U.S. patent application number 15/772226 was filed with the patent office on 2019-03-14 for selective mcl-1 binding peptides.
The applicant listed for this patent is MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Amy Keating, Raheleh Rezaei-Araghi.
Application Number | 20190077840 15/772226 |
Document ID | / |
Family ID | 58631249 |
Filed Date | 2019-03-14 |
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United States Patent
Application |
20190077840 |
Kind Code |
A1 |
Rezaei-Araghi; Raheleh ; et
al. |
March 14, 2019 |
SELECTIVE MCL-1 BINDING PEPTIDES
Abstract
Provided herein are peptides that bind Mcl-1. Also provided are
compositions containing these polypeptides and methods of using
such peptides in the treatment of cancer that include administering
to a subject one of the polypeptides.
Inventors: |
Rezaei-Araghi; Raheleh;
(Brookline, MA) ; Keating; Amy; (Arlington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MASSACHUSETTS INSTITUTE OF TECHNOLOGY |
Cambridge |
MA |
US |
|
|
Family ID: |
58631249 |
Appl. No.: |
15/772226 |
Filed: |
October 28, 2016 |
PCT Filed: |
October 28, 2016 |
PCT NO: |
PCT/US2016/059320 |
371 Date: |
April 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62248987 |
Oct 30, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1761 20130101;
A61P 35/00 20180101; C07K 14/62 20130101; C07K 14/4747 20130101;
A61K 38/00 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61P 35/00 20060101 A61P035/00; A61K 38/17 20060101
A61K038/17 |
Claims
1. A compound comprising the amino acid sequence: 1F 1G 2A 2B 2C 2D
2E 2F 2G 3A 3B 3C 3D 3E 3F 3G 4A 4B 4C 4D 4E 4F 4G 5A (SEQ ID NO:
1), wherein 1F is R or a conservative substitution or is missing;
1G is P or a conservative substitution or is missing; 2A is E or a
conservative substitution or is missing; 2B is I or a conservative
substitution; 2C is W or a conservative substitution; 2D is M or a
conservative substitution, or norleucine (B); 2E is T or a
conservative substitution, V or a conservative substitution,
2-aminoisobutyric acid (Aib); 2F is Q or a conservative
substitution; 2G is G or a conservative substitution; 3A is L or a
conservative substitution, F or a conservative substitution,
pentafluoro phenylalanine, cyclohexyl alanine (Cha), or
homo-cyclohexyl alanine (H-Cha); 3B is R or a conservative
substitution, W or a conservative substitution, Q or a conservative
substitution, D or a conservative substitution, Y or a conservative
substitution, Aib, D-phenyl glycine, .alpha.,.alpha.methyl leucine,
.alpha.,.alpha.methyl phenylalanine; 3C is R or a conservative
substitution; 3D is L or a conservative substitution; 3E is G or a
conservative substitution; 3F is D or a conservative substitution;
3G is E or a conservative substitution; 4A is I or a conservative
substitution; 4B is N or a conservative substitution; 4C is A or a
conservative substitution; 4D is Y or a conservative substitution;
4E is Y or a conservative substitution; 4F is A or a conservative
substitution; 4G is R or a conservative substitution or is missing;
5A is R or a conservative or is missing; provided that 2E, 3A, 3B,
4B and 4F are not T, L, R, N and A respectively, and wherein
optionally, the side chains of two amino acids separated by 3 or 6
amino acids are replaced by an intermolecular crosslink.
2. The compound of claim 1, wherein 1F, 1G and 2A are missing.
3. The compound of claim 1, wherein 2D, 4B and 4F are B, N and A
respectively.
4. The compound of claim 3, comprising at least one amino acid that
is not one of the 20 common, naturally-occurring amino acids at a
position selected from the group consisting of 2E, 3A, and 3B,
wherein every position not in that group is a common,
naturally-occurring amino acid.
5. The compound of claim 1, comprising an amino acid sequence
selected from the group consisting of: TABLE-US-00002 (SEQ ID NO:
8) IWBTQGChaRRLGDEINAYYARR, (SEQ ID NO: 9)
IWBTQGH-ChaRRLGDEINAYYARR, (SEQ ID NO: 10) IWBAibQGLRRLGDEINAYYARR.
(SEQ ID NO: 11) IWBAibQGChaRRLGDEINAYYARR, and (SEQ ID NO: 12)
IWBAibQGH-ChaRRLGDEINAYYARR,
wherein up to 3 of the amino acids are substituted by another amino
acid.
6.-19. (canceled)
20. The compound of claim 1, wherein the side chains of two amino
acids separated by 3 or 6 amino acids are substituted by an
intermolecular crosslink.
21.-22. (canceled)
23. The compound of claim 20, wherein the intermolecular crosslink
is an alkylene or alkenylene group.
24.-30. (canceled)
31. The compound of claim 1, comprising an amino acid sequence
selected from the group consisting of: TABLE-US-00003 (SEQ ID NO:
2) IWBTQGLRRLGDEIXAYYXRR, (SEQ ID NO: 3) IWBAibQGLRRLGDEIXAYYXRR,
(SEQ ID NO: 4) IWBAibQGChaRRLGDEIXAYYXRR, (SEQ ID NO: 5)
IWBAibQGLQRLGDEIXAYYXRR, (SEQ ID NO: 6) IWBAibQGLDRLGDEIXAYYXRR,
(SEQ ID NO: 7) IWBTQGLQRLGDEIXAYYXRR,
wherein X is an amino acid whose side chain is replaced with an
intermolecular crosslink to another amino acid and wherein up to 6
of the amino acids are substituted by another amino acid.
32.-48. (canceled)
49. The compound of claim 31, wherein X is .alpha.-4-Pentenyl
alanine.
50. The compound of claim 4, wherein the at least one amino acid
that is not one of the 20 common, naturally-occurring amino acids
is selected from the group consisting of: 4-hydroxyproline,
.alpha.-4-pentenyl alanine, aminoisobutyric acid (Aib), cyclohexyl
alanine (Cha), norleucine, desmosine, gamma-aminobutyric acid,
beta-cyanoalanine, norvaline,
4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,
1-amino-cyclopropanecarboxylic acid,
1-amino-2-phenyl-cyclopropanecarboxylic acid,
1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic
acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,
4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,
2,3-diaminopropionic acid, 2,4-diaminobutyric acid,
2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid,
4-aminobenzoic acid, ortho-, meta-substituted phenylalanines,
para-substituted phenylalanines, disubstituted phenylalanines,
substituted tyrosines and statine.
51.-58. (canceled)
59. The compound of claim 1, wherein at least one peptide bond is
replaced by a bond selected from the group consisting of a
retro-inverso bonds (C(O)--NH); a reduced amide bond
(NH--CH.sub.2); a thiomethylene bond (S--CH.sub.2 or CH.sub.2--S);
an oxomethylene bond (O--CH.sub.2 or CH.sub.2--O); an ethylene bond
(CH.sub.2--CH.sub.2); a thioamide bond (C(S)--NH); a trans-olefin
bond (CH.dbd.CH); a fluoro substituted trans-olefin bond
(CF.dbd.CH); a ketomethylene bond (C(O)--CHR) or CHR--C(O) wherein
R is H or CH.sub.3; and a fluoro-ketomethylene bond (C(O)--CFR or
CFR--C(O) wherein R is H or F or CH.sub.3.
60.-62. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/248,987, filed on Oct. 30, 2015. The entire
contents of the foregoing are incorporated herein by reference.
TECHNICAL FIELD
[0002] This disclosure relates to peptides that bind Mcl-1 and
methods of using such peptides in the treatment of cancer.
BACKGROUND
[0003] Mcl-1 is one of the most frequently amplified genes in
cancers and an important factor in resistance to chemotherapeutic
agents. Mcl-1 is a member of a family of anti-apoptotic proteins
that have homology to Bcl-2 and contain a so-called BH3 domain.
Mcl-1 and others members of the family (e.g., Bcl-xL, Bcl-2, Bcl-w,
Bfl-1 and Bcl-b) block apoptosis by interfering with the
homo-oligomerizing of Bak and Bax. The anti-apoptotic proteins
either bind directly to Bax and Bak or bind related pro-apoptotic
activator proteins (Bim, Bid and Puma), preventing activation of
Bax and Bak. Other proteins having BH3-domain, called sensitizers,
antagonize anti-apoptotic function by binding competitively with
Bax/Bak and activators.
[0004] Agents that selectively bind Mcl-1 compared to other members
of the Bcl-2 family of anti-apoptotic proteins, such as Bcl-xL or
Bcl-2, may be useful in treating a variety of cancers.
SUMMARY
[0005] The present disclosure describes peptides mimicking BH3
motifs that bind human Mcl-1. The peptides are relatively selective
for binding Mcl-1 in that they bind human Mcl-1 with greater
affinity than they bind one or more of several proteins considered
human homologs of Bcl-1, for example, Bfl-1, Bcl-w, Bcl-xL and
Bcl-2.
[0006] In some aspects, the present disclosure provides a compound
comprising, consisting of, or consisting essentially of the amino
acid sequence 1F 1G 2A 2B 2C 2D 2E 2F 2G 3A 3B 3C 3D 3E 3F 3G 4A 4B
4C 4D 4E 4F 4G 5A (SEQ ID NO: 1), wherein 1F is R or a conservative
substitution or is missing; 1G is P or a conservative substitution
or is missing; 2A is E or a conservative substitution or is
missing; 2B is I or a conservative substitution; 2C is W or a
conservative substitution; 2D is M or a conservative substitution,
or norleucine (B); 2E is T or a conservative substitution, V or a
conservative substitution, 2-aminoisobutyric acid (Aib), or X; 2F
is Q or a conservative substitution; 2G is G or a conservative
substitution; 3A is L or a conservative substitution, F or a
conservative substitution, pentafluoro phenylalanine, cyclohexyl
alanine (Cha), or homo-cyclohexyl alanine (H-Cha); 3B is R or a
conservative substitution, W or a conservative substitution, Q or a
conservative substitution, D or a conservative substitution, Y or a
conservative substitution, Aib, D-phenyl glycine,
.alpha.,.alpha.methyl leucine, .alpha.,.alpha.methyl phenylalanine,
or X; 3C is R or a conservative substitution; 3D is L or a
conservative substitution; 3E is G or a conservative substitution;
3F is D or a conservative substitution; 3G is E or a conservative
substitution; 4A is I or a conservative substitution; 4B is N or a
conservative substitution, or X; 4C is A or a conservative
substitution; 4D is Y or a conservative substitution; 4E is Y or a
conservative substitution; 4F is A or a conservative substitution,
or X; 4G is R or a conservative substitution or is missing; 5A is R
or a conservative or is missing; provided that 2E, 3A, 3B, 4B and
4F are not T, L, R, N and A respectively, wherein X is an amino
acid whose side chain is replaced with an intermolecular link to
another amino acid. In some cases 1F, 1G and 2A are missing. In
some cases, 2D, 4B and 4F are B, N and A respectively.
