U.S. patent application number 16/551102 was filed with the patent office on 2020-07-23 for stabilized anti-microbial peptides.
The applicant listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Gregory H. Bird, Rida Mourtada, Loren D. Walensky.
Application Number | 20200231638 16/551102 |
Document ID | / |
Family ID | 57609337 |
Filed Date | 2020-07-23 |
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United States Patent
Application |
20200231638 |
Kind Code |
A1 |
Walensky; Loren D. ; et
al. |
July 23, 2020 |
STABILIZED ANTI-MICROBIAL PEPTIDES
Abstract
The present invention provides methods of designing and making
structurally stabilized anti-microbial peptides for the prophylaxis
and treatment of infection. Methods are also disclosed for
designing stabilized anti-microbial peptides that are selectively
lytic/cytotoxic to bacteria, allowing for internal use of
anti-microbial peptides without mammalian membrane disruption and
cytotoxicity.
Inventors: |
Walensky; Loren D.; (Newton
Centre, MA) ; Mourtada; Rida; (Brookline, MA)
; Bird; Gregory H.; (Pelham, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
57609337 |
Appl. No.: |
16/551102 |
Filed: |
August 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15201235 |
Jul 1, 2016 |
10464975 |
|
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16551102 |
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62301518 |
Feb 29, 2016 |
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62188448 |
Jul 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/04 20180101;
C07K 14/463 20130101; A61P 31/00 20180101; A61K 38/1729 20130101;
A01N 43/90 20130101; A61K 38/00 20130101 |
International
Class: |
C07K 14/46 20060101
C07K014/46; A61K 38/17 20060101 A61K038/17; A01N 43/90 20060101
A01N043/90 |
Claims
1. A therapeutic compound comprising a cross-linked amino acid
sequence having the formula: ##STR00045## or a pharmaceutically
acceptable salt thereof, wherein: each R.sub.1 and R.sub.2 is
independently H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of
which is substituted or unsubstituted; each R.sub.3 is
independently alkylene, alkenylene, or alkynylene, any of which is
substituted or unsubstituted; each x is independently 2, 3, or 6;
each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20; z is 1 or 2; and each Xaa is
independently an amino acid, wherein the cross-linked amino acid
sequence has at least three and up to 9 amino acid substitutions
relative to the sequence set forth in SEQ ID NO:3; wherein two of
the at least three amino acid substitutions are substitutions of
two amino acids of SEQ ID NO:3 with stapling amino acids, and
wherein one of the at least three amino acid substitutions are
substitutions at any of positions 1, 2, 3, 7, 8, 9, 18, 19, 21, 22,
or 23 of SEQ ID NO: 3 and is selected from the group consisting of
G1K, 12K, G3K, G3E, H7K, H7E, S8K, S8E, A9K, G18E, E19K, M21B
(where B is norleucine), N22K, N22E, and S23K; wherein the
cross-linked amino acid sequence has an alpha helical conformation;
and wherein the compound or the pharmaceutically acceptable salt
thereof exhibits an increased antibacterial effect against at least
one bacterium relative to an un-cross-linked corresponding sequence
set forth in SEQ ID NO:3.
2-3. (canceled)
4. The therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 1, wherein x is 3; R.sub.3 is C.sub.8 alkylene,
C.sub.8 alkenylene, or C.sub.8 alkynylene; and the sum of x, w, and
y is at least 10.
5. The therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 1, wherein R.sub.3 is substituted with 1, 2, 3, or
4 R.sub.4, and each R.sub.4 is independently --NH.sub.3 or --OH,
wherein each --NH.sub.3 is optionally coupled with another chemical
entity.
6. The therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 5, wherein R.sub.3 is substituted with 2 R.sub.4,
and both R.sub.4 are --OH.
7. The therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 5, wherein R.sub.3 is substituted with 2 R.sub.4,
and one R.sub.4 is an optionally substituted --NH.sub.3 and the
other R.sub.4 is --OH.
8. The therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 1, wherein at least one Xaa is an amino acid that
is a non-natural amino acid.
9.-10. (canceled)
11. The therapeutic compound or the pharmaceutically acceptable
salt thereof of claim 1, wherein x is 6 and R.sub.3 spans from one
to two turns on an .alpha.-helix.
12.-23. (canceled)
24. The therapeutic compound or the pharmaceutically acceptable
salt thereof of claim 1, wherein the at least one bacterium is
Mycobacterium tuberculosis or Pseudomonas aeruginosa.
25.-53. (canceled)
54. The therapeutic compound or the pharmaceutically acceptable
salt thereof of claim 1, wherein the therapeutic compound comprises
a cross-linked amino acid sequence selected from the group
consisting of: TABLE-US-00017 (SEQ ID NO: 48) (a) Mag(i + 4)15(G1K)
KIGKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 47) (b) Mag(i + 4)15(I2K)
GKGKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 46) (c) Mag(i + 4)15(G3K)
GIRKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 43) (d) Mag(i + 4)15(H7K)
GIGKFLKSAKKFGKAXVGEXBNS; (SEQ ID NO: 42) (e) Mag(i + 4)15(S8K)
GIGKFLHKAKKFGKAXVGEXBNS; (SEQ ID NO: 41) (f) Mag(i + 4)15(A9K)
GIGKFLHSAKKFGKAXVGEXENS; (SEQ ID NO: 35) (g) Mag(i + 4)15(E19K)
GIGKFLHSAKKFGKAXVGKXBNS; (SEQ ID NO: 33) (h) Mag(i + 4)15(N22K)
GIGKFLHSAKKFGKAXVGEXBKS; (SEQ ID NO: 32) (i) Mag(i + 4)15(S23K)
GIGKFLHSAKKFGKAXVGEXBNK; (SEQ ID NO: 51) (j) Mag(i + 4)15(G3E)
GIBKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 55) (k) Mag(i + 4)15 (H7E)
GIGKFLESAKKFGKAXVGEXBNS; (SEQ ID NO: 56) (l) Mag(i + 4)15(S8E)
GIGKFLHEAKKFGKAXVGEXBNS; (SEQ ID NO: 65) (m) Mag(i + 4)15(G18E)
GIGKFLHSAKKFGKAXVEEXBNS; and (SEQ ID NO: 67) (n) Mag(i + 4)15(N22E)
GIGKFLHSAKKFGKAXVGEXBES;
wherein X is a stapling amino acid; and wherein B is
norleucine.
55. The therapeutic compound or the pharmaceutically acceptable
salt thereof of claim 54, wherein the stapling amino acid is an
.alpha.,.alpha.-disubstituted amino acid.
56. The therapeutic compound or the pharmaceutically acceptable
salt thereof of claim 54, wherein the stapling amino acid is S5
[(S)-2-(4-pentenyl)Alanine] or R5 [(R)-2-(4-pentenyl)Alanine].
57. The therapeutic compound or the pharmaceutically acceptable
salt thereof of claim 1, wherein the at least one bacterium is
Escherichia coli (E. coli).
58. A pharmaceutical composition comprising the therapeutic
compound or the pharmaceutically acceptable salt thereof of claim
54.
59. A method of treating an infection caused by a bacterium, the
method comprising administering a therapeutically-effective amount
of the therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 54 to a subject having, or at risk of having, the
infection caused by the bacterium.
60. The method of claim 59, wherein the subject is a human.
61. The method of claim 59, wherein the bacterium is
Gram-positive.
62. The method of claim 59, wherein the bacterium is
Gram-negative.
63. The method of claim 59, wherein the bacterium is Mycobacterium
tuberculosis or Pseudomonas aeruginosa.
64. The method of claim 59, wherein the infection is a bacterial
vaginal infection.
65. The method of claim 59, wherein the infection is a bacterial
pneumonia.
66. The method of claim 59, wherein the infection comprises a
bacterial biofilm.
67. A method of inhibiting the growth of a bacterium, the method
comprising contacting the bacterium with an effective amount of the
therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 54.
68. The method of claim 67, wherein the contacting occurs in a
subject harboring the bacterium.
69. A method of making an internally cross-linked peptide, the
method comprising: (i) providing a peptide having a sequence set
forth in any one of (a) through (n): TABLE-US-00018 (SEQ ID NO: 48)
(a) Mag(i + 4)15(G1K) KIGKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 47) (b)
Mag(i + 4)15(I2K) GKGKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 46) (c)
Mag(i + 4)15(G3K) GIRKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 43) (d)
Mag(i + 4)15(H7K) GIGKFLKSAKKFGKAXVGEXBNS; (SEQ ID NO: 42) (e)
Mag(i + 4)15(S8K) GIGKFLHKAKKFGKAXVGEXBNS; (SEQ ID NO: 41) (f)
Mag(i + 4)15(A9K) GIGKFLHSAKKFGKAXVGEXENS; (SEQ ID NO: 35) (g)
Mag(i + 4)15(E19K) GIGKFLHSAKKFGKAXVGKXBNS; (SEQ ID NO: 33) (h)
Mag(i + 4)15(N22K) GIGKFLHSAKKFGKAXVGEXBKS; (SEQ ID NO: 32) (i)
Mag(i + 4)15(S23K) GIGKFLHSAKKFGKAXVGEXBNK; (SEQ ID NO: 51) (j)
Mag(i + 4)15(G3E) GIBKFLHSAKKFGKAXVGEXBNS; (SEQ ID NO: 55) (k)
Mag(i + 4)15(H7E) GIGKFLESAKKFGKAXVGEXBNS; (SEQ ID NO: 56) (l)
Mag(i + 4)15(S8E) GIGKFLHEAKKFGKAXVGEXBNS; (SEQ ID NO: 65) (m)
Mag(i + 4)15(G18E) GIGKFLHSAKKFGKAXVEEXBNS; and (SEQ ID NO: 67) (n)
Mag(i + 4)15(N22E) GIGKFLHSAKKFGKAXVGEXBES;
wherein X is a stapling amino acid and B is norleucine; and (ii)
cross-linking the peptide.
70. The method of claim 69, wherein X is S5
[(S)-2-(4-pentenyl)Alanine] or R5 [(R)-2-(4-pentenyl)Alanine].
71. A method of treating an infection caused by a coronavirus, the
method comprising administering a therapeutically-effective amount
of the therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 1 to a subject having, or at risk of having, the
infection caused by the coronavirus.
72. A method of treating an infection caused by a coronavirus, the
method comprising administering a therapeutically-effective amount
of the therapeutic compound or the pharmaceutically acceptable salt
thereof of claim 54 to a subject having, or at risk of having, the
infection caused by the coronavirus.
Description
CROSS-REFERENCE
[0001] This application is a continuation and claims the benefit of
U.S. patent application Ser. No. 15/201,235, filed Jul. 1, 2016,
now issued U.S. Pat. No. 10,464,975, which claims the benefit of
U.S. Provisional Application No. 62/188,448 filed Jul. 2, 2015, and
U.S. Provisional Application No. 62/301,518 filed Feb. 29, 2016,
each of which are incorporated herein by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 26, 2019, is named 00530-0323002_SL.txt and is 173,650
bytes in size.
BACKGROUND
[0003] Anti-microbial peptides (AMPs) are an evolutionarily
conserved class of proteins that form an essential line of defense
against microbial invasion. These peptides are produced by many
disparate organisms and have been found to exhibit a wide spectrum
of activity against bacteria, fungi (including yeasts), protozoa
(including parasites), and viruses.
[0004] AMPs can be divided into four main structural groups:
stabilized .beta.-sheet peptides with two to four disulfide
bridges; loop peptides with a single disulfide bridge;
.alpha.-helical peptides; and extended structures rich in arginine,
glycine, proline, tryptophan, and histidine. Typically 12 to 50
amino acids in length, these peptides are usually cationic with an
amphipathic character. These biophysical properties allow them to
interact with bacterial membranes resulting in either disruption of
membrane integrity or translocation into bacterial cells and
disruption of intracellular processes.
[0005] The alpha-helical structural motif of AMPs can be important
to the ability of AMPs to interact with bacterial membranes. Upon
binding to the membrane, AMPs can either translocate or insert
themselves and permeabilize the membrane through a barrel-stove
mechanism, a carpet-like mechanism or a toroidal pore mechanism.
This process of permeabilization and disruption of membrane
integrity can account for the antimicrobial properties of
alpha-helical AMPs.
SUMMARY
[0006] The present disclosure provides structurally-stabilized
peptides related to (e.g., sharing sequence homology with)
anti-microbial peptides (AMPs), and methods for using such
stabilized peptides as therapeutic and/or prophylactic agents.
Methods are also provided for designing stabilized anti-microbial
peptides that are selectively lytic/cytotoxic to bacteria, allowing
for internal use of anti-microbial peptides without mammalian
membrane disruption and cytotoxicity.
[0007] More specifically, the document provides a compound having
the Formula (I):
##STR00001##
[0008] or a pharmaceutically acceptable salt thereof,
[0009] wherein;
[0010] each R.sub.1 and R.sub.2 is independently H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or
heterocyclylalkyl, any of which is substituted or
unsubstituted;
[0011] each R.sub.3 is independently alkylene, alkenylene, or
alkynylene, any of which is substituted or unsubstituted;
[0012] each x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10;
[0013] each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;
[0014] z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and
[0015] each Xaa is independently an amino acid;
[0016] wherein the compound exhibits an antimicrobial effect
against at least one microbe.
[0017] In some aspects, x is independently 2, 3, or 6.
[0018] Moreover, the document additionally provides an internally
cross-linked (ICL) anti-microbial peptide (AMP) containing amino
acids, the side chains of at least one pair (e.g., one or two
pairs) of amino acids separated by 2, 3, or 6 amino acids being
replaced, relative to the corresponding parent non-internally
cross-linked AMP, by the linking group, R.sub.3, which connects the
alpha carbons of the pair of amino acids such that: each R.sub.3 is
independently alkylene, alkenylene, or alkynylene (e.g., a C.sub.6,
C.sub.7, or C.sub.11 alkenylene) optionally substituted, e.g., with
1-6 R.sub.4; and
[0019] each R.sub.4 is independently --NH.sub.3 or --OH, wherein
each --NH.sub.3 is optionally substituted; and each R.sub.1 and
R.sub.2 is independently C.sub.1 to C.sub.10 alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or
heterocyclylalkyl, any of which is substituted or
unsubstituted.
[0020] In some aspects, the ICL AMP contains at least 18 contiguous
amino acids of any of SEQ ID NOs: 1-17 or a variant thereof having
1, 2, 3, 4, or 5 amino acid substitutions, or another polypeptide
sequence described herein except that: (a) within the 18 contiguous
amino acids the side chains of at least one pair (e.g., one or two
pairs) of amino acids separated by 2, 3, or 6 amino acids are
replaced, relative to the corresponding parent non-internally
cross-linked AMP, by the linking group, R.sub.3, which connects the
alpha carbons of the pair of amino acids as depicted in Formula (I)
and the alpha carbon of the second of the pair of amino acids is
substituted with R.sub.2 as depicted in Formula (I). In certain
aspects, the AMP variant comprises at least 18 contiguous amino
acids of any of SEQ ID NOs: 1-17 except that it includes (i) at
least one (e.g., 1, 2, 3) substitution of an amino acid to
histidine; and/or (ii) at least one (e.g., 1, 2, 3) substitution of
an amino acid to lysine; and/or (iii) at least one (e.g., 1, 2, 3)
substitution of an amino acid to D-alanine.
[0021] Moreover, the document additionally provides an internally
cross-linked (ICL) anti-microbial peptide (AMP) containing at least
20 amino acids, the side chains of at least one pair (e.g., one or
two pairs) of amino acids separated by 2, 3, or 6 amino acids being
replaced, relative to the corresponding parent non-internally
cross-linked AMP, by the linking group, R.sub.3, which connects the
alpha carbons of the pair of amino acids such that:
[0022] each R.sub.3 is independently alkylene, alkenylene, or
alkynylene (e.g., a C.sub.6, C.sub.7, or C.sub.11 alkenylene),
optionally substituted with 1-6 R.sub.4;
[0023] each R.sub.4 is independently --NH.sub.3 or --OH, wherein
each --NH.sub.3 is optionally substituted; and each R.sub.1 and
R.sub.2 is independently C.sub.1 to C.sub.10 alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or
heterocyclylalkyl, any of which is substituted or
unsubstituted.
[0024] Any of the above-described ICL AMPs can have R.sub.3
substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 R.sub.4. Each
R.sub.4 can be --OH. In some aspects, one R.sub.3 is --NH.sub.3 and
another is --OH.
[0025] Any of the above-described ICL AMPs can contain at least 18
contiguous amino acids of any of SEQ ID NOs: 1-17 (e.g., SEQ ID NO:
1) or a variant thereof having 1, 2, 3, 4, or 5 amino acid
substitutions, or another polypeptide sequence described herein
except that: (a) within the 18 contiguous amino acids the side
chains of at least one pair (e.g., one or two pairs) of amino acids
separated by 2, 3, or 6 amino acids are replaced, relative to the
corresponding parent non-internally cross-linked AMP, by the
linking group, R.sub.3, which connects the alpha carbons of the
pair of amino acids and the H of the alpha carbon of each pair of
amino acids having their side chains replaced by linking group
R.sub.3 is optionally, independently replaced by a C.sub.1 to
C.sub.10 alkyl, alkenyl, or alkynyl.
[0026] Any of the above-described ICL AMPs can contain an
.alpha.-helical region including a first surface hydrophobic patch,
such that the replacement with the linking group maintains or
results in, relative to the parent AMP without the replacement,
discontinuity between the first surface hydrophobic patch and an
additional surface hydrophobic patch or patches on the
.alpha.-helical region of the peptide. Moreover, the linking group
can contain a hydrophilizing modification, e.g., dihydroxylation.
The replacement with the linking group can be located in the first
surface hydrophobic patch, an additional surface hydrophobic patch,
or the first surface hydrophobic patch and an additional surface
hydrophobic patch on the AMP.
[0027] Any of the above-described ICL AMPs can contain the sequence
of any of SEQ ID NOs: 18-168 and 170-174. They can, e.g., contain
the sequence of Mag(i+4)1 (SEQ ID NO: 135), Mag(i+4)2 (SEQ ID NO:
136), Mag(i+4)4 (SEQ ID NO: 138), Mag(i+4)5 (SEQ ID NO: 139),
Mag(i+4)6 (SEQ ID NO: 140), Mag(i+4)11 (SEQ ID NO: 145), Mag(i+4)15
(SEQ ID NO: 149), Mag(i+4)16 (SEQ ID NO: 150), Mag(i+4)2,15(I2K,
A9K, G18H) (SEQ ID NO: 170), Mag(i+4)2,15(I2K, A9H) (SEQ ID NO:
171), Mag(i+4)2,15(I2K, A9H, N21E) (SEQ ID NO: 172),
Mag(i+4)2,15(I2K, A9H, G18H, N21E) (SEQ ID NO: 173), or
Mag(i+4)1,15(S8H, A9K, G18H, N21E) (SEQ ID NO: 174).
[0028] The document also features a method of treating or
preventing a microbial infection, the method including
administering an effective amount of any of the ICL AMPs described
above to a subject having, or at risk of having, an infection with
a microbial organism. The subject can be an animal or plant. The
animal can be a mammal, e.g., a human. The microbial organism can
be a bacterial organism, e.g., a Gram-positive bacterial organism
or a Gram-negative bacterial organism. The subject can have, or be
at risk of having, a bacterial vaginal infection. The bacterial
vaginal infection can include bacterial vaginosis. The bacterial
vaginal infection can include an infection with one or more
bacterial organisms that increase the likelihood of transmission of
a viral infection to the subject. The viral infection can be a
human immunodeficiency virus-1 (HIV-1) or human immunodeficiency
virus-2 (HIV-2) infection. Any of the above-described ICL AMPs can
be administered topically, e.g., to the vagina. Any of the
above-described ICL AMPs can be administered, e.g., to the lung.
The subject can include a bacterial biofilm. The subject can have,
or be at risk of having, cystic fibrosis.
[0029] The method can further include administering an effective
amount of at least one antibiotic. The established antibiotic can
act synergistically with the ICL AMP to inhibit or prevent
infection with the microbial organism. The ICL AMP and the
antibiotic can synergistically to overcome or prevent resistance to
the antibiotic.
[0030] Another aspect of the document is a composition containing
one or more of the any of the ICL AMPs described above. The
composition can further contain a food product or beverage. Any of
the above-described ICL AMPs can be added to the food product or
beverage prior to or during a fermentation or sterilization
process.
[0031] The composition can further contain a medical or hygienic
device. The one or more ICL AMPs can be coated onto or impregnated
into the medical or hygienic device. The composition can also
contain one or more antibiotics.
[0032] Also provided by the document is a method of inhibiting the
growth of, or killing, a microbial organism that involves
contacting the microbial organism with one or more of any of the
above-described ICL AMPs. The microbial organism can be an
extracellular microbial organism or an intracellular microbial
organism. The contacting can occur in a subject comprising the
microbial organism. Alternatively, the method can be an in vitro
method. It is understood that the method can be implemented using
any of the features described in the document (e.g., those
described above for a method of treating or preventing a microbial
infection). The microbial organism can be, e.g., Mycobacterium
tuberculosis.
[0033] Another feature of the document is a method of making any of
the above-described ICL AMPs, the method involving, with reference
to Formula (I): synthesizing an ICL AMP, determining the location
of an established surface hydrophobic patch in an .alpha.-helical
region of the peptide, and selecting integers w and y such that all
amino acids [Xaa].sub.x are located within the established surface
hydrophobic patch on the peptide. Alternatively, integers w and y
can be selected such that amino acids [Xaa].sub.x do not connect
two or more established hydrophobic patches in the .alpha.-helical
region of the peptide. Moreover, the method can include adding to
the linking group a hydrophilizing modification, including, e.g.,
dihydroxylation.
[0034] Another aspect of the document is a method of making any of
the above-described ICL AMPs can involve: synthesizing an ICL AMP
such that the ICL AMP comprises an .alpha.-helical region
comprising a first surface hydrophobic patch, the replacement with
the linking groups maintaining or resulting in, relative to the
corresponding parent non-internally crosslinked AMP, discontinuity
between the first hydrophobic patch and one or more additional
surface hydrophobic patches on internally cross-linked peptide.
Moreover, the method can include adding to the linking group a
hydrophilizing modification, including, e.g., dihydroxylation.
[0035] Yet another feature of the document is a method of designing
the any of the above-described ICL AMPs, involving:
[0036] creating one or more panels of ICL AMPs, each panel
containing a plurality of panel member ICL AMPs in each of which:
(a) the side chains of at least one pair of amino acids separated
by 2, 3, or 6 amino acids are replaced by the linking group,
R.sub.3, which connects the alpha carbons of the pair of amino
acids; and (b) in each member of each panel, the pair of amino
acids is at different positions as compared to the other members of
the relevant panel; and
[0037] testing each member of all panels for (i) the presence of
discontinuity between a first surface hydrophobic patch in an
.alpha.-helical region of the relevant member and one or more
additional surface hydrophobic patches on the .alpha.-helical
region of the member; and (ii) the ability of each member of each
panel for its ability to translocate into a microbial cell and lyse
or inhibit the growth of a mammalian cell. The method can further
involve manufacturing one or members of all the panels that have a
relatively high ability to translocate into a microbial cell and no
or a relatively low ability to lyse or inhibit the growth of a
mammalian cell.
[0038] Yet another feature of the document is a method of
identifying a microbial infection, involving:
[0039] contacting a microbial organism of the microbial infection
in a test medium with any of the above-described ICL AMPs; and
[0040] identifying the microbial organism by analyzing the nucleic
acids released from the microbial organism into the test medium.
The test medium can include a tissue sample, an organ sample, or a
bodily fluid sample from a subject with the microbial infection.
The medium can include culture medium to which a tissue sample, an
organ sample, or a bodily fluid sample from a subject with the
microbial infection had previously been added. The bodily fluid can
include, e.g., blood, urine, sputum, and/or feces. The contacting
can occur in vitro, or in a subject with the microbial infection.
The method can further include administering to the subject
containing the test medium or from which the test medium was
obtained, a treatment (e.g., including any of the above-described
ICL AMPs) appropriate for the identified microbial organism.
[0041] Yet another feature of the document is a method of
determining whether a test medium includes a microbial organism,
involving:
[0042] delivering to a medium suspected of containing a microbial
organism any of the above-described ICL AMPs; and
[0043] testing for the presence in the test medium of nucleic acids
that the microbial organism is known to include or express. The
test medium can include a tissue sample, an organ sample, or a
bodily fluid sample from a subject suspected of being infected with
the microbial organism. The test medium can include culture medium
to which a tissue sample, an organ sample, or a bodily fluid sample
from a subject suspected of being infected with the microbial
organism had previously been added. The bodily fluid can include,
e.g., blood, urine, sputum, and/or feces. The delivery can occur in
vitro, or in a subject suspected of being infected with the
microbial organism. The method can further include, if the test
medium is found to contain the microbial organism, administering to
the subject containing the test medium or from which the test
medium was obtained, a treatment (e.g., including any of the
above-described ICL AMPs) appropriate for the microbial
organism.
[0044] A non-limiting example of an agent that has a "relatively
high ability to inhibit the growth of a microbe" and a "relatively
low ability to lyse or inhibit the growth of a mammalian cell" can
have a minimum inhibitory concentration (MIC) between about 0.1
.mu.M and about 50 .mu.M, or between about 0.5 .mu.M and about 20
.mu.M. The MIC of a compound of the invention can be about 0.1
.mu.M, about 0.2 .mu.M, about 0.3 .mu.M, about 0.4 .mu.M, about 0.5
.mu.M, about 0.6 .mu.M, about 0.7 .mu.M, about 0.8 .mu.M, about 0.9
.mu.M, about 1 .mu.M, about 1.1 .mu.M, about 1.2 .mu.M, about 1.3
.mu.M, about 1.4 .mu.M, about 1.5 .mu.M, about 1.6 .mu.M, about 1.7
.mu.M, about 1.8 .mu.M, about 1.9 .mu.M, about 2 .mu.M, about 2.1
.mu.M, about 2.2 .mu.M, about 2.3 .mu.M, about 2.4 .mu.M, about 2.5
.mu.M, about 2.6 .mu.M, about 2.7 .mu.M, about 2.8 .mu.M, about 2.9
.mu.M, about 3 .mu.M, about 3.1 .mu.M, about 3.2 .mu.M, about 3.3
.mu.M, about 3.4 .mu.M, about 3.5 .mu.M, about 3.6 .mu.M, about 3.7
.mu.M, about 3.8 .mu.M, about 3.9 .mu.M, about 4 .mu.M, about 4.1
.mu.M, about 4.2 .mu.M, about 4.3 .mu.M, about 4.4 .mu.M, about 4.5
.mu.M, about 4.6 .mu.M, about 4.7 .mu.M, about 4.8 .mu.M, about 4.9
.mu.M, about 5 .mu.M, about 5.1 .mu.M, about 5.2 .mu.M, about 5.3
.mu.M, about 5.4 .mu.M, about 5.5 .mu.M, about 5.6 .mu.M, about 5.7
.mu.M, about 5.8 .mu.M, about 5.9 .mu.M, about 6 .mu.M, about 6.1
.mu.M, about 6.2 .mu.M, about 6.3 .mu.M, about 6.4 .mu.M, about 6.5
.mu.M, about 6.6 .mu.M, about 6.7 .mu.M, about 6.8 .mu.M, about 6.9
.mu.M, about 7 .mu.M, about 7.1 .mu.M, about 7.2 .mu.M, about 7.3
.mu.M, about 7.4 .mu.M, about 7.5 .mu.M, about 7.6 .mu.M, about 7.7
.mu.M, about 7.8 .mu.M, about 7.9 .mu.M, about 8 .mu.M, about 8.1
.mu.M, about 8.2 .mu.M, about 8.3 .mu.M, about 8.4 .mu.M, about 8.5
.mu.M, about 8.6 .mu.M, about 8.7 .mu.M, about 8.8 .mu.M, about 8.9
.mu.M, about 9 .mu.M, about 9.1 .mu.M, about 9.2 .mu.M, about 9.3
.mu.M, about 9.4 .mu.M, about 9.5 .mu.M, about 9.6 .mu.M, about 9.7
.mu.M, about 9.8 .mu.M, about 9.9 .mu.M, about 10 .mu.M, about 10.5
.mu.M, about 11 .mu.M, about 11.5 .mu.M, about 12 .mu.M, about 12.5
.mu.M, about 13 .mu.M, about 13.5 .mu.M, about 14 .mu.M, about 14.5
.mu.M, about 15 .mu.M, about 15.5 .mu.M, about 16 .mu.M, about 16.5
.mu.M, about 17 .mu.M, about 17.5 .mu.M, about 18 .mu.M, about 18.5
.mu.M, about 19 .mu.M, about 19.5 .mu.M, or about 20 .mu.M. At the
MIC, the compound has hemolytic activity against human red cells,
as measured in the in vitro hemolytic assay described in the
Methods herein, which can be 0-10%, 5-10%, 0-5%, about 0%, about
1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9%, about 10%, less than 10%, less than 9%, less
than 8%, less than 7%, less than 6%, less than 5%, less than 4%,
less than 3%, less than 2%, or less than 1%.
[0045] This document also features a method of designing an
antimicrobial peptide or stapled antimicrobial peptide (STAMP) that
has improved microbial activity and/or reduced hemolytic activity
relative to the unmodified antimicrobial peptide or stapled
antimicrobial peptide. The method involves one or more of: (i)
increasing the net positive charge of the antimicrobial peptide or
stapled antimicrobial peptide (e.g., by increasing the number of
basic residues, such as lysine, in the peptide, e.g., by amino acid
substitution); (ii) increasing the number of histidine residues,
e.g., by amino acid substitution; and/or (iii) reducing the rigid
helicity of an AMP or STAMP (e.g., substituting an amino acid with
D-alanine). In certain instances the AMP or STAMP has at least one
(e.g., 1, 2, 3, 4, 5) histidines and/or lysines relative to the
unmodified antimicrobial peptide.
[0046] As used herein, the terms "about" and "approximately" are
defined as being within plus or minus 10% of a given value or
state, preferably within plus or minus 5% of said value or
state.
[0047] 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.
[0048] 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
[0049] Staples are represented by the symbol "X" in the figures and
throughout the disclosure.
[0050] FIG. 1 is a chart depicting the maganin II amino acid
sequence (SEQ ID NO: 1) with the positions of various staples and
stitches indicated.
[0051] FIG. 2 is a depiction of the pexiganan sequence (SEQ ID NO:
2) with the positions of various staples and stitches
indicated.
[0052] FIG. 3A is a depiction of the magainin II amino acid
sequence aligned with the sequences of seven stapled derivatives to
show the position of the i, i+7 staple. The amino acids set forth
in this figure correspond to SEQ ID NOs: 134 and 178-184, numbered
consecutively.
[0053] FIG. 3B is a helical wheel projection of the magainin II
amino acid sequence (SEQ ID NO: 169) with staple positions denoted
by sequence numbers. Residues A15, N22, K4, K11, A18, H7, K14, G3,
M21, and K10 are the hydrophilic amino acids and residues A8, G1,
E19, F12, F5, S23, F16, A9, I2, I20, A13, L6, and V17 are the
hydrophobic amino acids.
[0054] FIG. 4A is a series of line graphs depicting the CD spectra
of magainin II and stapled analogues in aqueous solution.
[0055] FIG. 4B is a series of line graphs depicting the CD spectra
of magainin II and stapled analogues in TFE:Water (1:1) mixture. An
* indicates that the measured .alpha.-helicity exceeds the
calculated ideal .alpha.-helicity of an undecapeptide standard.
[0056] FIG. 5 is a series of line graphs depicting the hemolytic
activity of magainin II and stapled analogues. Peptides were
incubated with 1% (v/v) human red blood cells in phosphate-buffered
solution for 1 hour at 37.degree. C. and then the supernatant was
isolated and the amount of hemoglobin released was measured at 540
nm. % hemolysis was calculated relative to an untreated
control.
[0057] FIG. 6A is a depiction of the pexiganan amino acid sequence
aligned with the sequences of stapled derivatives to show the
positions of the i, i+7 staples. The amino acids set forth in this
figure correspond to SEQ ID NOs: 2 and 185-187, numbered
consecutively.
[0058] FIG. 6B is a helical wheel projection of the pexiganan amino
acid sequence (SEQ ID NO: 2) with staple positions denoted by
sequence numbers. Residues A15, K22, K4, K11, K18, K7, K14, G3,
K21, and K10 are the hydrophilic amino acids and residues K8, G1,
119, F12, F5, F16, A9, I2, L20, G13, L6, and V17 are the
hydrophobic amino acids.
[0059] FIG. 7A is a series of line graphs depicting the CD spectra
of pexiganan and stapled analogues in aqueous solution.
