyn software. The data were 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


Example 51: Aileron Peptide 1 Activation of the p53-Pathway in AML Cell Lines

The Molm13 cell line was treated with increasing amounts of Aileron peptide 1 (0.1 μM, 0.2 μM, 0.4 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, or 10.0 μM) (FIG. 1A). Lysates were subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, p53, p21, and 3-Actin. The results demonstrate that Aileron peptide 1 activates the p53-pathway in the Molm13 cell line.


The OCI/AML3 cell line was treated with increasing amounts of Aileron peptide 1 (0.1 μM, 0.2 μM, 0.4 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, or 10.0 μM) (FIG. 1B). Lysates were subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, p53, p21, and 3-Actin. The results demonstrate that Aileron peptide 1 activates the p53-pathway in the OCI/AML3 cell line.


The HL60 cell line was treated with increasing amounts of Aileron peptide 1 (0.1 μM, 0.2 μM, 0.4 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, or 10.0 μM) (FIG. 1C). Lysates were subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, p53, p21, and 3-Actin. The results demonstrate that Aileron peptide 1 does not activate the p53-pathway in the p53 null HL60 cell line.


The Molm13, OCI/AML3, Molm14 and ML2 cell lines were treated with vehicle or 1.0 μM Aileron peptide (FIG. 1B). Lysates were subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, p53, p21, and 3-Actin. The results demonstrate that Aileron peptide 1 activates the p53-pathway in these cell lines.


mRNA expression of p21, MDM2, Puma, Bax, and Gadd45a was also determined in Molm13 and Oci/AML3 cells lines following treatment with increasing amounts of Aileron peptide 1 (0.1 μM, 0.2 μM, 0.4 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, or 10.0 μM) (FIG. 2). Expression levels were normalized to GAPDH mRNA expression levels. The results demonstrate that Aileron peptide 1 activates the p53-pathway in these cell lines.


Example 52: Aileron Peptide 1 Activation of the p53-Pathway in Primary AML Cells

Two primary AML cell lines were treated with vehicle or 1.0 μM Aileron peptide 1 or 5.0 μM Aileron peptide 1 (FIG. 1C). Lysates were subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, p53, p21, and β-Actin. The results demonstrate that Aileron peptide 1 activates the p53-pathway in primary AML cells.


Example 53: Aileron Peptide 1 Stabilizes p53 in AML Cells

The Molm13, OCI/AML3, Molm14, HL60 and ML2 cell lines were treated with vehicle or increasing amounts of Aileron peptide 1 (0.1 μM, 0.2 μM, 0.4 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, or 10.0 μM) (FIGS. 3A and 3B) for 24 hrs, 48 hrs or 72 hrs. Lysates were subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, p53, p21, and β-Actin. The results demonstrate that Aileron peptide 1 stabilizes p53 in a time and dose dependant manner the AML p53 wild type cell lines tested.


Example 54: Immunoprecipitation Assays in AML Cells

AML p53 wild type cell lines were treated with vehicle or 10.0 μM Aileron peptide (FIGS. 4A, 4B, and 4C). Lysates were subjected to immunoprecipitation with a MDMX specific antibody (FIG. 4A), a p53 specific antibody (FIG. 4B), or a MDM2 specific antibody (FIG. 4C). Immunoprecipitates were washed and subjected to SDS-PAGE and probed by Western blotting with antibodies specific to MDM2, MDMX, p53, and/or β-Actin. The results demonstrate that Aileron peptide 1 inhibits the p53-MDMX and the p53-MDM2 interaction.


Example 55: Cellular Proliferation Assays of AML Cells

The Molm13, OCI/AML3, Molm14, HL60 and ML2 cell lines were plated at a known density (cells/mL) and treated with vehicle or increasing amounts of Aileron peptide 1 (0.1 μM, 0.2 μM, 0.4 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, or 10.0 μM) (FIGS. 5A-5D). Cell proliferation was measured over time by counting the number of live cells/mL at various time points and plotted. All p53 wild type cell lines treated with Aileron peptide 1 demonstrated reduced levels of cellular proliferation over time compared to vehicle alone.


Example 56: Clonogenicity Assay on AML Cell Lines

The Molm13, OCI/AML3, Molm14, HL60 and ML2 cell lines were treated with vehicle or 1.0 μM Aileron peptide 1. A clonogenicity assay was then performed and the number of clonies was counted (FIG. 6). The results demonstrated that Aileron peptide 1 treatment in the p53 wild type cell lines tested inhibited their clonogenic capacity.