[0007] In some aspects, the present disclosure provides a compound
comprising the amino acid sequence 1F 1G 2A 2B 2C 2D 2E 2F 2G 3A 3B
3C 3D 3E 3F 3G 4A 4B 4C 4D 4E 4F 4G 5A (SEQ ID NO: 1), wherein 1F
is R or a conservative substitution or is missing; 1G is P or a
conservative substitution or is missing; 2A is E or a conservative
substitution or is missing; 2B is I or a conservative substitution;
2C is W or a conservative substitution; 2D is M or a conservative
substitution, or norleucine (B); 2E is T or a conservative
substitution, V or a conservative substitution, 2-aminoisobutyric
acid (Aib), or X; 2F is Q or a conservative substitution; 2G is G
or a conservative substitution; 3A is L or a conservative
substitution, F or a conservative substitution, pentafluoro
phenylalanine, cyclohexyl alanine (Cha), or homo-cyclohexyl alanine
(H-Cha); 3B is R or a conservative substitution, W or a
conservative substitution, Q or a conservative substitution, D or a
conservative substitution, Y or a conservative substitution, Aib,
D-phenyl glycine, .alpha.,.alpha.methyl leucine,
.alpha.,.alpha.methyl phenylalanine, or X; 3C is R or a
conservative substitution; 3D is L or a conservative substitution;
3E is G or a conservative substitution; 3F is D or a conservative
substitution; 3G is E or a conservative substitution; 4A is I or a
conservative substitution; 4B is N or a conservative substitution,
or X; 4C is A or a conservative substitution; 4D is Y or a
conservative substitution; 4E is Y or a conservative substitution;
4F is A or a conservative substitution, or X; 4G is R or a
conservative substitution or is missing; 5A is R or a conservative
or is missing; provided that 2E, 3A, 3B, 4B and 4F are not T, L, R,
N and A respectively, and wherein the side chains of two amino
acids separated by 3 or 6 amino acids are optionally replaced by an
intramolecular cross-link. In some cases 1F, 1G and 2A are missing.
In some cases, 2D, 4B and 4F are B, N and A respectively.
[0008] In some cases the compound comprises at least one amino acid
that is not one of the 20 common, naturally-occurring amino acids
at a position selected from the group consisting of 2E, 3A, and 3B.
For example, an amino acid that is not one of the 20 common,
naturally-occurring amino acids may be 4-hydroxyproline,
.alpha.-4-pentenyl alanine, aminoisobutyric acid (Aib), cyclohexyl
alanine (Cha), norleucine, desmosine, gamma-aminobutyric acid,
beta-cyanoalanine, norvaline,
4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,
1-amino-cyclopropanecarboxylic acid,
1-amino-2-phenyl-cyclopropanecarboxylic acid,
1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic
acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,
4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,
2,3-diaminopropionic acid, 2,4-diaminobutyric acid,
2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid,
4-aminobenzoic acid, ortho-, meta- and/para-substituted
phenylalanines (e.g., substituted with --C(.dbd.O)C6H5; --CF3;
--CN; -halo; --NO2; CH3), disubstituted phenylalanines, substituted
tyrosines (e.g., further substituted with -Q=O)C6H5; --CF3; --CN;
-halo; --NO2; CH3), or statine.
[0009] Additionally, the amino acids can be derivatized to include
amino acid residues that are hydroxylated, phosphorylated,
sulfonated, acylated, and glycosylated.
[0010] In some embodiments, the compound comprises, consists of, or
consists essentially of the amino acid sequence
IWBTQGChaRRLGDEINAYYARR (SEQ ID NO: 8), wherein up to 6 (e.g., 1,
2, 3, 4, 5 or 6) of the amino acids are replaced by another amino
acid. In some cases, no more than 2 of the amino acids are replaced
by another amino acid. In some cases, none of the amino acids are
replaced by another amino acid. In some cases the substitution is a
conservative substitution. In some cases, neither B nor Cha is
substituted.
[0011] In some embodiments, the compound comprises, consists of, or
consists essentially of the amino acid sequence
IWBTQGH-ChaRRLGDEINAYYARR (SEQ ID NO: 9), wherein up to 6 (e.g., 1,
2, 3, 4, 5 or 6) of the amino acids are replaced by another amino
acid. In some cases, no more than 2 of the amino acids are replaced
by another amino acid. In some cases, none of the amino acids are
replaced by another amino acid. In some cases, the substitution is
a conservative substitution. In some cases, neither B nor H-Cha is
substituted
[0012] In some embodiments, the compound comprises, consists of, or
consists essentially of the amino acid sequence
IWBAibQGLRRLGDEINAYYARR (SEQ ID NO: 10), wherein up to 6 (e.g., 1,
2, 3, 4, 5 or 6) of the amino acids are replaced by another amino
acid. In some cases, no more than 2 of the amino acids are replaced
by another amino acid. In some cases, none of the amino acids are
replaced by another amino acid. In some cases, the substitution is
a conservative substitution. In some cases, neither B nor Aib is
substituted.
[0013] In some embodiments, the compound comprises, consists of, or
consists essentially of the amino acid sequence
IWBAibQGChaRRLGDEINAYYARR (SEQ ID NO: 11), wherein up to 6 (e.g.,
1, 2, 3, 4, 5 or 6) of the amino acids are replaced by another
amino acid. In some cases, no more than 2 of the amino acids are
replaced by another amino acid. In some cases, none of the amino
acids are replaced by another amino acid. In some cases, the
substitution is a conservative substitution. In some cases, neither
B nor Cha is substituted.
[0014] In some embodiments, the compound comprises, consists of, or
consists essentially of the amino acid sequence
IWBAibQGH-ChaRRLGDEINAYYARR (SEQ ID NO: 12), wherein up to 6 (e.g.,
1, 2, 3, 4, 5 or 6) of the amino acids are replaced by another
amino acid. In some cases, no more than 2 of the amino acids are
replaced by another amino acid. In some cases, none of the amino
acids are replaced by another amino acid. In some cases, the
substitution is a conservative substitution. In some cases, H-Cha
is not substituted
[0015] In some cases, the side chains of two amino acids separated
by 3 or 6 amino acids are replaced by an intermolecular crosslink.
In some cases, the two amino acids are separated by 3 amino acids.
In some cases, the two amino acids are separated by 6 amino
acids.
[0016] In some cases the intermolecular crosslink is an alkylene or
alkenylene group. In some cases, the intermolecular crosslink is an
alkylene group. In some cases, the alkylene group is C7, C8, C9,
C10, C11, C12 or C13. In some cases, the intermolecular crosslink
is an alkenylene group. In some cases, the alkenylene group is C7,
C8, C9, C10, C11, C12 or C13.
[0017] In some cases, the intermolecular crosslink is a lactam
bridge.
[0018] In some cases, the side chains of 4B and 4F form an
intermolecular crosslink. In some cases, the amino acid at 4B and
the amino acid at 4F are .alpha.-4-Pentenyl alanine.
[0019] In some cases, the compound comprises, consists of, or
consists essentially of the sequence IWBTQGLRRLGDEIXAYYXRR (SEQ ID
NO: 2), wherein X is an amino acid whose side chain is replaced
with an intermolecular link to another amino acid and wherein up to
6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acids are replaced by
another amino acid. In some cases, no more than two of the amino
acids are replaced by another amino acid. In some cases, none of
the amino acids are replaced by another amino acid. In some cases,
the substitution is a conservative substitution. In some cases, B
is not substituted
[0020] In some cases, the compound comprises, consists of, or
consists essentially of the sequence IWBAibQGLRRLGDEIXAYYXRR (SEQ
ID NO: 3), wherein X is an amino acid whose side chain is replaced
with an intermolecular link to another amino acid and wherein up to
6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acids are replaced by
another amino acid. In some cases, no more than two of the amino
acids are replaced by another amino acid. In some cases, the
substitution is a conservative substitution. In some cases, none of
the amino acids are replaced by another amino acid. In some cases,
neither B nor Aib is substituted.
[0021] In some cases, the compound comprises, consists of, or
consists essentially of the sequence IWBAibQGChaRRLGDEIXAYYXRR (SEQ
ID NO: 4), wherein X is an amino acid whose side chain is replaced
with an intermolecular link to another amino acid and wherein up to
6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acids are replaced by
another amino acid. In some cases, no more than two of the amino
acids are replaced by another amino acid. In some cases, none of
the amino acids are replaced by another amino acid. In some cases,
none B, Aib and Char are substituted.
[0022] In some cases, the compound comprises, consists of, or
consists essentially of the sequence IWBAibQGLQRLGDEIXAYYXRR (SEQ
ID NO: 5), wherein X is an amino acid whose side chain is replaced
with an intermolecular link to another amino acid and wherein up to
6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acids are replaced by
another amino acid. In some cases, no more than two of the amino
acids are replaced by another amino acid. In some cases, none of
the amino acids are replaced by another amino acid. In some cases,
the substitution is a conservative substitution. In some cases,
neither B nor Aib is substituted.
[0023] In some cases, the compound comprises, consists of, or
consists essentially of the sequence IWBAibQGLDRLGDEIXAYYXRR (SEQ
ID NO: 6), wherein X is an amino acid whose side chain is replaced
with an intermolecular link to another amino acid and wherein up to
6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acids are replaced by
another amino acid. In some cases, no more than two of the amino
acids are replaced by another amino acid. In some cases, none of
the amino acids are replaced by another amino acid. In some cases,
the substitution is a conservative substitution. In some cases,
neither B nor Aib is substituted.
[0024] In some cases, the compound comprises, consists of, or
consists essentially of the sequence IWBTQGLQRLGDEIXAYYXRR (SEQ ID
NO: 7), wherein X is an amino acid whose side chain is replaced
with an intermolecular link to another amino acid and wherein up to
6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acids are replaced by
another amino acid. In some cases, no more than two of the amino
acids are replaced by another amino acid. In some cases, none of
the amino acids are replaced by another amino acid. In some cases,
the substitution is a conservative substitution. In some cases, B
is not substituted.
[0025] In some cases of the compounds described herein, X is
.alpha.-4-Pentenyl alanine.
[0026] In some cases of the compounds described herein, at least
one amino acid that is not one of the 20 common,
naturally-occurring amino acids is selected from the group
consisting of: 4-hydroxyproline, .alpha.-4-pentenyl alanine,
aminoisobutyric acid (Aib), cyclohexyl alanine (Cha), norleucine,
desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline,
4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,
1-amino-cyclopropanecarboxylic acid,
1-amino-2-phenyl-cyclopropanecarboxylic acid,
1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic
acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,
4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,
2,3-di aminopropionic acid, 2,4-diaminobutyric acid,
2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid,
4-aminobenzoic acid, ortho-, meta-substituted phenylalanines,
para-substituted phenylalanines, disubstituted phenylalanines,
substituted tyrosines and statine.
[0027] In some cases of the compounds described herein, the peptide
includes of no more than 24 amino acids. In some cases the peptide
includes up to 50 amino acids (e.g., 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, etc.). In some cases, the compounds include at least
one amino acid that is not one of the 20 common,
naturally-occurring amino acids.
[0028] In some cases, the compounds described herein also comprise
a detectable label. In some cases, the detectable label is linked
to the peptide.
[0029] In some cases, the compounds described herein also comprise
a moiety linked to the peptide. In some cases, the moiety can be a
functional peptide, polyethylene glycol (PEG), alkyl groups (e.g.,
C1-C20 straight or branched alkyl groups), fatty acid radicals, and
combinations thereof. In some cases, the peptide is linked to
PEG.
[0030] In some cases of the compounds described herein, the peptide
is linked to a second peptide or functional moiety. In some cases
the second peptide or functional moiety modulates the activity of
the compound. For example, in some cases the second peptide or
functional moiety modulates the solubility of the compound,
modulates the stability of the compound, modulates the ability of
the compound to permeabilize a cell, acts to target the compound
within/to the cell, labels the compound, modulates the affinity of
the compound for MCL-1 and/or other members of the family (e.g.,
Bcl-xL, Bcl-2, Bcl-w, Bfl-1 and Bcl-b), modulates the specificity
of the compound for MCL-1 and/or other members of the family (e.g.,
Bcl-xL, Bcl-2, Bcl-w, Bfl-1 and Bcl-b), and/or any combination
thereof. Modulating the activity of the compounds described herein
can be increasing or decreasing the activity.