[0060] FIG. 7B is a series of line graphs depicting the CD spectra
of pexiganan and stapled analogues in TFE:Water (1:1) mixture.
[0061] FIG. 8 is a series of line graphs depicting the hemolytic
activity of pexiganan and stapled analogues. Peptides were
incubated with 1% (v/v) human red blood cells in phosphate-buffered
solution for 1 hour at 37.degree. C. and then the supernatant was
isolated and the amount of hemoglobin released was measured at 540
nm. % hemolysis was calculated relative to an untreated
control.
[0062] FIG. 9 is a depiction of the amino acid sequence of magainin
II and of various magainin stapled analogues using i+3, i+4, and
i+7 staples as single and/or double staples. The indicated staples
are comprised of, for example, two S.sub.5 stapling amino acids to
yield i, i+4 staples, one S.sub.5 and one R.sub.8 couple or one
R.sub.5 and one S.sub.8 couple to yield i, i+7 staples, and
R.sub.5/S.sub.5 or R.sub.3/S.sub.5 or S.sub.3/R.sub.5 pairs to
yield i, i+3 staples. The amino acids set forth in this figure
correspond to SEQ ID NOs: 134 and 188-252, numbered
consecutively.
[0063] FIG. 10 is a depiction of the amino acid sequences of
various pexiganan stapled analogues containing i+3, i+4, and i+7
staples (SEQ ID NOs: 253-258, respectively, in order of
appearance). The arrows denote the direction of the staple scan or
walk, in which the position of a staple spanning a fixed number of
amino acids (e.g., 2, 3, or 6) is shifted one amino acid at a time
down the length of the peptide.
[0064] FIG. 11 is a depiction of the amino acid sequences of the
members of a magainin II i+4 and i+7 stapled peptide library (SEQ
ID NOs: 135-168, respectively, in order of appearance). Amino acid
B stands for the unnatural amino acid norleucine. X,X represents
S.sub.5 pentenyl alanine pairs in i, i+4 stapled peptides and
S.sub.5 pentenyl and R.sub.8 octenyl alanine pairs in i, i+7
stapled peptides.
[0065] FIG. 12 is a bar graph depicting the minimum inhibitory
concentrations (MIC) of magainin II i, i+4 stapled peptides against
Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and
Staphylococcus aureus. MIC values over 50 .mu.g/mL were not
determined.
[0066] FIG. 13 is a bar graph depicting the MIC of magainin II 1+7
stapled peptides against Escherichia coli, Bacillus cereus,
Pseudomonas aeruginosa, and Staphylococcus aureus. MIC values over
50 .mu.g/mL were not determined.
[0067] FIG. 14 is a bar graph depicting the hemolytic activity of
magainin II i, i+4 and i, i+7 stapled peptides in 1% red blood cell
suspension for 1 hour at 37.degree. C. Activity was normalized to
total lysis with 1% Triton-X100 solution.
[0068] FIG. 15A is a model depicting the hydrophobic face of
magainin II. Regions colored in dark gray represent highly
hydrophobic residues (I, L, F, M, B); regions colored in medium
gray contain residues with relatively low hydrophobicity (G, A);
regions colored in light gray are charged/hydrophilic (H, K, E, N,
S).
[0069] FIG. 15B is a model depicting the hydrophilic face of
magainin II. Regions colored in dark gray represent highly
hydrophobic residues (I, L, F, M, B); regions colored in medium
gray contain residues with relatively low hydrophobicity (G, A);
regions colored in light gray are charged/hydrophilic (H, K, E, N,
S).
[0070] FIG. 15C is a model depicting the hydrophobic face of
Mag(i+4)1. Regions colored in dark gray represent highly
hydrophobic residues (I, L, F, M, B); regions colored in medium
gray contain residues with relatively low hydrophobicity (G, A);
regions colored in light gray are charged/hydrophilic (H, K, E, N,
S); regions that are circled depict the hydrophobic i+4 staple
position.
[0071] FIG. 15D is a model depicting the hydrophobic face of
Mag(i+4)9. Regions colored in dark gray represent highly
hydrophobic residues (I, L, F, M, B); regions colored in medium
gray contain residues with relatively low hydrophobicity (G, A);
regions colored in light gray are charged/hydrophilic (H, K, E, N,
S); regions that are circled depict the hydrophobic i+4 staple
position.
[0072] FIG. 15E is a model depicting the hydrophobic face of
Mag(i+4)16. Regions colored in dark gray represent highly
hydrophobic residues (I, L, F, M, B); regions colored in medium
gray contain residues with relatively low hydrophobicity (G, A);
regions colored in light gray are charged/hydrophilic (H, K, E, N,
S); regions that are circled depict the hydrophobic i+4 staple
position.
[0073] FIG. 15F is a model depicting the hydrophilic face of
Mag(i+4)6. Regions colored in dark gray represent highly
hydrophobic residues (I, L, F, M, B); regions colored in medium
gray contain residues with relatively low hydrophobicity (G, A);
regions colored in light gray are charged/hydrophilic (H, K, E, N,
S); regions that are circled depict the hydrophobic i+4 staple
position.
[0074] FIG. 16A is a plot depicting the specificity of the
interaction of magainin II stapled derivative Peptide 14 (SEQ ID
NO: 260) with liposomes simulating bacterial (e.g., E. coli) or
mammalian cell membranes using hydrogen-deuterium exchange mass
spectrometry.
[0075] FIG. 16B is a plot depicting the specificity of the
interaction of magainin II stapled derivative Peptide 15 (SEQ ID
NO: 261) with liposomes simulating bacterial (e.g., E. coli) or
mammalian cell membranes using hydrogen-deuterium exchange mass
spectrometry.
[0076] FIG. 16C is a plot depicting the specificity of the
interaction of magainin II (SEQ ID NO: 262) with liposomes
simulating bacterial (e.g., E. coli) or mammalian cell membranes
using hydrogen-deuterium exchange mass spectrometry.
[0077] FIG. 17 is a depiction of the amino acid sequences of the
members of a magainin II (i+4)15 lysine scan library (SEQ ID NOs:
32-48, respectively, in order of appearance). Amino acid B stands
for the non-natural amino acid norleucine.
[0078] FIG. 18 is a depiction of the amino acid sequences of the
members of a magainin II (i+4)15 glutamic acid scan library (SEQ ID
NOs: 49-68, respectively, in order of appearance). Amino acid B
stands for the non-natural amino acid norleucine.
[0079] FIG. 19 is a depiction of the amino acid sequences of the
members of a magainin II (i+4)15 histidine point mutations
sequences and G13 mutant analogues (SEQ ID NOs: 73-82,
respectively, in order of appearance). The following symbols are
defined as follows: B=Norleucine; !=2-aminoisobutyric acid;
&=Hydroxyproline; a=d-Alanine; k=d-Lysine.
[0080] FIG. 20 is a depiction of the amino acid sequences of the
members of a second generation of magainin II double staple
analogues (SEQ ID NOs: 170-174, respectively, in order of
appearance). Amino acid B stands for the non-natural amino acid
norleucine.
[0081] FIG. 21 is a depiction of sequences of pleurocidin-NH.sub.2
and two rationally designed double staple analogues (SEQ ID NOs:
175-177, respectively, in order of appearance).
[0082] FIG. 22A is a model of the hydrophobic face of
pleurocidin-NH.sub.2. Regions colored in dark gray have highly
hydrophobic residues (F, V, L, Y, W); regions colored in light gray
have relatively low hydrophobicity residues (G, A, T); regions
colored in very light gray represent charged/hydrophilic residues
(H, K, E, N, S); regions marked with dashed ellipses depict the
hydrophobic i+4 double staple positions; the region circled depicts
a lysine mutation. Surfaces were generated using PDB file 1Z64.
[0083] FIG. 22B is a model of the hydrophilic face of
Pleu(i+4)1,15. Regions colored in dark gray have highly hydrophobic
residues (F, V, L, Y, W); regions colored in light gray have
relatively low hydrophobicity residues (G, A, T); regions colored
in very light gray represent charged/hydrophilic residues (H, K, E,
N, S); regions marked with dashed ellipses depict the hydrophobic
i+4 double staple positions; the region circled depicts a lysine
mutation. Surfaces were generated using PDB file 1Z64.
[0084] FIG. 22C is a model of the hydrophobic face of
Pleu(i+4)1,15(A9K). Regions colored in dark gray have highly
hydrophobic residues (F, V, L, Y, W); regions colored in light gray
have relatively low hydrophobicity residues (G, A, T); regions
colored in very light gray represent charged/hydrophilic residues
(H, K, E, N, S); regions marked with dashed ellipses depict the
hydrophobic i+4 double staple positions; the region circled depicts
a lysine mutation. Surfaces were generated using PDB file 1Z64.
DETAILED DESCRIPTION
Stabilized Peptides
[0085] The present disclosure provides structurally-stabilized and
microbial-selective peptides related to anti-bacterial peptides
(AMP) (referred to at times as stabilized .alpha.-helices of AMP or
stabilized AMP or STAMP) comprising at least two modified amino
acids joined by an internal (intramolecular) cross-link (or
staple), wherein the at least two amino acids are separated by,
e.g., 2, 3, or 6 amino acids. Stabilized peptides herein include
stapled peptides, including peptides having, e.g., 1, 2, 3, 4, 5,
or more staples and/or stitched peptides.
[0086] A compound herein can exhibit helical stability by the
maintenance of .alpha.-helical structure by a compound of the
invention as measured by circular dichroism or NMR. For example, in
some aspects, the compound exhibits at least a 1.25, 1.5, 1.75 or
2-fold increase in .alpha.-helicity as determined by circular
dichroism compared to a corresponding un-cross-linked peptide. In
some aspects, the compound can exhibit about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, about 90%, about 95%, or about 100% helicity.
[0087] 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), unnatural alpha-amino acids (including, but not limited to
.alpha.,.alpha.-disubstituted and N-alkylated amino acids), natural
beta-amino acids (e.g., beta-alanine), and unnatural 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.
[0088] There are many known unnatural amino acids any of which may
be included in the peptides of the present invention. Some examples
of unnatural amino acids are 4-hydroxyproline, 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; --NO.sub.2; CH.sub.3), disubstituted
phenylalanines, substituted tyrosines (e.g., further substituted
with --C.dbd.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.
[0089] A "peptide" or "polypeptide" comprises a polymer of amino
acid residues linked together by peptide (amide) bonds. The terms,
as used herein, refer to proteins, polypeptides, and peptides of
any size, structure, or function. Typically, a peptide or
polypeptide will be at least three amino acids long. A peptide or
polypeptide may refer to an individual protein or a collection of
proteins. In some instances, peptides can include only natural
amino acids, although non-natural amino acids (i.e., compounds that
do not 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. A peptide or
polypeptide may also be a single molecule or may be a
multi-molecular complex, such as a protein. A peptide or
polypeptide may be just a fragment of a naturally occurring protein
or peptide. A peptide or polypeptide may be naturally occurring,
recombinant, or synthetic, or any combination thereof. "Dipeptide"
refers to two covalently linked amino acids.
[0090] In some aspects, the present disclosure provides internally
cross-linked (ICL) peptides comprising the amino acid sequence:
GIGKFLHZ.sub.1AKKFZ.sub.2KAFVZ.sub.3EIMNS (SEQ ID NO:1)
wherein:
[0091] Z.sub.1 is S or A and Z.sub.2 and Z.sub.3 are independently
A or G;
[0092] the side chains of two amino acids separated by two, three,
or six amino acids are replaced by an internal staple; the side
chains of three amino acids are replaced by an internal stitch; the
side chains of four amino acids are replaced by two internal
staples, or the side chains of five amino acids are replaced by the
combination of an internal staple and an internal stitch.
[0093] In some aspects, the present disclosure provides internally
cross-linked polypeptides comprising the amino acid sequence:
[0094] GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO:2)
[0095] wherein:
[0096] the side chains of two amino acids separated by two, three,
or six amino acids are replaced by an internal staple; the side
chains of three amino acids are replaced by an internal stitch; the
side chains of four amino acids are replaced by two internal
staples, or the side chains of five amino acids are replaced by the
combination of an internal staple and an internal stitch.
[0097] In some instances, one or more (e.g., 1, 2, 3, 4, or 5) V,
F, I, or L is replaced by a non-hydrophobic amino acid. In some
instances, internally cross-linked polypeptides of the disclosure
include an internal staple replacing the side chains of two amino
acids separated by two, three, or six amino acids comprises an
internal staple selected from those depicted in FIG. 1 or FIG. 2.
In some instances, the internal staples and/or the internal stitch
replacing the side chains of the three amino acids includes an
internal stitch selected from FIG. 1 and FIG. 2. In some instances,
the internal staples and/or the internal stitch comprises at least
two internal staples (replacing the side chains of 4 amino acids,
i.e., each staple is between two amino acids separated by 3 amino
acids). In some instances, the internal staples and/or the internal
stitch comprises a combination of at least one internal staple and
an internal stitch. In some instances, the internal stitch replaces
the side chain of a first amino acid and a second and a third amino
acid thereby cross-linking the first amino acid (which lies between
the second and third amino acids) to the second and third amino
acid via an internal cross-link, wherein the first and second amino
acid are separated by two, three, or six amino acids, the first and
the third amino acids are separated by two, three, or six amino
acids, and the second and third amino acids are distinct amino
acids. In some aspects, the internal stitch replacing the side
chains of the three amino acids cross-links a pair of amino acids
separated by two, three, or six amino acids. In some aspects, the
side chains of the four amino acids of the internally cross-linked
polypeptides of the disclosure are replaced by two distinct
internal staples. In some aspects, a first of the two distinct
internal staples cross-links a first pair of amino acids separated
by two, three, or six amino acids, and a second of the at least two
distinct internal staples cross-links a second pair of amino acids
separated by two, three, or six amino acids.
[0098] In some instances, peptides can include (e.g., comprise,
consist essentially of, or consist of) at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, or more contiguous amino acids of a sequence
selected from:
TABLE-US-00001 Magainin (SEQ ID NO: 3) GIGKFLHSAKKFGKAFVGEIMNS
Pexiganan (SEQ ID NO: 2) GIGKFLKKAKKFGKAFVKILKK
The following additional peptides can be modified by an internal
cross-link. In each case, an example of a cross-linked variant is
included (X indicates an amino acid whose side chain has been
replaced by an internal staple). An "--NH.sub.2" at the C-terminus
of a sequence indicates that the C-terminal amino acid is amidated.
A "--COOH" at the C-terminus of a sequence indicates that the
C-terminal amino acid is not modified.
TABLE-US-00002 Pleurocidin (Mucus Membrane of Winter Flounder) (SEQ
ID NO: 4) GWGSFFKKAAHVGKHVGKAALTHYL (SEQ ID NO: 18)
GWGSFFKKAAHXGKHVGKXALTHYL Pardaxin (Secretion from Red Sea
flatfish) (SEQ ID NO: 5) GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE (SEQ ID
NO: 19) GFFALIPKIISXPLFKTLXSAVGSALSSSGEQE Hagfish Intestinal
Antimicrobial Peptide (HFIAP) (SEQ ID NO: 6)
GFFKKAWRKVKHAGRRVLKKGVGRHYVNNWLK W = brominated Trp residue (SEQ ID
NO: 20) GFFKKAWRKVKHAXRRVLKKXVGRHYVNNWLK PGQ (Secretions from
Xenopus laevis) (SEQ ID NO: 7) GVLSNVIGYLKKLGTGALNAVLKQ (SEQ ID NO:
21) GVLSNVIGYLKKLXTGALNAXLKQ Buforin II (Stomach Secretion from
Asian Toad Bufo bufo garagrizans) (SEQ ID NO: 8)
TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 22) TRSSRAGLQFPXGRVHRLXRK
Dermaseptin (Skin secretion Phyllomedusa frogs) (SEQ ID NO: 9)
ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ-NH2 (SEQ ID NO: 23)
ALWKTMLKKLGTMXLHAGKAXLGAAADTISQGTQ-NH2 Caerin (Skin glands of Tree
Frog Litoria chloris) (SEQ ID NO: 10) GLFKVLGSVAKHLLPHVVPVIAEKL-NH2
(SEQ ID NO: 24) GLFKVLGSVAKHLXPHVVPVXAEKL-NH2 Melittin (SEQ ID NO:
11) GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 (SEQ ID NO: 25)
GIGAVLKVLTTGXPALISWXKRKRQQ-NH2 Cecropin A (SEQ ID NO: 12)
KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH2 (SEQ ID NO: 26)
KWKXFKKIEKXGQNIRDGIIKAGPAVAVVGQATQIAK-NH2 Lycotoxin I (SEQ ID NO:
13) KIKWFKTMKSIAKFIAKEQMKKHLGGE-COOH (SEQ ID NO: 27)
KIKWFKTXKSIAKFXAKEQMKKHLGGE-COOH Styelins B (SEQ ID NO: 14)
GFGPAFHSVSNFAKKHKTA-NH2 (SEQ ID NO: 28) GFGPXFHSVSNXAKKHKTA-NH2
Clavanin B (SEQ ID NO: 15) VFQFLGRIIHHVGNFVHGFSHVF-NH2 (SEQ ID NO:
29) VFQFXGRIIHHXGNFVHGFSHVF-NH2 Cathelicidin A (Example below is
CP-11, an indolicidin derivative from cow stomach) (SEQ ID NO: 16)
ILKKWPWWPWRRK-NH2 (SEQ ID NO: 30) IXKKWPWWWRRK-NH2 Dermcidin (SEQ
ID NO: 17) SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL- COOH
(SEQ ID NO: 31) SSLLEKGLDGXKKAVGGXGKLGKDAVEDLESVGKGAVHDVKDVLDSVL-
COOH
[0099] wherein the peptide has a reinforced or stabilized alpha
helical secondary structure (e.g., wherein the peptide includes at
least one internal crosslink).
[0100] The following additional peptides in Table 1 (SEQ ID
NOs:32-82, numbered consecutively from top to bottom, left column,
then from top to bottom, right column) are specific magainin
stapled analogues Mag(i+4)15, Mag(i+4)0, and Mag(i+4)18, and
magainin double stapled analogues Mag(i+4)1,15(A9K) and
Mag(i+4)2,15(A9K). The symbol "!" represents 2-aminoisobutyric
acid; the symbol "&" represents hydroxyproline; the symbol "B"
represents norleucine; the symbol "a" represents D-alanine; the
symbol "k" represents D-lysine.
TABLE-US-00003 TABLE 1 Sequences of specific magainin i + 4 stapled
or double stapled analogues Mag(i + 4)15(S23K)
GIGKFLHSAKKFGKAXVGEXBNK Mag(i + 4)15(K10E) GIGKFLHSAEKFGKAXVGEXBNS
Mag(i + 4)15(N22K) GIGKFLHSAKKFGKAXVGEXBKS Mag(i + 4)15(K11E)
GIGKFLHSAKEFGKAXVGEXBNS Mag(i + 4)15(B21K) GIGKFLHSAKKFGKAXVGEXKNS
Mag(i + 4)15(F12E) GIGKFLHSAKKEGKAXVGEXBNS Mag(i + 4)15(E19K)
GIGKFLHSAKKFGKAXVGKXBNS Mag(i + 4)15(G13E) GIGKFLHSAKKFEKAXVGEXBNS
Mag(i + 4)15(G18K) GIGKFLHSAKKFGKAXVKEXBNS Mag(i + 4)15(K14E)
GIGKFLHSAKKFGEAXVGEXBNS Mag(i + 4)15(V17K) GIGKFLHSAKKFGKAXKGEXBNS
Mag(i + 4)15(A15E) GIGKFLHSAKKFGKEXVGEXBNS Mag(i + 4)15(A15K)
GIGKFLHSAKKFGKKXVGEXBNS Mag(i + 4)15(V17E) GIGKFLHSAKKFGKAXEGEXBNS
Mag(i + 4)15(G13K) GIGKFLHSAKKFKKAXVGEXBNS Mag(i + 4)15(G18E)
GIGKFLHSAKKFGKAXVEEXBNS Mag(i + 4)15(F12K) GIGKFLHSAKKKGKAXVGEXBNS
Mag(i + 4)15(B21E) GIGKFLHSAKKFGKAXVGEXENS mag(i + 4)15(A9K)
GIGKFLHSKKKFGKAXVGEXBNS Mag(i + 4)15(N22E) GIGKFLHSAKKFGKAXVGEXBES
Mag(i + 4)15(S8K) GIGKFLHKAKKFGKAXVGEXBNS Mag(i + 4)15(S23E)
GIGKFLHSAKKFGKAXVGEXBNE Mag(i + 4)15(H7K) GIGKFLKSAKKFGKAXVGEXBNS
Mag(14-4)0 XIGKXLHSAKKFGKAFVGEIBNS Mag(i + 4)15(L6K)
GIGKFKHSAKKFGKAXVGEXBNS Mag(i + 4)18 GIGKFLHSAKKFGKAFVGXIBNX Mag(i
+ 4)15(F5K) GIGKKLHSAKKFGRAXVGEXBNS Mag(i + 4)1, 15(A9K)
GXGKFXHSKKKFGKAXVGEXBNS Mag(i + 4)15(G3K) GIKKFLHSAKKFGKAXVGEXBNS
Mag(i + 4)2, 15(A9K) GIXKFLXSKKKFGKAXVGEXBNS Mag(i + 4)15(I2K)
GKGKFLHSAKKFGKAXVGEXBNS Mag(i + 4)15(G3H) GIHKFLHSAKKFGKAXVGEXBNS
Mag(i + 4)15(G1K) KIGKFLHSAKKFGKAXVGEXBNS Mag(i + 4)15(S8H)
GIGKFLHHAKKFGKAXVGEXBNS Mag(i + 4)15(G1E) EIGKFLHSAKKFGKAXVGEXBNS
Mag(i + 4)15(A15H) GIGKFLHSAKKFGKHXVGEXBNS Mag(1 + 4)15(I2E)
GEGKFLHSAKKFGKAXVGEXBNS Mag(i + 4)15(G18H) GIGKFLHSAKKFGKAXVHEXBNS
Mag(i + 4)15(G3E) GIEKFLHSAKKFGKAXVGEXBNS Mag(i + 4)15(G13!)
GIGKFLHSAKKF!KAXVGEXBNS Mag(i + 4)15(K4E) GIGEFLHSAKKFGKAXVGEXBNS
Mag(i + 4)15(G13P) GIGKFLHSAKKFPKAXVGEXBNS Mag(i + 4)15(F5E)
GIGKELESAKKFGKAXVGEXBNS Mag(i + 4)15(G13&)
GIGKFLHSAKKF&KAXVGEXBNS Mag(i + 4)15(L6E)
GIGKFEHSAKKFGKAXVGEXBNS Mag(i + 4)15(G13A) GIGKFLHSAKKFAKAXVGEXBNS
Mag(i + 4)15(H7E) GIGKFLESAKKFGKAXVGEXBNS Mag(i + 4)15(G13a)
GIGKFLHSAKKFaKAXVGEXBNS Mag(i + 4)15(S8E) GIGKFLHEAKFFGKAXVGEXBNS
Mag(i + 4)15(G13k) GIGKFLHSAKKFkKAXVGEXBNS Mag(i + 4)15(A9E)
GIGKFLHSEKKFGKAXVGEXBNS
[0101] The following additional peptides in Table 2 (SEQ ID
NOs:83-133, numbered consecutively from top to bottom, left column,
then from top to bottom, right column) are specific magainin i+7
stapled analogues. The symbol "!" represents 2-aminoisobutyric
acid; the symbol "&" represents hydroxyproline; the symbol "B"
represents norleucine; the symbol "a" represents D-alanine; the
symbol "k" represents D-lysine.
TABLE-US-00004 TABLE 2 Sequences of specific magainin i + 7 stapled
or double stapled analogues Mag(i + 7)10(S23K)
GIGKFLHSAKXFGKAFVXEIBNK Mag(i + 7)10(K10E) GIGKFLHSAEXFGKAFVXEIBNS
Mag(i + 7)10(N22K) GIGKFLHSAKXFGKAFVXEIBKS Mag(i + 7)10(F12E)
GIGKFLHSAKXEGKAFVXEIBNS Mag(i + 7)10(B21K) GIGKFLHSAKXFGKAFVXEIKNS
Mag(i + 7)10(G13E) GIGKFLHSAKXFEKAFVXEIBNS Mag(i + 7)10(I20K)
GIGKFLHSAKXFGKAFVXEKBNS Mag(i + 7)10(K14E) GIGKFLHSAKXFGEAFVXEIBNS
Mag(i + 7)10(E19K) GIGKFLHSAKXFGKAFVXKIBNS Mag(i + 7)10(A15E)
GIGKFLHSAKXFGKEFVXEIBNS Mag(i + 7)10(V17K) GIGKFLHSAKXFGKAFKXEIBNS
Mag(i + 7)10(F16E) GIGKFLHSAKXFGKAEVXEIBNS Mag(i + 7)10(F15K)
GIGKFLHSAKXFGKAKVXEIBNS Mag(i + 7)10(V17E) GIGKFLHSAKXFGKAFEXEIBNS
Mag(i + 7)10(A14K) GIGKFLHSAKXFGKKFVXEIBNS Mag(i + 7)10(I20E)
GIGKFLHSAKXFGKAFVXEEBNS Mag(i + 7)10(G13K) GIGKFLHSAKXFKKAFVXEIBNS
Mag(i + 7)10(B21E) GIGKFLHSAKXFGKAFVXEIENS Mag(i + 7)10(F12K)
GIGKFLHSAKXKGKAFVXEIBNS Mag(i + 7)10(N22E) GIGKFLHSAKXFGKAFVXEIBES
Mag(i + 7)10(A9K) GIGKFLHSKKXFGKAFVXEIBNS Mag(i + 7)10(S23E)
GIGKFLHSAKXFGKAFVXEIBNE Mag(i + 7)10(S8K) GIGKFLHKAKXFGKAFVXEIBNS
Mag(i + 7)15 GIGKFLHSAKKFGKAXVGEIBNX Mag(i + 7)10(H7K)
GIGKFLKSAKXFGKAFVXEIBNS Mag(i + 7)0 XIGKFLHXAKKFGKAVFGEIBNS Mag(i +
7)10(L6K) GIGKFKHSAKXFGKAFVXEIBNS Mag(i + 4)1, (i + 7)10(A9K)
GXGKFXHSKKXFGKAFVXEIBNS Mag(i + 7)10(F5K) GIGKKLHSAKXFGKAFVXEIBNS
Mag(i + 4)2, (i + 7)10(A9K) GIXKFLXSKKXFGKAFVXEIBNS Mag(i +
7)10(G3K) GIKKFLHSAKXFGKAFVXEIBNS Mag(i + 7)10(G3H)
GIHKFLHSAKXFGKAFVXEIBNS Mag(i + 7)10(I2K) GKGKFLHSAKXFGKAFVXEIBNS
Mag(i + 7)10(S8H) GIGKFLHHAKXFGKAFVXEIBNS Mag(i + 7)10(G1K)
KIGKFLHSAKXFGKAFVXEIBNS Mag(i + 7)10(A15H) GIGKFLHSAKXFGKHFVXEIBNS
Mag(i + 7)10(G1E) EIGKFLHSAKXFGKAFVXEIBNS Mag(i + 7)10(G13!)
GIGKFLHSAKXF!KAFVXEIBNS Mag(i + 7)10(I2E) GEGKFLHSAKXFGKAFVXEIBNS
Mag(i + 7)10(13P) GIGKFLHSAKXFPKAFVXEIBNS Mag(i + 7)10(G3E)
GIEKFLHSAKXFGKAPVXEIBNS Mag(i + 7)10(G13&)
GIGKFLHSAKXF&KAFVXEIBNS Mag(i + 7)10(K4E)
GIGEFLHSAKXFGKAPVXEIBNS Mag(i + 7)10(G13A) GIGKFLHSAKXFAKAFVXEIBNS
Mag(i + 7)10(F5E) GIGKELHSAKXFGKAPVXEIBNS Mag(i + 7)10(G13a)
GIGKFLHSAKXFaKAFVXEIBNS Mag(i + 7)10(L6E) GIGKFEHSAKXFGKAPVXEIBNS
Mag(i + 7)10(G13k) GIGKFLHSAKXFkKAFVXEIBNS Mag(i + 7)10(H7E)
GIGKFLESAKXFGKAFVXEIBNS Mag(i + 7)10(S8E) GIGKFLHEAKXFGKAFVXEIBNS
Mag(i + 7)10A9E) GIGKFLHSEKXFGKAFVXEIBNX
[0102] As an example, the double stapled magainin analogue
Mag(i+4)1,15(A9K) shown in Table 1, above, displays especially
potent activity against Gram-negative bacteria (including, e.g., E.
coli, P aeruginosa), yet possesses little hemolytic activity even
at concentrations 8-fold higher than its MIC.
[0103] In some instances, the peptide has or can be induced to have
alpha helical secondary structure.
[0104] In some cases the peptide is a modified peptide that
includes 1, 2, or 3 conservative substitutions and/or 1 or 2
non-conservative substitutions and/or 1 or 2 insertions or
deletions compared to the sequence:
TABLE-US-00005 (SEQ ID NO: 1)
GIGKFLHZ.sub.1AKKFZ.sub.2KAFVZ.sub.3EIMNS
[0105] wherein Z.sub.1 is S or A; Z.sub.2 is G or A; and Z.sub.3 is
G or A;
[0106] wherein the peptide has a reinforced or stabilized alpha
helical secondary structure (e.g., wherein the peptide includes at
least one internal crosslink); and wherein the percent identity
calculation includes the cross-linked amino acids and the
cross-linked amino acids are considered non-conservative
substitutions. In some cases, the internal cross-link replaces the
side chains of two amino acids separated by 2 or 3 amino acids. In
some cases, the internal cross-link replaces the side chains of two
amino acids separated by 6 amino acids. In some cases, there are
two internal cross-links, each replacing the side chains of a pair
of amino acids separated by 3 amino acids and each cross-link being
on essentially the same face of the resulting essentially
alpha-helical peptide.
[0107] In some instances, the peptide is a modified peptide that
includes 1, 2, 3, 4, or 5 amino acid substitutions (e.g., 1, 2, 3,
4, or 5 amino acids are replaced with A or 1, 2, 3, 4, or 5 amino
acids are conservatively substituted).
[0108] In some instances, stabilized peptides can have at least 80%
(e.g., 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 100%) identity one
of SEQ ID NOs:1-17 or can include one of SEQ ID NOs:1-17 with one
or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, e.g., 1-2, 1-3, 1-4, or 1-5) conservative amino acid
substitutions. In some cases the side chain of an amino acid is
substituted by Formula I. In some cases, the stabilized peptide has
the sequence of one SEQ ID NOs: 1-17 with one or two staples (e.g.,
one staple between two amino acids separated by 2 or 3 (or 6) amino
acids or two staples each between two amino acids that are
separated by 2 or 3 (or 6) amino acids). In addition, 1, 2, 3, 4,
or 5 of the amino acids (whose side chains are not replaced with a
staple) in this stabilized peptide can be replaced by a
conservative substitution or can be replaced by A.
[0109] In some cases, the peptide is substituted to provide the
sequence:
[0110] ZZZKZZKKZKKZZKZZZKZZKK, where Z=the native amino acid of the
naturally occurring peptide (SEQ ID NO: 259).
[0111] In some instances, a "conservative amino acid substitution"
can include substitutions in which one amino acid residue is
replaced with another 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).
[0112] 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). The length
of a reference sequence aligned for comparison purposes can be at
least 30%, at least 40%, at least 50%, at least 60%, and 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
can be accomplished using, e.g., 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, e.g.,
in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).
[0113] As disclosed above, peptides herein include at least two
modified amino acids that together form an internal
(intramolecular) cross-link (or staple), wherein the at least two
modified amino acids are separated by: (A) two amino acids (i.e.,
i, i+3, shown in FIG. 1 and FIG. 2 as .diamond.), (B) three amino
acid (i.e., i, i+4, shown in FIG. 1 and FIG. 2 as .largecircle.),
or (C) six amino acids (i.e., i, i+7, shown in FIG. 1 and FIG. 2 as
.dagger.).
[0114] In the case of a cross-link between i and i+3, the
cross-link can be, e.g., a C.sub.7 alkylene or alkenylene. In the
case of a cross-link between i and i+4, the cross-link can be,
e.g., a C.sub.8 alkylene or alkenylene. In the case of a cross-link
between i and i+7, the cross-link can be, e.g., a C.sub.11,
C.sub.12, or C.sub.13 alkylene or alkenylene. When the cross-link
is an alkenylene, there can one or more double bonds.