Example 57: Cellular Proliferation Assays of AML Cell Lines

The OCI/AML3, HL60 and Kasumi-1 cell lines were plated at a known density (cells/mL) and treated with vehicle or 10.0 μM Aileron peptide 1 (FIGS. 7A and 7B). Cell proliferation was measured over time by counting the number of live cells/mL at various time points and plotted. The p53 wild type OCI/AML3, but not the p53 null HL60 or the p53R248Q Kasumi-1 cell lines treated with Aileron peptide 1 demonstrated reduced levels of cellular proliferation over time compared to vehicle alone.


Example 58: Apoptosis Assays of AML Cell Lines

The Molm13, OCI/AML3, Molm14, HL60 and ML2 cell lines were treated with vehicle or increasing amounts of Aileron peptide 1 (1.0 μM, 5.0 μM, or 10.0 μM) (FIGS. 8A-8E). Cells were probed with DAPI and a FITC-labeled anti-Annexin-V antibody. FACS analysis was performed to determine the number of viable cells and the number of cells in early apoptosis, late apoptosis and undergoing necrosis and the results were plotted. The results demonstrate that Aileron peptide 1 induces apoptotic cell death in p53 wild type AML cell lines tested.


Example 59: Cellular Proliferation in AML Cells Treated with Ara-C

AML cell lines were plated at a known density (cells/mL) and were treated with vehicle or increasing amounts of Ara-C alone (FIG. 9A); with vehicle, Aileron peptide 1 alone, or with Ara-C and Aileron peptide 1 (FIG. 9B); or with vehicle, Ara-C alone, or with Ara-C and increasing amounts of Aileron peptide 1 (FIG. 9C). Cell proliferation was measured over time by counting the number of live cells/mL at various time points and plotted. The results demonstrate that cytarabine (Ara-C) treatment inhibits proliferation of AML cell lines and that Ara-C synergizes with API to inhibit proliferation of AML cell lines.


Example 60: Cellular Proliferation Assays and Clonogenicity Assays of Primary AML Cells

Primary AML cell lines were plated at a known density (cells/mL) and treated with vehicle or increasing amounts of Aileron peptide 1 (1.0 μM, 5.0 μM, or 10.0 μM) (FIGS. 10A-10D). Cell proliferation was measured over time by counting the number of live cells/mL at various time points and plotted. The primary AML cell lines treated with Aileron peptide 1 demonstrated reduced levels of cellular proliferation over time compared to vehicle alone.


Example 61: Clonogenicity Assay on Primary AML Cell Lines

A primary AML cell line and a primary AML cell line from a patient in remission were treated with vehicle or increasing amounts of Aileron peptide 1 (0.1 μM, 0.25 μM, 0.5 μM, or 1.0 μM) (FIGS. 11A and 11B). A clonogenicity assay was then performed and the number of clonies was counted. The results demonstrated that Aileron peptide 1 treatment in the primary AM1 cell line tested inhibited its clonogenic capacity to a higher extent than the primary AML cell line from the patient in remission and cells from a healthy donor.


Example 62: Apoptosis Assays on Primary AML Cell Lines

A primary AML cell line was treated with vehicle or increasing amounts of Aileron peptide 1 (1.0 μM, 5.0 μM, or 10.0 μM) (FIG. 12). Cells were probed with DAPI and a FITC-labeled anti-Annexin-V antibody. FACS analysis was performed to determine the number of viable cells and the number of cells in early apoptosis, late apoptosis and undergoing necrosis and the results were plotted. The results demonstrate that Aileron peptide 1 induces apoptotic cell death in primary AML cells.