[0031] In some cases of the compounds described herein, the
peptides are modified. In some cases, the modification is selected
from the group consisting of acetylation, amidation, biotinylation,
cinnamoylation, farnesylation, fluoresceination, formylation,
myristoylation, palmitoylation, phosphorylation, stearoylation,
succinylation, sulfurylation, and combinations thereof.
[0032] In some cases of the compounds described herein, the
peptides include at least one peptide bond that is replaced by a
non-natural peptide bond. In some cases, the peptide bond is
replaced by a bond selected from the group consisting of a
retro-inverso bonds (C(O)--NH); a reduced amide bond (NH--CH2); a
thiomethylene bond (S--CH2 or CH2-S); an oxomethylene bond (O--CH2
or CH2-O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)--NH);
a trans-olefin bond (CH.dbd.CH); a fluoro substituted trans-olefin
bond (CF.dbd.CH); a ketomethylene bond (C(O)--CHR) or CHR--C(O)
wherein R is H or CH3; and a fluoro-ketomethylene bond (C(O)--CFR
or CFR--C(O) wherein R is H or F or CH3.
[0033] In some cases, the compounds described herein also comprise
a carrier protein.
[0034] In some aspects, the present disclosure provides a
pharmaceutical composition comprising the compounds described
herein. In some aspects, the present disclosure provides a method
of treating cancer comprising administering the compound described
herein.
[0035] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0036] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 a-g|Results of circular dichroism, competition
assays, BH3 profiling, and stability assays.
[0038] FIG. 2 a-b|Results of competition fluorescence anisotropy
binding experiments.
[0039] FIG. 3 a-c|Results of BH3 profiling assay of engineered MS1
and native BH3 peptides.
[0040] FIG. 4 a-b|Results of BH3 profiling assay of stapled BH3
peptides.
[0041] FIG. 5|Schematic of one bead one compound library
formation.
[0042] FIG. 6|Sequences of the Mcl-1 binding peptides and
structures of uncommon amino acids.
[0043] FIG. 7|Results of circular dichroism to determine
helicity.
[0044] FIG. 8|Results of competition assays to determine binding
affinity and specificity.
[0045] FIG. 9|Results of BH3 profiling assay of peptides with cell
lines dependent on Mcl-1, Bcl-xL, Bcl-2, or Bfl-1 (1:Aib,
2-aminoisobutyric acid; 2: Cha, cyclohexylalanine; and 3: hCha,
homo-cyclohexylalanine).
DETAILED DESCRIPTION
[0046] The present disclosure provides Mcl-1-binding peptides. In
some cases, the peptides include amino acids other than the 20
common, naturally-occurring amino acids. In some cases the peptides
have an internal (intramolecular) cross-link (or staple) that
replaces the side chains of two amino acids that are separated by 3
or 6 amino acids. In some cases the peptides have more than one
internal cross-link (e.g., the peptides include two staples or a
stitch).
[0047] Amino acids are the building blocks of the peptides herein.
The term "amino acid" refers to a molecule containing both an amino
group and a carboxyl group as well as a side chain. Amino acids
suitable for inclusion in the peptides disclosed herein include,
without limitation, natural alpha-amino acids such as D- and
L-isomers of the 20 common naturally-occurring alpha-amino acids
found in peptides (e.g., Ala (A), Arg (R), Asn (N), Cys (C), Asp
(D), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), leu (L), Lys (K),
Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and
Val (V), uncommon alpha-amino acids (including, but not limited to
.alpha.,.alpha.-disubstituted and N-alkylated amino acids), common
naturally-occurring beta-amino acids (e.g., beta-alanine), and
uncommon beta-amino acids. Amino acids used in the construction of
peptides of the present invention can be prepared by organic
synthesis, or obtained by other routes, such as, for example,
degradation of or isolation from a natural source. There are many
known amino acids beyond the 20 common naturally-occurring amino
acids, any of which may be included in the peptides of the present
invention. Some examples of uncommon amino acids are
4-hydroxyproline, .alpha.-4-pentenyl alanine, aminoisobutyric acid
(Aib), cyclohexyl alanine (Cha), norleucine, desmosine,
gamma-aminobutyric acid, beta-cyanoalanine, norvaline,
4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,
1-amino-cyclopropanecarboxylic acid,
1-amino-2-phenyl-cyclopropanecarboxylic acid,
1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic
acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,
4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,
2,3-diaminopropionic acid, 2,4-diaminobutyric acid,
2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid,
4-aminobenzoic acid, ortho-, meta- and/para-substituted
phenylalanines (e.g., substituted with --C(.dbd.O)C.sub.6H.sub.5;
--CF.sub.3; --CN; -halo; --NO2; CH.sub.3), disubstituted
phenylalanines, substituted tyrosines (e.g., further substituted
with -Q=O)C.sub.6H.sub.5; --CF.sub.3; --CN; -halo; --NO.sub.2;
CH.sub.3), and statine. Additionally, amino acids can be
derivatized to include amino acid residues that are hydroxylated,
phosphorylated, sulfonated, acylated, and glycosylated, to name a
few.
[0048] Useful amino acids include:
##STR00001##
[0049] In some instances, peptides include only common,
naturally-occurring amino acids, although uncommon amino acids
(i.e., compounds that do not commonly occur in nature but that can
be incorporated into a polypeptide chain) and/or amino acid analogs
as are known in the art may alternatively be employed. Also, one or
more of the amino acids in a peptide or polypeptide may be
modified, for example, by the addition of a chemical entity such as
a carbohydrate group, a hydroxyl group, a phosphate group, a
farnesyl group, an isofarnesyl group, a fatty acid group, a linker
for conjugation, functionalization, or other modification, etc.
[0050] In some instances, peptides can include (e.g., comprise,
consist essentially of, or consist of) at least seven (e.g., 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) contiguous
amino acids of any of SEQ ID NOs: 1-12. In some instances, the
peptides can comprise the amino acid sequence of any of SEQ ID NOs:
1-12 with 1, 2, 3, 4, 5 or 6 single amino acid substitutions, e.g.,
conservative substitutions. In some instances, the peptides can
comprise the amino acid sequence of any of SEQ ID NOs: 1-12 and
have the side chains of 2 amino acids separated by 3 or 6 amino
acids substituted by an internal cross-link and, in addition to the
cross-link, include 1, 2, 3, 4, 5 or 6 single amino acid
substitutions, e.g., conservative substitutions. In some instances,
the peptides can comprise the amino acid sequence of any of SEQ ID
NOs: 1-12 and have the side chains of more than 2 amino acids
separated by 3 or 6 amino acids substituted by an internal
cross-link and, in addition to the cross-link, include 1, 2, 3, 4,
5 or 6 single amino acid substitutions, e.g., conservative
substitutions.
[0051] In some instances, a "conservative amino acid substitution"
can include substitutions in which one amino acid residue is
replaced with another naturally-occurring 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., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0052] Methods for determining percent identity between amino acid
sequences are known in the art. For example, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 70%, 80%, 90%, or 100% of the
length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position. The determination of percent
identity between two amino acid sequences is accomplished using the
BLAST 2.0 program. Sequence comparison is performed using an
ungapped alignment and using the default parameters (Blossom 62
matrix, gap existence cost of 11, per residue gapped cost of 1, and
a lambda ratio of 0.85). The mathematical algorithm used in BLAST
programs is described in Altschul et al. (Nucleic Acids Res.
25:3389-3402, 1997).
[0053] As disclosed above, peptides herein include at least two
modified amino acids that together can form an internal
(intramolecular) cross-link (or staple), wherein the at least two
modified amino acids are separated by: (A) three amino acid (i.e.,
i, i+4) or (B) six amino acids (i.e., i, i+7). In the case of a
cross-between i and i+4 the cross-link can be a C8 alkylene or
alkenylene. In the case of a cross-link between i and i+7 the
cross-link can be a C11, C12 or C13 alkylene or alkenylene. When
the cross-link is an alkenylene there can one or more double bonds.
In the case of a cross-link between i and i+4 the cross-link can be
a C8 alkyl or alkene. In the case of a cross-link between i and i+7
the cross-link can be a C11, C12 or C13 alkyl or alkene (e.g., a
C11 alkene having a single double bond). When the cross-link is an
alkene there can be one or more double bonds.
[0054] "Peptide stapling" is a term coined from a synthetic
methodology wherein two olefin-containing side-chains (e.g.,
cross-linkable side chains) present in a polypeptide chain are
covalently joined (e.g., "stapled together") using a ring-closing
metathesis (RCM) reaction to form a cross-linked ring (Blackwell et
al., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int.
Ed. 37:3281, 1994). As used herein, the term "peptide stapling,"
includes the joining of two (e.g., at least one pair of) double
bond-containing side-chains, triple bond-containing side-chains, or
double bond-containing and triple bond-containing side chain, which
may be present in a polypeptide chain, using any number of reaction
conditions and/or catalysts to facilitate such a reaction, to
provide a singly "stapled" polypeptide. The term "multiply stapled"
polypeptides refers to those polypeptides containing more than one
individual staple, and may contain two, three, or more independent
staples of various spacings and compositions. Additionally, the
term "peptide stitching," as used herein, refers to multiple and
tandem "stapling" events in a single polypeptide chain to provide a
"stitched" (e.g., tandem or multiply stapled) polypeptide, in which
two staples, for example, are linked to a common residue. Peptide
stitching is disclosed in WO 2008121767 and in WO 2010/068684,
which are both hereby incorporated by reference. In some instances,
staples, as used herein, can retain the unsaturated bond or can be
reduced (e.g., as mentioned below in the stitching paragraph
description).
[0055] While many peptide staples have all hydrocarbon cross-links,
other type of cross-links or staples can be used. For example,
triazole-containing (e.g., 1, 4 triazole or 1, 5 triazole)
crosslinks can be used (Kawamoto et al. 2012 Journal of Medicinal
Chemistry 55:1137; WO 2010/060112).
[0056] Stapling of a peptide using all-hydrocarbon cross-link has
been shown to help maintain its native conformation and/or
secondary structure, particularly under physiologically relevant
conditions (Schafmiester et al., J. Am. Chem. Soc., 122:5891-5892,
2000; Walensky et al., Science, 305:1466-1470, 2004).
[0057] Stapling the polypeptide herein by an all-hydrocarbon
crosslink predisposed to have an alpha-helical secondary structure
can constrain the polypeptide to its native alpha-helical
conformation. The constrained secondary structure may, for example,
increase the peptide's resistance to proteolytic cleavage, may
increase the peptide's thermal stability, may increase the
peptide's hydrophobicity, may allow for better penetration of the
peptide into the target cell's membrane (e.g., through an
energy-dependent transport mechanism such as pinocytosis), and/or
may lead to an improvement in the peptide's biological activity
relative to the corresponding uncrosslinked (e.g., "unstitched" or
"unstapled") peptide.
[0058] Peptides herein may include at least two internally
cross-linked or stapled amino acids, wherein the at least two amino
acids are separated by three (i.e., i, i+4) or six (i.e., i, i+7)
amino acids. While at least two amino acids are required to support
an internal cross-link (e.g., a staple), additional pairs of
internally cross-linked amino acids can be included in a peptide,
e.g., to support additional internal cross-links (e.g., staples).
For example peptides can include 1, 2 or 3 staples.