[0115] In the case of a cross-link between i and i+3, the
cross-link can be, e.g., a C.sub.6, C.sub.7, or C.sub.8 alkylene or
alkenylene (e.g., a C.sub.6 alkenylene having one double bond). In
the case of a cross-link between i and i+4, the cross-link can be,
for example, a C.sub.8 alkylene or alkenylene. In the case of a
cross-link between i and i+7, the cross-link can be, e.g., a
C.sub.11, C.sub.12, or C.sub.13 alkylene or alkenylene (e.g., a
C.sub.11 alkenylene having one double bond). When the cross-link is
alkenlyene, there can be one or more double bonds. The cross-link
can be optionally substituted with 1-5 substituents selected from
--OH and --NH.sub.3.
[0116] "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 (see, e.g.,
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, e.g., in WO
2008121767 and WO 2010/068684, which are both hereby incorporated
by reference in their entirety. 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).
[0117] 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 (see, e.g., Kawamoto et al. 2012 J Med Chem. 55:1137;
WO 2010/060112).
[0118] Stapling of a peptide using an all-hydrocarbon cross-link
has been shown to help maintain its native conformation and/or
secondary structure, particularly under physiologically relevant
conditions (see, e.g., Schafmiester et al., J Am Chem Soc.,
122:5891-5892, 2000; Walensky et al., Science, 305:1466-1470,
2004).
[0119] Stapling the polypeptide herein by an all-hydrocarbon
crosslink predisposed to have an alpha-helical secondary structure
can improve stability and various pharmacokinetic properties.
[0120] Stabilized peptides herein include at least two internally
cross-linked or stapled amino acids, wherein the at least two amino
acids are separated, e.g., by two (i.e., i, i+3, shown in FIG. 1
and FIG. 2), three (i.e., i, i+4, shown FIG. 1 and FIG. 2), or six
(i.e., i, i+7, shown in FIG. 1 and FIG. 2) 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, 3, 4, 5, or more staples.
Examples of peptide staples are illustrated in the figures.
Cross-linked peptides (e.g., stapled and/or stitched peptides) are
generally referred to herein as STAMP peptides.
[0121] 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 (referred to here as the core or central
amino acid and shown in FIG. 1 and FIG. 2 as "i") forms an internal
cross-link (between alpha carbons) with each of the two flanking
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 2, 3, or
6 amino acids (e.g., i, i-3, i, i-4, i, i-7 (shown in FIG. 1 and
FIG. 2, i, i+3, i, i+4, i, i+7 (shown in FIG. 1 and FIG. 2, where
"i" is the core amino acid). 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.
[0122] In some aspects, peptides herein can include a combination
of at least one (e.g., 1, 2, 3, 4, or 5) staple and at least one
(e.g., 1, 2, 3, 4, or 5) stitch.
[0123] Cross-linked peptides (e.g., stapled and/or stitched
peptides) are generally referred to herein as STAMP peptides.
Peptides can include cross-linked amino acids at one or more of the
positions illustrated in FIG. 1 and FIG. 2.
[0124] In FIG. 1 and FIG. 2 positions of cross-links are indicated
by symbols and the letter "i". For example, i.sub.10 (C.sub.1) can
be linked via a i+3 staple to F.sub.1 or G.sub.0 (also called i-3)
or a i+4 staple to G.sub.1 or F.sub.0 (also called i-4) or a i+7
staple to C.sub.2 or C.sub.0 (also called i-7). Of course, i.sub.10
(C.sub.1) could be stitched to, for example F.sub.1 (i+3) and
C.sub.0 (i-7). Selection of amino acids for modification (e.g., to
support an internal cross-link) can also be facilitated by staple
scanning. The terms "staple scan" and "staple walk" refer
interchangeably to the synthesis of a library of stapled peptides
whereby the location of the i and i+3; i and i+4; and i and i+7
single and multiple staple, or stitches, are positioned
sequentially down the length of the peptide sequence, sampling all
possible positions, to identify desired, effective, suitable, or
optimal properties and activities for the stapled or stitched
constructs. Examples of staple scanning methods are illustrated in
the figures.
[0125] Suitable tethers are described herein and in, e.g.,
US2005/0250680, PCT/US2008/058575, WO 2009/108261, and WO
2010/148335.
[0126] Amino acid side chains suitable for use in the peptides
disclosed herein are known in the art. For example, suitable amino
acid side chains include methyl (as the alpha-amino acid side chain
for alanine is methyl), 4-hydroxyphenylmethyl (as the alpha-amino
acid side chain for tyrosine is 4-hydroxyphenylmethyl) and
thiomethyl (as the alpha-amino acid side chain for cysteine is
thiomethyl), etc. A "terminally unsaturated amino acid side chain"
refers to an amino acid side chain bearing a terminal unsaturated
moiety, such as a substituted or unsubstituted, double bond (e.g.,
olefinic) or a triple bond (e.g., acetylenic), that participates in
crosslinking reaction with other terminal unsaturated moieties in
the polypeptide chain. In certain aspects, a "terminally
unsaturated amino acid side chain" is a terminal olefinic amino
acid side chain. In certain aspects, a "terminally unsaturated
amino acid side chain" is a terminal acetylenic amino acid side
chain. In certain aspects, the terminal moiety of a "terminally
unsaturated amino acid side chain" is not further substituted.
[0127] 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+3, or 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+3; i and i+4; or i and i+7 are ideal candidates for chemical
modification and cross-linking (see FIG. 1 and FIG. 2). Thus, for
example, where a peptide has the sequence . . . Xaa.sub.1,
Xaa.sub.2, Xaa.sub.3, Xaa.sub.4, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9 . . . (wherein " . . . " indicates the
optional presence of additional amino acids), cross-links between
Xaa.sub.1 and Xaa.sub.4, or between Xaa.sub.1 and Xaa.sub.5, or
between Xaa.sub.1 and Xaa.sub.8 are useful as are cross-links
between Xaa.sub.2 and Xaa.sub.5, or between Xaa.sub.2 and
Xaa.sub.6, or between Xaa.sub.2 and Xaa.sub.9, etc.
[0128] Polypeptides can include more than one crosslink within the
polypeptide sequence to either further stabilize the sequence or
facilitate the stabilization of longer polypeptide stretches. If
the polypeptides are too long to be readily synthesized in one
part, independently synthesized, cross-linked peptides can be
conjoined by a technique called native chemical ligation (see,
e.g., Bang, et al., J. Am. Chem. Soc. 126:1377). Alternately, large
peptides are routinely synthesized using a convergent approach
whereby fully protected fragments are specifically and sequentially
reacted to form the full length desired product, after final
deprotection, such as in the industrial synthesis of Fuzeon.
Compounds
[0129] In some aspects, the stabilized peptides can have 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30 amino acids.
[0130] The invention features a modified polypeptide of Formula
(I),
##STR00002##
[0131] or a pharmaceutically-acceptable salt thereof,
[0132] wherein;
[0133] each R.sub.1 and R.sub.2 are independently H, or a C.sub.1
to C.sub.10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,
heteroarylalkyl, or heterocyclylalkyl;
[0134] each R.sub.3 is alkylene, alkenylene, or alkynylene (e.g., a
C.sub.6, C.sub.7, or C.sub.11 alkenylene) substituted with 1-6
R.sub.4;
[0135] each R.sub.4 is independently --NH.sub.3 or --OH, wherein
each --NH.sub.3 is optionally substituted;
[0136] wherein each R.sub.3 replaces, relative to the corresponding
parent (i.e., unmodified) non-internally cross-linked AMP, the side
chains of at least one pair (e.g., one or two pairs) of amino acids
separated by 2, 3, or 6 amino acids (i.e., x=2, 3, or 6).
[0137] As used above, and elsewhere in the present document, a
"corresponding parent (i.e., unmodified) non-internally
cross-linked AMP" can be a wild-type AMP, or any of the variants of
a wild-type AMP disclosed in the present document, except that such
a variant would not include an internal cross-link as described
herein.
[0138] In the case of Formula I, the following aspects are among
those disclosed.
[0139] In cases where x=2 (i.e., i+3 linkage), R.sub.3 can be, for
example, a C.sub.7 alkylene, alkenylene. Where it is an alkenylene,
there can one or more double bonds. In cases where x=6 (i.e., i+4
linkage), R.sub.3 can be, for example, a C.sub.11, C.sub.12, or
C.sub.13 alkylene or alkenylene. Where it is an alkenylene there
can be one or more double bonds. In cases where x=3 (i.e., i+4
linkage), R.sub.3 can be, for example, a C.sub.8 alkylene,
alkenylene. Where it is an alkenylene, there can one or more double
bonds.
[0140] In certain instances, the two alpha, alpha disubstituted
stereocenters (alpha carbons) are both in the R configuration or S
configuration (e.g., 1, 1+4 cross-link), or one stereocenter is R
and the other is S (e.g., 1, 1+7 cross-link). Thus, where Formula I
is depicted as
##STR00003##
the C' and C'' disubstituted stereocenters can both be in the R
configuration or they can both be in the S configuration, for
example when x is 3. When x is 6, the C' disubstituted stereocenter
is in the R configuration and the C'' disubstituted stereocenter is
in the S configuration or the C' disubstituted stereocenter is in
the S configuration and the C'' disubstituted stereocenter is in
the R configuration. An R.sub.3 double bond (based on the
definition above, R.sub.3 contains an alkane, alkene, or alkyne
moiety; in general, it is an alkene) can be in the E or Z
stereochemical configuration. Similar configurations are possible
for the carbons in Formula II corresponding to C' and C'' in the
formula depicted immediately above.
[0141] In some instances, the polypeptide includes an amino acid
sequence which, in addition to the amino acids side chains that are
replaced by a cross-link, have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
or 12 amino acid changes (e.g., conservative amino acid changes) in
any of SEQ ID NOs: 1-17.
[0142] In some aspects, a compound has the Formula (II):
##STR00004##
[0143] wherein: [0144] each A, C, D, and E is independently a
natural or non-natural amino acid; [0145] each B is independently a
natural or non-natural amino acid, amino acid analog,
##STR00005##
[0145] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0146] each R.sub.1 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted, or together with R.sub.1 and the atom to which both
R.sub.1 and L are bound forms a ring; [0147] each R.sub.2 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is
unsubstituted or substituted, or together with R.sub.2 and the atom
to which both R.sub.2 and L are bound forms a ring; [0148] each
R.sub.3 is independently hydrogen, alkyl, alkenyl, alkynyl,
arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, aryl, or heteroaryl, any of which is unsubstituted
or substituted; [0149] each L is independently a macrocycle-forming
linker; [0150] each L.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, heteroarylene, or [--R.sub.4--K--R.sub.4-]n, any of which
is unsubstituted or substituted; [0151] each R.sub.4 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any
of which is unsubstituted or substituted; [0152] each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, CONR.sub.3,
OSO.sub.2NR.sub.3, NR.sub.3q, CONR.sub.3q, OCONR.sub.3q, or
OSO.sub.2NR.sub.3q, wherein each R.sub.3q is independently a point
of attachment to R.sub.1 or R.sub.2; [0153] each n is independently
1, 2, 3, 4, or 5; [0154] each R.sub.7 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, any of
which is unsubstituted or substituted, or part of a cyclic
structure with a D residue; [0155] each R.sub.8 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, any of
which is unsubstituted or substituted, or part of a cyclic
structure with an E residue; [0156] each v and w is independently
an integer from 0-1000, from 1-1000, or 3-1000; [0157] each x, y,
and z is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and
[0158] U is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0159] In some aspects, each v and w is independently 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In
some aspects, each w is independently an integer from 3-1000, for
example, 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some
aspects, w is an integer from 3-10, for example 3-6, 3-8, 6-8, or
6-10. In some aspects, w is 3. In other aspects, w is 6. In some
aspects, each v is independently an integer from 1-1000, for
example 1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10. In some
aspects, v is 2. In one example, at least one of R.sub.1 and
R.sub.2 is alkyl that is unsubstituted or substituted with halo-.
In another example, both R.sub.1 and R.sub.2 are independently
alkyl that is unsubstituted or substituted with halo-. In some
aspects, at least one of R.sub.1 and R.sub.2 is methyl. In other
aspects, R.sub.1 and R.sub.2 are methyl.
[0160] In some aspects, x+y+z is at least 2 or at least 3. In other
aspects, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some aspects,
the sum of x+y+z is 3 or 6. In some aspects, the sum of x+y+z is 3.
In other aspects, the sum of x+y+z is 6.
[0161] Each occurrence of A, B, C, D, or E in a macrocycle or
macrocycle precursor is independently selected. For example, a
sequence represented by the formula [A].sub.x, when x is 3,
encompasses aspects where the amino acids are not identical, e.g.,
Gln-Asp-Ala, as well as aspects where the amino acids are
identical, e.g., Gln-Gln-Gln. This applies for any value of x, y,
or z in the indicated ranges. Similarly, when u is greater than 1,
each compound can encompass compounds that are the same or
different. For example, a compound can comprise compounds
comprising different linker lengths or chemical compositions.
[0162] In some aspects, the compound comprises a secondary
structure that is an .alpha.-helix where Its is --H, allowing for
intrahelical hydrogen bonding. In some aspects, at least one of A,
B, C, D, or E is an .alpha.,.alpha.-disubstituted amino acid. In
one example, B is an .alpha.,.alpha.-disubstituted amino acid. For
instance, at least one of A, B, C, D, or E is 2-aminoisobutyric
acid. In other aspects, at least one of A, B, C, D, or E is
##STR00006##
In other aspects, the length of the macrocycle-forming linker L as
measured from a first C.alpha. to a second C.alpha. is selected to
stabilize a desired secondary peptide structure, such as an
.alpha.-helix formed by residues of the compound including, but not
necessarily limited to, those between the first C.alpha. to a
second C.alpha..
[0163] In some aspects, a compound of Formula (II) has the Formula
(IIa):
##STR00007##
[0164] wherein: [0165] each A, C, D, and E is independently a
natural or non-natural amino acid; [0166] each B is independently a
natural or non-natural amino acid, amino acid analog,
##STR00008##
[0166] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0167] each L is independently a
macrocycle-forming linker; [0168] each L' is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
unsubstituted or substituted, or a bond, or together with R.sub.1
and the atom to which both R.sub.1 and L' are bound forms a ring;
[0169] each L'' is independently alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene, any of which is unsubstituted or substituted, or a
bond, or together with R.sub.2 and the atom to which both R.sub.2
and L'' are bound forms a ring; [0170] each R.sub.1 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is
unsubstituted or substituted, or together with L' and the atom to
which both R.sub.1 and L' are bound forms a ring; [0171] each
R.sub.2 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any
of which is unsubstituted or substituted, or together with L'' and
the atom to which both R.sub.2 and L'' are bound forms a ring;
[0172] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl,
or heteroaryl, any of which is unsubstituted or substituted; [0173]
each L.sub.4 is independently alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene,
heteroarylene, or [--R.sub.4--K--R.sub.4-]n, any of which is
unsubstituted or substituted; [0174] each R.sub.4 is alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
unsubstituted or substituted; [0175] each K is independently O, S,
SO, SO.sub.2, CO, CO.sub.2, CONR.sub.3, OSO.sub.2NR.sub.3,
NR.sub.3q, CONR.sub.3q, OCONR.sub.3q, or OSO.sub.2NR.sub.3q,
wherein each R.sub.3q is independently a point of attachment to
R.sub.1 or R.sub.2; [0176] each n is independently 1, 2, 3, 4, or
5; [0177] each R.sub.7 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, aryl, or heteroaryl, any of which is
unsubstituted or substituted, or part of a cyclic structure with a
D residue; [0178] each R.sub.8 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, any of
which is unsubstituted or substituted, or part of a cyclic
structure with an E residue; [0179] each v and w is independently
an integer from 0-1000, from 1-1000, or 3-1000; [0180] each x, y,
and z is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and
[0181] u is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0182] In some aspects, L is a macrocycle-forming linker of the
formula -L.sub.1-L.sub.2-. In some aspects, L.sub.1 and L.sub.2 are
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted.
[0183] In one example, at least one of R.sub.1 and R.sub.2 is alkyl
that is unsubstituted or substituted with halo-. In another
example, both R.sub.1 and R.sub.2 are independently alkyl that is
unsubstituted or substituted with halo-. In some aspects, at least
one of R.sub.1 and R.sub.2 is methyl. In other aspects, R.sub.1 and
R.sub.2 are methyl.
[0184] In some aspects, x+y+z is at least 2 or at least 3. In other
aspects, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence
of A, B, C, D, or E in a macrocycle or macrocycle precursor is
independently selected. For example, a sequence represented by the
formula [A].sub.x, when x is 3, encompasses aspects where the amino
acids are not identical, e.g. Gln-Asp-Ala as well as aspects where
the amino acids are identical, e.g. Gln-Gln-Gln. This applies for
any value of x, y, or z in the indicated ranges. Similarly, when u
is greater than 1, each compound may encompass moieties which are
the same or different. For example, a compound may comprise
moieties comprising different linker lengths or chemical
compositions.
[0185] In some aspects, the compound comprises a secondary
structure that is a helix where R.sub.8 is --H, allowing
intrahelical hydrogen bonding. In some aspects, at least one of A,
B, C, D, or E is an .alpha.,.alpha.-disubstituted amino acid. In
one example, B is an .alpha.,.alpha.-disubstituted amino acid. For
instance, at least one of A, B, C, D, or E is 2-aminoisobutyric
acid. In other aspects, at least one of A, B, C, D or E is
##STR00009##
[0186] In other aspects, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as a helix formed by residues of the compound including, but not
necessarily limited to, those between the first C.alpha. to a
second C.alpha..
[0187] In some aspects, the compound of Formula (II) has the
Formula (IIb):
##STR00010##
[0188] wherein: [0189] each A, C, D, and E is independently an
amino acid, wherein A, B, C, D, and E, taken together with the
crosslinked amino acids connected by the macrocycle-forming linkers
L and L', form the amino acid sequence of a target peptide; [0190]
each B is independently an amino acid,
##STR00011##
[0190] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0191] L is a macrocycle-forming linker of the
formula -L.sub.1-L.sub.2-; [0192] each R.sub.1 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is
unsubstituted or substituted, or together with L and the atom to
which both R.sub.1 and L are bound forms a ring; [0193] each
R.sub.2 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any
of which is unsubstituted or substituted, or together with L and
the atom to which both R.sub.2 and L are bound forms a ring; [0194]
R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl,
or heteroaryl, any of which is unsubstituted or substituted; [0195]
L' is a macrocycle-forming linker of the formula
-L.sub.1'-L.sub.2'-; [0196] each R.sub.1' is independently --H,
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted, or together with L' and the atom to which both
R.sub.1' and L' are bound forms a ring; [0197] each R.sub.2' is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is
unsubstituted or substituted, or together with L' and the atom to
which both R.sub.2' and L' are bound forms a ring; [0198] L.sub.1',
L.sub.2', and L.sub.4 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, heteroarylene, or [--R.sub.4--K--R.sub.4-].sub.n, any of
which is unsubstituted or substituted; [0199] each R.sub.4 is
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
unsubstituted or substituted; [0200] each K is independently O, S,
SO, SO.sub.2, CO, CO.sub.2, CONR.sub.3, OSO.sub.2NR.sub.3,
NR.sub.3q, CONR.sub.3q, OCONR.sub.3q, or OSO.sub.2NR.sub.3q,
wherein each R.sub.3q is independently a point of attachment to
R.sub.1, R.sub.2, R.sub.1', or R.sub.2'; [0201] each n is
independently 1, 2, 3, 4, or 5; [0202] each R.sub.7 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or
heteroaryl, any of which is unsubstituted or substituted, or part
of a cyclic structure with a D residue; [0203] each R.sub.8 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or
heteroaryl, any of which is unsubstituted or substituted, or part
of a cyclic structure with an E residue; [0204] each R.sub.7' is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or
heteroaryl, any of which is unsubstituted or substituted, or part
of a cyclic structure with a D residue; [0205] each R.sub.8' is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or
heteroaryl, any of which is unsubstituted or substituted, or part
of a cyclic structure with an E residue; [0206] each x, y and z is
independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0207] each x',
y' and z' is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0208] each v and w is independently an integer from 0-1000, from
1-1000, or 3-1000; [0209] each v' and w' is independently an
integer from 0-1000, from 1-1000, or 3-1000; and [0210] each n is
1, 2, 3, 4, or 5.
[0211] In some aspects, the sum of x'+y'+z' is 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10, for example 3 or 6, at least 2, or at least 3.
[0212] In some aspects, the compounds have the Formula (IIc):
##STR00012##
[0213] wherein: [0214] each A, C, D, and E is independently a
natural or non-natural amino acid; [0215] each B is independently a
natural or non-natural amino acid, amino acid analog,
##STR00013##
[0215] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0216] each R.sub.1 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted, or together with R.sub.1 and the atom to which both
R.sub.1 and L are bound forms a ring; [0217] each R.sub.2 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is
unsubstituted or substituted, or together with R.sub.2 and the atom
to which both R.sub.2 and L are bound forms a ring; [0218] each
R.sub.3 is independently hydrogen, alkyl, alkenyl, alkynyl,
arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, aryl, or heteroaryl, any of which is unsubstituted
or substituted; [0219] each L is independently macrocycle-forming
linker of the formula
[0219] ##STR00014## [0220] each L.sub.1, L.sub.2, and L.sub.3 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted; [0221] each R.sub.4 is alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene, any of which is unsubstituted or
substituted; [0222] each K is independently O, S, SO, SO.sub.2, CO,
CO.sub.2, CONR.sub.3, OSO.sub.2NR.sub.3, NR.sub.3q, CONR.sub.3q,
OCONR.sub.3q, or OSO.sub.2NR.sub.3q, wherein each R.sub.3q is
independently a point of attachment to R.sub.1 or R.sub.2; [0223]
each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, aryl, or heteroaryl, any of which is
unsubstituted or substituted, or part of a cyclic structure with a
D residue; [0224] each R.sub.8 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, any of
which is unsubstituted or substituted, or part of a cyclic
structure with an E residue; [0225] each v and w is independently
an integer from 0-1000, from 1-1000, or 3-1000; [0226] each x, y
and z is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0227]
u is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and [0228] each n is 1, 2,
3, 4, or 5.
[0229] In one example, at least one of R.sub.1 and R.sub.2 is alkyl
that is unsubstituted or substituted with halo-. In another
example, both R.sub.1 and R.sub.2 are independently alkyl that is
unsubstituted or substituted with halo-. In some aspects, at least
one of R.sub.1 and R.sub.2 is methyl. In other aspects, R.sub.1 and
R.sub.2 are methyl.
[0230] In some aspects, x+y+z is at least 2 or at least 3. In other
aspects, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence
of A, B, C, D, or E in a macrocycle or macrocycle precursor is
independently selected. For example, a sequence represented by the
formula [A].sub.x, when x is 3, encompasses aspects where the amino
acids are not identical, e.g. Gln-Asp-Ala as well as aspects where
the amino acids are identical, e.g. Gln-Gln-Gln. This applies for
any value of x, y, or z in the indicated ranges.
[0231] In some aspects, each of the first two amino acid
represented by E comprises an uncharged side chain or a negatively
charged side chain. In some aspects, each of the first three amino
acid represented by E comprises an uncharged side chain or a
negatively charged side chain. In some aspects, each of the first
four amino acid represented by E comprises an uncharged side chain
or a negatively charged side chain. In some aspects, one or more or
each of the amino acid that is 1+1, i+2, i+3, i+4, i+5, and/or i+6
with respect to E comprises an uncharged side chain or a negatively
charged side chain.
[0232] In some aspects, the first C-terminal amino acid and/or the
second C-terminal amino acid represented by E comprise a
hydrophobic side chain. For example, the first C-terminal amino
acid and/or the second C-terminal amino acid represented by E
comprises a hydrophobic side chain, for example a small hydrophobic
side chain. In some aspects, the first C-terminal amino acid, the
second C-terminal amino acid, and/or the third C-terminal amino
acid represented by E comprise a hydrophobic side chain. For
example, the first C-terminal amino acid, the second C-terminal
amino acid, and/or the third C-terminal amino acid represented by E
comprises a hydrophobic side chain, for example a small hydrophobic
side chain. In some aspects, one or more or each of the amino acid
that is 1+1, i+2, i+3, i+4, i+5, and/or i+6 with respect to E
comprises an uncharged side chain or a negatively charged side
chain.
[0233] In some aspects, each w is independently an integer from 1
to 1000. For example, the first amino acid represented by E
comprises a small hydrophobic side chain. In some aspects, w is
between 2 and 1000. For example, the second amino acid represented
by E comprises a small hydrophobic side chain. In some aspects, w
is between 3 and 1000. For example, the third amino acid
represented by E comprises a small hydrophobic side chain. For
example, the third amino acid represented by E comprises a small
hydrophobic side chain. In some aspects, w is between 4 and 1000.
In some aspects, w is between 5 and 1000. In some aspects, w is
between 6 and 1000. In some aspects, w is between 7 and 1000. In
some aspects, w is between 8 and 1000.
[0234] In some aspects, the compound comprises a secondary
structure that is a helix where R.sub.8 is --H, allowing
intrahelical hydrogen bonding. In some aspects, at least one of A,
B, C, D, or E is an .alpha.,.alpha.-disubstituted amino acid. In
one example, B is an .alpha.,.alpha.-disubstituted amino acid. For
instance, at least one of A, B, C, D, or E is 2-aminoisobutyric
acid. In other aspects, at least one of A, B, C, D, or E is
##STR00015##
[0235] In other aspects, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as a helix formed by residues of the compound including, but not
necessarily limited to, those between the first C.alpha. to a
second C.alpha..
[0236] In some aspects, L is a macrocycle-forming linker of the
formula
##STR00016##
[0237] In some aspects, L is a macrocycle-forming linker of the
formula
##STR00017##
or a tautomer thereof.
[0238] Exemplary aspects of the macrocycle-forming linker L are
shown below.
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028##
[0239] Amino acids that are used in the formation of triazole
crosslinkers are represented according to the legend indicated
below. Stereochemistry at the .alpha.-position of each amino acid
is S unless otherwise indicated. For azide amino acids, the number
of carbon atoms indicated refers to the number of methylene units
between the .alpha.-carbon and the terminal azide. For alkyne amino
acids, the number of carbon atoms indicated is the number of
methylene units between the .alpha.-position and the triazole
moiety plus the two carbon atoms within the triazole group derived
from the alkyne.
TABLE-US-00006 $5a5 .alpha.-Me alkyne 1,5 triazole (5 carbon) $5n3
.alpha.-Me azide 1,5 triazole (3 carbon) $4rn6 .alpha.-Me R-azide
1,4 triazole (6 carbon) $4a5 .alpha.-Me alkyne 1,4 triazole (5
carbon)
[0240] Where the macrocycle-forming linker spans approximately 1
turn of an .alpha.-helix, the linkage contains approximately 4
atoms to 12 atoms, approximately 6 atoms to 10 atoms, or
approximately 8 atoms. Where the macrocycle-forming linker spans
approximately 2 turns of the .alpha.-helix, the linkage contains
approximately 7 atoms to 15 atoms, approximately 9 atoms to 13
atoms, or approximately 11 atoms. Where the macrocycle-forming
linker spans approximately 3 turns of the .alpha.-helix, the
linkage contains approximately 13 atoms to 21 atoms, approximately
15 atoms to 19 atoms, or approximately 17 atoms. Where the
macrocycle-forming linker spans approximately 4 turns of the
.alpha.-helix, the linkage contains approximately 19 atoms to 27
atoms, approximately 21 atoms to 25 atoms, or approximately 23
atoms. Where the macrocycle-forming linker spans approximately 5
turns of the .alpha.-helix, the linkage contains approximately 25
atoms to 33 atoms, approximately 27 atoms to 31 atoms, or
approximately 29 atoms. Where the macrocycle-forming linker spans
approximately 1 turn of the .alpha.-helix, the resulting macrocycle
forms a ring containing approximately 17 members to 25 members,
approximately 19 members to 23 members, or approximately 21
members. Where the macrocycle-forming linker spans approximately 2
turns of the .alpha.-helix, the resulting macrocycle forms a ring
containing approximately 29 members to 37 members, approximately 31
members to 35 members, or approximately 33 members. Where the
macrocycle-forming linker spans approximately 3 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 44 members to 52 members, approximately 46 members to
50 members, or approximately 48 members. Where the
macrocycle-forming linker spans approximately 4 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 59 members to 67 members, approximately 61 members to
65 members, or approximately 63 members. Where the
macrocycle-forming linker spans approximately 5 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 74 members to 82 members, approximately 76 members to
80 members, or approximately 78 members.
[0241] In any aspect herein, each v, w, v', and w' can be,
independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50. In any aspect herein, each v, w, v', and w' can be,
independently, 0-1000, 0-500, 0-400, 0-300, 0-200, 0-100, 0-50,
0-40, 0-30, 0-25, 0-20, 0-15, 0-10, 0-8, 0-6, 0-5, 1-1000, 1-500,
1-400, 1-300, 1-200, 1-100, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15,
1-10, 1-8, 1-6, 1-5, 3-1000, 3-500, 3-400, 3-300, 3-200, 3-100,
3-50, 3-40, 3-30, 3-25, 3-20, 3-15, 3-10, 3-8, 3-6, or 3-5.
[0242] In one aspect, the compound of Formula (II) is:
##STR00029##
[0243] wherein each R.sub.1 and R.sub.2 is independently --H,
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted.
[0244] In related aspects, the compound comprises a structure of
Formula (II) which is:
##STR00030##
[0245] In some aspects, the compound of Formula (II) is:
##STR00031##
[0246] wherein each R.sub.1 and R.sub.2 is independently --H,
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with
halo-.
[0247] In related aspects, the compound of Formula (II) is:
##STR00032##
[0248] wherein each R.sub.1' and R.sub.2' is independently an amino
acid side chain.
[0249] In other aspects, the compound of Formula (II) is a compound
of any of the formulas shown below:
##STR00033## ##STR00034##
[0250] wherein "AA" represents any natural or non-natural amino
acid side chain, "" is [D].sub.v or [E].sub.w as defined above, and
n is an integer from 0 to 20, 50, 100, 200, 300, 400 or 500. In
some aspects, the substituent "n" shown in the preceding paragraph
is 0. In other aspects, the substituent "n" shown in the preceding
paragraph is less than 50, 40, 30, 20, 10, or 5.
[0251] Exemplary aspects of the macrocycle-forming linker L are
shown below.
##STR00035##
[0252] In other aspects, [D] and/or [E] in the compound of Formula
(II) are further modified in order to facilitate cellular uptake.
In some aspects, lipidating or PEGylating a compound facilitates
cellular uptake, increases bioavailability, increases blood
circulation, alters pharmacokinetics, decreases immunogenicity,
and/or decreases the needed frequency of administration.
[0253] In other aspects, at least one of [D] and [E] in the
compound of Formula (II) represents a moiety comprising an
additional macrocycle-forming linker such that the compound
comprises at least two macrocycle-forming linkers. In a specific
aspect, a compound comprises two macrocycle-forming linkers. In one
aspect, u is 2.
[0254] In some aspects, L is a macrocycle-forming linker of the
formula -L.sub.1-L.sub.2-. In some aspects, L.sub.1 and L.sub.2 are
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted; each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene, any of which is unsubstituted or substituted; each K
is independently O, S, SO, SO.sub.2, CO, CO.sub.2, CONR.sub.3,
OSO.sub.2NR.sub.3, NR.sub.3q, CONR.sub.3q, OCONR.sub.3q, or
OSO.sub.2NR.sub.3q, wherein each R.sub.3q is independently a point
of attachment to R.sub.1 or R.sub.2; and each n is independently 1,
2, 3, 4, or 5.
[0255] In an aspect of any of the Formulas described herein,
L.sub.1 and L.sub.2, either alone or in combination, form a
triazole or a thioether.
[0256] In an aspect of any of the Formulas described herein,
L.sub.1 and L.sub.2, either alone or in combination, do not form a
triazole or a thioether.
[0257] In other aspects, the length of the macrocycle-forming
linker L as measured from a first .alpha.-carbon to a second
.alpha.-carbon is selected to stabilize a desired secondary peptide
structure, such as a helix formed by residues of the compound
including, but not necessarily limited to, those between the first
.alpha.-carbon to a second .alpha.-carbon.
[0258] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some aspects, at least one of R.sub.1
and R.sub.2 is methyl. In other aspects, R.sub.1 and R.sub.2 are
methyl.
