Patent Application
20180094030
The present invention relates to cell penetrating peptides and methods of identifying cell penetrating peptides based upon hydrophobicity and polarity.
Cell-penetrating peptides (CPPs) such as the antennapedia-derived penetratin (Derossi et al., Biol. Chem., 269, 10444-10450, 1994) and the Tat peptide (Vives et al., J. Biol. Chem., 272, 16010-16017, 1997) are widely used tools for the delivery of cargo molecules such as peptides, proteins and oligonucleotides into cells (Fischer et al., Bioconjug. Chem., 12, 825-841, 2001). Areas of application range from purely cell biological to biomedical research (Dietz and Bahr, Mol. Cell., Neurosci, 27, 85-131, 2004). Initially, cellular uptake was believed to occur by direct permeation of the plasma membrane (Prochiantz, Curr. Opin. Cell Biol., 12, 400-406, 2000). In the past years, evidence has been accumulated that for several CPPs, endocytosis contributes at least significantly to the cellular uptake (for a review, see Fotin-Mleczek et al., Curr. Pharm. Design, 11, 3613-3628, 2005). Given these recent results, the specification of a peptide as a CPP therefore does not imply a specific cellular import mechanism, but rather refers to a function as a peptide that, when conjugated to a cargo, either covalently or non-covalently, enhances the cellular uptake of the cargo molecule.
Most cell penetrating peptides have many hydrophobic and/or positively charged residues, but their vast sequence diversity makes it difficult to predict whether any given peptide will be cell penetrating. Cruciani et al., J. Chemometrics, 2004; 18: 146-155, proposed a set of descriptors (PP1 [polarity] and PP2 [hydrophobicity]) for each of the 20 amino acids. However, despite these descriptors no method was proposed or exists that can reasonably predict the cell penetrating properties of a peptide based upon PP1 and PP2.
The present invention relates to cell penetrating peptides and methods of identifying cell penetrating peptides based upon hydrophobicity and polarity.
In one embodiment, the present invention relates to a method of identifying cell penetrating peptides among a group of peptides by: (1) determining the polarity (referred to as the “PP1”) of said peptides; (2) determining the hydrophobicity (referred to as the “PP2”) of said peptides; (3) identifying peptides within the group, wherein PP1<[(PP2*X1)+X], wherein X1 is 1.5 to 10 and X is 0.3 to −1.5; and (4) testing the peptides identified in step 3 in an in vitro or in vivo assay to confirm that said peptides are cell-penetrating.
In another embodiment, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-455 and compositions and conjugates containing the same. In particular, the present invention relates to the cell penetrating peptides of the present invention which are conjugated to small molecules, nucleic acids, fluorescent moieties, proteins, peptides, or other cargo for delivery to the inside of cells (such as the cytoplasm or nucleus) for various therapeutic and other applications.
In other embodiments, the present invention relates to an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-455. In other embodiments, the present invention relates to a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-455. The present invention also relates to methods of manufacturing and using such peptides, nucleotides, and vectors.
The present invention relates to cell penetrating peptides and methods of identifying cell penetrating peptides based upon hydrophobicity and polarity.
The polarity or PP1 of a peptide is the average polarity of all the amino acids in the peptide wherein the polarity of specific amino acids are set forth in Table 1. The hydrophobicity or PP2 of a peptide is the average hydrophobicity of all the amino acids in the peptide wherein the hydrophobicity of specific amino acids are set forth in Table 1.
Most cell penetrating peptides have many hydrophobic and/or positively charged residues, but their vast sequence diversity makes it difficult to predict whether any given peptide will be cell penetrating. Cruciani et al., J. Chemometrics, 2004; 18: 146-155, proposed a set of descriptors (PP1 [polarity] and PP2 [hydrophobicity]) for each of the 20 amino acids. However, despite these descriptors no method was proposed or exists that can reasonably predict the cell penetrating properties of a peptide based upon PP1 and PP2.
Thus, in one embodiment, the present invention relates to a method of identifying cell penetrating peptides among a group of peptides by (1) determining the polarity (or “PP1”) of said peptides; (2) determining the hydrophobicity (or “PP2”) of said peptides; (3) identifying peptides within the group, wherein PP1<[(PP2*X1)+X], wherein X1 is 1.5 to 10 and X is 0.3 to −1.5; and (4) testing the peptides identified in step 3 in an in vitro or in vivo assay to confirm that said peptides are cell-penetrating.