Claims
  • 1.-245. (canceled)
  • 246. A method of modulating the activity of p53 and/or MDM2 and/or MDMX in a subject with cancer comprising administering to the subject a therapeutically-effective amount of a peptidomimetic macrocycle or pharmaceutically acceptable salt thereof and at least one additional pharmaceutically active agent, 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:
  • 247. The method of claim 246, wherein the peptidomimetic macrocycle has a Formula:
  • 248. The method of claim 246, wherein w>2.
  • 249. The method of claim 249, wherein each of the first two amino acid represented by E comprises an uncharged side chain or a negatively charged side chain.
  • 250. The method of claim 249, wherein each E is independently an amino acid selected from Ala (alanine), D-Ala (D-alanine), Aib (u-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine).
  • 251. The method of claim 246, wherein the at least one additional pharmaceutically active agent is a nucleoside metabolic inhibitor, a microtubule inhibitor, a platinum-based drug, a hypomethylating agent, a protein kinase inhibitor, a bruton's tyrosine kinase inhibitor, a CDK4 and/or CDK6 inhibitor, a B-raf inhibitor, a K-ras inhibitor, a MEK-1 and/or MEK-2 inhibitor, an estrogen receptor antagonist, an HDAC inhibitor, an anti-CD20 monoclonal antibody, an anti-PD-1 monoclonal antibody, a hormonal antagonist, an agent the alleviates CDK2NA deletion, an agent that alleviates CDK9 abnormality, an AMT regulator, an agent that alleviates AKT activation, an agent that alleviates PTEN deletion, an agent that alleviates Wip-1Alpha overexpression, an agent that upregulates BIM, or an aromatase inhibitor.
  • 252. The method of claim 246, wherein the at least one additional pharmaceutically active agent is selected from the group consisting of venetoclax (ABT-199), clofarabine, cyclophosphamide, cytarabine, doxorubicin, imatinib mesylate, methotrexate, prednisone, vincristine, azacitadine, cyclophosphamide, cytarabine, dabrafenib, decitabine, doxorubicin, etoposide, vincristine, doxorubicin, methotrexate, capecitabine, cyclophosphamide, docetaxel, doxorubicin, eribulin mesylate, everolimus, exemestane, fluorouracil, fluorouracil, fulvestrant, gemcitabine, goserelin acetate, letrozole, megestrol acetate, methotrexate, paclitaxel, palbociclib, pertuzumab, tamoxifen citrate, trastuzumab, capecitabine, cetuximab, fluorouracil, irinotecan, ramucirumab, carboplatin, cisplatin, doxorubicin, megestrol acetate, paclitaxel, docetaxel, doxorubicin, fluorouracil, ramucirumab, trastuzumab, axitinib, everolimus, pazopanib, sorafenib tosylate, sorafenib tosylate, dacarbazine, paclitaxel, trametinib, vemurafenib, cisplatin, pemetrexed, bendamustine, bortezomib, brentuximab vedotin, chlorambucil, cyclophosphamide, dexamethasone, doxorubicin, ibrutinib, lenalidomide, methotrexate, prednisone, rituximab, vincristine, afatinib dimaleate, carboplatin, cisplatin, crizotinib, docetaxel, erlotinib, gemcitabine, methotrexate, paclitaxel, pemetrexed, ramucirumab, carboplatin, cisplatin, cyclophosphamide, gemcitabine, olaparib, paclitaxel, topotecan, abiraterone, cabazitaxel, docetaxel, enzalutamide, goserelin acetate, prednisone, doxorubicin, imatinib mesylate, romidepsin, obinutuzumab, pazopanib, selumetinib, midostaurin (PKC412), venetoclax and combinations thereof.
  • 253. The method of claim 246, wherein the at least one additional pharmaceutically active agent is a PD-1 antagonist or a PD-1 antagonist.
  • 254. The method of claim 246, wherein the at least one additional pharmaceutically active agent modulates the activity of CDK4 and/or CDK6, and/or inhibits CDK4 and/or CDK6.
  • 255. The method of claim 246, 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.
  • 256. The method of claim 246, wherein the subject comprises cancer cells that overexpress PD-L1, PD-1, miR-34, or any combination thereof.
  • 257. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 258. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 259. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 260. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 261. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 262. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 263. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 264. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 265. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 266. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 267. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 268. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 269. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 270. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 271. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 272. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 273. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 274. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 275. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 276. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 277. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 278. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 279. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 280. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 281. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 282. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 283. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 284. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 285. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 286. The method of claim 246, wherein the peptidomimetic macrocycle is
  • 287. A method of selecting a peptidomimetic macrocycle that reduces PD-L1 expression, comprising: (a) contacting a cancer cell line expressing a first level of PD-L1 with a peptidomimetic macrocycle comprising a polypeptide with a crosslinker connecting a first amino acid and a second amino acid;(b) incubating the cancer cell line for an incubation period;(c) measuring a second level of PD-L1 expression after the incubation period;(d) selecting the peptidomimetic macrocycle as a peptidomimetic macrocycle that reduces PD-L1 expression when the second level of PD-L1 expression is at least 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, or 100 fold lower than the first level of PD-L1 expression.
CROSS-REFERENCE

This application claims priority to U.S. Provisional Application No. 62/214,142, filed Sep. 3, 2015; U.S. Provisional Application No. 62/310,254, filed Mar. 18, 2016; U.S. Provisional Application No. 62/344,651, filed Jun. 2, 2016; and U.S. Provisional Application No. 62/344,791, filed Jun. 2, 2016, which are incorporated herein by reference in their entirety.

Provisional Applications (4)
Number Date Country
62214142 Sep 2015 US
62310254 Mar 2016 US
62344651 Jun 2016 US
62344791 Jun 2016 US