[0259] In some aspects, x+y+z is at least 2 or at least 3. In other
aspects, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence
of A, B, C, D, or E in a macrocycle or macrocycle precursor is
independently selected. For example, a sequence represented by the
formula [A].sub.x, when x is 3, encompasses aspects where the amino
acids are not identical, e.g. Gln-Asp-Ala, as well as aspects where
the amino acids are identical, e.g. Gln-Gln-Gln. This applies for
any value of x, y, or z in the indicated ranges. Similarly, when u
is greater than 1, each compound may encompass compounds which are
the same or different. For example, a compound may comprise
compounds comprising different linker lengths or chemical
compositions.
[0260] In some aspects, the compound comprises a secondary
structure that is a helix where R.sub.8 is --H, allowing
intrahelical hydrogen bonding. In some aspects, at least one of A,
B, C, D, or E is an .alpha.,.alpha.-disubstituted amino acid. In
one example, B is an .alpha.,.alpha.-disubstituted amino acid. For
instance, at least one of A, B, C, D, or E is 2-aminoisobutyric
acid. In other aspects, at least one of A, B, C, D or E is
##STR00036##
[0261] In some aspects, w is from 1 to 1000. For example, the first
amino acid represented by E comprises a small hydrophobic side
chain. In some aspects, w is from 2 to 1000. For example, the
second amino acid represented by E comprises a small hydrophobic
side chain. In some aspects, w is from 3 to 1000. For example, the
third amino acid represented by E can comprise a small hydrophobic
side chain. For example, the third amino acid represented by E can
comprise a small hydrophobic side chain. In some aspects, w is from
4 and 1000. In some aspects, w is from 5 and 1000. In some aspects,
w is from 6 and 1000. In some aspects, w is from 7 and 1000. In
some aspects, w is from 8 and 1000. In some aspects, w is an
integer from 3-10, for example 3-6, 3-8, 6-8, or 6-10. In some
aspects, w is 3. In other aspects, w is 6. In some aspects, v is an
integer from 1-10, for example 2-5. In some aspects, v is 2. In
some aspects, v is 3.
[0262] In some aspects, each of the first two amino acid
represented by E comprises an uncharged side chain or a negatively
charged side chain. In some aspects, each of the first three amino
acid represented by E comprises an uncharged side chain or a
negatively charged side chain. In some aspects, each of the first
four amino acid represented by E comprises an uncharged side chain
or a negatively charged side chain.
[0263] In some aspects, the first C-terminal amino acid and/or the
second C-terminal amino acid represented by E comprise a
hydrophobic side chain. For example, the first C-terminal amino
acid and/or the second C-terminal amino acid represented by E
comprises a hydrophobic side chain, for example a small hydrophobic
side chain. In some aspects, the first C-terminal amino acid, the
second C-terminal amino acid, and/or the third C-terminal amino
acid represented by E comprise a hydrophobic side chain. For
example, the first C-terminal amino acid, the second C-terminal
amino acid, and/or the third C-terminal amino acid represented by E
comprises a hydrophobic side chain, for example a small hydrophobic
side chain.
[0264] In some aspects, one or more or each of the amino acid that
is 1+1, i+2, i+3, i+4, i+5, and/or i+6 with respect to a first E
comprises an uncharged side chain or a negatively charged side
chain. In some aspects, each E is independently an amino acid
selected from the group consisting of Ala (alanine), D-Ala
(D-alanine), Aib (.alpha.-aminoisobutyric acid), Sar (N-methyl
glycine), and Ser (serine).
[0265] In other aspects, [D] and/or [E] in the compound of Formula
I, Ia, Ib, or Ic are further modified in order to facilitate
cellular uptake. In some aspects, lipidating or PEGylating a
compound facilitates cellular uptake, increases bioavailability,
increases blood circulation, alters pharmacokinetics, decreases
immunogenicity and/or decreases the needed frequency of
administration.
[0266] In other aspects, at least one of [D] and [E] in the
compound of Formula I, Ia, Ib, or Ic represents a moiety comprising
an additional macrocycle-forming linker such that the compound
comprises at least two macrocycle-forming linkers. In a specific
aspect, a compound comprises two macrocycle-forming linkers. In an
aspect, u is 2.
[0267] In other aspects, the invention provides compounds of
Formula (III):
##STR00037##
[0268] wherein: [0269] each A, C, D, and E is independently a
natural or non-natural amino acid; [0270] each B is independently a
natural or non-natural amino acid, amino acid analog,
##STR00038##
[0270] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0271] each R.sub.1 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted, or together with R.sub.1 and the atom to which R.sub.1
and L are bound forms a ring; [0272] each R.sub.2 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, any of which is
unsubstituted or substituted, or together with R.sub.2 and the atom
to which R.sub.2 and L are bound forms a ring; [0273] each R.sub.3
is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl,
or heteroaryl, any of which is unsubstituted or substituted; [0274]
each L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, heteroarylene or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted; [0275] each K is independently O, S, SO, SO.sub.2, CO,
CO.sub.2, CONR.sub.3, OSO.sub.2NR.sub.3, NR.sub.3q, CONR.sub.3q,
OCONR.sub.3q, or OSO.sub.2NR.sub.3q, wherein each R.sub.3q is
independently a point of attachment to R.sub.1 or R.sub.2; [0276]
each R.sub.4 is independently alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene, any of which is unsubstituted or substituted; [0277]
each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, aryl, or heteroaryl, any of which is
unsubstituted or substituted, or part of a cyclic structure with a
D residue; [0278] each R.sub.8 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, any of
which is unsubstituted or substituted, or part of a cyclic
structure with an E residue; [0279] each v and w is independently
an integer from 0-1000, from 1-1000, or 3-1000; [0280] each x, y
and z is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0281]
u is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and [0282] each n is
independently 1, 2, 3, 4, or 5.
[0283] In some aspects, the length of the macrocycle-forming linker
[-L.sub.1-S-L.sub.2-S-L.sub.3-] as measured from a first
.alpha.-carbon to a second .alpha.-carbon is selected to stabilize
a desired secondary peptide structure, such as a helix (including,
but not limited to a 3.sub.10 helix or an .alpha.-helix) formed by
residues of the compound including, but not necessarily limited to,
those between the first .alpha.-carbon to a second .alpha.-carbon.
In some aspects, the thiol moieties are the side chains of the
amino acid residues L-cysteine, D-cysteine, .alpha.-methyl-L
cysteine, .alpha.-methyl-D-cysteine, L-homocysteine,
D-homocysteine, .alpha.-methyl-L-homocysteine, or
.alpha.-methyl-D-homocysteine. A bis-alkylating reagent is of the
general formula X-L.sub.2-Y, wherein L.sub.2 is a linker moiety and
X and Y are leaving groups that are displaced by --SH moieties to
form bonds with L.sub.2. In some aspects, X and Y are halogens,
such as I, Br, or Cl.
[0284] In other aspects, the invention provides compounds of
Formula (IV) or (IVa):
##STR00039##
[0285] wherein: [0286] each A, C, D, and E is independently a
natural or non-natural amino acid; [0287] each B is independently a
natural or non-natural amino acid, amino acid analog,
##STR00040##
[0287] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0288] each R.sub.1 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted, or part of a cyclic structure with an E residue;
[0289] each R.sub.2 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, any of which is unsubstituted or substituted, or
part of a cyclic structure with an E residue; [0290] each R.sub.3
is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl,
or heteroaryl, any of which is unsubstituted or substituted; [0291]
each L is independently a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-; [0292] each L.sub.1, L.sub.2, and L.sub.4 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted; [0293] each R.sub.4 is independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
unsubstituted or substituted; [0294] each K is independently O, S,
SO, SO.sub.2, CO, CO.sub.2, CONR.sub.3, OSO.sub.2NR.sub.3,
NR.sub.3q, CONR.sub.3q, OCONR.sub.3q, or OSO.sub.2NR.sub.3q,
wherein each R.sub.3q is independently a point of attachment to
R.sub.1 or R.sub.2; [0295] each R.sub.7 is independently --H,
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, any of
which is unsubstituted or substituted; [0296] each v and w is
independently integers from 0-1000, from 1-1000, or 3-1000; [0297]
each x, y, and z is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10; [0298] u is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and [0299] each n
is independently 1, 2, 3, 4, 5.
[0300] In one example, L.sub.1 and L.sub.2, either alone or in
combination, do not form a triazole or a thioether.
[0301] In one example, at least one of R.sub.1 and R.sub.2 is alkyl
that is unsubstituted or substituted with halo-. In another
example, both R.sub.1 and R.sub.2 are independently alkyl that is
unsubstituted or substituted with halo-. In some aspects, at least
one of R.sub.1 and R.sub.2 is methyl. In other aspects, R.sub.1 and
R.sub.2 are methyl.
[0302] In some aspects, x+y+z is at least 1. In other aspects,
x+y+z is at least 2. In other aspects, x+y+z is 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10. Each occurrence of A, B, C, D, or E in a macrocycle
or macrocycle precursor is independently selected. For example, a
sequence represented by the formula [A].sub.x, when x is 3,
encompasses aspects where the amino acids are not identical, e.g.
Gln-Asp-Ala, as well as aspects where the amino acids are
identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or
z in the indicated ranges.
[0303] In some aspects, the compound comprises a secondary
structure which is an .alpha.-helix and R.sub.8 is --H, allowing
intrahelical hydrogen bonding. In some aspects, at least one of A,
B, C, D or E is an .alpha.,.alpha.-disubstituted amino acid. In one
example, B is an .alpha.,.alpha.-disubstituted amino acid. For
example, at least one of A, B, C, D, or E is 2-aminoisobutyric
acid. In other aspects, at least one of A, B, C, D, or E is
##STR00041##
[0304] In other aspects, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the compound including,
but not necessarily limited to, those between the first C.alpha. to
a second C.alpha..
[0305] In some aspects, the compound has the Formula (V) or Formula
(Va):
##STR00042##
[0306] wherein: [0307] each A.sub.a, C.sub.a, D.sub.a, E.sub.a,
A.sub.b, C.sub.b, and D.sub.b is independently a natural or
non-natural amino acid; [0308] each B.sub.a and B.sub.b is
independently a natural or non-natural amino acid,
##STR00043##
[0308] [--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-]; [0309] each R.sub.a1 is independently alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted; or H; or R.sub.a1 forms a macrocycle-forming linker L'
connected to the alpha position of one of the D.sub.a or E.sub.a
amino acids; or together with L.sub.a forms a ring that is
unsubstituted or substituted; [0310] each R.sub.a2 is independently
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted; or H; or R.sub.a2 forms a macrocycle-forming linker L'
connected to the alpha position of one of the D.sub.a or E.sub.a
amino acids; or together with L.sub.a forms a ring that is
unsubstituted or substituted; [0311] each R.sub.b1 is independently
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, any of which is unsubstituted or
substituted; or H; or R.sub.b1 forms a macrocycle-forming linker L'
connected to the alpha position of one of the D.sub.b amino acids;
or together with L.sub.b forms a ring that is unsubstituted or
substituted; [0312] each R.sub.3 is independently alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, any of which is
unsubstituted or substituted, or H; [0313] each L.sub.a is
independently a macrocycle-forming linker, and optionally forms a
ring with R.sub.a1 or R.sub.a2 that is unsubstituted or
substituted; [0314] each L.sub.b is independently a
macrocycle-forming linker, and optionally forms a ring with
R.sub.b1 that is unsubstituted or substituted; [0315] each L' is
independently a macrocycle-forming linker; [0316] each L.sub.4 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene,
heterocycloarylene, or [--R.sub.4--K--R.sub.4-].sub.n, any of which
is unsubstituted or substituted; [0317] each R.sub.4 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any
of which is unsubstituted or substituted; [0318] each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, OCO.sub.2,
NR.sub.3, CONR.sub.3, OCONR.sub.3, OSO.sub.2NR.sub.3, NR.sub.3q,
CONR.sub.3q, OCONR.sub.3q, or OSO.sub.2NR.sub.3q, wherein each
R.sub.3q is independently a point of attachment to R.sub.a1, Rae,
or R.sub.b1; [0319] each R.sub.a7 is independently alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, any of which is
unsubstituted or substituted; or H; or part of a cyclic structure
with a D.sub.a amino acid; [0320] each R.sub.b7 is independently
alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
any of which is unsubstituted or substituted; or H; or part of a
cyclic structure with a D.sub.b amino acid; [0321] each R.sub.a8 is
independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or
heterocycloaryl, any of which is unsubstituted or substituted; or
H; or part of a cyclic structure with an E.sub.a amino acid; [0322]
each R.sub.b8 is independently alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, any of which is unsubstituted or
substituted; or H; or an amino acid sequence of 1-1000 amino acid
residues; [0323] each va and vb is independently an integer from
0-1000; [0324] each wa and wb is independently an integer from
0-1000; [0325] each ua and ub is independently 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10, wherein ua+ub is at least 1; [0326] each xa and xb
is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0327] each
ya and yb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0328] each za and zb is independently 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10; and [0329] each n is independently 1, 2, 3, 4, or 5,
[0330] or a pharmaceutically-acceptable salt thereof.
[0331] In some aspects, the compound of the invention has the
formula defined above, wherein: [0332] each L.sub.a is
independently a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-, and optionally forms a ring with R.sub.a1 or
R.sub.a2 that is unsubstituted or substituted; [0333] each L.sub.b
is independently a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-, and optionally forms a ring with R.sub.b1 that
is unsubstituted or substituted; [0334] each L' is independently a
macrocycle-forming linker of the formula -L.sub.1-L.sub.2-; [0335]
each L.sub.1 and L.sub.2 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted; [0336] each R.sub.4 is independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
unsubstituted or substituted; [0337] each K is independently O, S,
SO, SO.sub.2, CO, CO.sub.2, OCO.sub.2, NR.sub.3, CONR.sub.3,
OCONR.sub.3, OSO.sub.2NR.sub.3, NR.sub.3q, CONR.sub.3q,
OCONR.sub.3q, or OSO.sub.2NR.sub.3q, wherein each R.sub.3q is
independently a point of attachment to R.sub.a1, R.sub.a2, or
R.sub.b1;
[0338] or a pharmaceutically-acceptable salt thereof.
[0339] In some aspects, the compound has the formula defined above
wherein each L.sub.a and L.sub.b is independently a
triazole-containing macrocycle-forming linker. In some aspects, the
compound has the formula defined above, wherein: [0340] each
L.sub.a and L.sub.b is independently a macrocycle-forming linker of
the formula:
[0340] ##STR00044## [0341] each L.sub.1, L.sub.2, and L.sub.3 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene,
heterocycloarylene, or [--R.sub.4--K--R.sub.4-].sub.n, any of which
is unsubstituted or substituted; [0342] each R.sub.4 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any
of which is unsubstituted or substituted; [0343] each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, OCO.sub.2,
NR.sub.3, CONR.sub.3, OCONR.sub.3, OSO.sub.2NR.sub.3, NR.sub.3q,
CONR.sub.3q, OCONR.sub.3q, or OSO.sub.2NR.sub.3q, wherein each
R.sub.3q is independently a point of attachment to R.sub.a1,
R.sub.a2, or R.sub.b1; and [0344] each n is independently 1, 2, 3,
4, or 5,
[0345] or a pharmaceutically-acceptable salt thereof.
[0346] In some aspects, the compound has the formula defined above,
wherein: [0347] each L.sub.a and L.sub.b is independently a
macrocycle-forming linker of the formula
-L.sub.1-SR.sub.9R.sub.10-L.sub.2-SR.sub.11R.sub.12-L.sub.3-,
wherein each L.sub.1, L.sub.2, and L.sub.3 is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4-].sub.n, any of which is unsubstituted or
substituted; and each R.sub.9, R.sub.10, R.sub.11, and R.sub.12 is
independently absent or 0; [0348] each R.sub.4 is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
unsubstituted or substituted; [0349] each K is independently O, S,
SO, SO.sub.2, CO, CO.sub.2, OCO.sub.2, NR.sub.3, CONR.sub.3,
OCONR.sub.3, OSO.sub.2NR.sub.3, NR.sub.3q, CONR.sub.3q,
OCONR.sub.3q, or OSO.sub.2NR.sub.3q, wherein each R.sub.3q is
independently a point of attachment to R.sub.a1, R.sub.a2, or
R.sub.b1; and [0350] each n is independently 1, 2, 3, 4, or 5,
[0351] or a pharmaceutically-acceptable salt thereof.
[0352] In some aspects, the compound has the formula defined above
wherein one or both L.sub.a and L.sub.b is independently a
bis-thioether-containing macrocycle-forming linker. In some
aspects, each L.sub.a and L.sub.b is independently a
macrocycle-forming linker of the formula
-L.sub.1-S-L.sub.2-S-L.sub.3-.
[0353] In some aspects, the compound has the formula defined above
wherein one or both L.sub.a and L.sub.b is independently a
bis-sulfone-containing macrocycle-forming linker. In some aspects,
each L.sub.a and L.sub.b is independently a macrocycle-forming
linker of the formula
-L.sub.1-SO.sub.2-L.sub.2-SO.sub.2-L.sub.3-.
[0354] In some aspects, the compound has the formula defined above
wherein one or both L.sub.a and L.sub.b is independently a
bis-sulfoxide-containing macrocycle-forming linker. In some
aspects, each L.sub.a and L.sub.b is independently a
macrocycle-forming linker of the formula
-L.sub.1-S(O)-L.sub.2-S(O)-L.sub.3-.
[0355] In some aspects, a compound of the invention comprises one
or more secondary structures. In some aspects, the compound
comprises a secondary structure that is an .alpha.-helix. In some
aspects, the compound comprises a secondary structure that is a
.beta.-hairpin turn.
[0356] In some aspects, u.sub.a is 0. In some aspects, u.sub.a is
0, and L.sub.b is a triazole-containing macrocycle-forming linker
that crosslinks an .alpha.-helical secondary structure. In some
aspects, u.sub.a is 0, and L.sub.b is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
secondary structure.
[0357] In some aspects, u.sub.b is 0. In some aspects, u.sub.b is
0, and L.sub.a is a triazole-containing macrocycle-forming linker
that crosslinks an .alpha.-helical secondary structure. In some
aspects, u.sub.b is 0, and L.sub.a is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
secondary structure.
[0358] In some aspects, the compound comprises only .alpha.-helical
secondary structures.
[0359] In other aspects, the compound comprises a combination of
secondary structures, wherein the secondary structures are
.alpha.-helical and .beta.-hairpin structures. In some aspects,
L.sub.a and L.sub.b are a combination of hydrocarbon-, triazole, or
sulfur-containing macrocycle-forming linkers. In some aspects, the
compound comprises L.sub.a and L.sub.b, wherein L.sub.a is a
hydrocarbon-containing macrocycle-forming linker that crosslinks a
.beta.-hairpin structure, and L.sub.b is a triazole-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
structure. In some aspects, the compound comprises L.sub.a and
L.sub.b, wherein L.sub.a is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
structure, and L.sub.b is a triazole-containing macrocycle-forming
linker that crosslinks a .beta.-hairpin structure. In some aspects,
the compound comprises L.sub.a and L.sub.b, wherein L.sub.a is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical structure, and L.sub.b is a hydrocarbon-containing
macrocycle-forming linker that crosslinks a .beta.-hairpin
structure. In some aspects, the compound comprises L.sub.a and
L.sub.b, wherein L.sub.a is a triazole-containing
macrocycle-forming linker that crosslinks a .beta.-hairpin
structure, and L.sub.b is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
structure.
[0360] In some aspects, u.sub.a+u.sub.b is at least 1. In some
aspects, u.sub.a+u.sub.b=2.
[0361] In some aspects, u.sub.a is 1, and u.sub.b is 1. In some
aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure, and L.sub.b is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure. In some aspects, u.sub.a is 1,
u.sub.b is 1, L.sub.a is a triazole-containing macrocycle-forming
linker that crosslinks an .alpha.-helical secondary structure, and
L.sub.b is a triazole-containing macrocycle-forming linker that
crosslinks a .beta.-hairpin secondary structure. In some aspects,
u.sub.a is 1, u.sub.b is 1, L.sub.a is a triazole-containing
macrocycle-forming linker that crosslinks a .beta.-hairpin
secondary structure, and L.sub.b is a triazole-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
secondary structure.
[0362] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure, and L.sub.b is a
hydrocarbon-containing macrocycle-forming linker that crosslinks an
.alpha.-helical structure. In some aspects, u.sub.a is 1, u.sub.b
is 1, L.sub.a is a triazole-containing macrocycle-forming linker
that crosslinks an .alpha.-helical secondary structure, and L.sub.b
is a hydrocarbon-containing macrocycle-forming linker that
crosslinks a .beta.-hairpin structure. In some aspects, u.sub.a is
1, u.sub.b is 1, L.sub.a is a triazole-containing
macrocycle-forming linker that crosslinks a .beta.-hairpin
secondary structure, and L.sub.b is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
structure.
[0363] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
hydrocarbon-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure, and L.sub.b is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure. In some aspects, u.sub.a is 1,
u.sub.b is 1, L.sub.a is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
secondary structure, and L.sub.b is a triazole-containing
macrocycle-forming linker that crosslinks a .beta.-hairpin
secondary structure. In some aspects, u.sub.a is 1, u.sub.b is 1,
L.sub.a is a hydrocarbon-containing macrocycle-forming linker that
crosslinks a .beta.-hairpin secondary structure, and L.sub.b is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure.
[0364] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
triazole-containing macrocycle-forming linker that crosslinks an
.alpha.-helical secondary structure, and L.sub.b is a
sulfur-containing macrocycle-forming linker.
[0365] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
sulfur-containing macrocycle-forming linker, and L.sub.b is a
triazole-containing macrocycle-forming linker with an
.alpha.-helical secondary structure.
[0366] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
hydrocarbon-containing macrocycle-forming linker with an
.alpha.-helical secondary structure, and L.sub.b is a
sulfur-containing macrocycle-forming linker.
[0367] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
sulfur-containing macrocycle-forming linker, and L.sub.b is a
hydrocarbon-containing macrocycle-forming linker with an
.alpha.-helical secondary structure.
[0368] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
sulfur-containing macrocycle-forming linker, and L.sub.b is a
sulfur-containing macrocycle-forming linker.
[0369] In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is a
hydrocarbon-containing macrocycle-forming linker that crosslinks an
.alpha.-helical structure, and L.sub.b is a hydrocarbon-containing
macrocycle-forming linker that crosslinks an .alpha.-helical
structure. In some aspects, u.sub.a is 1, u.sub.b is 1, L.sub.a is
a hydrocarbon-containing macrocycle-forming linker that crosslinks
an .alpha.-helical structure, and L.sub.b is a
hydrocarbon-containing macrocycle-forming linker that crosslinks a
.beta.-hairpin structure. In some aspects, u.sub.a is 1, u.sub.b is
1, L.sub.a is a hydrocarbon-containing macrocycle-forming linker
that crosslinks a .beta.-hairpin structure, and L.sub.b is a
hydrocarbon-containing macrocycle-forming linker that crosslinks an
.alpha.-helical structure.
[0370] In some aspects, R.sub.b1 is H.
[0371] In some aspects, each v and w is 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some aspects,
w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50,
3-30, 3-20, or 3-10. In some aspects, the sum of x+y+z is 3 or 6.
In some aspects, the sum of x+y+z is 3. In other aspects, the sum
of x+y+z is 6.
[0372] Unless otherwise stated, any compounds (including compounds,
macrocycle precursors, and other compositions) are also meant to
encompass compounds which differ only in the presence of one or
more isotopically enriched atoms. For example, compounds having the
described structures except for the replacement of a hydrogen by a
deuterium or tritium, or the replacement of a carbon by .sup.13C-
or .sup.14C-enriched carbon are within the scope of this
disclosure.
[0373] In some aspects, the compounds disclosed herein can contain
unnatural proportions of atomic isotopes at one or more of atoms
that constitute such compounds. For example, the compounds can be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). In other
aspects, one or more carbon atoms are replaced with a silicon atom.
All isotopic variations of the compounds disclosed herein, whether
radioactive or not, are contemplated herein.
[0374] A compound described herein can be at least 1% pure, at
least 2% pure, at least 3% pure, at least 4% pure, at least 5%
pure, at least 6% pure, at least 7% pure, at least 8% pure, at
least 9% pure, at least 10% pure, at least 11% pure, at least 12%
pure, at least 13% pure, at least 14% pure, at least 15% pure, at
least 16% pure, at least 17% pure, at least 18% pure, at least 19%
pure, at least 20% pure, at least 21% pure, at least 22% pure, at
least 23% pure, at least 24% pure, at least 25% pure, at least 26%
pure, at least 27% pure, at least 28% pure, at least 29% pure, at
least 30% pure, at least 31% pure, at least 32% pure, at least 33%
pure, at least 34% pure, at least 35% pure, at least 36% pure, at
least 37% pure, at least 38% pure, at least 39% pure, at least 40%
pure, at least 41% pure, at least 42% pure, at least 43% pure, at
least 44% pure, at least 45% pure, at least 46% pure, at least 47%
pure, at least 48% pure, at least 49% pure, at least 50% pure, at
least 51% pure, at least 52% pure, at least 53% pure, at least 54%
pure, at least 55% pure, at least 56% pure, at least 57% pure, at
least 58% pure, at least 59% pure, at least 60% pure, at least 61%
pure, at least 62% pure, at least 63% pure, at least 64% pure, at
least 65% pure, at least 66% pure, at least 67% pure, at least 68%
pure, at least 69% pure, at least 70% pure, at least 71% pure, at
least 72% pure, at least 73% pure, at least 74% pure, at least 75%
pure, at least 76% pure, at least 77% pure, at least 78% pure, at
least 79% pure, at least 80% pure, at least 81% pure, at least 82%
pure, at least 83% pure, at least 84% pure, at least 85% pure, at
least 86% pure, at least 87% pure, at least 88% pure, at least 89%
pure, at least 90% pure, at least 91% pure, at least 92% pure, at
least 93% pure, at least 94% pure, at least 95% pure, at least 96%
pure, at least 97% pure, at least 98% pure, at least 99% pure, at
least 99.1% pure, at least 99.2% pure, at least 99.3% pure, at
least 99.4% pure, at least 99.5% pure, at least 99.6% pure, at
least 99.7% pure, at least 99.8% pure, or at least 99.9% pure on a
chemical, optical, isomeric, enantiomeric, or diastereomeric basis.
Purity can be assessed, e.g., by HPLC, MS, LC/MS, melting point, or
NMR.
[0375] Two or more peptides can share a degree of homology. A pair
of peptides can have, for example, up to about 20% pairwise
homology, up to about 25% pairwise homology, up to about 30%
pairwise homology, up to about 35% pairwise homology, up to about
40% pairwise homology, up to about 45% pairwise homology, up to
about 50% pairwise homology, up to about 55% pairwise homology, up
to about 60% pairwise homology, up to about 65% pairwise homology,
up to about 70% pairwise homology, up to about 75% pairwise
homology, up to about 80% pairwise homology, up to about 85%
pairwise homology, up to about 90% pairwise homology, up to about
95% pairwise homology, up to about 96% pairwise homology, up to
about 97% pairwise homology, up to about 98% pairwise homology, up
to about 99% pairwise homology, up to about 99.5% pairwise
homology, or up to about 99.9% pairwise homology. A pair of
peptides can have, for example, at least about 20% pairwise
homology, at least about 25% pairwise homology, at least about 30%
pairwise homology, at least about 35% pairwise homology, at least
about 40% pairwise homology, at least about 45% pairwise homology,
at least about 50% pairwise homology, at least about 55% pairwise
homology, at least about 60% pairwise homology, at least about 65%
pairwise homology, at least about 70% pairwise homology, at least
about 75% pairwise homology, at least about 80% pairwise homology,
at least about 85% pairwise homology, at least about 90% pairwise
homology, at least about 95% pairwise homology, at least about 96%
pairwise homology, at least about 97% pairwise homology, at least
about 98% pairwise homology, at least about 99% pairwise homology,
at least about 99.5% pairwise homology, at least about 99.9%
pairwise homology.
[0376] Various methods and software programs can be used to
determine the homology between two or more peptides, such as NCBI
BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or
another suitable method or algorithm.
[0377] In some aspects, the compound comprises at least one helical
motif, such as a 3.sub.10 or an .alpha.-helix motif. For example,
A, B, and/or C in the compound of Formula I, II, or III include one
or more helices. As a general matter, helices include from 3 to 4
amino acid residues per turn. In some aspects, the helix of the
compound includes 1 to 5 turns and, therefore, 3 to 20 amino acid
residues. In specific aspects, the helix includes 1 turn, 2 turns,
3 turns, 4 turns, or 5 turns. In some aspects, the
macrocycle-forming linker stabilizes a helix motif included within
the compound. Thus, in some aspects, the length of the
macrocycle-forming linker L from a first .alpha.-carbon to a second
.alpha.-carbon is selected to increase the stability of a helix. In
some aspects, the macrocycle-forming linker spans from 1 turn to 5
turns of the helix. In some aspects, the macrocycle-forming linker
spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns
of the helix. In some aspects, the length of the macrocycle-forming
linker is approximately 5 .ANG. to 9 .ANG. per turn of the helix,
or approximately 6 .ANG. to 8 .ANG. per turn of the helix. Where
the macrocycle-forming linker spans approximately 1 turn of a
helix, the length is equal to approximately 5 carbon-carbon bonds
to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to
11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds.
Where the macrocycle-forming linker spans approximately 2 turns of
a helix, the length is equal to approximately 8 carbon-carbon bonds
to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to
14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds.
Where the macrocycle-forming linker spans approximately 3 turns of
a helix, the length is equal to approximately 14 carbon-carbon
bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon
bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon
bonds. Where the macrocycle-forming linker spans approximately 4
turns of a helix, the length is equal to approximately 20
carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22
carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 5 turns of a helix, the length is equal to
approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds,
approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or
approximately 30 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 1 turn of a helix, the linkage contains
approximately 4 atoms to 12 atoms, approximately 6 atoms to 10
atoms, or approximately 8 atoms. Where the macrocycle-forming
linker spans approximately 2 turns of the helix, the linkage
contains approximately 7 atoms to 15 atoms, approximately 9 atoms
to 13 atoms, or approximately 11 atoms. Where the
macrocycle-forming linker spans approximately 3 turns of the helix,
the linkage contains approximately 13 atoms to 21 atoms,
approximately 15 atoms to 19 atoms, or approximately 17 atoms.
Where the macrocycle-forming linker spans approximately 4 turns of
the helix, the linkage contains approximately 19 atoms to 27 atoms,
approximately 21 atoms to 25 atoms, or approximately 23 atoms.
Where the macrocycle-forming linker spans approximately 5 turns of
the helix, the linkage contains approximately 25 atoms to 33 atoms,
approximately 27 atoms to 31 atoms, or approximately 29 atoms.
Where the macrocycle-forming linker spans approximately 1 turn of
the helix, the resulting macrocycle forms a ring containing
approximately 17 members to 25 members, approximately 19 members to
23 members, or approximately 21 members. Where the
macrocycle-forming linker spans approximately 2 turns of the helix,
the resulting macrocycle forms a ring containing approximately 29
members to 37 members, approximately 31 members to 35 members, or
approximately 33 members. Where the macrocycle-forming linker spans
approximately 3 turns of the helix, the resulting macrocycle forms
a ring containing approximately 44 members to 52 members,
approximately 46 members to 50 members, or approximately 48
members. Where the macrocycle-forming linker spans approximately 4
turns of the helix, the resulting macrocycle forms a ring
containing approximately 59 members to 67 members, approximately 61
members to 65 members, or approximately 63 members. Where the
macrocycle-forming linker spans approximately 5 turns of the helix,
the resulting macrocycle forms a ring containing approximately 74
members to 82 members, approximately 76 members to 80 members, or
approximately 78 members.
[0378] 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 aspects substantially free means that a
composition contains at least about 90% by weight of a preferred
enantiomer. In other aspects 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, EX.
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.
[0379] 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.
[0380] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine. The term "alkyl" refers to a hydrocarbon chain
that may be a straight chain or branched chain, containing the
indicated number of carbon atoms. For example, C.sub.1-C.sub.10
indicates that the group may have from 1 to 10 (inclusive) carbon
atoms in it. In the absence of any numerical designation, "alkyl"
is a chain (straight or branched) having 1 to 20 (inclusive) carbon
atoms in it. The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0381] The term "alkenyl" refers to a hydrocarbon chain that may be
a straight chain or branched chain having one or more carbon-carbon
double bonds in either Z or E geometric configurations. The alkenyl
moiety contains the indicated number of carbon atoms. For example,
C.sub.2-C.sub.10 indicates that the group may have from 2 to 10
(inclusive) carbon atoms in it. The term "lower alkenyl" refers to
a C.sub.2-C.sub.8 alkenyl chain. In the absence of any numerical
designation, "alkenyl" is a chain (straight or branched) having 2
to 20 (inclusive) carbon atoms in it.