In particular embodiments, X1 is 1.7 and X is 0.3 (as shown in
In other particular embodiments, X1 is 8 and X is −0.4 to 0.1. In other particular embodiments, X1 is 6 and X is −0.4 to 0.1. In other particular embodiments, X1 is 4 and X is −0.4 to 0.1. In other particular embodiments, X1 is 2 and X is −0.4 to 0.1. In other particular embodiments, X1 is 1.7 and X is −0.4 to 0.1. In other particular embodiments, X1 is 1.7 and X is 0.1. In other particular embodiments X1 is 1.7 and X is 0. In other particular embodiments, X1 is 1.7 and X is −0.1. In other particular embodiments, X1 is 1.7 and X is −0.2. In other particular embodiments, X1 is 1.7 and X is −0.3. In other particular embodiments, X1 is 1.7 and X is −0.4.
In another embodiment, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-455 and compositions and conjugates containing the same. In particular, the present invention relates to the cell penetrating peptides of the present invention which are conjugated to small molecules, nucleic acids, fluorescent moieties, proteins, peptides, or other cargo for delivery to the inside of cells (such as the cytoplasm or nucleus) for various therapeutic and other applications.
In other embodiments, the present invention relates to an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-455. In other embodiments, the present invention provides a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-455. The present invention also relates to methods of manufacturing and using such peptides, nucleotides, and vectors.
In one preferred embodiment, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9 and compositions and conjugates containing the same. In another preferred embodiment, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 15, 16, 17 and 18 and compositions and conjugates containing the same.
In one particular embodiment, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19 and compositions and conjugates containing the same.
In other particular embodiments, the present invention relates to an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
In other particular embodiments, the present invention provides a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-30 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-30.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-40 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-40.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-50 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-50.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-60 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-60.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-70 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-70.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-80 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-80.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-90 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-90.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 91-100 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 91-100.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-110 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-110.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-120 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-120.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 121-130 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 121-130.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 131-140 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 131-140.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-150 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-150.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 151-160 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 151-160.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161-170 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161-170.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 171-180 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 171-180.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 181-190 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 181-190.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191-200 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191-200.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-210 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-210.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 211-220 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 211-220.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 221-230 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 221-230.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 231-240 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 231-240.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 241-250 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 241-250.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 251-260 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 251-260.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 261-270 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 261-270.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 271-280 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 271-280.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 281-290 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 281-290.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 291-300 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 291-300.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 301-310 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 301-310.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 311-320 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 311-320.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 321-330 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 321-330.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 331-340 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 331-340.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 341-350 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 341-350.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 351-360 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 351-360.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 361-370 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 361-370.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 371-380 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 371-380.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 381-390 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 381-390.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-400 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-400.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 401-410 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 401-410.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 411-420 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 411-420.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 421-430 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 421-430.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 431-440 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 431-440.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 441-450 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 441-450.
In other particular embodiments, the present invention relates to a cell penetrating peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 451-455 and compositions and conjugates containing the same. In other embodiments, the present invention relates to a an isolated nucleotide or a vector comprising an isolated nucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 451-455.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.6 to −0.85. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.6 to −0.85.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.6. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.6.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.65. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.65.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.7. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.7.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.75. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.75.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.8. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.8.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.85. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 1.7 to 2.3 and X is −0.85.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.60. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.60.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.65. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.65.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.7. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.7.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.75. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.75.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.8. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.8.
In other particular embodiments, the present invention relates to a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.85. In other embodiments, the present invention relates to an isolated nucleotide or a vector comprising an isolated nucleotide encoding a cell penetrating peptide wherein the PP1 of the peptide is <[(the PP2 of the peptide*X1)+X], wherein X1 is 2.0 and X is −0.85.
All peptide sequences mentioned herein are written according to the usual convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right, unless noted otherwise. A short line between two amino acid residues indicates a peptide bond. Where the amino acid has isomeric forms, it is the L form of the amino acid that is represented unless otherwise expressly indicated.
For convenience in describing this invention, the conventional and nonconventional abbreviations for the various amino acids residues are used. These abbreviations are familiar to those skilled in the art, but for clarity are listed below:
Asp-D-Aspartic Acid; Ala=A=Alanine; Arg-R=Arginine; Asn=N=Asparagine; Gly-G=Glycine; Glu=E=Glutamic Acid; Gln=Q-Glutamine; His-H=Histidine; Ile=I=Isoleucine; Leu=L=Leucine; Lys=K=Lysine; Met=M=Methionine; Phe=F=Phenylalanine; Pro=P=Proline; Ser-S-Serine; Thr=T=Threonine; Trp=W=Tryptophan; Tyr=Y=Tyrosine; and Val=V=Valine.