[0059] Alternatively or in addition, peptides can include three
internally cross-linked or stitched amino acids, e.g., yielding two
staples arising from a common origin. A peptide stitch includes at
least three internally cross-linked amino acids, wherein the middle
of the three amino acids forms an internal cross-link (between
alpha carbons) with each of the two flanking (not immediately
adjacent) modified amino acids. The alpha carbon of the core amino
acid has side chains that are internal cross-links to the alpha
carbons of other amino acids in the peptide, which can be saturated
or not saturated. Amino acids cross-linked to the core amino acid
can be separated from the core amino acid in either direction by 3
or 6 amino acids. The number of amino acids on either side of the
core (e.g., between the core amino acid and an amino acid
cross-linked to the core) can be the same or different.
[0060] As noted above an internal tether or cross-link can extend
across the length of one helical turn (i.e., about 3.4 amino acids
(i.e., i, i+4) or two helical turns (i.e., about 7 amino acids
(i.e., i, i+7). Accordingly, amino acids positioned at i and i+4;
or i and i+7 are candidates for chemical modification and
cross-linking. Thus, for example, where a peptide has the sequence
. . . X.sub.aa1, X.sub.aa2, X.sub.aa3, X.sub.aa4, X.sub.aa5,
X.sub.aa6, X.sub.aa7, X.sub.aa8, X.sub.aa9 . . . , cross-links
between X.sub.aa1 and X.sub.aa5, or between X.sub.aa1 and X.sub.aa8
are useful as are cross-links between X.sub.aa2 and X.sub.aa6, or
between X.sub.aa2 and X.sub.aa9, etc.
[0061] As disclosed above, peptides herein may include at least one
amino acid that is not one of the 20 common, naturally-occurring
amino acids, wherein every position not in that group is a common,
naturally-occurring amino acid. In the case of at least one amino
acid that is not one of the 20 common, naturally-occurring amino
acids, the peptide may not have an internal cross-link (or staple).
These uncommon amino acids may help maintain the native
conformation and/or secondary structure, particularly under
physiologically relevant conditions.
[0062] In some instances, peptides can include (e.g., comprise,
consist essentially of, or consist of) at least one amino acid,
that is not one of the 20 common, naturally-occurring amino acids,
in the at least seven (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22) contiguous amino acids of any of SEQ ID
Nos: 1-12.
[0063] Peptides can contain one or more asymmetric centers and thus
occur as racemates and racemic mixtures, single enantiomers,
individual diastereomers and diastereomeric mixtures and geometric
isomers (e.g. Z or cis and E or trans) of any olefins present. For
example, peptides disclosed herein can exist in particular
geometric or stereoisomeric forms, including, for example, cis- and
trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,
(L)-isomers, the racemic mixtures thereof, and other mixtures
thereof. Enantiomers can be free (e.g., substantially free) of
their corresponding enantiomer, and/or may also be optically
enriched. "Optically enriched," as used herein, means that the
compound is made up of a significantly greater proportion of one
enantiomer. In certain embodiments substantially free means that a
composition contains at least about 90% by weight of a preferred
enantiomer. In other embodiments the compound is made up of at
least about 95%, 98%, or 99% by weight of a preferred enantiomer.
Preferred enantiomers may be isolated from racemic mixtures using
techniques known in the art, including, but not limited to, for
example, chiral high pressure liquid chromatography (HPLC) and the
formation and crystallization of chiral salts or prepared by
asymmetric syntheses (see, e.g., Jacques, et al, Enantiomers,
Racemates and Resolutions (Wiley Interscience, New York, 1981);
Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E X.
Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962);
Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p.
268 (EX. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind.
1972). All such isomeric forms of these compounds are expressly
included in the present invention.
[0064] Peptides can also be represented in multiple tautomeric
forms, in such instances, the invention expressly includes all
tautomeric forms of the compounds described herein (e.g., isomers
in equilibrium (e.g., keto-enol), wherein alkylation at multiple
sites can yield regioisomers), regioisomers, and oxidation products
of the compounds disclosed herein (the invention expressly includes
all such reaction products). All such isomeric forms of such
compounds are included as are all crystal forms.
[0065] In some instances, the hydrocarbon tethers (i.e., cross
links) described herein can be further manipulated. In one
instance, a double bond of a hydrocarbon alkenyl tether, (e.g., as
synthesized using a ruthenium-catalyzed ring closing metathesis
(RCM)) can be oxidized (e.g., via epoxidation or dihydroxylation)
to provide one of compounds below.
##STR00002##
[0066] Either the epoxide moiety or one of the free hydroxyl
moieties can be further functionalized. For example, the epoxide
can be treated with a nucleophile, which provides additional
functionality that can be used, for example, to attach a tag (e.g.,
a radioisotope or fluorescent tag). The tag can be used to help
direct the compound to a desired location in the body or track the
location of the compound in the body. Alternatively, an additional
therapeutic agent can be chemically attached to the functionalized
tether (e.g., an anti-cancer agent such as rapamycin, vinblastine,
taxol, etc.). Such derivatization can alternatively be achieved by
synthetic manipulation of the amino or carboxy-terminus of the
polypeptide or via the amino acid side chain. Other agents can be
attached to the functionalized tether, e.g., an agent that
facilitates entry of the polypeptide into cells.
[0067] While hydrocarbon tethers have been described, other tethers
are also envisioned. For example, the tether can include one or
more of an ether, thioether, ester, amine, or amide moiety. In some
cases, a common, naturally-occurring amino acid side chain can be
incorporated into the tether. For example, a tether can be coupled
with a functional group such as the hydroxyl in serine, the thiol
in cysteine, the primary amine in lysine, the acid in aspartate or
glutamate, or the amide in asparagine or glutamine. Accordingly, it
is possible to create a tether using common, naturally-occurring
amino acids rather than using a tether that is made by coupling two
uncommon amino acids. It is also possible to use a single uncommon
amino acid together with a common, naturally-occurring amino
acid.
[0068] It is further envisioned that the length of the tether can
be varied. For instance, a shorter length of tether can be used
where it is desirable to provide a relatively high degree of
constraint on the secondary alpha-helical structure, whereas, in
some instances, it is desirable to provide less constraint on the
secondary alpha-helical structure, and thus a longer tether may be
desired.
[0069] Additionally, while examples of tethers spanning from amino
acids i to i+3, i to i+4; and i to i+7 have been described in order
to provide a tether that is primarily on a single face of the alpha
helix, the tethers can be synthesized to span any combinations of
numbers of amino acids.
[0070] In some instances, alpha disubstituted amino acids are used
in the polypeptide to improve the stability of the alpha helical
secondary structure. However, alpha disubstituted amino acids are
not required, and instances using mono-alpha substituents (e.g., in
the tethered amino acids) are also envisioned.
[0071] The stapled polypeptides can include a drug, a toxin, a
derivative of polyethylene glycol; a second polypeptide; a
carbohydrate, etc. Whcrc a polymer or other agent is linked to the
stapled polypeptide is can be desirable for the composition to be
substantially homogeneous.
[0072] The addition of polyethelene glycol (PEG) molecules can
improve the pharmacokinetic and pharmacodynamic properties of the
polypeptide. For example, PEGylation can reduce renal clearance and
can result in a more stable plasma concentration. PEG is a water
soluble polymer and can be represented as linked to the polypeptide
as formula:
[0073] XO--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--Y where n
is 2 to 10,000 and X is H or a terminal modification, e.g., a
C.sub.1-4 alkyl; and Y is an amide, carbamate or urea linkage to an
amine group (including but not limited to, the epsilon amine of
lysine or the N-terminus) of the polypeptide. Y may also be a
maleimide linkage to a thiol group (including but not limited to,
the thiol group of cysteine). Other methods for linking PEG to a
polypeptide, directly or indirectly, are known to those of ordinary
skill in the art. The PEG can be linear or branched. Various forms
of PEG including various functionalized derivatives are
commercially available.
[0074] PEG having degradable linkages in the backbone can be used.
For example, PEG can be prepared with ester linkages that are
subject to hydrolysis. Conjugates having degradable PEG linkages
are described in WO 99/34833; WO 99/14259, and U.S. Pat. No.
6,348,558.
[0075] In certain embodiments, macromolecular polymer (e.g., PEG)
is attached to an agent described herein through an intermediate
linker. In certain embodiments, the linker is made up of from 1 to
20 amino acids linked by peptide bonds, wherein the amino acids are
selected from the 20 common, naturally-occurring amino acids. Some
of these amino acids may be glycosylated, as is well understood by
those in the art. In other embodiments, the 1 to 20 amino acids are
selected from glycine, alanine, proline, asparagine, glutamine, and
lysine. In other embodiments, a linker is made up of a majority of
amino acids that are sterically unhindered, such as glycine and
alanine. Non-peptide linkers are also possible. For example, alkyl
linkers such as --NH(CH.sub.2).sub.nC(O)--, wherein n=2-20 can be
used. These alkyl linkers may further be substituted by any
non-sterically hindering group such as lower alkyl (e.g.,
C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br), CN, NH.sub.2,
phenyl, etc. U.S. Pat. No. 5,446,090 describes a bifunctional PEG
linker and its use in forming conjugates having a peptide at each
of the PEG linker termini.
[0076] The stapled and non-stapled peptides can also be modified,
e.g., to further facilitate cellular uptake or increase in vivo
stability, in some embodiments. For example, acylating 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.
[0077] In some embodiments, the peptides disclosed herein have an
enhanced ability to penetrate cell membranes.
[0078] Methods of synthesizing the compounds of the described
herein are known in the art. Nevertheless, the following exemplary
method may be used. It will be appreciated that the various steps
may be performed in an alternate sequence or order to give the
desired compounds. Synthetic chemistry transformations and
protecting group methodologies (protection and deprotection) useful
in synthesizing the compounds described herein are known in the art
and include, for example, those such as described in R. Larock,
Comprehensive Organic Transformations, VCH Publishers (1989); T. W.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,
3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser,
Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and
Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for
Organic Synthesis, John Wiley and Sons (1995), and subsequent
editions thereof.
[0079] The peptides of this invention can be made by chemical
synthesis methods, which are well known to the ordinarily skilled
artisan. See, for example, Fields et al., Chapter 3 in Synthetic
Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New
York, N.Y., 1992, p. 77. Hence, peptides can be synthesized using
the automated Merrifield techniques of solid phase synthesis with
the .alpha.-NH.sub.2 protected by either t-Boc or Fmoc chemistry
using side chain protected amino acids on, for example, an Applied
Biosystems Peptide Synthesizer Model 430A or 431.
[0080] One manner of making of the peptides described herein is
using solid phase peptide synthesis (SPPS). The C-terminal amino
acid is attached to a cross-linked polystyrene resin via an acid
labile bond with a linker molecule. This resin is insoluble in the
solvents used for synthesis, making it relatively simple and fast
to wash away excess reagents and by-products. The N-terminus is
protected with the Fmoc group, which is stable in acid, but
removable by base. Any side chain functional groups are protected
with base stable, acid labile groups.
[0081] Longer peptides could be made by conjoining individual
synthetic peptides using native chemical ligation. Alternatively,
the longer synthetic peptides can be synthesized by well-known
recombinant DNA techniques. Such techniques are provided in
well-known standard manuals with detailed protocols. To construct a
gene encoding a peptide of this invention, the amino acid sequence
is reverse translated to obtain a nucleic acid sequence encoding
the amino acid sequence, preferably with codons that are optimum
for the organism in which the gene is to be expressed. Next, a
synthetic gene is made, typically by synthesizing oligonucleotides
which encode the peptide and any regulatory elements, if necessary.