[0382] The term "alkynyl" refers to a hydrocarbon chain that may be
a straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group may have from 2 to 10 (inclusive) carbon atoms in it. The
term "lower alkynyl" refers to a C.sub.2-C.sub.8 alkynyl chain. In
the absence of any numerical designation, "alkynyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0383] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, 4, or 5 atoms of
each ring may be substituted by a substituent. Examples of aryl
groups include phenyl, naphthyl and the like. The term "arylalkyl"
or the term "aralkyl" refers to alkyl substituted with an aryl. The
term "arylalkoxy" refers to an alkoxy substituted with aryl.
[0384] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, preferably 3 to 8 carbons, and more preferably 3 to 6
carbons, wherein the cycloalkyl group additionally may be
optionally substituted. Preferred cycloalkyl groups include,
without limitation, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl,
cycloheptyl, cycloheptadienyl, cycloheptatrienyl, cyclooctyl,
cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, and
cyclooctynyl.
[0385] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Examples of heteroaryl groups include
pyrrolyl, pyridyl, furyl or furanyl, imidazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, benzimidazolyl, pyridazyl, pyrimidyl, thiophenyl,
quinolinyl, indolyl, thiazolyl, oxazolyl, isoxazolyl and the like.
The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an
alkyl substituted with a heteroaryl. The term "heteroarylalkoxy"
refers to an alkoxy substituted with heteroaryl.
[0386] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, or 3 atoms of each ring may be
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, aziridinyl, oxiryl,
thiiryl, morpholinyl, tetrahydrofuranyl, and the like.
[0387] The term "substituents" refers to a group "substituted" on
an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at
any atom of that group. Suitable substituents include, without
limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl,
alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino,
alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl,
azido, and cyano groups.
[0388] 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 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 naturally occurring amino
acids rather than using a tether that is made by coupling two
non-naturally occurring amino acids. It is also possible to use a
single non-naturally occurring amino acid together with a naturally
occurring amino acid.
[0389] 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.
[0390] 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.
[0391] 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.
[0392] The stapled polypeptides can include a drug, a toxin, a
derivative of polyethylene glycol; a second polypeptide; a
carbohydrate, etc. Where a polymer or other agent is linked to the
stapled polypeptide, it can be desirable for the composition to be
substantially homogeneous.
[0393] The addition of polyethelene glycol (PEG) moieties 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:
[0394] 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.
[0395] 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, e.g., in WO 99/34833, WO 99/14259, and U.S. Pat. No.
6,348,558.
[0396] In certain aspects, macromolecular polymer (e.g., PEG) is
attached to an agent described herein through an intermediate
linker. In certain aspects, 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 naturally occurring amino acids. Some of these
amino acids may be glycosylated, as is well understood by those in
the art. In other aspects, the 1 to 20 amino acids are selected
from glycine, alanine, proline, asparagine, glutamine, and lysine.
In other aspects, 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.
[0397] 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, e.g., 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.
[0398] The peptides of this invention can be made by chemical
synthesis methods, which are well known to the ordinarily skilled
artisan. See, e.g., 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.
[0399] 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.
[0400] 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.
[0401] 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.
[0402] 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.
[0403] 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.,
C.sub.1-C.sub.20 straight or branched alkyl groups); fatty acid
radicals; and combinations thereof.
[0404] .alpha., .alpha.-Disubstituted non-natural amino acids
containing olefinic side chains of varying length can be
synthesized by known methods (see, e.g., 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: a) one S.sub.5 amino acid and one
R.sub.8 is used or b) one S.sub.8 amino acid and one R.sub.5 amino
acid is used. R.sub.8 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).
[0405] 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 (see, e.g.,
Williams et al., Org. Synth., 80:31, 2003).
[0406] The compounds of this invention may be modified by appending
appropriate functionalities to enhance selective biological
properties (including, e.g., hydrophobicity and/or the
position/occurrence of hydrophobic patches). Such modifications are
known in the art and include those which increase biological
penetration into a given biological compartment (e.g., blood,
lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism, and alter rate of excretion.
[0407] A stabilized AMP selective for microbial versus mammalian
membranes (i.e., a peptide able to kill or inhibit the growth of a
microbe while also having a relatively low ability to lyse or
inhibit the growth of a mammalian cell) may, e.g., possess a MIC
for one or more microbes more than about 1.5-fold lower, more than
about 2-fold lower, more than about 2.5-fold lower, more than about
3-fold lower, more than about 4-fold lower, more than about 5-fold
lower, more than about 6-fold lower, more than about 7-fold lower,
more than about 8-fold lower, more than about 9-fold lower, more
than about 10-fold lower, more than about 15-fold lower, or more
than about 20-fold lower than the MIC of the corresponding parent
(i.e., unmodified) non-internally cross-linked peptide for the same
one or more microbes. An antimicrobial peptide selective for
microbial versus mammalian membranes can have a MIC of, e.g., about
1 .mu.g/ml, about 2 .mu.g/ml, about 3 .mu.g/ml, about 4 .mu.g/ml,
about 5 .mu.g/ml, about 6 .mu.g/ml, about 7 .mu.g/ml, about 8
.mu.g/ml, about 9 .mu.g/ml, about 10 .mu.g/ml, about 12 .mu.g/ml,
about 14 .mu.g/ml, about 16 .mu.g/ml, about 18 .mu.g/ml, about 20
.mu.g/ml, about 22 .mu.g/ml, about 24 .mu.g/ml, about 26 .mu.g/ml,
about 28 .mu.g/ml, or about 30 .mu.g/ml. An antimicrobial peptide
selective for microbial versus mammalian membranes may lyse, e.g.,
less than about 50%, less than about 40%, less than about 30%, less
than about 25%, less than about 20%, less than about 15%, less than
about 10%, less than about 5%, less than about 2.5%, less than
about 2%, or less than about 1% of red blood cells (RBCs) in a RBC
hemolytic activity assay when administered at a concentration,
e.g., greater than or approximately equal to 1-fold, 1.5-fold,
2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,
9-fold, or 10-fold its MIC for one or more microbes.
[0408] To avoid mammalian cell lytic properties and generate
microbial-selective stabilized (e.g., stapled) AMPs, any of the
stabilized (e.g., stapled) AMPs of this document can include an
.alpha.-helical region that contains a first surface hydrophobic
patch. In these stabilized AMPs the replacement of a relevant pair
of amino acids by a linking group (e.g., R.sub.3 in Formula (I))
results in discontinuity between the first surface hydrophobic
patch and an additional one or more (e.g., 2, 3, 4, 5, 6, 8, or 10)
surface hydrophobic patches in .alpha.-helical region. See, e.g.,
Example 11. Referring to Formula (I), such stabilized AMPs can be
made, e.g., by determining the location of an established surface
hydrophobic patch in an .alpha.-helical region of the stabilized
AMP, and selecting integers w and y such that all amino acids
[Xaa].sub.x are located within the established surface hydrophobic
patch. In an alternative method for generating microbial-selective
structurally-stabilized AMPs, again referring to Formula (I), the
location of two or more (e.g., 3, 4, 5, 6, 8, or 10) established
surface hydrophobic patches in an .alpha.-helical region of the
stabilized AMP is determined and integers w and y are selected such
that amino acids [Xaa].sub.x do not connect two or more (e.g., 3,
4, 5, 6, 8, or 10) established surface hydrophobic patches in the
.alpha.-helical region of the stabilized AMP.
[0409] Another method of making the above-described selective and
internally cross-linked (ICL) anti-microbial peptide (AMP) involves
synthesizing the ICL AMP such that the ICL AMP includes an
.alpha.-helical region comprising a first surface hydrophobic
patch, and the replacement of a relevant pair of amino acids by a
linking group (e.g., R.sub.3 in Formula (I)) maintains or results
in, relative to the corresponding parent non-internally crosslinked
AMP, discontinuity between the first hydrophobic patch and one or
more (e.g., 2, 3, 4, 5, 6, 8, or 10) additional surface hydrophobic
patches on the ICL AMP.
[0410] The document also includes a method of designing an
internally cross-linked (ICL) anti-microbial peptide (AMP) that
includes: (a) creating one or more panels of ICL AMPs, each panel
containing a plurality of panel member ICL AMPs in each of which:
(a) the side chains of at least one pair of amino acids separated
by 2, 3, or 6 amino acids are replaced by the linking group,
R.sub.3 (see Formula (I)), which connects the alpha carbons of the
pair of amino acids; and (b) in each member of each panel, the pair
of amino acids is at different positions as compared to the other
members of the relevant panel; and (b) testing each member of all
panels for (i) the presence of discontinuity between a first
surface hydrophobic patch in an .alpha.-helical region of the
relevant member and one or more additional surface hydrophobic
patches on the .alpha.-helical region of the member; and (ii) the
ability of each member of each panel for its ability to kill or
inhibit the growth of a microbe (e.g., any of those disclosed
herein such as a bacterium) and lyse or inhibit the growth of a
mammalian cell. This method can further include manufacturing one
or members of all the panels that have a relatively high ability to
kill or inhibit the growth of a microbe (e.g., a bacterium) and a
relatively low ability to lyse or inhibit the growth of a mammalian
(e.g., human) cell. Methods of measuring the ability of chemical
and biological agents to kill and/or inhibit the growth of
microbial organisms and to lyse or inhibit the growth of human
cells (e.g., red blood cells, leukocytes, or epithelial cells) are
well known in the art (see, e.g., the Examples herein).
[0411] Hydrophobic patches within a peptide or protein may be
identified using techniques generally known in the art, including,
e.g., computational prediction/simulation (e.g., using ExPASy
ProtScale, Scooby-domain prediction, PSIPRED, Kyte Doolittle
plotting, and/or SPLIT,) and/or experimental determination (e.g.,
using techniques involving NMR spectroscopy, electron microscopy,
homology modeling, small-angle X-ray and/or neutron scattering
(SAXS/SANS), and/or X-ray crystallography) of the structure of the
peptide or protein.
[0412] Pharmaceutically-acceptable salts of the compounds of this
invention include those derived from pharmaceutically-acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, benzoate, benzenesulfonate,
butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate,
glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,
hydrobromide, hydroiodide, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
palmoate, phosphate, picrate, pivalate, propionate, salicylate,
succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate,
and undecanoate. Salts derived from appropriate bases include
alkali metal (e.g., sodium), alkaline earth metal (e.g.,
magnesium), ammonium and N-(alkyl).sub.4.sup.+ salts. This
invention also envisions the quaternization of any basic
nitrogen-containing groups of the compounds disclosed herein. Water
or oil-soluble or dispersible products may be obtained by such
quaternization.
[0413] 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., Meth Enzymol.,
446:369-386 (2008); Bird et al, Curr Protoc Chem Biol., 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) and are described
herein (see, e.g., Example 1).
[0414] In some aspects, the peptides are substantially free of
non-stapled peptide 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 aspect, 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 aspect, the
resin is washed with NMP. Shaking and bubbling an inert gas into
the solution may be performed.
[0415] Properties of the cross-linked polypeptides of the invention
can be assayed, for example, using the methods described below.
[0416] Assays to Determine .alpha.-Helicity:
[0417] Compounds are dissolved in an aqueous solution (e.g. 5 mM
potassium phosphate solution at pH 7, or distilled H.sub.2O, to
concentrations of 25-50 .mu.M). Circular dichroism (CD) spectra are
obtained on a spectropolarimeter (e.g., Jasco J-710, Aviv) using
standard measurement parameters (e.g. temperature, 20.degree. C.;
wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec;
accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length,
0.1 cm). The .alpha.-helical content of each peptide is calculated
by dividing the mean residue ellipticity by the reported value for
a model helical decapeptide (Yang et al., Methods Enzymol. 130:208
(1986)).
[0418] Assays to Determine Melting Temperature (Tm):
[0419] Cross-linked or the unmodified template peptides are
dissolved in distilled H.sub.2O or other buffer or solvent (e.g. at
a final concentration of 50 .mu.M) and Tm is determined by
measuring the change in ellipticity over a temperature range (e.g.
4 to 95.degree. C.) on a spectropolarimeter (e.g., Jasco J-710,
Aviv) using standard parameters (e.g. wavelength 222 nm; step
resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response,
1 sec; bandwidth, 1 nm; temperature increase rate: 1.degree.
C./min; path length, 0.1 cm).
[0420] In Vitro Protease Resistance Assays:
[0421] The amide bond of the peptide backbone is susceptible to
hydrolysis by proteases, thereby rendering peptidic compounds
vulnerable to rapid degradation in vivo. Peptide helix formation,
however, typically buries and/or twists and/or shields the amide
backbone and therefore may prevent or substantially retard
proteolytic cleavage. The compounds of the present invention may be
subjected to in vitro enzymatic proteolysis (e.g. trypsin,
chymotrypsin, pepsin) to assess for any change in degradation rate
compared to a corresponding uncrosslinked or alternatively stapled
polypeptide. For example, the compound and a corresponding
uncrosslinked polypeptide are incubated with trypsin agarose and
the reactions quenched at various time points by centrifugation and
subsequent HPLC injection to quantitate the residual substrate by
ultraviolet absorption at 280 nm. Briefly, the compound and
peptidomimetic precursor (5 mcg) are incubated with trypsin agarose
(Pierce) (S/E.about.125) for 0, 10, 20, 90, and 180 minutes.
Reactions are quenched by tabletop centrifugation at high speed;
remaining substrate in the isolated supernatant is quantified by
HPLC-based peak detection at 280 nm. The proteolytic reaction
displays first order kinetics and the rate constant, k, is
determined from a plot of ln[S] versus time.
[0422] Compounds and/or a corresponding uncrosslinked polypeptide
can be each incubated with fresh mouse, rat and/or human serum
(e.g. 1-2 mL) at 37.degree. C. for, e.g., 0, 1, 2, 4, 8, and 24
hours. Samples of differing macrocycle concentration may be
prepared by serial dilution with serum. To determine the level of
intact compound, the following procedure may be used: The samples
are extracted, for example, by transferring 100 .mu.L of sera to 2
ml centrifuge tubes followed by the addition of 10 .mu.L of 50%
formic acid and 500 .mu.L acetonitrile and centrifugation at 14,000
RPM for 10 min at 4+/-2.degree. C. The supernatants are then
transferred to fresh 2 ml tubes and evaporated on Turbovap under
N.sub.2<10 psi, 37.degree. C. The samples are reconstituted in
100 .mu.L of 50:50 acetonitrile:water and submitted to LC-MS/MS
analysis. Equivalent or similar procedures for testing ex vivo
stability are known and may be used to determine stability of
macrocycles in serum.
[0423] In Vivo Protease Resistance Assays:
[0424] A key benefit of peptide stapling is the translation of in
vitro protease resistance into markedly improved pharmacokinetics
in vivo. Structurally-stabilized AMPs with potent and selective
antimicrobial activity are screened for protease stability in vivo,
e.g., using previously published methods (see, e.g., Bird et al.,
PNAS, 2010).
Pharmaceutical Compositions
[0425] One or more of the stabilized peptides disclosed herein
(e.g., one or more of SEQ ID NOs: 1-17) 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 Data Standards
Manual (DSM).
[0426] The pharmaceutical compositions of this invention may be
administered, e.g., orally, parenterally, by inhalation spray or
nebulizer, topically, rectally, nasally, buccally, vaginally, via
an implanted reservoir, by injection (e.g., intravenously,
intra-arterially, subdermally, intraperitoneally, intramuscularly,
and/or subcutaneously), in an ophthalmic preparation, or via
transmucosal administration. Suitable dosages may range from about
0.001 to about 100 mg/kg of body weight, or according to the
requirements of the particular drug. 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,
intracutaneous, intravenous, intramuscular, intraarticular,
intra-arterial, intrasynovial, intrasternal, intrathecal,
intralesional and intracranial injection or infusion techniques.
Alternatively or in addition, the present invention may be
administered according to any of the methods as described in the
FDA DSM.
[0427] As used herein, the compounds of this invention, including
the compounds of formulae described herein, are defined to include
pharmaceutically-acceptable derivatives or prodrugs thereof. A
"pharmaceutically-acceptable derivative or prodrug" means any
pharmaceutically-acceptable salt, ester, salt of an ester, or other
derivative of a compound or agent disclosed herein which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
derivatives and prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal (e.g., by allowing an orally
administered compound to be more readily absorbed into the blood)
or which enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system) relative to the
parent species. Preferred prodrugs include derivatives where a
group which enhances aqueous solubility or active transport through
the gut membrane is appended to the structure of formulae described
herein.
[0428] In some instances, pharmaceutical compositions can include
an effective amount of one or more stabilized 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 infection).
[0429] The methods herein contemplate administration of an
effective amount of compound or compound composition to achieve the
desired or stated effect. Typically, the pharmaceutical
compositions of this invention will be administered from about 1 to
about 6 times per day or, alternatively, as a continuous infusion.
Such administration can be used as a chronic or acute therapy. The
amount of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. A
typical preparation will contain from about 5% to about 95% active
compound (w/w). Alternatively, such preparations contain from about
20% to about 80% active compound.
[0430] Dosing can be determined using various techniques. The
selected dosage level can depend upon a variety of factors,
including, e.g., the activity of the particular compound employed,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular compound being
employed, the duration of the treatment, other drugs, compounds,
and/or materials used in combination with the particular compound
employed, the age, sex, weight, condition, general health, and/or
prior medical history of the patient being treated, and like
factors well known in the medical arts. The dosage values can also
vary with the severity of the condition to be alleviated. For any
particular subject, specific dosage regimens can be adjusted over
time according to the individual need and the professional judgment
of the person administering or supervising the administration of
the compositions.
[0431] In some aspects, a suitable daily dose of a compound of the
disclosure can be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above. The
precise time of administration and amount of any particular
compound that will yield the most effective treatment in a given
patient will depend upon the activity, pharmacokinetics, and
bioavailability of a particular compound, physiological condition
of the patient (including age, sex, disease type and stage, general
physical condition, responsiveness to a given dosage and type of
medication), route of administration, and the like.
[0432] A physician or veterinarian can prescribe the effective
amount of the pharmaceutical composition required. For example, the
physician or veterinarian could start doses of the compounds of the
disclosure employed in the pharmaceutical composition at levels
lower than that required in order to achieve the desired
therapeutic effect and gradually increase the dosage until the
desired effect is achieved.
[0433] Pharmaceutical compositions described herein can be in unit
dosage forms suitable for single administration of precise dosages.
In unit dosage form, the formulation is divided into unit doses
containing appropriate quantities of one or more compounds. The
unit dosage can be in the form of a package containing discrete
quantities of the formulation. Non-limiting examples are liquids in
vials or ampoules. Aqueous suspension compositions can be packaged
in single-dose non-reclosable containers. Multiple-dose reclosable
containers can be used, for example, in combination with a
preservative. Formulations for parenteral injection can be
presented in unit dosage form, for example, in ampoules, or in
multi dose containers with a preservative.
[0434] A compound described herein can be present in a composition
in a range of from about 1 mg to about 2000 mg; from about 100 mg
to about 2000 mg; from about 10 mg to about 2000 mg; from about 5
mg to about 1000 mg, from about 10 mg to about 500 mg, from about
50 mg to about 250 mg, from about 100 mg to about 200 mg, from
about 1 mg to about 50 mg, from about 50 mg to about 100 mg, from
about 100 mg to about 150 mg, from about 150 mg to about 200 mg,
from about 200 mg to about 250 mg, from about 250 mg to about 300
mg, from about 300 mg to about 350 mg, from about 350 mg to about
400 mg, from about 400 mg to about 450 mg, from about 450 mg to
about 500 mg, from about 500 mg to about 550 mg, from about 550 mg
to about 600 mg, from about 600 mg to about 650 mg, from about 650
mg to about 700 mg, from about 700 mg to about 750 mg, from about
750 mg to about 800 mg, from about 800 mg to about 850 mg, from
about 850 mg to about 900 mg, from about 900 mg to about 950 mg, or
from about 950 mg to about 1000 mg.
[0435] A compound described herein can be present in a composition
in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg,
about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg,
about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg,
about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg,
about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg,
about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250
mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about
500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg,
about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950
mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg,
about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about
1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600
mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg,
about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.
[0436] In some aspects, a dose can be expressed in terms of an
amount of the drug divided by the mass of the subject, for example,
milligrams of drug per kilograms of subject body mass. In some
aspects, a compound is administered in an amount ranging from about
5 mg/kg to about 50 mg/kg, 250 mg/kg to about 2000 mg/kg, about 10
mg/kg to about 800 mg/kg, about 50 mg/kg to about 400 mg/kg, about
100 mg/kg to about 300 mg/kg, or about 150 mg/kg to about 200
mg/kg.
[0437] Dosage can be based on the amount of the compound per kg
body weight of the patient. Alternatively, the dosage of the
subject disclosure can be determined by reference to the plasma
concentrations of the compound. For example, the maximum plasma
concentration (C.sub.max) and the area under the plasma
concentration-time curve from time 0 to infinity (AUC) can be
used.
[0438] In some aspects, the subject is a human subject and the
amount of the compound administered is 0.01-100 mg per kilogram
body weight of the human subject. For example, in various examples,
the amount of the compound administered is about 0.01-50 mg/kg,
about 0.01-20 mg/kg, about 0.01-10 mg/kg, about 0.1-100 mg/kg,
about 0.1-50 mg/kg, about 0.1-20 mg/kg, about 0.1-10 mg/kg, about
0.5-100 mg/kg, about 0.5-50 mg/kg, about 0.5-20 mg/kg, about 0.5-10
mg/kg, about 1-100 mg/kg, about 1-50 mg/kg, about 1-20 mg/kg, about
1-10 mg/kg body weight of the human subject. In one aspect, about
0.5 mg-10 mg of the compound per kilogram body weight of the human
subject is administered. In some examples the amount of the
compound administered is about 0.16 mg, about 0.32 mg, about 0.64
mg, about 1.28 mg, about 3.56 mg, about 7.12 mg, about 14.24 mg, or
about 20 mg per kilogram body weight of the human subject. In some
examples the amount of the compound administered is about 0.16 mg,
about 0.32 mg, about 0.64 mg, about 1.28 mg, about 3.56 mg, about
7.12 mg, or about 14.24 mg per kilogram body weight of the human
subject. In some examples the amount of the compound administered
is about 0.16 mg per kilogram body weight of the human subject. In
some examples the amount of the compound administered is about 0.32
mg per kilogram body weight of the human subject. In some examples
the amount of the compound administered is about 0.64 mg per
kilogram body weight of the human subject. In some examples the
amount of the compound administered is about 1.28 mg per kilogram
body weight of the human subject. In some examples the amount of
the compound administered is about 3.56 mg per kilogram body weight
of the human subject. In some examples the amount of the compound
administered is about 7.12 mg per kilogram body weight of the human
subject. In some examples the amount of the compound administered
is about 14.24 mg per kilogram body weight of the human
subject.
[0439] In some aspects about 0.5-about 20 mg or about 0.5-about 10
mg of the compound per kilogram body weight of the human subject is
administered two times a week. For example about 0.5-about 1 mg,
about 0.5-about 5 mg, about 0.5-about 10 mg, about 0.5-about 15 mg,
about 1-about 5 mg, about 1-about 10 mg, about 1-about 15 mg, about
1-about 20 mg, about 5-about 10 mg, about 1-about 15 mg, about
5-about 20 mg, about 10-about 15 mg, about 10-about 20 mg, or about
15-about 20 mg of the compound per kilogram body weight of the
human subject is administered about twice a week. In some examples,
about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg,
about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg,
about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg,
about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 10.5 mg,
about 11 mg, about 11.5 mg, about 12 mg, about 12.5 mg, about 13
mg, about 13.5 mg, about 14 mg, about 14.5 mg, about 15 mg, about
15.5 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg,
about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, or about 20
mg of the compound per kilogram body weight of the human subject is
administered two times a week. In some examples, the amount of the
compound administered is about 1.25 mg, about 2.5 mg, about 5 mg,
about 10 mg, or about 20 mg per kilogram body weight of the human
subject and the compound is administered two times a week. In some
examples, the amount of the compound administered is about 1.25 mg,
about 2.5 mg, about 5 mg or about 10 mg per kilogram body weight of
the human subject. The compound can be administered once a week,
two times a week, three, four, five, six, or seven times a week.
The compound can be administered once every 3 weeks.
[0440] In some aspects, the compound is administered gradually over
a period of time. A desired amount of compound can, for example can
be administered gradually over a period of from about 0.1 h-24 h.
In some cases, a desired amount of compound is administered
gradually over a period of 0.1 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3
h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13
h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or
24 h. In some examples, a desired amount of compound is
administered gradually over a period of 0.25-12 h, for example over
a period of 0.25-1 h, 0.25-2 h, 0.25-3 h, 0.25-4 h, 0.25-6 h,
0.25-8 h, or 0.25-10 h. In some examples, a desired amount of
compound is administered gradually over a period of 0.25-2 h. In
some examples, a desired amount of compound is administered
gradually over a period of 0.25-1 h. In some examples, a desired
amount of compound is administered gradually over a period of 0.25
h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h,
1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, or 2.0 h.
In some examples, a desired amount of compound is administered
gradually over a period of 1 h. In some examples, a desired amount
of compound is administered gradually over a period of 2 h.
[0441] Administration of the compounds can continue as long as
necessary. In some aspects, one or more compound of the disclosure
is administered for more than 1 day, more than 1 week, more than 1
month, more than 2 months, more than 3 months, more than 4 months,
more than 5 months, more than 6 months, more than 7 months, more
than 8 months, more than 9 months, more than 10 months, more than
11 months, more than 12 months, more than 13 months, more than 14
months, more than 15 months, more than 16 months, more than 17
months, more than 18 months, more than 19 months, more than 20
months, more than 21 months, more than 22 months, more than 23
months, or more than 24 months. In some aspects, one or more
compound of the disclosure is administered for less than 1 week,
less than 1 month, less than 2 months, less than 3 months, less
than 4 months, less than 5 months, less than 6 months, less than 7
months, less than 8 months, less than 9 months, less than 10
months, less than 11 months, less than 12 months, less than 13
months, less than 14 months, less than 15 months, less than 16
months, less than 17 months, less than 18 months, less than 19
months, less than 20 months, less than 21 months, less than 22
months, less than 23 months, or less than 24 months.
[0442] In some aspects, the compound is administered on day 1, 8,
15, and 28 of a 28 day cycle. In some aspects, the compound is
administered on day 1, 8, 15, and 28 of a 28 day cycle and
administration is continued for two cycles. In some aspects, the
compound is administered on day 1, 8, 15, and 28 of a 28 day cycle
and administration is continued for three cycles. In some aspects,
the compound is administered on day 1, 8, 15, and 28 of a 28 day
cycle and administration is continued for 4, 5, 6, 7, 8, 9, 10, or
more cycles.
[0443] In some aspects, the compound is administered on day 1, 8,
11, and 21 of a 21-day cycle. In some aspects, the compound is
administered on day 1, 8, 11, and 21 of a 21-day cycle and
administration is continued for two cycles. In some aspects, the
compound is administered on day 1, 8, 11, and 21 of a 21-day cycle
and administration is continued for three cycles. In some aspects,
the compound is administered on day 1, 8, 11, and 21 of a 21-day
cycle and administration is continued for 4, 5, 6, 7, 8, 9, 10, or
more cycles.
[0444] In some aspects, one or more compound of the disclosure is
administered chronically on an ongoing basis. In some aspects
administration of one or more compound of the disclosure is
continued until documentation of disease progression, unacceptable
toxicity, or patient or physician decision to discontinue
administration.
[0445] 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. Such additional therapeutic agents may include
antimicrobial agents (e.g., antibiotics) known in the art. When
co-administered, stapled AMPs of the invention operate in
conjunction with antimicrobial agents to produce mechanistically
additive or synergistic antimicrobial effects. For example, this
can be due to structurally-stabilized AMPs piercing otherwise
resistant bacterial membranes to allow for entry of alternative
antibiotics and drug modalities. It is understood that the same
additional therapeutic agents may be administered as a complex
chemically bound (covalently or non-covalently) to an appropriate
stapled peptide.
[0446] Examples of antibiotics suitable for co-administration with
the stapled peptides disclosed herein include, but are not limited
to, quinolones (e.g., levofloxacin, norfloxacin, ofloxacin,
ciprofloxacin, perfloxacin, lomefloxacin, fleroxacin, sparfloxacin,
grepafloxacin, trovafloxacin, clinafloxacin, gemifloxacin,
enoxacin, sitafloxacin, nadifloxacin, tosulfloxacin, cinnoxacin,
rosoxacin, miloxacin, moxifloxacin, gatifloxacin, cinnoxacin,
enoxacin, fleroxacin, lomafloxacin, lomefloxacin, miloxacin,
nalidixic acid, nadifloxacin, oxolinic acid, pefloxacin, pirimidic
acid, pipemidic acid, rosoxacin, rufloxacin, temafloxacin,
tosufloxacin, trovafloxacin, besifloxacin); .beta.-lactams
including cephalosporins (e.g., cefacetrile, cefixime, cefadroxil,
cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin,
cefcapene, cefdaloxime, cefdinir, cefditoren, cefatrizine,
cefetamet, cefazaflur, cefazedone, cefazolin, cefaradine,
cefroxadine, ceftezole, cefteram, ceftibuten, ceftiofur,
ceftiolene, ceftizoxime, cefaclor, cefprozil, cefuroxime,
cefuzonam, cefmenoxime, cefodizime, cefotaxime, cefovecin,
cefpimizole, cefpirome, cefquinome, ceftobiprole, cefpodoxime,
ceftazidime, ceftaroline, cefclidine, cefepime, cefluprenam,
cefoselis, cefozopran, cephalexin, cephaloridine, cefamandole,
cefsulodin, cefonicid, cefoperazine, cefoperazone, cefoprozil,
ceftriaxone), penicillins and penicillin derivatives (e.g.,
penicillin G, penicillin V, procaine penicillin, benzathine
penicillin, benzathine benzylpenicillin, ampicillin, epicillin,
amoxicillin, benzylpenicillin, clometocillin,
phenoxymethylpenicillin, oxacillin, methicillin, dicloxacillin,
flucloxacillin, temocillin, azlocillin, carbenicillin, ricarcillin,
mezlocillin, piperacillin, apalcillin, hetacillin, bacampicillin,
sulbenicillin, mecicilam, pevmecillinam, ciclacillin, talapicillin,
aspoxicillin, azidocillin, cloxacillin, nafcillin, pivampicillin,
penamecillin, mecillinam, propicillin, pheneticillin, ticarcillin
temocillin), carbapenems (e.g., thienamycin, tomopenem, lenapenem,
tebipenem, razupenem, imipenem, meropenem, ertapenem, doripenem,
panipenem (betamipron), biapenem), carbacephems (e.g., loracarbef),
penems (e.g., faropenem), cephamycins (e.g., cefbuperazone,
cefmetazole, cefminox, cefotetan, cefoxitin), monobactams (e.g.,
aztreonam, nocardicin A, tabtoxin, tigemonam), and oxacephems
(e.g., flomoxef, latamoxef); lipopeptide antibiotics (e.g.,
amphomycin, aspartocin, brevistin, cerexin A, cerexin B,
glumamycin, laspartomycin, tsushimycin, zaomycin, daptomycin);
polymyxin antibiotics (e.g., polymyxin B, colistin (polymyxin E),
polymyxin M); aminoglycosides (e.g., gentamicin, amikacin,
tobramycin, debekacin, kanamycin, neomycin, netilmicin,
paromomycin, sisomycin, spectinomycin, streptomycin); glycopeptides
(e.g., vancomycin, teicoplanin, telavancin, ramoplanin, daptomycin,
decaplanin, bleomycin); macrolides (e.g., azithromycin,
clarithromycin, erythromycin, fidaxomicin, telithromycin,
carbomycin A, josamycin, kitasamycin,
midecamycin/midecamycinacetate, oleandomycin, solithromycin,
spiramycin, troleandomycin, tylosin/tylocine, roxithromycin,
dirithromycin, troleandomycin, spectinomycin, methymycin,
neomethymycin, erythronolid, megalomycin, picromycin, narbomycin,
oleandomycin, triacetyl-oleandomycin, laukamycin, kujimycin A,
albocyclin, cineromycin B); ansamycins (e.g., streptovaricin,
geldanamycin, herbimycin, rifamycin, rifampin, rifabutin,
rifapentine, rifamixin); linezolid; pristinamycin; and sulfonamides
(e.g., sulfanilamide, sulfacetarnide, sulfapyridine, sulfathiazole,
sulfadiazine, sulfamerazine, sulfadimidine, sulfasomidine,
sulfasalazine, mafenide, sulfamethoxazole, sulfamethoxypyridazine,
sulfadimethoxine, sulfasymazine, sulfadoxine, sulfametopyrazine,
sulfaguanidine, succinylsulfathiazole, phthalylsulfathiazole).