Also for convenience, and readily known to one skilled in the art, the following abbreviations or symbols are used to represent the moieties, reagents and the like used herein:
In general, the peptides of the present invention may be readily synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids. Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acid or fragment thereof having its carboxyl group and other reactive groups protected and the free primary carboxyl group of another amino acid or fragment thereof having its amino group or other reactive groups protected.
Such conventional procedures for synthesizing the peptides of the present invention include, for example, any solid phase peptide synthesis method. In such a method the synthesis of the peptides can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods. Such methods are disclosed in, for example, Merrifield, R. B., J. Amer. Chem. Soc. 85, 2149-2154 (1963); Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980), which are incorporated herein by reference.
During the synthesis of peptides, it may be desired that certain reactive groups on the amino acid, for example, the alpha-amino group, a hydroxyl group, and/or reactive side chain groups, be protected to prevent a chemical reaction therewith. This may be accomplished, for example, by reacting the reactive group with a protecting group which may later be removed. For example, the alpha amino group of an amino acid or fragment thereof may be protected to prevent a chemical reaction therewith while the carboxyl group of that amino acid or fragment thereof reacts with another amino acid or fragment thereof to form a peptide bond. This may be followed by the selective removal of the alpha amino protecting group to allow a subsequent reaction to take place at that site, for example with the carboxyl group of another amino acid or fragment thereof.
Alpha amino groups may, for example, be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as allyloxycarbony, benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); and aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, and allyloxycarbonyl. In an embodiment, Fmoc is used for alpha amino protection.
Hydroxyl groups (OH) of the amino acids may, for example, be protected by a suitable protecting group selected from benzyl (Bzl), 2,6-dichlorobenzyl (2,6 diCl-Bzl), and tert-butyl (t-Bu). In an embodiment wherein a hydroxyl group of tyrosine, serine, or threonine is intended to be protected, t-Bu may, for example, be used.
Epsilon-amino acid groups may, for example, be protected by a suitable protecting group selected from 2-chloro-benzyloxycarbonyl (2-Cl—Z), 2-bromo-benzyloxycarbonyl (2-Br—Z), allycarbonyl and t-butyloxycarbonyl (Boc). In an embodiment wherein an epsilon-amino group of lysine is intended to be protected, Boc may, for example, be used.
Beta- and gamma-amide groups may, for example, be protected by a suitable protecting group selected from 4-methyltrityl (Mtt), 2,4,6-trimethoxybenzyl (Tmob), 4,4′-dimethoxydityl (Dod), bis-(4-methoxyphenyl)-methyl and Trityl (Trt). In an embodiment wherein an amide group of asparagine or glutamine is intended to be protected, Trt may, for example, be used.
Indole groups may, for example, be protected by a suitable protecting group selected from formyl (For), Mesityl-2-sulfonyl (Mts) and t-butyloxycarbonyl (Boc). In an embodiment wherein the indole group of tryptophan is intended to be protected, Boc may, for example, be used.
Imidazole groups may, for example, be protected by a suitable protecting group selected from Benzyl (Bzl), t-butyloxycarbonyl (Boc), and Trityl (Trt). In an embodiment wherein the imidazole group of histidine is intended to be protected, Trt may, for example, be used.
Solid phase synthesis may be commenced from the C-terminal end of the peptide by coupling a protected alpha-amino acid to a suitable resin. Such a starting material can be prepared by attaching an alpha-amino-protected amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, or by an amide bond between an Fmoc-Linker, such as p-((R,S)-?-(1-(9H-fluoren-9-yl)-methoxyformamido)-2,4-dimethyloxybenzyl)-phenoxyacetic acid (Rink linker), and a benzhydrylamine (BHA) resin. Preparation of the hydroxymethyl resin is well known in the art. Fmoc-Linker-BHA resin supports are commercially available and generally used when the desired peptide being synthesized has an unsubstituted amide at the C-terminus.