The synthetic gene is inserted in a suitable cloning vector and
transfected into a host cell. The peptide is then expressed under
suitable conditions appropriate for the selected expression system
and host. The peptide is purified and characterized by standard
methods.
[0082] The peptides can be made in a high-throughput, combinatorial
fashion, e.g., using a high-throughput multiple channel
combinatorial synthesizer available from Advanced Chemtech.
[0083] Peptide bonds can be replaced, e.g., to increase
physiological stability of the peptide, by: a retro-inverso bonds
(C(O)--NH); a reduced amide bond (NH--CH.sub.2); a thiomethylene
bond (S--CH.sub.2 or CH.sub.2--S); an oxomethylene bond
(O--CH.sub.2 or CH.sub.2--O); an ethylene bond
(CH.sub.2--CH.sub.2); a thioamide bond (C(S)--NH); a trans-olefin
bond (CH.dbd.CH); a fluoro substituted trans-olefin bond
(CF.dbd.CH); a ketomethylene bond (C(O)--CHR) or CHR--C(O) wherein
R is H or CH.sub.3; and a fluoro-ketomethylene bond (C(O)--CFR or
CFR--C(O) wherein R is H or F or CH.sub.3.
[0084] The polypeptides can be further modified by: acetylation,
amidation, biotinylation, cinnamoylation, farnesylation,
fluoresceination, formylation, myristoylation, palmitoylation,
phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and
sulfurylation. As indicated above, peptides can be conjugated to,
for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20
straight or branched alkyl groups); fatty acid radicals; and
combinations thereof.
[0085] .alpha.,.alpha.-Disubstituted amino acids containing
olefinic side chains of varying length can be synthesized by known
methods (Williams et al. J. Am. Chem. Soc., 113:9276, 1991;
Schafmeister et al., J. Am. Chem Soc., 122:5891, 2000; and Bird et
al., Methods Enzymol., 446:369, 2008; Bird et al, Current Protocols
in Chemical Biology, 2011). For peptides where an i linked to i+7
staple is used (two turns of the helix stabilized) either one S5
amino acid and one R8 is used or one S8 amino acid and one R5 amino
acid is used. R8 is synthesized using the same route, except that
the starting chiral auxiliary confers the R-alkyl-stereoisomer.
Also, 8-iodooctene is used in place of 5-iodopentene. Inhibitors
are synthesized on a solid support using solid-phase peptide
synthesis (SPPS) on MBHA resin (see, e.g., WO 2010/148335).
[0086] Fmoc-protected .alpha.-amino acids (other than the olefinic
amino acids Fmoc-S.sub.5--OH, Fmoc-R.sub.8--OH, Fmoc-R.sub.8--OH,
Fmoc-S.sub.8--OH and Fmoc-R.sub.5--OH),
2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially
available from, e.g., Novabiochem (San Diego, Calif.).
Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP),
N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA),
1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and
piperidine are commercially available from, e.g., Sigma-Aldrich.
Olefinic amino acid synthesis is reported in the art (Williams et
al., Org. Synth., 80:31, 2003).
[0087] In some instances, peptides can include a detectable label.
As used herein, a "label" refers to a moiety that has at least one
element, isotope, or functional group incorporated into the moiety
which enables detection of the peptide to which the label is
attached. Labels can be directly attached (ie, via a bond) or can
be attached by a linker (e.g., such as, for example, a cyclic or
acyclic, branched or unbranched, substituted or unsubstituted
alkylene; cyclic or acyclic, branched or unbranched, substituted or
unsubstituted alkenylene; cyclic or acyclic, branched or
unbranched, substituted or unsubstituted alkynylene; cyclic or
acyclic, branched or unbranched, substituted or unsubstituted
heteroalkylene; cyclic or acyclic, branched or unbranched,
substituted or unsubstituted heteroalkenylene; cyclic or acyclic,
branched or unbranched, substituted or unsubstituted
heteroalkynylene; substituted or unsubstituted arylene; substituted
or unsubstituted heteroarylene; or substituted or unsubstituted
acylene, or any combination thereof, which can make up a linker).
Labels can be attached to a peptide at any position that does not
interfere with the biological activity or characteristic of the
inventive polypeptide that is being detected.
[0088] Labels can include: labels that contain isotopic moieties,
which may be radioactive or heavy isotopes, including, but not
limited to, .sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N,
.sup.31P, .sup.32P, .sup.35S, .sup.67Ga, .sup.99mTc (Tc-99m),
.sup.111In, .sup.123I, .sup.125I, .sup.169Yb, and .sup.186Re;
labels that include immune or immunoreactive moieties, which may be
antibodies or antigens, which may be bound to enzymes {e.g., such
as horseradish peroxidase); labels that are colored, luminescent,
phosphorescent, or include fluorescent moieties (e.g., such as the
fluorescent label FITC); labels that have one or more photoaffinity
moieties; labels that have ligand moieties with one or more known
binding partners (such as biotin-streptavidin, FK506-FKBP,
etc.).
[0089] In some instances, labels can include one or more
photoaffinity moieties for the direct elucidation of intermolecular
interactions in biological systems. A variety of known photophores
can be employed, most relying on photoconversion of diazo
compounds, azides, or diazirines to nitrenes or carbenes (see,
e.g., Bayley, H., Photogenerated Reagents in Biochemistry and
Molecular Biology (1983), Elsevier, Amsterdam, the entire contents
of which are incorporated herein by reference). In certain
embodiments of the invention, the photoaffinity labels employed are
o-, m- and p-azidobenzoyls, substituted with one or more halogen
moieties, including, but not limited to
4-azido-2,3,5,6-tetrafluorobenzoic acid.
[0090] Labels can also be or can serve as imaging agents. Exemplary
imaging agents include, but are not limited to, those used in
positron emissions tomography (PET), computer assisted tomography
(CAT), single photon emission computerized tomography, x-ray,
fluoroscopy, and magnetic resonance imaging (MRI); anti-emetics;
and contrast agents. Exemplary diagnostic agents include but are
not limited to, fluorescent moieties, luminescent moieties,
magnetic moieties; gadolinium chelates (e.g., gadolinium chelates
with DTPA, DTPA-BMA, DOTA and HP-DO3A), iron chelates, magnesium
chelates, manganese chelates, copper chelates, chromium chelates,
iodine-based materials useful for CAT and x-ray imaging, and
radionuclides. Suitable radionuclides include, but are not limited
to, .sup.123I, .sup.125I, .sup.130I, .sup.131I, .sup.133I,
.sup.135I, .sup.47Sc, .sup.72As, .sup.72Se, .sup.90Y, .sup.88Y,
.sup.97Ru, .sup.100Pd, .sup.101mRh, .sup.119Sb, .sup.128Ba,
.sup.197Hg, .sup.211At, .sup.212Bi, .sup.212Pb, .sup.109Pd,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.75Br, .sup.77Br,
.sup.99mTc, .sup.14C, .sup.13N, .sup.15O, .sup.32P, .sup.33P, and
.sup.18F.
[0091] Fluorescent and luminescent moieties include, but are not
limited to, a variety of different organic or inorganic small
molecules commonly referred to as "dyes," "labels," or
"indicators." Examples include, but are not limited to,
fluorescein, rhodamine, acridine dyes, Alexa dyes, cyanine dyes,
etc. Fluorescent and luminescent moieties may include a variety of
naturally-occurring proteins and derivatives thereof, e.g.,
genetically engineered variants. For example, fluorescent proteins
include green fluorescent protein (GFP), enhanced GFP, red, blue,
yellow, cyan, and sapphire fluorescent proteins, reef coral
fluorescent protein, etc. Luminescent proteins include luciferase,
aequorin and derivatives thereof. Numerous fluorescent and
luminescent dyes and proteins are known in the art (see, e.g., U.S.
Patent Publication 2004/0067503; Valeur, B., "Molecular
Fluorescence: Principles and Applications," John Wiley and Sons,
2002; and Handbook of Fluorescent Probes and Research Products,
Molecular Probes, 9th edition, 2002).
[0092] Again, methods suitable for obtaining (e.g., synthesizing),
stapling, and purifying the peptides disclosed herein are also
known in the art (see, e.g., Bird et. al., Methods in Enzymol.,
446:369-386 (2008); Bird et al, Current Protocols in Chemical
Biology, 2011; Walensky et al., Science, 305:1466-1470 (2004);
Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000); U.S.
patent application Ser. No. 12/525,123, filed Mar. 18, 2010; and
U.S. Pat. No. 7,723,468, issued May 25, 2010, each of which are
hereby incorporated by reference in their entirety).
[0093] In some embodiments, the peptides are substantially free of
contaminants or are isolated. Methods for purifying peptides
include, for example, synthesizing the peptide on a solid-phase
support. Following cyclization, the solid-phase support may be
isolated and suspended in a solution of a solvent such as DMSO,
DMSO/dichloromethane mixture, or DMSO/NMP mixture. The
DMSO/dichloromethane or DMSO/NMP mixture may comprise about 30%,
40%, 50% or 60% DMSO. In a specific embodiment, a 50%/50% DMSO/NMP
solution is used. The solution may be incubated for a period of 1,
6, 12 or 24 hours, following which the resin may be washed, for
example with dichloromethane or NMP. In one embodiment, the resin
is washed with NMP. Shaking and bubbling an inert gas into the
solution may be performed.
Pharmaceutical Compositions
[0094] One or more of the peptides disclosed herein (e.g., one or
more of SEQ ID NOs: 1-12) can be formulated for use as or in
pharmaceutical compositions. Such compositions can be formulated or
adapted for administration to a subject via any route, e.g., any
route approved by the Food and Drug Administration (FDA). Exemplary
methods are described in the FDA's CDER Data Standards Manual,
version number 004 (which is available at
fda.give/cder/dsm/DRG/drg00301.htm). For example, compositions can
be formulated or adapted for administration by inhalation (e.g.,
oral and/or nasal inhalation (e.g., via nebulizer or spray)),
injection (e.g., intravenously, intra-arterial, subdermally,
intraperitoneally, intramuscularly, and/or subcutaneously); and/or
for oral administration, transmucosal administration, and/or
topical administration (including topical (e.g., nasal) sprays
and/or solutions). In some instances, pharmaceutical compositions
can include an effective amount of one or more peptides. The terms
"effective amount" and "effective to treat," as used herein, refer
to an amount or a concentration of one or more compounds or a
pharmaceutical composition described herein utilized for a period
of time (including acute or chronic administration and periodic or
continuous administration) that is effective within the context of
its administration for causing an intended effect or physiological
outcome (e.g., treatment of cancer).
[0095] Pharmaceutical compositions of this invention can include
one or more peptides and any pharmaceutically acceptable carrier
and/or vehicle. In some instances, pharmaceuticals can further
include one or more additional therapeutic agents in amounts
effective for achieving a modulation of disease or disease
symptoms.
[0096] The term "pharmaceutically acceptable carrier or adjuvant"
refers to a carrier or adjuvant that may be administered to a
patient, together with a compound of this invention, and which does
not destroy the pharmacological activity thereof and is nontoxic
when administered in doses sufficient to deliver a therapeutic
amount of the compound.
[0097] Pharmaceutically acceptable carriers, adjuvants and vehicles
that may be used in the pharmaceutical compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, self-emulsifying drug delivery systems
(SEDDS) such as d-.alpha.-tocopherol polyethyleneglycol 1000
succinate, surfactants used in pharmaceutical dosage forms such as
Tweens or other similar polymeric delivery matrices, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat. Cyclodextrins such as .alpha.-, .beta.-, and
.gamma.-cyclodextrin, may also be advantageously used to enhance
delivery of compounds of the formulae described herein.