[0447] When the compositions of this invention comprise a
combination of a compound of the formulae described herein and one
or more additional therapeutic or prophylactic agents, both the
compound and the additional agent should be present at dosage
levels of between about 1% to about 100%, or between about 5% to
about 95% of the dosage normally administered in a monotherapy
regimen. The additional agents may be administered separately, as
part of a multiple dose regimen, from the compounds of this
invention. Alternatively, those agents may be part of a single
dosage form, mixed together with the compounds of this invention in
a single composition.
[0448] 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.
[0449] 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 can also be advantageously used to enhance
delivery of compounds of the formulae described herein.
[0450] 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 parenteral, epidural, subcutaneous, intracutaneous,
intra-venous, intra-muscular, intra-articular, intra-arterial,
intra-synovial, intrasternal, intra-thecal, intra-lesional and
intra-cranial injection or infusion techniques.
[0451] An effective amount of a compound of the disclosure can be
administered in either single or multiple doses by any of the
accepted modes of administration. Regardless of the route of
administration selected, the compounds of the present disclosure,
and/or the pharmaceutical compositions of the present disclosure,
are formulated into pharmaceutically-acceptable dosage forms. The
compounds according to the disclosure can be formulated for
administration in any convenient way for use in human or veterinary
medicine, by analogy with other pharmaceuticals. In one aspect, the
disclosure provides pharmaceutical formulation comprising a
therapeutically-effective amount of one or more of the compounds
described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
In one aspect, one or more of the compounds described herein are
formulated for parenteral administration for parenteral
administration, one or more compounds disclosed herein can be
formulated as aqueous or non-aqueous solutions, dispersions,
suspensions, or emulsions or sterile powders which can be
reconstituted into sterile injectable solutions or dispersions just
prior to use. Such formulations can comprise sugars, alcohols,
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents. These compositions can also
contain adjuvants such as preservatives, wetting agents,
emulsifying agents and dispersing agents. Prevention of the action
of microorganisms upon the subject compounds can be ensured by the
inclusion of various antibacterial and antifungal agents, for
example, paraben, chlorobutanol, phenol sorbic acid, and the like.
It can also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form can be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin. If desired,
the formulation can be diluted prior to use with, e.g., an isotonic
saline solution or a dextrose solution. In some examples, the
compound is formulated as an aqueous solution and is administered
intravenously.
[0452] Pharmaceutical compositions can be in the form of a solution
or powder for injection. 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.
[0453] 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.
[0454] The pharmaceutical compositions of this invention may also
be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating excipient
that is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycols.
[0455] 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.
[0456] 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.
[0457] 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 aspects, the association is
covalent. In other aspects, 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.
[0458] 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
[0459] The disclosure includes methods of using the peptides herein
for the prophylaxis and/or treatment of infection. The terms
"treat", "treating", or "treatment" as used herein, refers to
partially or completely alleviating, inhibiting, ameliorating,
and/or relieving the disease or condition from which the subject is
suffering. This means any manner in which one or more of the
symptoms of a disease or disorder (e.g., cancer) are ameliorated or
otherwise beneficially altered. As used herein, amelioration of the
symptoms of a particular disorder (e.g., infection) refers to any
lessening, whether permanent or temporary, lasting or transient
that can be attributed to or associated with treatment by the
compositions and methods of the present invention. In some aspects,
treatment can promote or result in, for example, a decrease in the
number of microbial cells or organisms (e.g., in a subject)
relative to the number of microbial cells or organisms prior to
treatment; a decrease in the viability (e.g., the average/mean
viability) of microbial cells or organisms (e.g., in a subject)
relative to the viability (e.g., the average/mean viability) of
microbial cells or organisms (e.g., in the subject) prior to
treatment; and/or reductions in one or more symptoms associated
with one or more infections in a subject relative to the subject's
symptoms prior to treatment.
[0460] Examples of bacteria internally cross-linked AMPs are active
against include, without limitation, Staphylococci (e.g., S.
aureus, S. intermedius, S. epidermidis, and other coagulase
negative Staphylococci), Neisseriae (e.g., N. gonorrheae and N.
meningitidis), Streptococci (e.g., Group A Streptococcus (e.g., S.
pyogenes), Group B Streptococcus (e.g., S. agalactiae), Group C
Streptococcus, Group G Streptococcus, S. pneumoniae, and viridans
Streptococci), Chlamydia trachomatis, Treponemae (e.g., T.
pallidum, T. pertenue, and T. cerateum), Haemophilus bacteria
(e.g., H. ducreyi, H. influenzae, and H. aegyptius), Bordetellae
(e.g., B. pertussis, B. parapertussis, and B. bronchiseptica),
Gardnerella vaginalis, Bacillus (e.g., B. anthracis and B. cereus),
Mycobacteria (e.g., M. tuberculosis and M. leprae), Listeria
monocytogenes, Borrelia burgdorferi, Actinobacillus
pleuropneumonias, Helicobacter pylori, Clostridium (e.g. C.
perfringens, C. septicum, C. novyi, and C. tetani), Escherichia
coli, Porphyromonas gingivalis, Vibrio cholerae, Salmonella
bacteria (e.g., S. enteriditis, S. typhimurium, and S. typhi),
Shigella bacteria, Francisella bacteria, Yersinia bacteria (e.g. Y.
pestis and Y. enterocolitica), Burkholderia bacteria, Pseudomonas
bacteria, and Brucella bacteria. Mycoplasmal organisms AMPs are
active against include, e.g., M. pneumoniae, M. fermentans, M.
hominis, and M. penetrans.
[0461] Examples of fungal (including yeast) organisms internally
cross-linked AMPs are active against include, but are not limited
to, Candida albicans, other Candida species, Cryptococcus
neoformans, Histoplasma capsulatum, and Pneumocystis carinii.
[0462] Examples of protozoan parasites internally cross-linked AMPs
are active against include, without limitation, Trichomonas
vaginalis, Plasmodium falciparum, P. vivax, P. ovale, P. malariae,
Entamoeba histolytica, Toxoplasma brucei, Toxoplasma gondii, and
Leishmania major.
[0463] Examples of viruses internally cross-linked AMPs may be
employed against include, but are not limited to, human
immunodeficiency virus (HIV) 1 and 2, human lymphotropic virus
(HTLV), measles virus, rabies virus, hepatitis virus A, B, and C,
rotaviruses, rhinoviruses, influenza virus, parainfluenza virus,
respiratory syncytial virus, adenoviruses, parvoviruses (e.g.,
parvovirus B19), roseola virus, enteroviruses, papilloma viruses,
retroviruses, herpesviruses (e.g., herpes simplex virus, varicella
zoster virus, Epstein Barr virus (EBV), human cytomegalovirus
(CMV), human herpesvirus 6, 7 and 8), poxviruses (e.g., variola
major and variola minor, vaccinia, and monkeypox virus), feline
leukemia virus, feline immunodeficiency virus, and simian
immunodeficiency virus.
[0464] Disorders that can be treated by the compositions,
formulations, and/or methods described herein include, but are not
limited to, infectious diseases. Infectious diseases can be caused
by pathogens, such as bacteria, viruses, fungi or parasites. In
some aspects, an infectious disease can be passed from person to
person. In some aspects, an infectious disease can be transmitted
by bites from insects or animals. In some aspects, an infectious
disease can be acquired by ingesting contaminated food or water or
being exposed to organisms in the environment. Some infectious
diseases can be prevented by vaccines.
[0465] In specific aspects, infectious diseases that can be treated
by the compositions, formulations, and/or methods described herein
include, but are not limited to, Acinetobacter infections,
Actinomycosis, African sleeping sickness (African trypanosomiasis),
AIDS (Acquired immunodeficiency syndrome), Amebiasis, Anaplasmosis,
Angiostrongyliasis, Anisakiasis, Anthrax, Arcanobacterium
haemolyticum infection, Argentine hemorrhagic fever, Ascariasis,
Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus
infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides
infection, Balantidiasis, Bartonellosis, Baylisascaris infection,
BK virus infection, Black piedra, Blastocystosis, Blastomycosis,
Bolivian hemorrhagic fever, Botulism (and Infant botulism),
Brazilian hemorrhagic fever, Brucellosis, Bubonic plague,
Burkholderia infection, Buruli ulcer, Caicivirus infection
(Norovirus and Sapovirus), Campylobacteriosis, Candidiasis
(Moniliasis; Thrush), Capillariasis, Carrion's disease, Cat-scratch
disease, Cellulitis, Chagas Disease (American trypanosomiasis),
Chancroid, Chickenpox, Chikungunya, Chlamydia, Chlamydophila
pneumoniae infection (Taiwan acute respiratory agent or TWAR),
Cholera, Chromoblastomycosis, Chytridiomycosis, Clonorchiasis,
Clostridium difficile colitis, Coccidioidomycosis, Colorado tick
fever (CTF), Common cold (Acute viral rhinopharyngitis; Acute
coryza), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic
fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneous larva
migrans (CLM), Cyclosporiasis, Cysticercosis, Cytomegalovirus
infection, Dengue fever, Desmodesmus infection, Dientamoebiasis,
Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic
fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm
infection), Enterococcus infection, Enterovirus infection, Epidemic
typhus, Erythema infectiosum (Fifth disease), Exanthem subitum
(Sixth disease), Fasciolasis, Fasciolopsiasis, Fatal familial
insomnia (FFI), Filariasis, Food poisoning by Clostridium
perfringens, Free-living amebic infection, Fusobacterium infection,
Gas gangrene (Clostridial myonecrosis), Geotrichosis,
Gerstmann-Straussler-Scheinker syndrome (GSS), Giardiasis,
Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale
(Donovanosis), Group A streptococcal infection, Group B
streptococcal infection, Haemophilus influenzae infection, Hand,
foot, and mouth disease (HFMD), Hantavirus Pulmonary Syndrome
(HPS), Heartland virus disease, Helicobacter pylori infection,
Hemolytic-uremic syndrome (HUS), Hemorrhagic fever with renal
syndrome (HFRS), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis
D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection,
Human bocavirus infection, Human ewingii ehrlichiosis, Human
granulocytic anaplasmosis (HGA), Human metapneumovirus infection,
Human monocytic ehrlichiosis, Human papillomavirus (HPV) infection,
Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr
Virus Infectious Mononucleosis (Mono), Influenza (flu),
Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae
infection, Kuru, Lassa fever, Legionellosis (Legionnaires'
disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy,
Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis),
Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis,
Malaria, Marburg hemorrhagic fever (MHF), Measles, Middle East
respiratory syndrome (MERS), Melioidosis (Whitmore's disease),
Meningitis, Meningococcal disease, Metagonimiasis,
Microsporidiosis, Molluscum contagiosum (MC), Monkeypox, Mumps,
Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma
(disambiguation), Myiasis, Neonatal conjunctivitis (Ophthalmia
neonatorum), Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),
Nocardiosis, Onchocerciasis (River blindness), Opisthorchiasis,
Paracoccidioidomycosis (South American blastomycosis),
Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice),
Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice,
Crab lice), Pelvic inflammatory disease (PID), Pertussis (Whooping
cough), Plague, Pneumococcal infection, Pneumocystis pneumonia
(PCP), Pneumonia, Poliomyelitis, Prevotella infection, Primary
amoebic meningoencephalitis (PAM), Progressive multifocal
leukoencephalopathy, Psittacosis, Q fever, Rabies, Relapsing fever,
Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus
infection, Rickettsial infection, Rickettsialpox, Rift Valley fever
(RVF), Rocky Mountain spotted fever (RMSF), Rotavirus infection,
Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome),
Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary
dysentery), Shingles (Herpes zoster), Smallpox (Variola),
Sporotrichosis, Staphylococcal food poisoning, Staphylococcal
infection, Strongyloidiasis, Subacute sclerosing panencephalitis,
Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber's
itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis
(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manum
(Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete's foot),
Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis
versicolor), Toxocariasis (Ocular Larva Migrans (OLM)),
Toxocariasis (Visceral Larva Migrans (VLM)), Trachoma,
Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis (Whipworm
infection), Tuberculosis, Tularemia, Typhoid fever, Typhus fever,
Ureaplasma urealyticum infection, Valley fever, Venezuelan equine
encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus
infection, Vibrio parahaemolyticus enteritis, Viral pneumonia, West
Nile Fever, White piedra (Tinea blanca), Yersinia
pseudotuberculosis infection, Yersiniosis, Yellow fever, and
Zygomycosis.
[0466] The compositions, formulations, and/or methods described
herein can be used to treat a pathogen. In some aspects, the
pathogen can be a virus, bacterium, prion, a fungus, or a parasite.
In specific aspects, the pathogen described herein include, but are
not limited to, Acinetobacter baumannii, Actinomyces israelii,
Actinomyces gerencseriae and Propionibacterium propionicus,
Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba
histolytica, Anaplasma species, Angiostrongylus, Anisakis, Bacillus
anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris
lumbricoides, Aspergillus species, Astroviridae family, Babesia
species, Bacillus cereus, bacterial vaginosis microbiota,
Bacteroides species, Balantidium coli, Bartonella, Baylisascaris
species, BK virus, Piedraia hortae, Blastocystis species,
Blastomyces dermatitidis, Machupo virus, Clostridium botulinum,
Sabia, Brucella species, Enterobacteriaceae, Burkholderia cepacia
and other Burkholderia species, Mycobacterium ulcerans,
Caliciviridae family, Campylobacter species, Candida albicans and
other Candida species, Capillaria philippinensis, Capillaria
hepatica, Capillaria aerophila, Bartonella bacilliformis,
Bartonella henselae, Group A Streptococcus and Staphylococcus,
Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus
(VZV), Alphavirus, Chlamydia trachomatis, Chlamydophila pneumoniae,
Vibrio cholera, Fonsecaea pedrosoi, Batrachochytrium dendrabatidis,
Clonorchis sinensis, Clostridium difficile, Coccidioides immitis
and Coccidioides posadasii, Colorado tick fever virus (CTFV),
rhinoviruses and coronaviruses, PRNP, Crimean-Congo hemorrhagic
fever virus, Cryptococcus neoformans, Cryptosporidium species,
Ancylostoma braziliense; multiple other parasites, Cyclospora
cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses
(DEN-1, DEN-2, DEN-3 and DEN-4)--Flaviviruses, Green algae
Desmodesmus armatus, Dientamoeba fragilis, Corynebacterium
diphtheria, Diphyllobothrium, Dracunculus medinensis, Ebolavirus
(EBOV), Echinococcus species, Ehrlichia species, Enterobius
vermicularis, Enterococcus species, Enterovirus species, Rickettsia
prowazekii, Parvovirus B19, Human herpesvirus 6 (HHV-6) and Human
herpesvirus 7 (HHV-7), Fasciola hepatica and Fasciola gigantica,
Fasciolopsis buski, PRNP, Filarioidea superfamily, Clostridium
perfringens, Fusobacterium species, Clostridium perfringens, other
Clostridium species, Geotrichum candidum, Giardia lamblia,
Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma
hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,
Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus
influenza, Enteroviruses, mainly Coxsackie A virus and Enterovirus
71 (EV71), Sin Nombre virus, Heartland virus, Helicobacter pylori,
Escherichia coli O157:H7, O111 and O104:H4, Bunyaviridae family,
Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis
D Virus, Hepatitis E virus, Herpes simplex virus 1 and 2 (HSV-1 and
HSV-2), Histoplasma capsulatum, Ancylostoma duodenale and Necator
americanus, Human bocavirus (HBoV), Ehrlichia ewingii, Anaplasma
phagocytophilum, Human metapneumovirus (hMPV), Ehrlichia
chaffeensis, Human papillomavirus (HPV), Human parainfluenza
viruses (HPIV), Hymenolepis nana and Hymenolepis diminuta,
Epstein-Barr Virus (EBV), Orthomyxoviridae family, Isospora belli,
Kingella kingae, Lassa virus, Legionella pneumophila, Leishmania
species, Mycobacterium leprae, Mycobacterium lepromatosis,
Leptospira species, Listeria monocytogenes, Borrelia burgdorferi,
Borrelia garinii, Borrelia afzelii, Wuchereria bancrofti, Brugia
malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium
species, Marburg virus, Measles virus, Middle East respiratory
syndrome coronavirus, Burkholderia pseudomallei, Neisseria
meningitides, Metagonimus yokagawai, Microsporidia phylum,
Molluscum contagiosum virus (MCV), Monkeypox virus, Mumps virus,
Rickettsia typhi, Mycoplasma pneumoniae, Actinomycetoma,
Eumycetoma, parasitic dipterous fly larvae, Chlamydia trachomatis,
Neisseria gonorrhoeae, Nocardia asteroides, Nocardia species,
Onchocerca volvulus, Opisthorchis viverrini and Opisthorchis
felineus, Paracoccidioides brasiliensis, Pediculus humanus capitis,
Phthirus pubis, Bordetella pertussis, Yersinia pestis,
Streptococcus pneumoniae, Pneumocystis jirovecii, Poliovirus,
Prevotella species, Naegleria fowleri, JC virus, Chlamydophila
psittaci, Coxiella burnetii, Rabies virus, Borrelia hermsii,
Borrelia recurrentis, Borrelia species, Respiratory syncytial virus
(RSV), Rhinosporidium seeberi, Rhinovirus, Rickettsia species,
Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsia,
Rotavirus, Rubella virus, Salmonella species, SARS coronavirus,
Sarcoptes scabiei, Schistosoma species, Shigella species, Varicella
zoster virus (VZV), Variola major, Variola minor, Sporothrix
schenckii, Staphylococcus species, Strongyloides stercoralis,
Measles virus, Treponema pallidum, Taenia species, Clostridium
tetani, Trichophyton species, Trichophyton tonsurans,
Epidermophyton floccosum, Trichophyton rubrum, Trichophyton
mentagrophytes, Trichophyton rubrum, Hortaea werneckii,
Trichophyton species, Malassezia species, Toxocara canis, Toxocara
cati, Chlamydia trachomatis, Toxoplasma gondii, Trichinella
spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium
tuberculosis, Francisella tularensis, Salmonella enterica subsp.
enterica, serovar typhi, Rickettsia, Ureaplasma urealyticum,
Coccidioides immitis, Coccidioides posadasii, Venezuelan equine
encephalitis virus, Guanarito virus, Vibrio vulnificus, Vibrio
parahaemolyticus, multiple viruses, West Nile virus, Trichosporon
beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica,
Yellow fever virus, Mucorales order (Mucormycosis), and
Entomophthorales order (Entomophthoramycosis).
[0467] All the methods of treatment and prophylaxis described
herein may be applied to at least any or all the above-listed
microbial organisms.
[0468] In some aspects, the compounds of the invention can be toxic
to one microbe. In some aspects, the compounds of the invention can
be toxic to two microbes. In some aspects, the compounds of the
invention can be toxic to three microbes. In some aspects, the
compounds of the invention can be toxic to four microbes. In some
aspects, the compounds of the invention can be toxic to five
microbes.
[0469] In some aspects, the compounds of the invention can be used
to treat a microbe without damaging the host subject. In some
aspects, the compounds of the invention can be used to treat two
microbes without damaging the host subject. In some aspects, the
compounds of the invention can be used to treat three microbes
without damaging the host subject. In some aspects, the compounds
of the invention can be used to treat four microbes without
damaging the host subject. In some aspects, the compounds of the
invention can be used to treat five microbes without damaging the
host subject.
[0470] In general, methods include selecting a subject and
administering to the subject an 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 microbial infection and can be
administered, e.g., orally, intravenously or topically.
[0471] 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.
[0472] An effective amount can be administered in one or more
administrations, applications or dosages. A therapeutically
effective amount of a therapeutic compound (i.e., an effective
dosage) depends on the therapeutic compounds selected. The
compositions can be administered one from one or more times per day
to one or more times per week; including once every other day. The
skilled artisan will appreciate that certain factors may influence
the dosage and timing required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present.
[0473] Moreover, treatment of a subject with a therapeutically
effective amount of the therapeutic compounds described herein can
include a single treatment or a series of treatments. For example,
effective amounts can be administered at least once. Upon
improvement of a patient's condition, a maintenance dose of a
compound, composition or combination of this invention may be
administered, if necessary. Subsequently, the dosage or frequency
of administration, or both, may be reduced, as a function of the
symptoms, to a level at which the improved condition is retained.
Patients may, however, require intermittent treatment on a
long-term basis upon any recurrence of disease symptoms.
[0474] In some instances, the peptides herein can further be
co-administered with one or more additional therapeutic agents in
amounts effective for achieving a modulation of disease or disease
symptoms. Such additional therapeutic agents may include
conventional antimicrobial agents (e.g., antibiotics) known in the
art. When co-administered, stapled AMPs of the invention operate in
conjunction with conventional antimicrobial agents to produce
mechanistically additive or synergistic antimicrobial effects.
Without being limited by any particular mechanism of action,
certain internally cross-linked (e.g., stapled) AMPs having the
ability to produce "pores" in the membranes of certain microbial
organisms (including, e.g., Gram-negative bacteria) can act to
facilitate and/or enhance the passage of appropriate conventional
antimicrobial agents to the interiors of relevant microbial cells.
For the same purpose, the internally cross-linked AMPs can be
conjugated (covalently or non-covalently) to appropriate
antimicrobial agents, the resulting conjugates being administered
to appropriate subjects.
[0475] The ability of internally cross-linked AMPs to produce
"pores" in the membranes of microbial organisms provides the basis
for another utility for them. Thus, e.g., relevant microbial
organisms (e.g., any of those disclosed herein) can be contacted
either in a subject or in vitro to a internally cross-linked AMP
with the ability to produce "pores" or even lysis of the microbial
organism. As result of this activity, nucleic acids (e.g., DNA
and/or RNA) are released from microbial organisms into their
surroundings. This phenomenon can be used as a basis for accurate,
rapid, and inexpensive identification of the microbial organism.
Where the contacting occurs in a subject, any of a variety of
bodily fluids (e.g., blood, lymph, urine, feces, mucus, or tears)
or body lavages can be tested. Where the contacting occurs in
vitro, culture medium can be tested.
Application to Medical or Hygienic Devices
[0476] The antimicrobial peptides of the invention can be applied
to, or incorporated into, various medical and/or hygienic devices
(e.g., as a coating, or impregnated within a biodegradable device
for exposure or release as the device degrades or dissolves after
the device is inserted into a bodily canal of a vertebrate subject,
inserted into a bodily cavity of a vertebrate subject, or applied
to a tissue or organ of a vertebrate animal) to prevent or inhibit
microbial (e.g., bacterial or biofilm) growth. Medical or hygienic
devices suitable for use with the stapled peptides disclosed herein
include, but are not limited to, devices that are inserted into a
bodily canal of a vertebrate subject, inserted into a bodily cavity
of a vertebrate subject, or applied to a tissue or organ of a
vertebrate animal for the purpose of: (a) wound protection; (b)
preventing or reducing unwanted, or overcoming restricted, release
from the body of the vertebrate subject of a bodily fluid, bodily
secretion, or excreta (e.g., blood, menses, urine, lymphatic fluid,
cerebrospinal fluid, semen, saliva, vaginal secretions, mucus, or
feces); (c) delivering a drug or some other therapeutic or
prophylactic agent to a subject; (d) replacing absent or
supplementing defective organ functions; or (e) maintaining the
patency of a bodily canal (e.g., a blood vessel). Specific examples
of medical or hygienic devices include, without limitation:
peripheral IVs, central lines, portacaths, dialysis catheters;
rectal devices such as suppositories, enemas, and catheters; nasal,
tracheal, or esophageal delivery devices; vaginal devices such as
vaginal tampons and contraceptive devices (e.g., diaphragms or
intrauterine devices (IUDs)); venous, arterial, intracranial and
other needles, catheters and stents; renal dialysis accesses;
surgical bandages, sutures, or dressings; ostomy devices; natural
and synthetic implantable tissue matrices (see, e.g., U.S. Pat. No.
5,885,829, incorporated herein by reference in its entirety); pace
makers and pace maker wires and leads; synthetic and natural
prostheses such as hip and knee and joint prostheses and heart
valves; osmotic pumps (e.g., mini osmotic pumps) that are implanted
in body cavity (e.g., the peritoneal cavity) and provide slow
delivery of a drug or some other therapeutic or prophylactic
agent.
Treatment of Biofilms and Biofilm-Associated Infections
[0477] Stapled AMPs of the invention can be used to treat or
sterilize bacterial biofilms in vivo or in vitro. For example,
effective amounts can be administered to the lung for treating
cystic fibrosis or (topically) to the vagina for treating bacterial
vaginosis (BV). BV is a syndrome in which the vaginal flora becomes
altered such that Lactobacillus species no longer dominate [Forsum
et al. (2005) APMIS 113:81-90]. BV is characterized by overgrowth
of organisms such as Gardnerella vaginalis, some anaerobes, and
Mycoplasma hominis. Vaginal organisms such as Atopobium species may
be associated with BV [Verstraelen et al. (2004) Am J. Obstet. and
Gynecol. 191:1130-1132]. The anaerobes associated with BV include
Bacteroides, Prevotella, Peptostreptococcus, and Morbiluncus
species [Forsum et al. (2005), supra]. In studies of pregnant
women, both asymptomatic and symptomatic BV patients were found to
have a 10-fold or higher increase in these organisms, particularly
Gardnerella. Symptomatic women have 100- to 1000-fold increases in
Gardnerella bacteria and anaerobes. In such patients there is a
concomitant drop in lactobacilli, and for unknown reasons, the
lactobacilli that are present make less hydrogen peroxide that
their normal counterparts. The amine product trimethylamine is a
metabolic product of bacterial overgrowth and its fishy odor is
indicative of BV. Factors that have been associated with BV include
sexual activity, particularly new sexual partners, antibiotic use,
reduction for unknown reasons of pH, and use of IUDs (intrauterine
devices) [Hawes et al. (1996) J. Infect. Dis. 190:1374-1381].
Approximately one half of BV patients are asymptomatic. Persistent
vaginal inflammation is associated with BV.
[0478] The clinical diagnosis of BV is based on having three of the
four of the following characteristics in vaginal discharges: (1) pH
above 4.5; (2) a thin skim milk appearance; (3) a fishy amine odor
when 10% potassium hydroxide is placed on the discharge; and (4)
clue cells [Amsel et al. (1983) Am. J. Med. 74:14-22]. Clue cells
are vaginal cells that are so covered with bacteria that their
borders are obscured. On microscopic examination of vaginal
discharges from patients with BV, long lactobacilli morphotypes are
seen to be diminished.
[0479] Pregnant women with BV have 50% to 100% increases in
preterm, low birth-weight deliveries, amniotic fluid infections,
and chorioamnion infections [Hillier et al. (1995) N. Engl. J. Med.
333:1737-1742]. The high concentration of potentially virulent
microbes also predisposes the upper genital tract to infections,
including postpartum endometritis after cesarean delivery, pelvic
inflammatory disease following therapeutic abortion, and vaginal
cuff cellulitis following abdominal hysterectomy.
[0480] Treatment options for BV include metronidazole (orally) and
clindamycin (topically) in non-pregnant women and metronidazole in
symptomatic pregnant women as first line treatment regimens. For
recurrent BV, regular treatment and then biweekly suppressive doses
of metronidazole are recommended. Lactobacillus given orally or
intravaginally may help, though its effectiveness is still in
debate.
Further Applications
[0481] Stapled peptides as disclosed herein can also be used to
prevent or reduce a likelihood of viral (e.g., HIV) infection. For
example, stapled AMPS can be administered intravaginally (e.g.,
topically) to eliminate distinct vaginal bacterial flora that pose
an inflammatory risk shown to increase the risk of viral (e.g.,
HIV-1 or HIV-2) transmission and/or infection.
[0482] The peptides herein can be applied in the food or beverage
processing context (e.g., to food and beverage (e.g., beer)
products in sterilization and/or fermentation processes) to reduce
or eliminate the risk of microbial (e.g., bacterial) contamination.
Currently, the naturally occurring antibacterial peptide nisin is
used in food processing to eradicate pathogenic Gram-positive
bacteria. However, no effective antimicrobial agent is available to
eradicate Gram-negative bacteria in the food processing context.
Unlike nisin, the synthetic peptides of the invention display
broad-spectrum activity against both Gram-positive and
Gram-negative bacteria.
[0483] Stapled AMPS of the invention can also be applied in
veterinary and/or agricultural applications to prevent and/or treat
microbial infection in, e.g., an animal or a plant suffering from
an infection or at risk of infection. Examples of suitable animals
for treatment are generally known in the art and include (but are
not limited to), e.g., poultry and other birds (including chickens,
turkeys, ducks, ostrich, emu, quail), ruminants (including goats,
sheep, and cattle), fish, pigs, rabbits, mice, rats, horses,
donkeys, monkeys, apes, felines (including cats), hamsters,
ferrets, guinea pigs, and canines (including dogs). Examples of
suitable plants for treatment are generally known in the art and
include (but are not limited to), e.g., almond, apple, amaranth,
artichoke, asparagus, avocado, banana and plantain, barley, beet,
berries (including blueberry, blackberry, strawberry, and
raspberry), breadfruit and jackfruit, brussels sprout, cabbage,
carrot, cassava, cauliflower and broccoli, celery, chayote, cherry,
coconut, collard and kale, corn (maize), cucumber and zucchini,
dandelion, eggplant, endive and chicory, garlic, kohlrabi, grape,
legume, lettuce, melons (including honeydew, cantaloupe, and
watermelon), mustard, oat, oca, olive, okra, onion, orange and
grapefruit, oyster plant, pear, peach, pemmican, pepper, potato and
other tubers, quinoa, radish, rice, rhubarb, rye, sago, sorghum,
soybean, spinach, pumpkin and other squashes, sunchoke, taro, teff,
tomato, turnip, ulluco, vanilla, watercress, wheat, yam, and
yautia. Examples of suitable foods for treatment are generally
known in the art and include (but are not limited to), e.g., algae,
mushrooms, and products derived from animals (e.g., beef, butter,
eggs, (ice) cream, gravy, milk, pork, veal, yogurt) and/or plants
(e.g., beer, bread, cereal, chocolate, coffee, ketchup, mustard
sauce, oatmeal, juice, monosodium glutamate, salad, soda, soft
drinks, soymilk, soy sauce, tea, tofu, fries, vinegar, wine) as
described above.
[0484] The peptides herein can also be applied in the personal care
and/or consumer products context (e.g., to health or beauty
products in sterilization processes) to reduce or eliminate the
risk of microbial (e.g., bacterial) contamination. Examples of
suitable products for treatment are generally known in the art and
include (but are not limited to), e.g., brushes, conditioners,
clips, clippers, curling irons, shampoos, soaps, lotions, topical
acne ointments, oils, colorants, dyes, perfumes, pins, fragrances,
razors, shaving devices, deodorants, cosmetics, kitchen and/or
dining devices (e.g., cutting boards, racks, containers, pots,
pans, utensils), and cleaning products (e.g., brooms, mops,
dustpans, sweepers) and cleaning solutions.
EXAMPLES
Example 1: Synthesis of Stapled Magainin II Analogues
[0485] Magainin II is a well characterized AMP that was used as
scaffold for the design of compounds. A panel of stapled magainin
II peptides was generated (FIG. 3A). For the initial panel, seven
different locations using an i, i+7 staple that spans two
.alpha.-helical turns were sampled. The staple locations on the
hydrophobic and hydrophilic face of the peptide and on the two
interfaces were tested (FIG. 3B). Three mutations (S8A, G13A, G18A)
were introduced since alanine canpromote helicity within a peptide
sequence.
Example 2: Helical Characterization of Stapled Magainin II
Analogues
[0486] To determine the helicity of the panel of stapled magainin
II analogues, the peptides were dissolved in water using CD
spectroscopy in the presence and absence of trifluoroethanol (TFE;
50% v/v), a helix-promoting solvent. In the absence of TFE, the
stapled analogues displayed much higher levels of helicity compared
to unstapled magainin II, which was partially disordered (FIG. 4A).
Levels of helicity ranged from approximately 34% for MagStap 2 to
100% for MagStap 1 and 3, as compared to an idealized
.alpha.-helical peptide. These results suggest that the location of
the hydrocarbon staple has significant impact on inducing
.alpha.-helical structure. When the helicity in the presence of TFE
was measured, most of the stapled analogues were 50-60% helical
except for MagStap 3, which retained 100% .alpha.-helical content.
In contrast, the induced .alpha.-helicity for magainin II did not
exceed 32%, even in the presence of TFE (FIG. 4B).
Example 3: Antimicrobial Activity of Stapled Magainin II
Peptides
[0487] The panel of stapled magainin II peptides was tested on two
Gram-negative bacterial strains, E. coli and S. marcescens and two
Gram-positive strains, B. cereus and E. durans. The results of this
analysis are present in Table 3.