In an embodiment, peptide synthesis is microwave assisted. Microwave assisted peptide synthesis is an attractive method for accelerating the solid phase peptide synthesis. This may be performed using Microwave Peptide Synthesizer, for example a Liberty peptide synthesizer (CEM Corporation, Matthews, N.C.). Microwave assisted peptide synthesis allows for methods to be created that control a reaction at a set temperature for a set amount of time. The synthesizer automatically regulates the amount of power delivered to the reaction to keep the temperature at the set point.
Typically, the amino acids or mimetic are coupled onto the Fmoc-Linker-BHA resin using the Fmoc protected form of amino acid or mimetic, with 2-5 equivalents of amino acid and a suitable coupling reagent. After coupling, the resin may be washed and dried under vacuum. Loading of the amino acid onto the resin may be determined by amino acid analysis of an aliquot of Fmoc-amino acid resin or by determination of Fmoc groups by UV analysis. Any unreacted amino groups may be capped by reacting the resin with acetic anhydride and diispropylethylamine in methylene chloride.
The resins are carried through several repetitive cycles to add amino acids sequentially. The alpha amino Fmoc protecting groups are removed under basic conditions. Piperidine, piperazine or morpholine (20-40% v/v) in DMF may be used for this purpose. In an embodiment, 20% piperidine in DMF is utilized.
Following the removal of the alpha amino protecting group, the subsequent protected amino acids are coupled stepwise in the desired order to obtain an intermediate, protected peptide-resin. The activating reagents used for coupling of the amino acids in the solid phase synthesis of the peptides are well known in the art. For example, appropriate reagents for such syntheses are benzotriazol-1-yloxy-tri-(dimethylamino) phosphonium hexafluorophosphate (BOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylcarbodiimide (DIC). In an embodiment, the reagent is HBTU or DIC. Other activating agents are described by Barany and Merrifield (in The Peptides, Vol 2, J. Meienhofer, ed., Academic Press, 1979, pp 1-284). Various reagents such as 1 hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (HOSu) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT) may be added to the coupling mixtures in order to optimize the synthetic cycles. In an embodiment, HOBT is added.
Following synthesis of the peptide, the blocking groups may be removed and the peptide cleaved from the resin. For example, the peptide-resins may be treated with 100 L ethanedithiol, 100 l dimethylsulfide, 300 L anisole, and 9.5 mL trifluoroacetic acid, per gram of resin, at room temperature for 180 min. Alternatively, the peptide-resins may be treated with 1.0 mL triisopropyl silane and 9.5 mL trifluoroacetic acid, per gram of resin, at room temperature for 90 min. The resin may then be filtered off and the peptide precipitated by addition of chilled ethyl ether. The precipitates may then be centrifuged and the ether layer decanted.
Purification of the crude peptide may be, for example, performed on a Shimadzu LC-8A system by high performance liquid chromatography (HPLC) on a reverse phase C18 Column (50×250 mm, 300 Å, 10 m). The peptides may be dissolved in a minimum amount of water and acetonitrile and injected on to a column. Gradient elution may be generally started at 2%-70% B over 70 minutes, (buffer A: 0.1% TFA/H2O, buffer B: 0.1% TFA/CH3CN) at a flow rate of 60 ml/min. UV detection set at 220/280 nm. The fractions containing the products may be separated and their purity judged on Shimadzu LC-10AT analytical system using reverse phase Pursuit C18 column (4.6×50 mm) at a flow rate of 2.5 ml/min., gradient (2-70%) over 10 min.[buffer A: 0.1% TFA/H2O, buffer B: 0.1% TFA/CH3CN)]. Fractions judged to be of high purity may then be pooled and lyophilized.