[0098] The pharmaceutical compositions of this invention may
contain any conventional non-toxic pharmaceutically-acceptable
carriers, adjuvants or vehicles. In some cases, the pH of the
formulation may be adjusted with pharmaceutically acceptable acids,
bases or buffers to enhance the stability of the formulated
compound or its delivery form. The term parenteral as used herein
includes subcutaneous, intra-cutaneous, intra-venous,
intra-muscular, intra-articular, intra-arterial, intra-synovial,
intra-sternal, intra-thecal, intra-lesional and intra-cranial
injection or infusion techniques.
[0099] Pharmaceutical compositions can be in the form of a solution
or powder for inhalation and/or nasal administration. Such
compositions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents (such as, for
example, Tween 80) and suspending agents. The sterile injectable
preparation may also be a sterile injectable solution or suspension
in a non-toxic parenterally acceptable diluent or solvent, for
example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are mannitol, water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose, any bland fixed oil
may be employed including synthetic mono- or diglycerides. Fatty
acids, such as oleic acid and its glyceride derivatives are useful
in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, or carboxymethyl cellulose or similar dispersing agents
which are commonly used in the formulation of pharmaceutically
acceptable dosage forms such as emulsions and or suspensions. Other
commonly used surfactants such as Tweens or Spans and/or other
similar emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0100] Pharmaceutical compositions can be orally administered in
any orally acceptable dosage form including, but not limited to,
capsules, tablets, emulsions and aqueous suspensions, dispersions
and solutions. In the case of tablets for oral use, carriers which
are commonly used include lactose and corn starch. Lubricating
agents, such as magnesium stearate, are also typically added. For
oral administration in a capsule form, useful diluents include
lactose and dried corn starch. When aqueous suspensions and/or
emulsions are administered orally, the active ingredient may be
suspended or dissolved in an oily phase is combined with
emulsifying and/or suspending agents. If desired, certain
sweetening and/or flavoring and/or coloring agents may be
added.
[0101] Alternatively or in addition, pharmaceutical compositions
can be administered by nasal aerosol or inhalation. Such
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and may be prepared as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other solubilizing or dispersing agents known
in the art.
[0102] In some embodiments, the present disclosure provides methods
for using any one or more of the peptides or pharmaceutical
compositions (indicated below as `X`) disclosed herein in the
following methods:
[0103] Substance X for use as a medicament in the treatment of one
or more diseases or conditions disclosed herein (e.g., cancer,
referred to in the following examples as `Y`). Use of substance X
for the manufacture of a medicament for the treatment of Y; and
substance X for use in the treatment of Y.
[0104] In some instances, one or more peptides disclosed herein can
be conjugated, for example, to a carrier protein. Such conjugated
compositions can be monovalent or multivalent. For example,
conjugated compositions can include one peptide disclosed herein
conjugated to a carrier protein. Alternatively, conjugated
compositions can include two or more peptides disclosed herein
conjugated to a carrier.
[0105] As used herein, when two entities are "conjugated" to one
another they are linked by a direct or indirect covalent or
non-covalent interaction. In certain embodiments, the association
is covalent. In other embodiments, the association is non-covalent.
Non-covalent interactions include hydrogen bonding, van der Waals
interactions, hydrophobic interactions, magnetic interactions,
electrostatic interactions, etc. An indirect covalent interaction
is when two entities are covalently connected, optionally through a
linker group.
[0106] Carrier proteins can include any protein that increases or
enhances immunogenicity in a subject. Exemplary carrier proteins
are described in the art (see, e.g., Fattom et al., Infect. Immun.,
58:2309-2312, 1990; Devi et al., Proc. Natl. Acad. Sci. USA
88:7175-7179, 1991; Li et al., Infect. Immun. 57:3823-3827, 1989;
Szu et al., Infect. Immun. 59:4555-4561, 1991; Szu et al., J. Exp.
Med. 166:1510-1524, 1987; and Szu et al., Infect. Immun.
62:4440-4444, 1994). Polymeric carriers can be a natural or a
synthetic material containing one or more primary and/or secondary
amino groups, azido groups, or carboxyl groups. Carriers can be
water soluble.
Methods of Treatment
[0107] The disclosure includes methods of using the peptides herein
for the prophylaxis and/or treatment of cancer. The terms "treat"
or "treating," as used herein, refers to partially or completely
alleviating, inhibiting, ameliorating, and/or relieving the disease
or condition from which the subject is suffering.
[0108] In general, methods include selecting a subject and
administering to the subject an effective amount (a therapeutically
effective amount) of one or more of the peptides herein, e.g., in
or as a pharmaceutical composition, and optionally repeating
administration as required for the prophylaxis or treatment of a
cancer.
[0109] Specific dosage and treatment regimens for any particular
patient will depend upon a variety of factors, including the
activity of the specific compound employed, the age, body weight,
general health status, sex, diet, time of administration, rate of
excretion, drug combination, the severity and course of the
disease, condition or symptoms, the patient's disposition to the
disease, condition or symptoms, and the judgment of the treating
physician.
EXAMPLES
[0110] Described below are studies characterizing variants of MS-1,
a previously described peptide that binds Mcl-1 with increased
specificity and affinity relative to Bfl-1, Bcl-xL, Bcl-2, and
Bcl-w (Foight et al. ACS Chem. Biol. 2014, 9, 1962-1968). The
variants were characterized for secondary structure, specificity
for binding to Mcl-1, affinity of binding to Mcl-1, peptide
stability, and functionality in cells. Further, a library was
created as a tool to discover novel BH3-like peptides.
Example 1: Characterization of Stapled Variants of MS1
[0111] To improve the binding and helicity of MS1 while minimizing
its molecular weight, we truncated the peptide and generated a
stapled variant by replacing Asn at 4b and Ala at 4f with amino
acids containing olefin tethers and performing ruthenium-catalyzed
olefin metathesis to yield M1d (see Table 1 for sequences and
position notation). Introduction of the hydrocarbon staple at this
position of the BH3 domain, which we call site "d" in keeping with
prior work, has been shown to promote binding to Mcl-1 (Stewart, M.
L.; Fire, E.; Keating, A. E.; Walensky, L. D. Nat Chem Biol 2010, 6
(8), 595). The crystal structure of a peptide stapled at position d
shows direct hydrophobic contacts made between the staple atoms and
the edge of the Mcl-1 binding groove.
[0112] The secondary structure and binding of M1d were evaluated in
solution using fluorescence polarization assays and circular
dichroism (FIGS. 1a and c). M1d is .about.3-fold more helical in
solution than MS1. The hydrocarbon stapling strategy also improved
Mcl-1-binding affinity and increased peptide resistance to
proteolysis, in comparison to MS1 (FIGS. 1a and d). Although M1d is
able to compete effectively with a peptide corresponding to the BH3
region of Bim, which is a native partner for Mcl-1, structural
modeling suggested that M1d may not be maximally exploiting the
available binding opportunities at three helix positions: 2e, 3a
and 3b. Furthermore, modifying a peptide by introducing a staple
changes its properties, such that re-optimization of the sequence
may be beneficial. However, the virtually unlimited sequence
variations that could be studied present a challenge. To date,
hydrocarbon stapled peptides have typically been rationally
optimized by iterative mutagenesis. Obtaining tight-binding and
biologically active molecules often requires many rounds of
laborious synthesis and stapling of different peptidic
candidates.
[0113] The binding curves from representative experiments are shown
in FIG. 1. Competition assay of stapled peptides with fluorescently
labeled 21mer Bim-BH3 was performed to measure the binding to Mcl-1
(see FIG. 1 (a)). The standard errors reported are over three
experiments. FIG. 1 (b) shows the kinetic analysis of M1d, M2d and
M3d binding to Mcl-1, using biolayer interferometry. FIG. 1 (c)
shows the circular dichroism analysis of unmodified and stapled
variants, in 20 mM tris buffer, pH 7.4. HPLC was used to measure
the half-lives of unstapled and stapled peptides exposed to
chymotrypsin and the half-lives are shown in FIG. 1 (d). BH3
profiling of stapled peptides M1d, M2d, and M3d in Mcl-1 2640 cells
are shown in FIG. 1 (e). Structural overlay of structures of Mcl-1
bound to SAHBd (yellow, PDB 3MK8) and M3d (magenta, model based on
3MK8) can be seen in FIG. 1 (f) and the overlaid structures of MS1
(yellow, model based on 3MK8) and M3d (magenta, also a model based
on 3MK8) comparing position 2e is seen in FIG. 1 (g).
TABLE-US-00001 TABLE 1 Mcl-1 binding peptide 1F 1G 2A 2B 2C 2D 2E
2F 2G 3A 3B 3C 3D 3E 3F 3G 4A 4B 4C 4D 4E 4F 4G 5A MS1 R P E I W M
T Q G L R R L G D E I N A Y Y A R M1d -- -- -- I W B X Q G L X R L
G D E I N A Y Y A R R SEQ ID NO: 2 M2d -- -- -- I W B Aib Q G L R R
L G D E I X A Y Y X R R SEQ ID NO: 3 M3d -- -- -- I W B Aib Q G Cha
R R L G D E I X A Y Y X R R SEQ ID NO: 4 M4d -- -- -- I W B Aib Q G
L Q R L G D E I X A Y Y X R R SEQ ID NO: 5 M5d -- -- -- I W B Aib Q
G L D R L G D E I X A Y Y X R R SEQ ID NO: 6 M6d -- -- -- I W B T Q
G L Q R L G D E I X A Y Y X R R SEQ ID NO: 7 M1 -- -- -- I W B T Q
G L R R L G D E I N A Y Y A R R Ma-Leu3aCha -- -- -- I W B T Q G
Cha R R L G D E I N A Y Y A R R SEQ ID NO: 8 Ma-Leu3aHCha -- -- --
I W B T Q G H-Cha R R L G D E I N A Y Y A R R SEQ ID NO: 9
M1-Thr2eAib -- -- -- I W B Aib Q G L R R L G D E I N A Y Y A R R
SEQ ID NO: 10 M1-Leu3aCha/Thr2eAib -- -- -- I W B Aib Q G Cha R R L
G D E I N A Y Y A R R SEQ ID NO: 11 M1-Leu3a HCha/Thr2eAib -- -- --
I W B Aib Q G H-Cha R R L G D E I N A Y Y A R R SEQ ID NO: 12 Table
1. X = an amino acid whose side chain is replaced with an
intermolecular link to another amino acid (e.g., .alpha.-4-pentenyl
alanine), B = Norleucine, Aib = Aminoisobutyric acid, Cha =
cyclohexyl alanine, and H-Cha = homo cyclohexyl alanine.
Example 2: Identification of Novel Variants Using One Bead One
Compound Libraries
[0114] To accelerate the discovery of therapeutic peptides, we
extended the application of one-bead-one-compound libraries (Xiao,
W.; Bononi, F. C.; Townsend, J.; Li, Y.; Liu, R.; Lam, K. S. Comb.
Chem. High Throughput Screen. 2013, 16 (6), 441) by applying
on-bead ring-closing metathesis (RCM) to make libraries of BH3-like
stapled peptides (FIG. 5). Installation of a staple into a peptide
requires incorporation of two appropriately spaced
.alpha.-methyl-.alpha.-alkenyl residues, with defined
stereochemical configuration and alkene chain length, followed by
RCM on the resin (Kim, Y. W.; Grossman, T. N.; Verdine, G. L. Nat.