TABLE-US-00007 TABLE 3 Minimum inhibitory concentrations (MICs) of
magainin II and stapled derivatives against Gram-negative and
Gram-positive bacterial strains Antimicrobial Activity MIC
(.mu.g/ml) Peptide E. coli S. marcescens B. cereus E. durans
Magainin II 50 >50 >50 >50 MagStap 1 12.5 >50 12.5 25
MagStap 2 3.1 >50 3.1 12.5 Magstap 3 3.1 >50 6.2 25 MagStap 4
12.5 >50 6.2 25 MagStap 5 12.5 >50 12.5 25 MagStap 6 3.1
>50 3.1 12.5 MagStap 7 6.2 >50 6.2 25
[0488] The peptides in Table 3 are SEQ ID NOs: 134 and 178-184,
numbered consecutively from top to bottom.
[0489] The minimum inhibitory concentration (MIC) of the stapled
peptides in E. coli was more than 3-fold lower than the MIC of
magainin II; with select compounds, like MagStap 2 and 6,
exhibiting greater than a 15-fold increase in potency. Even though
the MICs for all the peptides tested in S. marcescens were greater
than the range of concentrations tested, treatment with the stapled
peptides resulted in partial inhibition of growth when compared to
magainin II. The MICs of the panel, when tested on B. cereus, were
very similar to the values obtained with E. coli. These results
suggest that the double membrane structure of Gram-negatives does
not protect the pathogens from AMP toxicity. Our MIC results with
E. durans exhibited a 2 to 3-fold increase in antimicrobial
activity. Of note, E. durans MIC measurements were conducted in the
presence of 5% lysed horse blood for optimal E. durans growth.
Since the solubility experiments demonstrated that the addition of
bovine serum albumin (BSA) attenuates peptide activity, the serum
proteins present in the horse blood may be binding to the AMPs and
impeding their activity.
[0490] Understanding how the staple location affects the
antimicrobial activity of AMPs can be crucial to refining peptide
design. When comparing MIC values of the stapled panel, staple
positions 2 and 6 consistently resulted in a much lower MIC value.
However, staple positions 1 and 5 had the highest MIC value among
the peptides in the panel across all 4 strains. Thus, when the
staple location is on the hydrophobic face of the peptide, a marked
increase in antimicrobial potency can be achieved. On the other
hand, when the staple location was installed on the hydrophilic
face, the gains in potency were lower.
Example 4: Hemolytic Activity of Stapled Magainin II Peptides
[0491] The ability of AMPs to distinguish between eukaryotic and
bacterial membranes is central to their activity as it helps
prevent injury to the host organism's cells. Nevertheless, a big
obstacle in the field of AMP therapy is increasing AMP potency
while minimizing injury to mammalian (e.g., human) cells. While
increases in helicity and hydrophobicity often result in favorable
gains in antimicrobial activity, increased helicity and
hydrophobicity can also result in much lower membrane selectivity.
In order to assess how selective the initial panel of stapled
peptides were; the ability to lyse red blood cells isolated from
healthy patient blood samples was measured (FIG. 5). Magainin II
displayed no hemolytic activity across the span of concentrations
tested, while the stapled analogues were all highly hemolytic even
at concentrations below their MICs. Of the stapled analogues,
MagStap 5 displayed the lowest hemolytic activity, while MagStap 4
had the highest hemolytic activity. One possible explanation for
these results is that the hydrocarbon staple resulted in a
significant increase in hydrophobicity as evidenced by the elution
times on a C-18 HPLC column (see, e.g., Example 11).
Example 5: Synthesis of Stapled Pexiganan Analogues
[0492] Three Pexiganan analogues were synthesized where the staple
was placed in the position corresponding to that used in MagStap2,
MagStap5, and MagStap6 to test the locations that displayed the
most and least activity (FIG. 6A and FIG. 6B).
Example 6: Helical Characterization of Stapled Pexiganan
Analogues
[0493] The helicity of Pexiganan and its stapled analogues in water
in the presence and absence of the helicity inducer, TFE (50% v/v),
were measured. In the absence of TFE, Pexiganan displayed little
secondary structure in solution while the stapled analogues showed
very low amounts of helical conformation (FIG. 7A). Upon the
addition of TFE, Pexiganan, PexStap2, and PexStap6 displayed
similar levels of helical conformation in the range of 25-35%.
These results suggest that the electrostatic repulsion made the
helical conformation less favorable (FIG. 7B). Furthermore, the
PexStap5 analogue, where the staple was added to the hydrophilic
side and replaced two lysine residues, displayed a much higher
degree of helicity around 83%. This result further underscores the
importance of electrostatic repulsion between residues on the
hydrophilic side in determining the relative amount of helicity an
AMP can adopt.
Example 7: Antimicrobial Activity of Stapled Pexiganan
Analogues
[0494] Pexiganan and its stapled analogues were tested against the
following strains of bacteria: E. coli, S. marcescens, B. cereus,
S. aureus, and P. aeruginosa. The results of this study are
presented in Table 4.
TABLE-US-00008 TABLE 4 Minimum inhibitory concentrations of
pexiganan and stapled derivatives against Gram-negative and
Gram-positive bacterial strains Antimicrobial Activity MIC
(.mu.g/ml) S. B. S. P. Peptide E. coli marcescens cereus aureus
aeruginosa Pexiganan 3.3 >50 3.1 6.3 1.6 PexStap 2 3.1 >50
3.1 3.1 3.1 PexStap 5 1.6 >50 1.6 3.1 1.6 PexStap 6 3.1 >50
6.3 3.1 1.6 PexDiol 5 1.6 >50 3.1 3.1 1.6
[0495] The stapled pexiganan analogues did not exhibit a
significant increase in antimicrobial activity. Their activity was
mostly maintained across the strains that were tested and in the
case of S. marcescens, the stapled analogues displayed partial
growth inhibition when compared to pexiganan. Due to the mechanism
of action of AMPs that requires the presence of a certain
concentration of peptide for pore formation to occur, an upper
limit in terms of peptide potency may have been reached.
Nevertheless, the protease resistance and favorable pharmacokinetic
profiles afforded by peptide stapling, represent potential benefits
for stapled pexiganan AMPs compared to the unmodified peptide.
Example 8: Hemolytic Activity of Stapled Pexiganan Analogues
[0496] When human red blood cells were incubated in the presence of
stapled pexiganan peptides, a significant amount of hemolytic
activity was detected as opposed to no hemolysis in the case of
pexiganan (FIG. 8). However, when compared to the stapled magainin
II panel, the pexiganan analogues displayed lower levels of
hemolytic activity across the range of concentrations tested. The
increase in cationic residues resulted in a decrease in the overall
hydrophobicity of the peptides, which partially restored their
membrane selectivity. In addition, PexStap5 displayed higher levels
of hemolytic activity when compared to PexStap2 and PexStap6,
providing further evidence that increased hydrophobicity reduces
membrane selectivity. Interestingly, PexStap5 is more hemolytic
than MagStap5 even though it is less hydrophobic. In terms of
helicity, a certain threshold appears to exist, which when exceeded
can drastically increase the hemolytic activity of an AMP (see,
e.g., Example 11).
Example 9: Dihydroxylation of the Hydrocarbon Staple
[0497] While the addition of the hydrocarbon staple has resulted in
significant improvements in antimicrobial activity there was also
an increase in hemolytic activity was observed, which can be due to
the hydrophobic nature of the hydrocarbon staple. Thus, one
approach to retain antimicrobial potency enhancement, while
mitigating hydrophobicity that can predispose to lysis, is to
modulate the hydrophobicity of the staple itself; thus, the
Sharpless dihydroxylation reaction was used to introduce two
alcohol groups into the alkene group present in the staple. The
presence of these two hydrophilic groups on the staple can reduce
its hydrophibicity resulting in lower hemolytic activity. To test
this hypothesis, a dihydroxylated form of PexStap5, referred to as
PexDiol5, was synthesized.
[0498] First, the degree of helical folding in PexDiol 5 was
measured and similar levels of helicity were found in the presence
and absence of TFE (50% v/v) when compared to PexStap5 (FIG. 7).
Second, the antimicrobial activity of PexDiol5 was tested against
our panel of bacterial pathogens. Antimicrobial activity was
maintained in the presence of the diol moiety on the staple (Table
3). Finally, the hemolytic activity of PexDiol5 was tested. Though
PexDiol5 displayed significant hemolytic activity across the range
of concentrations tested, it was notably lower that the activity of
PexStap5 (FIG. 8). This result validated the reasoning that the
staple's hydrophobicity played a key role in the decrease in
membrane selectivity.
Example 10: Aminohydroxylation of the Hydrocarbon Staple
[0499] While the diol moiety partially decreased the overall
hydrophobicity of the peptides, adding a point charge through the
addition of an amine moiety can further decrease the hydrophobicity
of the staple. Furthermore, an amine handle on the staple provides
an opportunity to attach other chemical groups to the staple that
could further enhance antimicrobial activity. To this end,
Sharpless aminohydroxylation reaction was used to modify the alkene
group in PexStap2, PexStap5, and PexStap6 and generate PexAminol2,
PexAminol5, and PexAminol6.
Example 11: Design Principles for Generating Stapled AMPs with
Microbial Vs. Mammalian Membrane Selectivity
[0500] A peptide library based on the sequence of magainin II was
generated and all possible i, i+4, or i, i+7 staple insertion
points were surveyed by sequentially moving the staple across the
peptide sequence from its N- to C-terminus (FIG. 11). The
antimicrobial activity of this library in four different bacterial
pathogens that included Gram-positive and Gram-negative species
were tested (FIG. 12, FIG. 13), and counter-screened for mammalian
membrane lysis using a red blood cell (RBC) hemolytic activity
assay (FIG. 14).
[0501] In general, the incorporation of a staple into the magainin
II sequence resulted in greater antimicrobial activity compared to
the unmodified sequence, with activity varying for differentially
stapled species. Although the i+7 stapled derivatives were more
active than the i+4 stapled compounds, the i+7 analogs were
likewise lytic in the RBC hemolytic activity assay, consistent with
the greater hydrophobic content of the i, i+7 staple. In contrast,
the i+4 panel demonstrated a striking pattern of differential RBC
lysis activity based on the periodicity of the staple insertion
site (FIG. 14). Specifically, analysis of the topographic landscape
of alpha-helical magainin II revealed that RBC lysis by i, i+4
stapled magainin peptides depended on whether the staple localized
within an established hydrophobic patch of the helical surface or
extended beyond this region, or even linking previously separated
hydrophobic patches to yield a new continuous hydrophobic surface
(FIG. 15A, B). For example, in the case of stapled derivative
Mag(i+4)1, antimicrobial activity was increased substantially in E.
coli and P. aeruginosa (both Gram-negative pathogens) displaying
MICs of 6.2 and 12.5 .mu.g/mL, respectively, compared to greater
than or equal to 50 .mu.g/mL for the unmodified peptide (Tables 5,
6, 7, FIG. 15C).
TABLE-US-00009 TABLE 5 MIC of magainin II and stapled derivatives
against Gram-negative and Gram-positive bacterial strains %
Hemolysis at Peptide Sequence E.coli B.cereus P.aeruginosa S.aureus
25 .mu.g/ml Magainin II GIGKFLHSAKKFGKAFVGEIBNS 5.0 >50 >50
>50 2.2 Mag(i + 4)1 GXGKFXHSAKKFGKAFVGEIBNS 3.1 25 12.5 >50
4.4 Mag(i + 4)6 GIGKFLXSAKXFGKAFVGEIBNS 6.2 12.5 25 25 7.8 Mag(i +
4)8 GIGKFLHSXKKFXKAFVGEIBNS 3.1 3.1 3.1 12.5 74.6 Mag(i +_4)16
GIGKFLHSAKKFGKAFXGEIXNS 3.1 12.5 6.2 50 7.8
[0502] The peptides in Table 5 are SEQ ID NOs: 134, 135, 140, 142,
and 150, numbered consecutively from top to bottom.
TABLE-US-00010 TABLE 6 MIC of magainin II and additional stapled
derivatives against Gram-negative and Gram-positive bacterial
strains Antimicrobial Activity MIC (.mu.g/ml) % Hemolysis at
Peptide E. coli B. cereus P. aeruginosa S. aureus 25 .mu.g/ml
Magainin II 50 >50 >50 >50 2.2 Mag(i + 4)0 3.1 12.5 50
>50 4.8 Mag(i + 4)1 3.1 25 12.5 >50 4.4 Mag(i + 4)2 3.1 12.5
12.5 12.5 19.6 Mag(i + 4)3 6.2 12.5 25 6.2 41.6 Mag(i + 4)4 3.1 6.2
12.5 25 17.8 Mag(i + 4)5 12.5 12.5 50 25 13.3 Mag(i + 4)6 6.2 12.5
25 25 7.8 Mag(i + 4)7 3.1 6.2 6.2 6.2 62.5 Mag(i + 4)8 3.1 3.1 3.1
12.5 74.6 Mag(i + 4)9 6.2 12.5 >50 >50 66.9 Mag(i + 4)10 3.1
6.2 12.5 6.2 23.6 Mag(i + 4)11 3.1 6.2 6.2 12.5 17.5 Mag(i + 4)12
3.1 3.1 3.1 6.2 76.8 Mag(i + 4)13 3.1 3.1 6.2 6.2 59.7 Mag(i + 4)14
1.6 3.1 3.1 3.1 92.1 Mag(i + 4)15 1.6 6.2 6.2 12.5 11.9 Mag(i +
4)16 3.1 12.5 6.2 50 7.8 Mag(i + 4)17 1.6 1.6 6.2 3.1 56.0 Mag(i +
4)18 1.6 3.1 3.1 1.6 96.5 Mag(i + 4)1, 15(A9K) 1.6 25 3.1 50 2.4
Mag(i + 4)2, 15(A9K) 1.6 3.1 3.1 3.1 15.4
[0503] The peptides in Table 6 are SEQ ID NOs: 134, 69, and
135-154, numbered consecutively from top to bottom. The sequence of
Mag(i+4)18 (SEQ ID NO: 152) is GIGKFLHSAKKFGKAFVGXIBNX; the
sequence of Mag(i+4)1,15(A9K) (SEQ ID NO: 153) is
GXGKFXHSKKKFGKAXVGEXBNS; the sequence of Mag(i+4)2,15(A9K) (SEQ ID
NO: 154) is GIXKFLXSKKKFGKAXVGEXBNS.
[0504] Yet, even when the dose was increased to 25 .mu.g/mL in the
RBC hemolytic activity assay, only 5% hemolysis was observed for
the stapled peptide, comparing favorably with the 2% hemolysis
observed for the unmodified peptide at the same dose (Table 5, FIG.
15C). Thus, the properties of Mag(i+4)1 reflect a suitable
therapeutic window for bacterial vs. mammalian membrane
selectivity. In contrast, when the staple was moved to a location
that imposes hydrophobicity at a prior site of hydrophilicity, such
as in Mag(i+4)8, indiscriminate lytic activity was observed (Tables
5, 6, FIG. 15D). However, when the staple again occupies a
previously hydrophobic region, microbial-selective lytic activity
is restored (FIG. 15E). The properties of Mag(i+4)6 provide yet
another example that highlights the importance of maintaining
discontinuity in hydrophobic surface patches to achieve membrane
selectivity: although the staple replaces two cationic amino acids
in the sequence, the hemolytic activity at 25 .mu.g/mL remains low
at 11% (FIG. 15F). Whereas Mag(i+4)6 increases the overall
hydrophobic surface area to a similar extent as seen in Mag(i+4)8
hemolysis is not increased in the case of Mag(i+4)6 because
hydrophobic patch discontinuity is maintained.
[0505] Based on these results, other stapled peptides (e.g., AIVIPs
not based on the sequence of magainin II) with at least two
internally cross-linked or stapled amino acids, wherein the at
least two amino acids are separated by two (i.e., i, i+3, shown in
FIG. 1 and FIG. 2), three (i.e., i, i+4, shown in FIG. 1 and FIG.
2), or six (i.e., i, i+7, shown in FIG. 1 and FIG. 2; also see
Table 7, below) amino acids were synthesized and assayed.
TABLE-US-00011 TABLE 7 MIC of magainin II and i + 7 stapled
derivatives against Gram-negative and Gram-positive bacterial
strains Antimicrobial Activity % MIC (.mu.g/ml) Hemolysis Peptide
E. coli B. cereus P. aeruginosa S. aureus at 25 .mu.g/ml Magainin
II 50 >50 >50 >50 2.2 Mag(i + 7)1 3.1 3.1 3.1 12.5 49.9
Mag(i + 7)2 3.1 6.2 12.5 12.5 69.3 Mag(i + 7)3 6.2 12.5 25 12.5
52.5 Mag(i + 7)4 1.6 6.2 3.1 25 42.6 Mag(i + 7)5 3.1 3.1 3.1 6.2
87.0 Mag(i + 7)6 3.1 6.2 25 6.2 47.1 Mag(i + 7)7 3.1 3.1 6.2 3.1
95.5 Mag(i + 7)8 1.6 3.1 3.1 6.2 85.4 Mag(i + 7)9 3.1 3.1 3.1 3.1
94.6 Mag(i + 7)10 3.1 6.2 6.2 3.1 53.1 Mag(i + 7)11 1.6 3.1 1.6 3.1
84.8 Mag(i + 7)12 1.6 3.1 3.1 12.5 80.3 Mag(i + 7)13 1.6 3.1 6.2
12.5 63.6 Mag(i + 7)14 1.6 1.6 6.2 1.6 68.1
[0506] The peptides in Table 7 are SEQ ID NOs: 134 and 155-168,
numbered consecutively from top to bottom.
[0507] Hydrogen-deuterium exchange mass spectrometry experiments
assessing the interaction of magainin II, Mag(i+4)14, and
Mag(i+4)15 with liposomes simulating mammalian (DOPC:cholesterol
(9:1)) or bacterial (DOPC:DOPG (8:2)) cell membranes show that
Mag(i+4)14 interacts indiscriminately with mammalian and bacterial
membranes (since co-incubation with liposomes simulating those
membranes protects the Mag(i+4)14 peptide from deuteration). See
FIG. 16. In contrast, Mag(i+4)15 selectively interacts with
bacterial membranes, but not mammalian membranes; only
co-incubation with liposomes simulating bacterial membranes shields
Mag(i+4)15 peptides from deuteration. The parent peptide (magainin
II) is deuterated in all conditions, as it interacts poorly with
both bacterial and mammalian membranes at the concentration
used.
Example 12: The Effect of Charge Distribution on Antimicrobial
Peptide Selectivity and Activity
[0508] The positive charges on Magainin align on the hydrophilic
side near the N-terminus, and a glutamic acid residue is conserved
near the C-terminus. To determine the effect of moving a positive
charge and a negative charge on the activity and selectivity of
stapled AMPs (STAMPs), a library of peptides was generated, where a
lysine or glutamic acid residue was installed at various positions
(FIGS. 17 and 18). Mag(i+4)15 was chosen as a model stapled
scaffold due to the large increase in antimicrobial activity and
modest increase in hemolytic activity compared to the unstapled
peptide.
[0509] From the lysine scan library, a general trend of decreased
hemolytic activity was observed no matter where the lysine residue
was installed (Table 8). This trend suggests that non-selective
toxicity, for example, lysis of mammalian membranes in addition to
bacterial membranes, is predominantly due to the hydrophobic patch
of the surface of the peptide.
[0510] Antimicrobial activity was highly dependent on the position
of the positive charge placement. Gram-positive activity was more
easily disrupted than was Gram-negative activity when a hydrophobic
residue was mutated to lysine. In some instances, such as in
Mag(i+4)15(A9K), Gram-positive activity was greatly attenuated
while Gram-negative activity was unaffected (Table 8).
[0511] In comparison to the parent STAMP template, the glutamic
acid scan library exhibited an overall lower hemolytic activity, as
was observed from the lysine scan library (Table 9). However, the
antimicrobial activity was attenuated in the majority of cases, and
highlights the role of positive charge in the antimicrobial
activity of AMPs. Whenever a lysine residue was mutated,
antimicrobial activity was reduced across the bacterial panel.
These data are consistent with the observation that any reduction
in the positive charge of magainin II notably impairs antimicrobial
activity. The sensitivity of Gram-positive pathogens to particular
glutamic acid mutations was more pronounced than forGram-negative
bacteria, as observed from the lysine scan library. For example,
when a negative charge was added near the C-terminus, such as
Mag(i+4)15(G18E) and Mag(i+4)15(N22E), only a slight reduction in
peptide activity was observed (Table 9). This result supports the
assertion that a dipole moment within magainin II helps maintain
selective lytic activity.
TABLE-US-00012 TABLE 8 MIC of magainin II-lysine derivatives
against Gram-negative and Gram-positive bacterial strains
Antimicrobial Activity MIC (.mu.g/ml) % E. B. P. S. Hemolysis
Peptide coli cereus aeruginosa aureus at 25 .mu.g/ml Mag(i + 4)15
1.6 6.2 6.2 12.5 11.9 Mag(i + 4)15(S23K) 1.6 6.2 3.1 50 5.0 Mag(i +
4)15(N22K) 1.6 3.1 3.1 12.5 12.0 Mag(i + 4)15(B21K) 1.6 25 6.2
>50 3.7 Mag(i + 4)15(E19K) 1.6 6.2 3.1 12.5 7.1 Mag(i +
4)15(G18K) 1.6 3.1 3.1 12.5 10.1 Mag(i + 4)15(V17K) 3.1 50 12.5
>50 2.2 Mag(i + 4)15(A15K) 3.1 6.2 3.1 25 4.1 Mag(i + 4)15(G13K)
3.1 >50 12.5 >50 1.8 Mag(i + 4)15(F12K) 3.1 >50 12.5
>50 2.0 Mag(i + 4)15(A9K) 3.1 >50 12.5 >50 1.9 Mag(i +
4)15(S8K) 1.6 3.1 6.2 6.2 3.4 Mag(i + 4)15(H7K) 1.6 3.1 3.1 6.2 3.1
Mag(i + 4)15(L6K) 3.1 >50 12.5 >50 1.9 Mag(i + 4)15(F5K) 3.1
>50 12.5 >50 1.7 Mag(i + 4)15(G3K) 1.6 6.2 3.1 12.5 3.8 Mag(i
+ 4)15(I2K) 1.6 12.5 3.1 23 2.3 Mag(i + 4)15(G1K) 1.6 12.5 3.1 25
4.5
[0512] The peptides in Table 8 are SEQ ID NOs: 149 and 32-48,
numbered consecutively from top to bottom.
TABLE-US-00013 TABLE 9 MIC of magainin II-glutamic acid derivatives
against Gram-negative and Gram-positive bacterial strains
Antimicrobial Activity MIC (.mu.g/ml) % E. B. P. S. Hemolysis
Peptide coli cereus aeruginosa aureus at 25 .mu.g/ml Mag(i + 4)15
1.6 6.2 6.2 12.5 11.9 Mag(i + 4)15(G1E) 12.5 >50 50 >50 4.1
Mag(i + 4)15(I2E) 12.5 >50 >50 >50 3.6 Mag(i + 4)15(G3E)
3.1 12.5 12.5 50 3.9 Mag(i + 4)15(K4E) 12.5 50 >50 >50 2.2
Mag(i + 4)15(F5E) 50 >50 >50 >50 0.5 Mag(i + 4)15(L6E) 50
>50 >50 >50 1.1 Mag(i + 4)15(H7E) 3.1 6.2 12.5 25 2.9
Mag(i + 4)15(S8E) 3.1 12.5 12.5 50 2.9 Mag(i + 4)15(A9E) 50 >50
>50 >50 0.6 Mag(i + 4)15(K10E) 6.2 >50 >50 >50 2.4
Mag(i + 4)15(K11E) 6.2 12.5 >50 50 2.9 Mag(i + 4)15(F12E) 50
>50 >50 >50 0.6 Mag(i + 4)15(G13E) 25 >50 >50 >50
2.2 Mag(i + 4)15(E14E) 12.5 50 >50 >50 3.3 Mag(i + 4)15(A15E)
3.1 25 25 50 3.8 Mag(i + 4)15(V17E) 50 >50 >50 >50 0.5
Mag(i + 4)15(G18E) 3.1 6.2 12.5 12.5 4.8 Mag(i + 4)15(B21E) 25
>50 >50 >50 0.8 Mag(i + 4)15(N22E) 3.1 12.5 12.5 25 3.4
Mag(i + 4)15(S23E) 3.1 12.5 12.5 25 4.9
The peptides in Table 9 are SEQ ID NOs: 149 and 49-68, numbered
consecutively from top to bottom.
[0513] In addition to the positive and negative charge scan study,
selective histidine mutations of the AMP compounds were generated.
The positive charge of the histidine side chain is "softer" than
the lysine side chain due to resonance within the imidazole. In
addition, the pK.sub.a of the imidazole side chain is close to 6,
such that at physiological conditions, the residue is not
protonated as extensively as is the side chain of lysine. Due to
the negatively charged environment close to a bacterial membrane,
the pH adjacent to the bacterial membrane is likely below 6, and
can cause histidine residues to be protonated. In addition,
antimicrobial peptides, such as histatins, rely on the charge
switching behavior of histidine to modulate peptide activity and
selectivity. The effect of installing histidines to regions close
to the hydrophilic face of magainin II that would extend the
positively charged region in the context of a bacterial membrane
and increase the likelihood of membrane penetration was tested
(Table 10). In this exemplary peptide library, residues G3, S8,
A15, and G18 were mutated. Overall, the mutations slightly reduced
hemolytic activity and resulted in modest gains in antimicrobial
activity. The most effective mutant of the group was
Mag(i+4)15(S8H), where an increase in activity across the panel of
bacteria tested was observed with a modest decrease in hemolytic
activity. Thus, the results demonstrate the utility of installing
histidine residues to selectively increase antimicrobial activity,
while preserving selectivity, of AMP drug candidates.
Example 13: The Effect of the Helical Bend on Antimicrobial
Activity of AMPs
[0514] The .alpha.-helical nature of AMPs promotes interaction with
membranes to induce bacterial lysis. However, some AMP sequences
possess a helix disruptor residue, such as glycine or proline,
close to the center of the peptides. To determine the role of
helical disruptors in AMP sequences, a library of peptides in which
the residue G13 was mutated into helix promoting residues like
alanine and 2-aminoisobutyric acid (Aib) or helix disruptors, such
as proline, hydroxyproline, D-alanine and D-lysine were synthesized
(Table 10). The helix promoters resulted in an increase in
antimicrobial activity in both Gram-positive and Gram-negative
pathogens. Yet, a concurrent increase in hemolytic activity was
observed, which is likely due to the hydrophobic nature of alanine
and Aib. In contrast, the proline and hydroxyproline mutations
resulted in an overall decrease in lytic activity of both bacterial
and mammalian membranes. For the d-alanine mutant stapled AMP, an
increase in antimicrobial activity was observed with a concurrent
decrease in hemolytic activity. In terms of hydrophobicity, the two
enantiomeric forms of alanine are identical. However, d-alanine can
disrupt a helix. These results suggest that AMP selectivity can be
achieved even in compounds that do not possess a high level of
helical rigidity.
TABLE-US-00014 TABLE 10 MIC of magainin II with helical disruptors
against Gram-negative and Gram-positive bacterial strains
Antimicrobial Activity % MIC (.mu.g/ml) Hemolysis E. B. P. S. at
Peptide coli cereus aeruginosa aureus 25 .mu.g/ml Mag(i + 4)15 1.6
6.2 6.2 12.5 11.9 Mag(i + 4)15(G3H) 1.6 6.2 12.5 25 5.1 Mag(i +
4)15(S8H) 1.6 3.1 3.1 3.1 8.7 Mag(i + 4)15(A15H) 3.1 6.2 12.5 12.5
4.7 Mag(i + 4)15(G18H) 1.6 3.1 6.2 6.2 6.8 Mag(i + 4)15(G13!) 1.6
3.1 3.1 3.1 71.5 Mag(i + 4)15(G13P) 3.1 25 25 >50 2.0 Mag(i +
4)15(G13&) 12.5 >50 50 >50 1.3 Mag(i + 4)15(G13A) 1.6 3.1
6.2 3.1 20.5 Mag(i + 4)15(G13a) 1.6 6.2 3.1 6.2 4.3 Mag(i +
4)15(G13k) 3.1 50 25 >50 2.1
The peptides in Table 10 are SEQ ID NOs: 149 and 73-82, numbered
consecutively from top to bottom.
Example 14: Integrating Insights from the Charge Scan Libraries and
Histidine Mutants
[0515] A series of compounds based on the results of the above
staple scanning and mutation scanning libraries were designed.
Using the double stapledSTAMP candidates, Mag(i+4)1,15 (A9K) and
Mag(i+4)2,15 (A9K), a panel of peptide derivatives was generated in
which mutant positions were chosen based on previous data (Table
11). For example, a glutamic acid mutation (N21E) was installed in
these double-stapled STAMPs with the goal of increasing selectivity
of Mag(i+4)2,15, one of the most potent double stapled AMPs. Other
mutations were incorporated to make up for potential losses in
antimicrobial activity and/or to increase membrane selectivity. In
this panel, Mag(i+4)2,15 (I2K, A9H) exhibited only mildly
attenuated antimicrobial activity, but the previously observed 16%
hemolytic activity was completely eliminated. Enhancing positive
charge by installing additional histidine mutations was shown to
maintain or improve antimicrobial activity while ensuring that
selectivity was preserved or improved.
TABLE-US-00015 MIC of magainin II derivatives against Gram-negative
and Gram-positive bacterial strains Antimicrobial Activity MIC
(.mu.g/ml) % Hemolysis at Peptide E. coli B. cereus P. aeruginosa
S. aureus 25 .mu.g/ml Mag(i + 4)15 1.6 6.2 6.2 12.5 11.9 Mag(i +
4)2, 15(I2K, A9K, G18H) 3.1 12.5 3.1 25 2.2 Mag(i + 4)2, 15(I2X,
A9H) 3.1 6.2 6.2 12.5 0.6 Mag(i + 4)2, 15(I2K, A9H, N21E) 3.1 25
12.5 50 1.8 Mag(i + 4)2, 15(I2X, A9H, G18H, N21E) 3.1 6.2 6.2 25
4.0 Mag(i + 4)1, 15(S8H, A9K, G18H, N21E) 3.1 50 12.5 >50
4.2
The peptides in Table 11 are SEQ ID NOs: 149 and 170-174, numbered
consecutively from top to bottom.
Example 15: Validation of STAMP Hydrophobicity Patch
[0516] To study the significance of the hydrophobicity patch,
antimicrobial peptide pleurocidin, which is found on the skin of
winter flounder, was selected. Pleurocidin exhibits antimicrobial
activity against both Gram-positive and Gram-negative pathogens
with low hemolytic activity. However, the unstructured nature of
this peptide is a has prevented in vivo utility. Using the NMR
structure of pleurocidin in the presence of negatively charge
micelles, the hydrophobic surface areas within the .alpha.-helical
folded structure was identified (FIG. 22A). A double staple was
then inserted within the confines of those hydrophobic regions
(FIG. 22B). To create a discontinuous hydrophobic face, thereby
reducing the risk of mammalian cell lysis while preserving
antimicrobial activity (based on the experiments described above),
the A9 residue was mutated to a lysine (FIG. 22C). When tested
against Gram-positive and Gram-negative pathogens, the STAMP
constructs exhibited improved antimicrobial activities that were
one dilution range better than the parent unstapled peptide. This
improvement in antimicrobial activity of the double stapled AMP,
Pleu(i+4)1,15 (A9K), was achieved while maintaining low hemolytic
activity similar to that of Mag(i+4)2,15 (A9K) (Table 12).
TABLE-US-00016 TABLE 12 MIC of pleurocidin derivatives against
Gram-negative and Gram-positive bacterial strains Antimicrobial
Activity MIC (.mu.g/ml) % Hemolysis at Peptide E. coli B. cereus P.
aeruginosa S. aureus 25 .mu.g/ml Pleurocidin-NH2 3.1 6.2 3.1 6.2
0.5 Pleu(i + 4)1, 15 1.6 3.1 1.6 1.6 88.2 Pleu(i + 4)1, 15(A9K) 1.6
3.1 1.6 3.1 16.3
The peptides in Table 12 are SEQ ID Nos: 175-177, numbered
consecutively from top to bottom.