In particular embodiments, the cell penetrating peptides of the present invention (including SEQ ID NOs. 1-455) are conjugated to small molecules, nucleic acids, fluorescent moieties, proteins, peptides, or other cargo for delivery to the inside of cells (such as the cytoplasm or nucleus) for various therapeutic and other applications. Examples of such cargo include but are not limited to the cargo disclosed in U.S. Patent Application Publication No. 2008/0234183 incorporated herein by reference in its entirety. Using CPPs for delivering conjugated cargo to the inside of cells and methods of conjugating cargo such as small molecules, nucleic acids, fluorescent moieties, proteins, peptides and/or other cargo are well known in the art. See for example id. (U.S. Patent Application Publication No. 2008/0234183); Rhee et al., 201. C105Y, a Novel Cell Penetrating Peptide Enhances Gene Transfer of Sec-R Targeted Molecular Conjugates, Molecular Therapy (2005) 11, S79-S79; Johnson et al., Cell-penetrating Peptide for Enhanced Delivery of Nucleic Acids and Drugs to Ocular Tissues Including Retina and Cornea, Molecular Therapy (2007) 16 (1), 107-114; El-Andaloussi et al., A Novel Cell-penetrating Peptide, M918, for Efficient Delivery of Proteins and Peptide Nucleic Acids, Molecular Therapy (2007) 15 (10), 1820-1826; and Crombez et al., A New Potent Secondary Amphipathic Cell-Penetrating Peptide for siRNA Delivery Into Mammalian Cells, Molecular Therapy (2008) 17 (1), 95-103; Sasaki, Y. et al., Cell-penetrating peptide-conjugated XIAP-inhibitory cyclic hexapeptides enter into Jurkat cells and inhibit cell proliferation FEBS Journal (2008) 275 (23), 6011-6021; Kolluri, S. K. et al., A Short Nur77-Derived Peptide Converts Bcl-2 from a Protector to a Killer, Cancer Cell (2008) 14 (4), 285-298; Avbelj, M., The Role of Intermediary Domain of MyD88 in Cell Activation and Therapeutic Inhibition of TLRs J. Immunology (2011), 1; 187(5):2394-404.
In addition, the foregoing examples demonstrate the conjugation of SEQ ID NOs. 1-19 to fluorescein isothiocyanate (FITC) and their subsequent cell penetration as summarized in the cell assay section (also below).
The peptides in the specific examples below were prepared by solid state synthesis. See Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968). A preferred method is the Merrifield process. Merrifield, Recent Progress in Hormone Res., 23:451 (1967). In addition, the peptides in the specific examples below were synthesized by tagging the N-terminus of the peptide with FITC as a green fluorescent dye. Examples 1-9 were prepared by C S Bio Company, Inc. and Examples 10-19 were prepared by HYBIO Pharmaceutical Co., Ltd.
Material:
All chemicals and solvents such as DMF (Dimethylformamide), DCM (Methylene Chloride), DIEA (Diisopropylethylamine), and piperidine were purchased from VWR and Aldrich, and used as purchased without further purification. Mass spectra were recorded with Electrospray ionization mode. The automated stepwise assembly of protected amino acids was constructed on a CS 336X series peptide synthesizer (C S Bio Company, Menlo Park, Calif., USA) with Rink Amide MBHA resin as the polymer support. N-(9-fluorenyl)methoxycarbonyl (Fmoc) chemistry was employed for the synthesis. The protecting groups for Fmoc amino acids (AAs) were as follows, Arg: (Pbf), Asn/Gln/Cys/His: (Trt), Asp/Glu: (OtBu), Lys/Trp: (Boc), Ser/Thr/Tyr: (tBu).
Synthesis:
The above peptide (SEQ ID NO. 1) as conjugated to FITC was synthesized using Fmoc chemistry. The synthesis route started from deFmoc of pre-loaded Rink Amide resin and coupling/de-protecting of desired AAs according to the given sequences for all the orders. Coupling reagent was DIC/HOBt, and reaction solvents were DMF and DCM. The ratio of peptidyl resin/AA/DIC/HOBT was 1/4/4/4 (mol/mol). After coupling program, DeFmoc was executed using 20% piperidine in DMF. For example, a 0.4 mmol synthesis was performed till the last AA was attached. After deFmoc, the resin was coupled with Fmoc-Ahx-OH, followed by deFmoc and FITC attachment.
Fmoc-Rink Amide Resin (0.85 g, 0.4 mmol, sub: 0.47 mm/g, Lot#110810, C S Bio) was mixed in a 25 mL reaction vessel (RV) with DMF (10 mL), and swollen for 10-30 min. The RV was mounted on a CS336 peptide automated synthesizer and the amino acids were loaded onto amino acid (AA) wheel according to the given peptide sequence. HOBt (0.5M in DMF) and DIC (0.5M in DMF) were all pre-dissolved separately in transferable bottles under N2. Fmoc-amino acids (AAs, 4 eq) were weighed and prelocated as powder on the AA wheel. For example, 0.4 mmol synthesis needed 1.6 mmol of AA. The preset program started from AA dissolving in the AA tube and the solution was pumped thru M-VA to T-VA. HOBt solution was later mixed with AA. N2 bubbling was used to assist mixing. While DIC solution was combined with the AA/HOBt solution, the whole mixture was transferred into the RV with drained resin in 5 min and the coupling started at the same time.