Protoc. 2011, 6 (6), 761). To evaluate conditions that would
produce a high-quality stapled peptide library suitable for
analysis without purification, and to establish an efficient
on-bead screening procedure, we synthesized a first-generation
stapled library based on the sequence of Bim BH3 (library LA). This
library incorporated a number of mutations that have been studied
previously to serve as controls. The library was synthesized on
Tentagel macrobeads and screened for binding to Mcl-1 and
Bcl-x.sub.L. Bcl-x.sub.L is a paralog of Mcl-1 and an undesired
interaction target for candidate inhibitors. The screen entailed
bead blocking, incubation with biotinylated Mcl-1 (.sup.BioMcl-1),
washing, incubation with streptavidin-coated quantum dots
(Qdots-SA605), and visualization using a fluorescence microscope.
173 hit peptides, which were selected manually under the
microscope, were cleaved from the beads and identified using mass
spectrometry. Both the stapling chemistry and subsequent tests for
binding were carried out with peptides bound to resin, with one
unique peptide per bead. Selected hit peptides were confirmed to
bind to Mcl-1 in solution.
Example 3: Optimization of One Bead One Compound Library
[0115] To apply this method to the optimization of M1d, a library
of 108 stapled peptides (library LB) was designed to be enriched in
Mcl-1 binders. Positions 2e, 3a and 3b were diversified with 3, 4
and 9 amino ac-ids, respectively, varying the residue size,
hydrophobicity and/or charge (Table 1). Many of the side chains
were chosen to be hydrophobic to favor hydrophobic contacts with
Mcl-1. We also introduced known helix-inducing side chains to
improve the helical content of stapled peptides, which is expected
to be poor at the peptide N-terminus because of two Gly residues in
the MS1 template. To choose residues for the mutation sites, we
used the large amount of mutational data from SPOT arrays and
side-chain scanning of BH3 peptides (Dutta, S.; Gulla, S.; Chen, T.
S.; Fire, E.; Grant, R. A.; Keating, A. E. J. Mol. Biol. 2010, 398
(5), 747; Foight, G. W.; Ryan, J. A.; Gulla, S. V; Letai, A.;
Keating, A. E. ACS Chem. Biol. 2014, 9 (9), 1962; Bocrsma, M. D.;
Sadowsky, J. D.; Tomita, Y. A.; Gellman, S. H. Protein Sci. 2008,
17 (7), 1232). We also analyzed structures of Mcl-1 bound to
different BH3 variants using Bioluminate (version 1.9, Schrodinger,
LLC, New York, N.Y., 2015) to select side chains for the library.
We incorporated functionalities, beyond those found in the 20
common, naturally-occurring amino acids, that we predicted could
access crevices in Mcl-1 that are too distant to be reached by the
sidechains found in the 20 common, naturally-occurring amino acids.
LB was sorted for binding to Mcl-1 in two rounds of competition
screening in which beads were selected that could bind to Mcl-1 in
the presence of 12.5- or 50-fold higher concentration of
Bcl-xL.
Example 4: Screening of Optimized Library
[0116] LB was initially screened under conditions optimized for the
first generation library, LA: 0.2 .mu.M .sup.BioMcl-1 in presence
of 2.5 .mu.M myc-tagged Bcl-xL (.sup.mycBcl-xL). However, for LB we
used a Complex Object Parametric Analyzer and Sorter (COPAS) for
cell sorting that permits fluorescence-based sorting of up to 300
beads/s. The 5% of beads with highest fluorescence intensities were
sorted into 96-well plates. Beads were washed and then
post-screened using more stringent conditions: 0.05 .mu.M
.sup.BioMcl-1 plus 2.5 .mu.M .sup.mycBcl-xl. After incubation with
streptavidin-coated quantum dots, beads were further washed and
visualized using a fluorescence microscope. 170 brightest beads
from a pool of .about.10,000 were isolated manually. MALDI mass
spectrometry confirmed that the selected beads included at least 6
different sequences out of the possible 108 stapled BH3 peptides.
Sequences M1d (32 beads), M2d (48 beads), and M3d (38 beads) were
chosen for further analysis (Table 1). Interestingly, M2d and M3d
incorporate previously untested side chains at positions 2e and 3a,
which are highly conserved in known BH3 motifs.
Example 5: Characterization of M2d and M3d Peptide Binding to
Mcl-1
[0117] M2d and M3d were tested in solution in competition with a
fluorescently labeled Bim BH3 peptide for binding to five human
Bcl-2 paralogs (FIG. 1 and FIG. 2). Unlabeled peptides were mixed
with fluoresceinated Bim BH3 and one of Mcl-1, Bfl-1, Bcl-w,
Bcl-x.sub.L, or Bcl-2. The competition experiments indicated that
M2d and M3d are both highly selective for Mcl-1 over 4 other
anti-apoptotic members and are both considerably tighter binders of
Mcl-1 than is M1d. These peptides competed with fluoresceinated Bim
21mer for Mcl-1 binding with half-maximal inhibitory concentrations
(IC.sub.50 values) of 106.+-.12 nM and 72.+-.11 nM, for M2d and M3d
respectively (compare with 350 nM for M1d and 811 nM for MS1).
Position 2e is usually conserved as small (alanine, glycine,
serine) in natural BH3 sequences. However, previous studies have
shown that Mcl-1 can bind BH3 peptides with bulkier threonine and
leucine at this site (Foight, G, W.; Ryan, J. A.; Gulla, S. V;
Letai, A.; Keating, A. E. ACS Chem. Biol. 2014, 9(9), 1962 and
Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. Nat Chem
Biol 2010, 6 (8), 595). Our results show that, likewise,
introducing the larger, branched 2-aminoisobutyric acid (Aib) at
the 2e position in M2d is not only well tolerated but increases
binding affinity for Mcl-1. Computational modeling of Aib at 2e in
Mcl-1 complexes (2PQK and 3MK8) shows how two methyl groups can be
accommodated in the Mcl-1 interface (FIG. 1(g)). Interestingly, the
largest increase in potency was observed for M3d, which includes
Aib at 2e and Cha at 3a. The selection of Cha at 3a was not easily
anticipated from the inspection of Mcl-1 complexed with BH3
peptides, and the stabilizing effect was somewhat surprising given
that leucine is highly conserved at this position in native BH3
domains. Molecular modeling using Bioluminate predicts that the
flexible cyclohexyl moiety of M3d penetrates deep into the
previously defined P2 binding pocket of Mcl-1 (FIG. 1(f)),
explaining the increase in potency of M3d for Mcl-1 over other
Bcl-2 paralogs (FIG. 1(a))(Burke, J. P.; Bian, Z.; Shaw, S.; Zhao,
B.; Goodwin, C. M.; Belmar, J.; Browning, C. F.; Vigil, D.;
Friberg, A.; Camper, D. V; Rossanese, O. W.; Lee, T.; Olejniczak,
E. T.; Fesik, S. W. J. Med. Chem. 2015, 58 (9), 3794). Discovery of
unpredictable chemical moieties strongly improving Mcl-1 binding
clearly demonstrate the effectiveness of our combinatorial
approach.
[0118] We used biolayer interferometry to measure the kinetics of
binding of stapled peptides to Mcl-1 (FIG. 1(b)). We generated
biotinylated stapled peptides and attached these to a
streptavidin-modified probe surface. Binding of M2d and M3d to
Mcl-1 was slower compared to binding of M1d, likely due to
rearrangements required for packing of the bulky side chains of Aib
and Cha. However, consistent with their higher affinity,
dissociation of M2d and M3d from Mcl-1 was even slower compared to
M1d. Peptide M3d had the lowest k.sub.diss (1.8.times.10.sup.-4)
and K.sub.d (5 nM) values.
Example 6: Structural Characterization of M1d, M2d and M3d
Peptides
[0119] The secondary structure of selected stapled peptides was
assessed by circular dichroism (CD) spectroscopy (FIG. 1(c)).
Interestingly, the observed trend for binding affinities was
paralleled by the measured helicity. M2d and M3d showed
significantly higher helicity than M1d, with mean residue
ellipticity (MRE) at 222 nm of 15,450 and 21,000 deg cm.sup.2
dmol.sup.-1, respectively. Therefore, the increases in helicity can
be attributed to both helix-inducing substitutions: Aib at 2e and
Cha at 3a (Armstrong, K. M.; Fairman, R.; Baldwin, R. L. J. Mol
Biol. 1993, 230 (1), 284. and Venkatraman, J.; Shankaramma, S. C.;
Balaram, P. Chem. Rev. 2001, 101 (10), 3131).
Example 7: Characterization of M1d, M2d and M3d Peptide
Stability
[0120] All of the stapled MS1 variants that we tested were more
protease resistant than un-metathesized M1d, which is a key
pharmacologic advantage of the stapling approach. Unstapled M1d has
a half-life 2-6-fold shorter than the corresponding stapled
peptides (FIG. 1(d)). Notably, the protease resistance analysis
revealed longer half-lives for both M2d and M3d compared to M1d,
indicating the utility of introducing helix-promoting amino acids
into a stapled peptide for maximizing protease resistance.
Example 8: BH3 Profiling of Peptide Variants
[0121] We performed BH3 profiling to test the function of stapled
variants of MS1 in cells. A whole-cell BH3 profiling experiment
quantifies the dependence of cancer cell mitochondrial integrity on
specific anti-apoptotic proteins and can be predictive of cellular
responses to chemotherapy (Ryan, J. A.; Brunelle, J. K.; Letai, A.
Proc. Natl. Acad. Sci. 2010, 107 (29), 12895 and Ryan, J. A; Letai,
A. Methods 2013, 61(2), 156). In this assay, permeabilized cells
are stained with dye JC1 to monitor mitochondrial membrane
integrity in response to increasing doses of BH3 peptides. We used
this assay to test the specificity of our Mcl-1-binding stapled
peptides in cell lines with different established dependencies on
Bcl-x.sub.L and Mcl-1. Mcl-1/Myc 2640 is an engineered murine
leukemia cell line overexpressing murine Mcl-1 and Myc, and MDA-MB
231 is a human breast cancer cell line with a primed
Bcl-xL-dependent pro-file (Brunelle, J. K.; Ryan, J.; Yecies, D.;
Opferman, J. T.; Letai, A. J Cell Biol. 2009, 187 (3), 429 and
Ryan, J. A.; Brunelle, J. K.; Letai, A. Proc. Natl. Acad. Sci.
2010, 107 (29), 12895). By BH3 profiling using native BH3 peptides
from BAD and NOXAA, we confirmed cell line dependencies on these
anti-apoptotic proteins (FIG. 3). FIG. 3 shows the BH3 profiling of
cell lines using engineered (MS1) and native BH3 peptides (Bim,
NoxA). Mcl-1 2640 cell line is dependent on Mcl-1 whereas
MDA-MB-231 is dependent on Bcl-xL, as indicated by response to NoxA
and Bad, respectively. BBDL is Bax/Bak deficient leukocyte (error
bars indicate the standard deviation over 3 or more replicates).
Both M1d and M2d showed Bax/Bak independent activity in Bax/Bak
deficient cells, indicating non-specific toxicity (FIG. 4).
However, peptide M3d showed no activity in Bax/Bak negative cells,
consistent with an on-target mechanism, (Deng, J.; Carlson, N.;
Takeyama, K.; Dal Cin, P.; Shipp, M.; Letai, A. Cancer Cell 2015,
12 (2), 171) and was remarkably potent when tested on Mcl-1/Myc
2640, with an EC.sub.50 value of 30 nM (FIGS. 1(e) and 4(b). The
specificity of M3d was confirmed by the much higher EC.sub.50
(estimated as >5 mM) observed for the Bcl-x.sub.L dependent cell
line MDA-MB 231 (FIG. 4(a)).