After shaking for 3-6 hr, reaction mixture was filtered off and the resin was washed with DMF three times, followed by deFmoc according to the preset program using 20% Pip in DMF. The next AA was attached following the same route. Seven washing steps were done with DMF/DCM alternatively after deFmoc. The coupling process was repeated with the respective building blocks according to the given sequence till the last AA was coupled. Coupling Time: 3-6 hrs for each AA attachment. After deFmoc of last AA, the resin was coupled with Fmoc-Ahx-OH (3 eq) using DIC/HOBt. After deFmoc, FITC (3 eq) was attached in DMF with 1-2 eq of DIEA.
Cleavage:
The final peptidyl resin (1-1.5 g) was mixed with TFA cocktail (TFA/EDT/TIS/H2O) and the mixture was shaken at room temperature for 4 hr. The cleaved peptide was filtered and the resin was washed by TFA. After ether precipitation and washing, the crude peptide was obtained in a yield of 50-90%. The crude peptide was directly purified without lyophilization.
Purification:
100 mg of FITC peptide were dissolved in Buffer A 0.1% TFA in water and ACN, and the peptide solution was loaded onto a C18 column (2 inch) with a prep HPLC purification system. With a flow rate of 25-40 mL/min, the purification was finished in a TFA (0.1%) buffer system with a 60 min gradient. Fractions (peptide purity>95%) containing the expected MW were collected. The prep HPLC column was then washed for at least three void column volumes by 80% Buffer B and equilibrated to 5% Buffer B before next loading.
Lyophilzation:
The fractions (purity>90%) were combined and transferred to 1 L lyophilization jars which were deeply frozen by liquid nitrogen. After freezing, the jars were placed onto Lyophilizer (Virtis Freezemobile 35EL) and dried overnight. The vacuum was below 500 mT and chamber temperature was below −60° C. The lyophilisation was completed in 12-18 hrs at room temperature (environment temperature).
Results:
Starting from 0.2 mm synthesis, purification was done in a TFA system and the final yield was 15 mg (2.8%) of product. (ES)+-LCMS m/e calculated (“calcd”) for C130H167N35O22S2 found 2636.1.
The above peptide (SEQ ID NO. 2) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 19 mg (4.0%) of the above peptide. (ES)+-LCMS m/c calculated (“calcd”) for C108H173N35O22S found 2345.84.
The above peptide (SEQ ID NO. 3) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 17 mg (3.3%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C113H162N42O25S found 2540.86.
The above peptide (SEQ ID NO. 4) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 52 mg (8.5%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C146H238N38O31S found 3053.79.
The above peptide (SEQ ID NO. 5) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 75 mg (12.2%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C143H226N42O29S2 found 3061.76.
The above peptide (SEQ ID NO. 6) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 12 mg (2.1%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C134H196N36O30S2 found 2855.38.
The above peptide (SEQ ID NO. 7) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 15 mg (3.1%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C124H179N25S22S found 2404.
The above peptide (SEQ ID NO. 8) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 78 mg (12.5%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C144H242N50O26S found 3121.89.
The above peptide (SEQ ID NO. 9) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.2 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 1 to yield 17 mg (2.6%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C154H231N51O30S found 3308.91.
The above peptide (SEQ ID NO. 10) as conjugated to FITC was synthesized using Fmoc chemistry. The synthesis was carried out on a 0.15 mmole scale using the Fmoc-Linker-Rink amide resin (0.5 g, Sub=0.3 mmol/g). 0.5 g dry resin was placed in a peptide synthesis reactor column (20×150 mm), swelled and washed with DMF. 20% piperidine was then added, agitated for 5 min and drained, then, 20% piperidine was added again, agitated for 7 min, and then the resin was washed with DMF. 0.75 mmol (5 eq) Fmoc-Arg(Pbf)-OH, 0.75 mmol HOBt, 0.75 mmol HBTU, and 0.75 mmol DIPEA were added into the reaction column, and agitated gently for 2 hours with nitrogen. Some resin sample was subjected to a color test, and then the Fmoc group was deprotected. The steps above were repeated until all the amino acids were coupled. At the end of the synthesis, the resin was transferred to a reaction vessel on a shaker for cleavage. The peptide was cleaved from the resin using a 20.0 mL cleavage cocktail (TFA:TIS:H2O:EDT=91:3:3:3(v/v)) for 120 minutes at room temperature avoiding light. The deprotection solution was added to 1000 mL cold Et2O to precipitate the peptide. The peptide was centrifuged in 250 mL polypropylene tubes. The precipitates from the individual tubes were combined in a single tube and washed 3 times with cold Et2O and dried in a desiccator under house vacuum.
The crude material was purified by preparative HPLC on a C18-Column (250×46 mm, 10?m particle size) and eluted with a linear gradient of 5-95% B (buffer A: 0.1% TFA/H2O; buffer B:ACN) in 30 min., with a flow rate 19 mL/min, with detection at 220 nm. The fractions were collected and were checked by analytical HPLC. Fractions containing pure product were combined and lyophilized to a white amorphous powder.
FITC coupling: 0.15 mmol of peptidyl resin was placed in the reaction vessel, followed by addition of 0.165 mmol FITC, with a reagent mixture of Pyridine:DMF:DCM=12:7:5 (V/V). The mixture was reacted for 2 hours in N2. After that, the peptide was cleaved from the resin.
The yield was 80 mg (18%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C132H181N29O21 S2 found 2574.78.
The above peptide (SEQ ID NO. 11) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 90 mg (18%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C129H179N35O25S found 2652.12.
The above peptide (SEQ ID NO. 12) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 50 mg (12%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C122H168N22O24S2 found 2389.5.
The above peptide (SEQ ID NO. 13) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 60 mg (12%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C125H181N39O23 S found 2630.12.
The above peptide (SEQ ID NO. 14) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 55 mg (11%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C123H182N32O23S3 found 2573.6.
The above peptide (SEQ ID NO. 15) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 50 mg (11%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C128H172N28O23S found 2501.7.
The above peptide (SEQ ID NO. 16) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 20 mg (4%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C121H165N27O21S found 2461.6.
The above peptide (SEQ ID NO. 17) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 25 mg (6%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C123H171N27O23S found 2427.8.
The above peptide (SEQ ID NO. 18) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 80 mg (19%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C123H176N26O23S found 2417.8.
The above peptide (SEQ ID NO. 19) as conjugated to FITC was synthesized using Fmoc chemistry. Fmoc Rink Amide MBHA resin (0.15 mmol) was subjected to solid phase synthesis and purification by following the procedure in example 10 to yield 90 mg (21%) of the above peptide. (ES)+-LCMS m/e calculated (“calcd”) for C119H178N32O22 S found 2439.8.
The peptides of Examples 1-19 were tested for cell penetration in H460 and HeLa cell lines as follows.
Materials:
The H460 cell line and HeLa (ATCC) were maintained in growth media then passaged every 2-3 days. Growth media for H460 was RPMI 1640, 10% fetal calf serum, sodium pyruvate, antibiotics and glutamine (GIBCO). Growth media for HeLa cells was DMEM supplemented with 10% heat-inactivated fetal calf serum, antibiotics and glutamine (GIBCO).
Methods and Procedures:
Cells were plated onto Whatman glass-bottom 96-well plates or Perkin Elmer glass-bottom 96-well plates and cultured overnight. Peptide stocks were prepared in DMSO and were diluted in cell growth media for cellular uptake studies. After 2 and 24 h of peptide incubation at various concentrations, media was removed followed by three washes of acidic saline. Formaldehyde fixation, with or without Hoechst 33342 dye solution (to stain nuclei), was followed by PBS washes. Plates were imaged on the Operetta High Content Imaging system in confocal fluorescence mode using the 40× water immersion high NA objective.
The results for the peptides of Examples 1-9 in H460 cells are shown in
Using the method of the present invention, additional peptides were identified that are predicted to be cell-penetrating. For example, the peptides of SEQ ID NOS. 20-455 are peptides wherein PP1<[(PP2*X1)+X], wherein X1 is 1.5 to 10 and X is 0.3 to −1.5, and therefore are predicted to be cell-penetrating. See Table 2.
Table 2 shows the peptides of SEQ ID NOS. 20-455 identified within larger sequences or proteins which are predicted to be cell-penetrating according to the method of the present invention of identifying cell penetrating peptides.
| Number | Date | Country | |
|---|---|---|---|
| 61664210 | Jun 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14410930 | Dec 2014 | US |
| Child | 15625219 | US |