Example 9: Characterization of Novel Non-Stapled Peptides with
Amino Acids that are not the 20 Common, Naturally-Occurring Amino
Acids
[0122] We identified two substitutions that--both in the presence
or absence of a staple modification--improve binding to Mcl-1 and
preserve a high degree of specificity. These modifications are:
replacement of a leucine residue at position "3a" with either
cyclohexyl phenylalanine or homo-cyclohexyl phenylalanine and
replacement of a threonine at position "2c" with 2-amino isobutyric
acid (see Table 1). Both are non-conservative mutations, and the
leucine that is substituted in our peptides is highly conserved in
natural peptides that bind to Mcl-1, making it somewhat surprising
that a change to the larger cyclohexyl phenylalanine or
homo-cyclohexyl phenylalanine groups is beneficial. FIG. 6 shows
the sequences of both stapled and non-stapled peptides, along with
the structures of various uncommon amino acid examples.
[0123] FIGS. 7 and 8, along with the following describe the binding
and biophysical characterization of the new staple-less
peptides.
[0124] The secondary structure of selected non-stapled peptides
with uncommon amino acids was assessed by circular dichroism (CD)
spectroscopy at 22.degree. C. in H.sub.2O (FIG. 7). Single point
mutations were compared with their parent construct (M1). Increase
in helical content can be attributed to the addition of
helix-inducing amino acids.
[0125] The binding affinity and specificity for the non-stapled
peptides with uncommon amino acids were assessed using a
competition assay. IC.sub.50 values for binding to Mcl-1 (target)
or Bcl-xL (undesired competitor) were obtained from competition
assays with fluoresceinated Bim peptide (FIG. 8). All single point
mutations incorporated conferred higher binding affinity to Mcl-1
and preserve selectivity over other Bcl-2 family proteins. Data are
the mean and s.d. for at least duplicate experiments.
Example 10: BH3 Profiling of Novel Non-Stapled Peptides
[0126] We measured the depolarization of the mitochondrial membrane
of p185+B-ALL cell lines dependent on Mcl-1, Bcl-xL, Bcl-2 or Bfl-1
in response to treatment with unstapled peptides in comparison with
MS1. The results for the peptides compared to MS1 at 10 nM are
shown in the top panel of FIG. 9. The results of known BH3 peptides
at a higher concentration (10 .mu.M) are shown in the bottom panel
of FIG. 9. In the sequences listed, 1=Aib, 2-aminoisobutyric acid;
2=Cha, cyclohexylalanine; and 3=h-Cha, homo-cyclohexylalanine.
[0127] The unstapled peptides in the top panel of FIG. 9 were
highly potent and selective for inducing MOMP in Mcl-1 dependent,
but not Bcl-xL, Bcl-2 or Bfl-1 dependent permeabilized cells at 10
nM. The known peptides (BIM, PUMA, BAD, NOXAA) were less potent and
were tested at 10 .mu.M, as shown in the bottom panel of FIG. 9;
these peptides were less selective. The over-expressing cell lines
are described in: Koss et al. "Defining specificity and on-target
activity of BH3-mimetics using engineered B-ALL cell lines."
Oncotarget (2016) vol. 7 (10) pp. 11500-11, which is incorporated
herein in its entirety.
Other Embodiments
[0128] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
20124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Arg, a conservative substitution or
absentMOD_RES(2)..(2)Pro, a conservative substitution or
absentMOD_RES(3)..(3)Glu, a conservative substitution or
absentMOD_RES(4)..(4)Ile or a conservative
substitutionMOD_RES(5)..(5)Trp or a conservative
substitutionMOD_RES(6)..(6)Met, a conservative substitution or
norleucineMOD_RES(7)..(7)Thr, Val, a conservative substitution,
2-aminoisobutyric acid or any amino acid available for
crosslinkingMOD_RES(8)..(8)Gln or a conservative
substitutionMOD_RES(9)..(9)Gly or a conservative
substitutionMOD_RES(10)..(10)Leu, Phe, a conservative substitution,
pentafluoro phenylalanine, cyclohexylalanine or homo-
cyclohexylalanineMOD_RES(11)..(11)Arg, Trp, Gln, Asp, Tyr, a
conservative substitution, 2-aminoisobutyric acid, D-phenyl
glycine, alpha-alpha methyl leucine, alpha-alpha methyl
phenylalanine or any amino acid available for
crosslinkingMOD_RES(12)..(12)Arg or a conservative
substitutionMOD_RES(13)..(13)Leu or a conservative
substitutionMOD_RES(14)..(14)Gly or a conservative
substitutionMOD_RES(15)..(15)Asp or a conservative
substitutionMOD_RES(16)..(16)Glu or a conservative
substitutionMOD_RES(17)..(17)Ile or a conservative
substitutionMOD_RES(18)..(18)Asn, a conservative substitution or
any amino acid available for crosslinkingMOD_RES(19)..(19)Ala or a
conservative substitutionMOD_RES(20)..(20)Tyr or a conservative
substitutionMOD_RES(21)..(21)Tyr or a conservative
substitutionMOD_RES(22)..(22)Ala, a conservative substitution or
any amino acid available for crosslinkingMOD_RES(23)..(23)Arg, a
conservative substitution or absentMOD_RES(24)..(24)Arg, a
conservative substitution or absentSee specification as filed for
detailed description of substitutions and preferred embodiments
1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(15)..(15)Any amino acid
available for crosslinkingMOD_RES(19)..(19)Any amino acid available
for crosslinkingSee specification as filed for detailed description
of substitutions and preferred embodiments 2Ile Trp Xaa Thr Gln Gly
Leu Arg Arg Leu Gly Asp Glu Ile Xaa Ala 1 5 10 15 Tyr Tyr Xaa Arg
Arg 20 321PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(15)..(15)Any amino acid available for
crosslinkingMOD_RES(19)..(19)Any amino acid available for
crosslinkingSee specification as filed for detailed description of
substitutions and preferred embodiments 3Ile Trp Xaa Xaa Gln Gly
Leu Arg Arg Leu Gly Asp Glu Ile Xaa Ala 1 5 10 15 Tyr Tyr Xaa Arg
Arg 20 421PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(7)..(7)CyclohexylalanineMOD_RES(15)..(15)Any amino acid
available for crosslinkingMOD_RES(19)..(19)Any amino acid available
for crosslinkingSee specification as filed for detailed description
of substitutions and preferred embodiments 4Ile Trp Xaa Xaa Gln Gly
Xaa Arg Arg Leu Gly Asp Glu Ile Xaa Ala 1 5 10 15 Tyr Tyr Xaa Arg
Arg 20 521PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(15)..(15)Any amino acid available for
crosslinkingMOD_RES(19)..(19)Any amino acid available for
crosslinkingSee specification as filed for detailed description of
substitutions and preferred embodiments 5Ile Trp Xaa Xaa Gln Gly
Leu Gln Arg Leu Gly Asp Glu Ile Xaa Ala 1 5 10 15 Tyr Tyr Xaa Arg
Arg 20 621PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(15)..(15)Any amino acid available for
crosslinkingMOD_RES(19)..(19)Any amino acid available for
crosslinkingSee specification as filed for detailed description of
substitutions and preferred embodiments 6Ile Trp Xaa Xaa Gln Gly
Leu Asp Arg Leu Gly Asp Glu Ile Xaa Ala 1 5 10 15 Tyr Tyr Xaa Arg
Arg 20 721PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)NorleucineMOD_RES(15)..(15)Any
amino acid available for crosslinkingMOD_RES(19)..(19)Any amino
acid available for crosslinkingSee specification as filed for
detailed description of substitutions and preferred embodiments
7Ile Trp Xaa Thr Gln Gly Leu Gln Arg Leu Gly Asp Glu Ile Xaa Ala 1
5 10 15 Tyr Tyr Xaa Arg Arg 20 821PRTArtificial SequenceDescription
of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(7)..(7)CyclohexylalanineSee
specification as filed for detailed description of substitutions
and preferred embodiments 8Ile Trp Xaa Thr Gln Gly Xaa Arg Arg Leu
Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg Arg 20
921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(7)..(7)Homo-cyclohexylalanineSee
specification as filed for detailed description of substitutions
and preferred embodiments 9Ile Trp Xaa Thr Gln Gly Xaa Arg Arg Leu
Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg Arg 20
1021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidSee specification as filed for detailed description of
substitutions and preferred embodiments 10Ile Trp Xaa Xaa Gln Gly
Leu Arg Arg Leu Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg
Arg 20 1121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(7)..(7)CyclohexylalanineSee specification as filed for
detailed description of substitutions and preferred embodiments
11Ile Trp Xaa Xaa Gln Gly Xaa Arg Arg Leu Gly Asp Glu Ile Asn Ala 1
5 10 15 Tyr Tyr Ala Arg Arg 20 1221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(7)..(7)Homo-cyclohexylalanineSee specification as filed
for detailed description of substitutions and preferred embodiments
12Ile Trp Xaa Xaa Gln Gly Xaa Arg Arg Leu Gly Asp Glu Ile Asn Ala 1
5 10 15 Tyr Tyr Ala Arg Arg 20 1323PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Arg
Pro Glu Ile Trp Met Thr Gln Gly Leu Arg Arg Leu Gly Asp Glu 1 5 10
15 Ile Asn Ala Tyr Tyr Ala Arg 20 1421PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acid, Thr, Val or AlaMOD_RES(7)..(7)Norleucine, Leu, modified Leu
or absentMOD_RES(8)..(8)Arg, Asp or Gln 14Ile Trp Xaa Xaa Gln Gly
Xaa Xaa Arg Leu Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg
Arg 20 1521PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acid, Thr, Val, or AlaMOD_RES(7)..(7)Norleucine, Leu, modified Leu
or absentMOD_RES(8)..(8)Arg, Asp or
GlnMOD_RES(15)..(15)Alpha-4-pentenyl
alanineMOD_RES(19)..(19)Alpha-4-pentenyl alanine 15Ile Trp Xaa Xaa
Gln Gly Xaa Xaa Arg Leu Gly Asp Glu Ile Xaa Ala 1 5 10 15 Tyr Tyr
Xaa Arg Arg 20 1621PRTArtificial SequenceDescription of Artificial
Sequence Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(7)..(7)Cyclohexylalanine 16Ile Trp Xaa Xaa Gln Gly Xaa
Arg Arg Leu Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg Arg
20 1721PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(4)..(4)2-aminoisobutyric
acidMOD_RES(7)..(7)Homo-cyclohexylalanine 17Ile Trp Xaa Xaa Gln Gly
Xaa Arg Arg Leu Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg
Arg 20 1821PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(7)..(7)Cyclohexylalanine
18Ile Trp Xaa Thr Gln Gly Xaa Arg Arg Leu Gly Asp Glu Ile Asn Ala 1
5 10 15 Tyr Tyr Ala Arg Arg 20 1921PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)NorleucineMOD_RES(7)..(7)Homo-cyclohexylalanine
19Ile Trp Xaa Thr Gln Gly Xaa Arg Arg Leu Gly Asp Glu Ile Asn Ala 1
5 10 15 Tyr Tyr Ala Arg Arg 20 2021PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)Norleucine 20Ile Trp Xaa Thr Gln Gly Leu Arg
Arg Leu Gly Asp Glu Ile Asn Ala 1 5 10 15 Tyr Tyr Ala Arg Arg
20
* * * * *