Methods Used in Examples
[0517] Solid Phase Peptide Synthesis:
[0518] Fmoc-based solid-phase peptide synthesis was used to
synthesize the antimicrobial peptides and their stapled
derivatives. To achieve the various staple lengths, .alpha.-methyl,
.alpha.-alkenyl amino acids were used flanking two, three, or six
residues. The R.sub.5 residue was incorporated at position i and
S.sub.5 at position i+3, while two S.sub.5 residues were used at
the i and i+4 locations, and an R.sub.8 at position i and S.sub.5
at i+7 [29]. Alternative stapling amino acid couples can also be
used to generate the corresponding staples (e.g., R.sub.3/S.sub.5
or S.sub.3/R.sub.5 for i, 1+3; R.sub.5/R.sub.5 for i, 1+4;
S.sub.8/R.sub.5 for i, i+7). For the stapling reaction, Grubbs
first-generation ruthenium catalyst dissolved in dichloroethane was
added to the peptides while still on resin. To ensure maximal
conversion, three to five rounds of stapling were performed. Once
stapled, the peptides were cleaved off the resin using
trifluoroacetic acid, then precipitated using a hexane:ether (1:1)
mixture, and afterwards they were air dried and purified using
LC-MS. We performed amino acid analysis both to precisely determine
the amount of peptide purified and to ensure the correct sequence
was made
Circular Dichroism Spectroscopy:
[0519] Compounds were dissolved in an aqueous solution (e.g. 5 mM
potassium phosphate solution at pH 7, or distilled H.sub.2O, to
concentrations of 25-50 .mu.M). Circular dichroism (CD) spectra
were obtained on a spectropolarimeter (Aviv) using standard
measurement parameters (e.g., temperature, 37.degree. C.;
wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec;
accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length,
0.1 cm). The .alpha.-helical content of each peptide was calculated
by dividing the mean residue ellipticity by the reported value for
a model helical decapeptide (see, e.g., Yang et al., Meth Enzymol.
130:208 (1986)).
[0520] Antimicrobial Activity Assay:
[0521] The following microbroth dilution protocol was adapted to
determine the minimum inhibitory concentration (MIC) of each
peptide. First, Mueller-Hinton broth (MHB) was passed through an
anion exchange column to remove polyanionic species and generate
refined MHB. This refined broth was then used in the standard
microbroth dilution protocol devised by Hancock and coworkers for
96 well plates (note: No BSA was used in the protocol because
initial studies revealed that it could interfere with peptide
activity). Briefly, bacterial cells was grown overnight in refined
MHB at 37.degree. C. and then diluted and allowed to grow again for
several hours. Serial dilutions of peptide stocks in water (10
.mu.l) were prepared using clear round-bottom polypropylene 96-well
plates. Then 90 .mu.l of bacteria in refined MHB was added to give
a final inoculum of 5.times.10.sup.5 CFU/ml. The plates were then
covered with porous tape to reduce evaporation, and incubated for
20-24 hours at 37.degree. C. The MIC is the minimum peptide
concentration at which no visible growth was observed.
[0522] Hemolytic Activity Assay:
[0523] For the determination of hemolytic activity, human blood
samples were centrifuged to isolate red blood cells (RBCs), which
are then washed and suspended in phosphate-buffered saline to yield
a 1% (v/v) suspension. The suspension was then added to serial
dilutions of peptide stocks in water in clear round-bottom
polypropylene 96-well plates and the plates were incubated for 1
hour at 37.degree. C. After incubation, the plates were centrifuged
and the supernatant was isolated to determine the amount of
hemoglobin released using a spectrophotometer (570 nm), according
to the equation: % Hemolysis=(Treated Absorbance-Untreated Control
Absorbance)/(1% Triton-X100 Treated Absorbance-Untreated Control
Absorbance). The minimum hemolytic concentration (MHC) is the
peptide concentration at which there is less than 1% hemoglobin
release.
OTHER ASPECTS
[0524] 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.
[0525] All patent applications, patents, and other publications
cited herein are incorporated by reference in their entireties.
Sequence CWU 1
1
262123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Ser or AlaMOD_RES(13)..(13)Ala or
GlyMOD_RES(18)..(18)Ala or Gly 1Gly Ile Gly Lys Phe Leu His Xaa Ala
Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Xaa Glu Ile Met Asn Ser
20222PRTUnknownDescription of Unknown Pexiganan peptide 2Gly Ile
Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val
Lys Ile Leu Lys Lys 20323PRTUnknownDescription of Unknown Magainin
peptide 3Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys
Ala Phe1 5 10 15Val Gly Glu Ile Met Asn Ser
20425PRTPseudopleuronectes americanus 4Gly Trp Gly Ser Phe Phe Lys
Lys Ala Ala His Val Gly Lys His Val1 5 10 15Gly Lys Ala Ala Leu Thr
His Tyr Leu 20 25533PRTPardachirus marmoratus 5Gly Phe Phe Ala Leu
Ile Pro Lys Ile Ile Ser Ser Pro Leu Phe Lys1 5 10 15Thr Leu Leu Ser
Ala Val Gly Ser Ala Leu Ser Ser Ser Gly Glu Gln 20 25
30Glu632PRTMyxine glutinosa 6Gly Phe Phe Lys Lys Ala Trp Arg Lys
Val Lys His Ala Gly Arg Arg1 5 10 15Val Leu Lys Lys Gly Val Gly Arg
His Tyr Val Asn Asn Trp Leu Lys 20 25 30724PRTXenopus laevis 7Gly
Val Leu Ser Asn Val Ile Gly Tyr Leu Lys Lys Leu Gly Thr Gly1 5 10
15Ala Leu Asn Ala Val Leu Lys Gln 20821PRTBufo gargarizans 8Thr Arg
Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His1 5 10 15Arg
Leu Leu Arg Lys 20934PRTPhyllomedusa sp. 9Ala Leu Trp Lys Thr Met
Leu Lys Lys Leu Gly Thr Met Ala Leu His1 5 10 15Ala Gly Lys Ala Ala
Leu Gly Ala Ala Ala Asp Thr Ile Ser Gln Gly 20 25 30Thr
Gln1025PRTLitoria chloris 10Gly Leu Phe Lys Val Leu Gly Ser Val Ala
Lys His Leu Leu Pro His1 5 10 15Val Val Pro Val Ile Ala Glu Lys Leu
20 251126PRTUnknownDescription of Unknown Melittin peptide 11Gly
Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu1 5 10
15Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20
251237PRTUnknownDescription of Unknown Cecropin A polypeptide 12Lys
Trp Lys Leu Phe Lys Lys Ile Glu Lys Val Gly Gln Asn Ile Arg1 5 10
15Asp Gly Ile Ile Lys Ala Gly Pro Ala Val Ala Val Val Gly Gln Ala
20 25 30Thr Gln Ile Ala Lys 351327PRTUnknownDescription of Unknown
Lycotoxin I peptide 13Lys Ile Lys Trp Phe Lys Thr Met Lys Ser Ile
Ala Lys Phe Ile Ala1 5 10 15Lys Glu Gln Met Lys Lys His Leu Gly Gly
Glu 20 251419PRTUnknownDescription of Unknown Styelins B peptide
14Gly Phe Gly Pro Ala Phe His Ser Val Ser Asn Phe Ala Lys Lys His1
5 10 15Lys Thr Ala1523PRTUnknownDescription of Unknown Clavanin B
peptide 15Val Phe Gln Phe Leu Gly Arg Ile Ile His His Val Gly Asn
Phe Val1 5 10 15His Gly Phe Ser His Val Phe
201613PRTUnknownDescription of Unknown Cathelicidin A peptide 16Ile
Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys1 5
101748PRTUnknownDescription of Unknown Dermcidin polypeptide 17Ser
Ser Leu Leu Glu Lys Gly Leu Asp Gly Ala Lys Lys Ala Val Gly1 5 10
15Gly Leu Gly Lys Leu Gly Lys Asp Ala Val Glu Asp Leu Glu Ser Val
20 25 30Gly Lys Gly Ala Val His Asp Val Lys Asp Val Leu Asp Ser Val
Leu 35 40 451825PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(12)..(12)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(19)..(19)Any amino acid whose side chain has been
replaced by an internal staple 18Gly Trp Gly Ser Phe Phe Lys Lys
Ala Ala His Xaa Gly Lys His Val1 5 10 15Gly Lys Xaa Ala Leu Thr His
Tyr Leu 20 251933PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(12)..(12)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(19)..(19)Any amino acid whose side chain has been
replaced by an internal staple 19Gly Phe Phe Ala Leu Ile Pro Lys
Ile Ile Ser Xaa Pro Leu Phe Lys1 5 10 15Thr Leu Xaa Ser Ala Val Gly
Ser Ala Leu Ser Ser Ser Gly Glu Gln 20 25 30Glu2032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(7)..(7)Brominated TrpMOD_RES(14)..(14)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(30)..(30)Brominated Trp 20Gly
Phe Phe Lys Lys Ala Trp Arg Lys Val Lys His Ala Xaa Arg Arg1 5 10
15Val Leu Lys Lys Xaa Val Gly Arg His Tyr Val Asn Asn Trp Leu Lys
20 25 302124PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(14)..(14)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Any amino acid whose side chain has been
replaced by an internal staple 21Gly Val Leu Ser Asn Val Ile Gly
Tyr Leu Lys Lys Leu Xaa Thr Gly1 5 10 15Ala Leu Asn Ala Xaa Leu Lys
Gln 202221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal staple 22Thr
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Xaa Gly Arg Val His1 5 10
15Arg Leu Xaa Arg Lys 202334PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(14)..(14)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Any amino acid whose side chain has been
replaced by an internal staple 23Ala Leu Trp Lys Thr Met Leu Lys
Lys Leu Gly Thr Met Xaa Leu His1 5 10 15Ala Gly Lys Ala Xaa Leu Gly
Ala Ala Ala Asp Thr Ile Ser Gln Gly 20 25 30Thr
Gln2425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(14)..(14)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(21)..(21)Any amino
acid whose side chain has been replaced by an internal staple 24Gly
Leu Phe Lys Val Leu Gly Ser Val Ala Lys His Leu Xaa Pro His1 5 10
15Val Val Pro Val Xaa Ala Glu Lys Leu 20 252526PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(13)..(13)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal staple 25Gly Ile Gly
Ala Val Leu Lys Val Leu Thr Thr Gly Xaa Pro Ala Leu1 5 10 15Ile Ser
Trp Xaa Lys Arg Lys Arg Gln Gln 20 252637PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(4)..(4)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(11)..(11)Any amino acid whose
side chain has been replaced by an internal staple 26Lys Trp Lys
Xaa Phe Lys Lys Ile Glu Lys Xaa Gly Gln Asn Ile Arg1 5 10 15Asp Gly
Ile Ile Lys Ala Gly Pro Ala Val Ala Val Val Gly Gln Ala 20 25 30Thr
Gln Ile Ala Lys 352727PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(8)..(8)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(15)..(15)Any amino acid whose side chain has been
replaced by an internal staple 27Lys Ile Lys Trp Phe Lys Thr Xaa
Lys Ser Ile Ala Lys Phe Xaa Ala1 5 10 15Lys Glu Gln Met Lys Lys His
Leu Gly Gly Glu 20 252819PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(5)..(5)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(12)..(12)Any amino acid whose side chain has been
replaced by an internal staple 28Gly Phe Gly Pro Xaa Phe His Ser
Val Ser Asn Xaa Ala Lys Lys His1 5 10 15Lys Thr
Ala2923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(5)..(5)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(12)..(12)Any amino acid
whose side chain has been replaced by an internal staple 29Val Phe
Gln Phe Xaa Gly Arg Ile Ile His His Xaa Gly Asn Phe Val1 5 10 15His
Gly Phe Ser His Val Phe 203013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(2)..(2)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(9)..(9)Any amino acid whose side chain has been
replaced by an internal staple 30Ile Xaa Lys Lys Trp Pro Trp Trp
Xaa Trp Arg Arg Lys1 5 103148PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(11)..(11)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal staple 31Ser Ser Leu Leu Glu Lys Gly Leu
Asp Gly Xaa Lys Lys Ala Val Gly1 5 10 15Gly Xaa Gly Lys Leu Gly Lys
Asp Ala Val Glu Asp Leu Glu Ser Val 20 25 30Gly Lys Gly Ala Val His
Asp Val Lys Asp Val Leu Asp Ser Val Leu 35 40 453223PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 32Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Lys
203323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 33Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Lys Ser
203423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal staple 34Gly
Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10
15Val Gly Glu Xaa Lys Asn Ser 203523PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 35Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Lys Xaa Xaa Asn Ser
203623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 36Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Lys Glu Xaa Xaa Asn Ser
203723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 37Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Lys Gly Glu Xaa Xaa Asn Ser
203823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 38Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Lys Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
203923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 39Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Lys Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 40Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Lys Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 41Gly Ile Gly Lys Phe Leu His Ser
Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 42Gly Ile Gly Lys Phe Leu His Lys
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 43Gly Ile Gly Lys Phe Leu Lys Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 44Gly Ile Gly Lys Phe Lys His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 45Gly Ile Gly Lys Lys Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204623PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 46Gly Ile Lys Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 47Gly Lys Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 48Lys Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
204923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 49Glu Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 50Gly Glu Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 51Gly Ile Glu Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 52Gly Ile Gly Glu Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 53Gly Ile Gly Lys Glu Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 54Gly Ile Gly Lys Phe Glu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 55Gly Ile Gly Lys Phe Leu Glu Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 56Gly Ile Gly Lys Phe Leu His Glu
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 57Gly Ile Gly Lys Phe Leu His Ser
Glu Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 58Gly Ile Gly Lys Phe Leu His Ser
Ala Glu Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
205923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 59Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Glu Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
206023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 60Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Glu Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
206123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 61Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Glu Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
206223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 62Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Glu Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
206323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 63Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Glu Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Ser
206423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 64Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Glu Gly Glu Xaa Xaa Asn Ser
206523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 65Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Glu Glu Xaa Xaa Asn Ser
206623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal staple 66Gly
Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10
15Val Gly Glu Xaa Glu Asn Ser 206723PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 67Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Glu Ser
206823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 68Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn Glu
206923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(5)..(5)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 69Xaa Ile Gly Lys Xaa Leu His Ser
Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn Ser
207023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(19)..(19)Any amino acid whose side chain
has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 70Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val
Gly Xaa Ile Xaa Asn Xaa 207123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(2)..(2)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(6)..(6)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(16)..(16)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 71Gly Xaa
Gly Lys Phe Xaa His Ser Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(3)..(3)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(7)..(7)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(16)..(16)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 72Gly Ile
Xaa Lys Phe Leu Xaa Ser Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207323PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 73Gly Ile
His Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207423PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 74Gly Ile
Gly Lys Phe Leu His His Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207523PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 75Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys His Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207623PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 76Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val
His Glu Xaa Xaa Asn Ser 207723PRTArtificial SequenceDescription of
Artificial Sequence Synthetic
peptideMOD_RES(13)..(13)2-aminoisobutyric acidMOD_RES(16)..(16)Any
amino acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 77Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Xaa Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207823PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 78Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Pro Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 207923PRTArtificial SequenceDescription of
Artificial Sequence Synthetic
peptideMOD_RES(13)..(13)HydroxyprolineMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 79Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Xaa Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 208023PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 80Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 208123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic
peptideMOD_RES(13)..(13)D-AlanineMOD_RES(16)..(16)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 81Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Xaa Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 208223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic
peptideMOD_RES(13)..(13)D-LysineMOD_RES(16)..(16)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 82Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Xaa Lys Ala Xaa1 5 10 15Val
Gly Glu Xaa Xaa Asn Ser 208323PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(11)..(11)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 83Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val
Xaa Glu Ile Xaa Asn Lys 208423PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(11)..(11)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 84Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val
Xaa Glu Ile Xaa Lys Ser 208523PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(11)..(11)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal staple 85Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Lys Asn Ser
208623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 86Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Lys Xaa Asn Ser
208723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 87Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Lys Ile Xaa Asn Ser
208823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 88Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Lys Xaa Glu Ile Xaa Asn Ser
208923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 89Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Lys1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 90Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Lys Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 91Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Lys Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 92Gly Ile Gly Lys Phe Leu His Ser
Ala Lys Xaa Lys Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 93Gly Ile Gly Lys Phe Leu His Ser
Lys Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 94Gly Ile Gly Lys Phe Leu His Lys
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 95Gly Ile Gly Lys Phe Leu Lys Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 96Gly Ile Gly Lys Phe Lys His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 97Gly Ile Gly Lys Lys Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 98Gly Ile Lys Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
209923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 99Gly Lys Gly Lys Phe Leu His Ser
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn Ser
2010023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 100Lys Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 101Glu Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 102Gly Glu Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 103Gly Ile Glu Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 104Gly Ile Gly Glu Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 105Gly Ile Gly Lys Glu Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 106Gly Ile Gly Lys Phe Glu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 107Gly Ile Gly Lys Phe Leu Glu
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 108Gly Ile Gly Lys Phe Leu His
Glu Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2010923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 109Gly Ile Gly Lys Phe Leu His
Ser Glu Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 110Gly Ile Gly Lys Phe Leu His
Ser Ala Glu Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 111Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Glu Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 112Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Glu Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 113Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Glu Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 114Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Glu Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 115Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Glu1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2011623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 116Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Glu Xaa Glu Ile Xaa Asn
Ser 2011723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side
chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 117Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Glu Xaa Asn
Ser 2011823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal staple
118Gly Ile Gly Lys Phe Leu His Ser Ala Lys Xaa Phe Gly Lys Ala Phe1
5 10 15Val Xaa Glu Ile Glu Asn Ser 2011923PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(11)..(11)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(18)..(18)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 119Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Glu
Ser 2012023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 120Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Glu 2012123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 121Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val
Gly Glu Ile Xaa Asn Xaa 2012223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(8)..(8)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 122Xaa
Ile Gly Lys Phe Leu His Xaa Ala Lys Lys Phe Gly Lys Ala Phe1 5 10
15Val Gly Glu Ile Xaa Asn Ser 2012323PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(6)..(6)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(11)..(11)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(18)..(18)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 123Gly Xaa Gly Lys Phe Xaa His
Ser Lys Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2012423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(11)..(11)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(18)..(18)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 124Gly Ile Xaa Lys Phe Leu Xaa
Ser Lys Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2012523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 125Gly Ile His Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2012623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 126Gly Ile Gly Lys Phe Leu His
His Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2012723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 127Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys His Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2012823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal
stapleMOD_RES(13)..(13)2-aminoisobutyric acidMOD_RES(18)..(18)Any
amino acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 128Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Xaa Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2012923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 129Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Pro Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2013023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal
stapleMOD_RES(13)..(13)HydroxyprolineMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 130Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Xaa Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2013123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 131Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2013223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal
stapleMOD_RES(13)..(13)D-AlanineMOD_RES(18)..(18)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 132Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Xaa Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2013323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal
stapleMOD_RES(13)..(13)D-LysineMOD_RES(18)..(18)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 133Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Xaa Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2013423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(21)..(21)Norleucine 134Gly Ile Gly Lys Phe
Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile
Xaa Asn Ser 2013523PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side
chain has been replaced by an internal stapleMOD_RES(6)..(6)Any
amino acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 135Gly Xaa Gly Lys Phe Xaa His
Ser Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2013623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 136Gly Ile Xaa Lys Phe Leu Xaa
Ser Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2013723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(8)..(8)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 137Gly Ile Gly Xaa Phe Leu His
Xaa Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2013823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(5)..(5)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 138Gly Ile Gly Lys Xaa Leu His
Ser Xaa Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2013923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(6)..(6)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(10)..(10)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 139Gly Ile Gly Lys Phe Xaa His
Ser Ala Xaa Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(7)..(7)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(11)..(11)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 140Gly Ile Gly Lys Phe Leu Xaa
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(12)..(12)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 141Gly Ile Gly Lys Phe Leu His
Xaa Ala Lys Lys Xaa Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(13)..(13)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 142Gly Ile Gly Lys Phe Leu His
Ser Xaa Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(10)..(10)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(14)..(14)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 143Gly Ile Gly Lys Phe Leu His
Ser Ala Xaa Lys Phe Gly Xaa Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(15)..(15)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 144Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Xaa Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 145Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Xaa Gly Lys Ala Xaa1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2014623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(13)..(13)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(17)..(17)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 146Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Xaa Gly Glu Ile Xaa Asn
Ser 2014723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(14)..(14)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 147Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Phe Gly Xaa Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2014823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(15)..(15)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 148Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Phe Gly Lys Xaa Phe1 5 10 15Val Gly Xaa Ile Xaa Asn
Ser 2014923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 149Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn
Ser 2015023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(17)..(17)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(21)..(21)Any amino
acid whose side chain has been replaced by an internal staple
150Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe1
5 10 15Xaa Gly Glu Ile Xaa Asn Ser 2015123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(22)..(22)Any amino acid
whose side chain has been replaced by an internal staple 151Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val
Xaa Glu Ile Xaa Xaa Ser 2015223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 152Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val
Gly Xaa Ile Xaa Asn Xaa
2015323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 153Gly Xaa Gly Lys Phe Xaa His
Ser Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn
Ser 2015423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 154Gly Ile Xaa Lys Phe Leu Xaa
Ser Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn
Ser 2015523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 155Gly Xaa Gly Lys Phe Leu His
Ser Xaa Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2015623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(10)..(10)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 156Gly Ile Xaa Lys Phe Leu His
Ser Ala Xaa Lys Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2015723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(11)..(11)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 157Gly Ile Gly Xaa Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2015823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(5)..(5)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(12)..(12)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 158Gly Ile Gly Lys Xaa Leu His
Ser Ala Lys Lys Xaa Gly Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2015923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(6)..(6)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(13)..(13)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 159Gly Ile Gly Lys Phe Xaa His
Ser Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2016023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(7)..(7)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(14)..(14)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 160Gly Ile Gly Lys Phe Leu Xaa
Ser Ala Lys Lys Phe Gly Xaa Ala Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2016123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(15)..(15)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 161Gly Ile Gly Lys Phe Leu His
Xaa Ala Lys Lys Phe Gly Lys Xaa Phe1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2016223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(16)..(16)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 162Gly Ile Gly Lys Phe Leu His
Ser Xaa Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Ile Xaa Asn
Ser 2016323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(10)..(10)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(17)..(17)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 163Gly Ile Gly Lys Phe Leu His
Ser Ala Xaa Lys Phe Gly Lys Ala Phe1 5 10 15Xaa Gly Glu Ile Xaa Asn
Ser 2016423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 164Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2016523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 165Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Xaa Gly Lys Ala Phe1 5 10 15Val Gly Xaa Ile Xaa Asn
Ser 2016623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(13)..(13)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 166Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Gly Glu Xaa Xaa Asn
Ser 2016723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(14)..(14)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(21)..(21)Any amino
acid whose side chain has been replaced by an internal staple
167Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Ala Xaa Ala Phe1
5 10 15Val Gly Glu Ile Xaa Asn Ser 2016823PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(15)..(15)Any amino acid whose side chain has been
replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(22)..(22)Any amino acid
whose side chain has been replaced by an internal staple 168Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Ala Lys Xaa Phe1 5 10 15Val
Gly Glu Ile Xaa Xaa Ser 2016923PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 169Gly Ile Gly Lys Phe Leu
His Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Met
Asn Ser 2017023PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side
chain has been replaced by an internal stapleMOD_RES(7)..(7)Any
amino acid whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 170Gly Lys Xaa Lys Phe Leu Xaa
Ser Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val His Glu Xaa Xaa Asn
Ser 2017123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 171Gly Lys Xaa Lys Phe Leu Xaa
Ser His Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Asn
Ser 2017223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 172Gly Lys Xaa Lys Phe Leu Xaa
Ser His Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val Gly Glu Xaa Xaa Glu
Ser 2017323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 173Gly Lys Xaa Lys Phe Leu Xaa
Ser His Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val His Glu Xaa Xaa Glu
Ser 2017423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 174Gly Xaa Gly Lys Phe Xaa His
Ser Lys Lys Lys Phe Gly Lys Ala Xaa1 5 10 15Val His Glu Xaa Xaa Glu
Ser 2017525PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 175Gly Trp Gly Ser Phe Phe Lys Lys Ala Ala His
Val Gly Lys His Val1 5 10 15Gly Lys Ala Ala Leu Thr His Tyr Leu 20
2517625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal staple 176Gly Xaa Gly
Ser Phe Xaa Lys Lys Ala Ala His Val Gly Lys His Xaa1 5 10 15Gly Lys
Ala Xaa Leu Thr His Tyr Leu 20 2517725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(6)..(6)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal staple 177Gly Xaa Gly
Ser Phe Xaa Lys Lys Lys Ala His Val Gly Lys His Xaa1 5 10 15Gly Lys
Ala Xaa Leu Thr His Tyr Leu 20 2517823PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(7)..(7)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(14)..(14)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 178Gly Ile Gly Lys Phe Leu Xaa
Ala Ala Lys Lys Phe Ala Xaa Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2017923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(15)..(15)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 179Gly Ile Gly Lys Phe Leu His
Xaa Ala Lys Lys Phe Ala Lys Xaa Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2018023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(16)..(16)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 180Gly Ile Gly Lys Phe Leu His
Ala Xaa Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2018123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(10)..(10)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(17)..(17)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 181Gly Ile Gly Lys Phe Leu His
Ala Ala Xaa Lys Phe Ala Lys Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn
Ser 2018223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 182Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2018323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 183Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Xaa Ala Lys Ala Phe1 5 10 15Val Ala Xaa Ile Xaa Asn
Ser 2018423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(13)..(13)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 184Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Ala Glu Xaa Xaa Asn
Ser 2018522PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(15)..(15)Any amino acid
whose side chain has been replaced by an internal staple 185Gly Ile
Gly Lys Phe Leu Lys Xaa Ala Lys Lys Phe Gly Lys Xaa Phe1 5 10 15Val
Lys Ile Leu Lys Lys 2018622PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(11)..(11)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal staple 186Gly Ile Gly Lys Phe Leu Lys Lys
Ala Lys Xaa Phe Gly Lys Ala Phe1 5 10 15Val Xaa Ile Leu Lys Lys
2018722PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal staple
187Gly Ile Gly Lys Phe
Leu Lys Lys Ala Lys Lys Xaa Gly Lys Ala Phe1 5 10 15Val Lys Xaa Leu
Lys Lys 2018823PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(7)..(7)Any amino acid whose side
chain has been replaced by an internal stapleMOD_RES(14)..(14)Any
amino acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 188Gly Ile Gly Lys Phe Leu Xaa
Ala Ala Lys Lys Phe Ala Xaa Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2018923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(15)..(15)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 189Gly Ile Gly Lys Phe Leu His
Xaa Ala Lys Lys Phe Ala Lys Xaa Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2019023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(16)..(16)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 190Gly Ile Gly Lys Phe Leu His
Ala Xaa Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2019123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(10)..(10)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(17)..(17)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 191Gly Ile Gly Lys Phe Leu His
Ala Ala Xaa Lys Phe Ala Lys Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn
Ser 2019223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 192Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2019323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 193Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Xaa Ala Lys Ala Phe1 5 10 15Val Ala Xaa Ile Xaa Asn
Ser 2019423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(13)..(13)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 194Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Ala Glu Xaa Xaa Asn
Ser 2019523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(14)..(14)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(21)..(21)Any amino
acid whose side chain has been replaced by an internal staple
195Gly Ile Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Xaa Ala Phe1
5 10 15Val Ala Glu Ile Xaa Asn Ser 2019623PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(15)..(15)Any amino acid whose side chain has been
replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(22)..(22)Any amino acid
whose side chain has been replaced by an internal staple 196Gly Ile
Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Xaa Phe1 5 10 15Val
Ala Glu Ile Xaa Xaa Ser 2019723PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 197Gly Ile
Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val
Ala Glu Ile Xaa Asn Xaa 2019823PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(13)..(13)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 198Gly
Ile Gly Lys Phe Xaa His Ala Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10
15Val Ala Glu Ile Xaa Asn Ser 2019923PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(5)..(5)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(12)..(12)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 199Gly Ile Gly Lys Xaa Leu His
Ala Ala Lys Lys Xaa Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(11)..(11)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 200Gly Ile Gly Xaa Phe Leu His
Ala Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(10)..(10)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 201Gly Ile Xaa Lys Phe Leu His
Ala Ala Xaa Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 202Gly Xaa Gly Lys Phe Leu His
Ala Xaa Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(8)..(8)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 203Xaa Ile Gly Lys Phe Leu His
Xaa Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(5)..(5)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 204Xaa Ile Gly Lys Xaa Leu His
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 205Gly Xaa Gly Lys Phe Xaa His
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 206Gly Ile Xaa Lys Phe Leu Xaa
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(8)..(8)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 207Gly Ile Gly Xaa Phe Leu His
Xaa Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(5)..(5)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 208Gly Ile Gly Lys Xaa Leu His
Ala Xaa Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2020923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(6)..(6)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(10)..(10)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 209Gly Ile Gly Lys Phe Xaa His
Ala Ala Xaa Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(7)..(7)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(11)..(11)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 210Gly Ile Gly Lys Phe Leu Xaa
Ala Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(12)..(12)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 211Gly Ile Gly Lys Phe Leu His
Xaa Ala Lys Lys Xaa Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(13)..(13)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 212Gly Ile Gly Lys Phe Leu His
Ala Xaa Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(10)..(10)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(14)..(14)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 213Gly Ile Gly Lys Phe Leu His
Ala Ala Xaa Lys Phe Ala Xaa Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(15)..(15)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 214Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Xaa Phe Ala Lys Xaa Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 215Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Xaa Ala Lys Ala Xaa1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2021623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(13)..(13)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(17)..(17)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 216Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn
Ser 2021723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(14)..(14)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 217Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Xaa Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2021823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(15)..(15)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 218Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Lys Xaa Phe1 5 10 15Val Ala Xaa Ile Xaa Asn
Ser 2021923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 219Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val Ala Glu Xaa Xaa Asn
Ser 2022023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(17)..(17)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(21)..(21)Any amino
acid whose side chain has been replaced by an internal staple
220Gly Ile Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1
5 10 15Xaa Ala Glu Ile Xaa Asn Ser 2022123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(18)..(18)Any amino acid whose side chain has been
replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(22)..(22)Any amino acid
whose side chain has been replaced by an internal staple 221Gly Ile
Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val
Xaa Glu Ile Xaa Xaa Ser 2022223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 222Gly Ile
Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val
Ala Xaa Ile Xaa Asn Xaa 2022323PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(4)..(4)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 223Xaa
Ile Gly Xaa Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5
10
15Val Ala Glu Ile Xaa Asn Ser 2022423PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(5)..(5)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 224Gly Xaa Gly Lys Xaa Leu His
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2022523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 225Gly Ile Xaa Lys Phe Xaa His
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2022623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 226Gly Ile Gly Xaa Phe Leu Xaa
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2022723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(5)..(5)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(8)..(8)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 227Gly Ile Gly Lys Xaa Leu His
Xaa Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2022823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(6)..(6)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 228Gly Ile Gly Lys Phe Xaa His
Ala Xaa Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2022923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(7)..(7)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(10)..(10)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 229Gly Ile Gly Lys Phe Leu Xaa
Ala Ala Xaa Lys Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(11)..(11)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 230Gly Ile Gly Lys Phe Leu His
Xaa Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(12)..(12)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 231Gly Ile Gly Lys Phe Leu His
Ala Xaa Lys Lys Xaa Ala Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(10)..(10)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(13)..(13)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 232Gly Ile Gly Lys Phe Leu His
Ala Ala Xaa Lys Phe Xaa Lys Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(11)..(11)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(14)..(14)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 233Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Xaa Phe Ala Xaa Ala Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(15)..(15)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 234Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Xaa Ala Lys Xaa Phe1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(13)..(13)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(16)..(16)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 235Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Xaa Lys Ala Xaa1 5 10 15Val Ala Glu Ile Xaa Asn
Ser 2023623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(14)..(14)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(17)..(17)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 236Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Xaa Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn
Ser 2023723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(15)..(15)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(18)..(18)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 237Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Lys Xaa Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2023823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(16)..(16)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(19)..(19)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 238Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val Ala Xaa Ile Xaa Asn
Ser 2023923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(17)..(17)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 239Gly Ile Gly Lys Phe Leu His
Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Xaa Ala Glu Xaa Xaa Asn
Ser 2024023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(18)..(18)Any amino acid whose side chain
has been replaced by an internal stapleMOD_RES(21)..(21)Any amino
acid whose side chain has been replaced by an internal staple
240Gly Ile Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1
5 10 15Val Xaa Glu Ile Xaa Asn Ser 2024123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(19)..(19)Any amino acid whose side chain has been
replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(22)..(22)Any amino acid
whose side chain has been replaced by an internal staple 241Gly Ile
Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val
Ala Xaa Ile Xaa Xaa Ser 2024223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(20)..(20)Any amino
acid whose side chain has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 242Gly Ile
Gly Lys Phe Leu His Ala Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Val
Ala Glu Xaa Xaa Asn Xaa 2024323PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(2)..(2)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(5)..(5)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(12)..(12)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(19)..(19)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 243Gly
Xaa Gly Lys Xaa Leu His Ala Ala Lys Lys Xaa Ala Lys Ala Phe1 5 10
15Val Ala Xaa Ile Xaa Asn Ser 2024423PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(6)..(6)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(11)..(11)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(18)..(18)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 244Gly Ile Xaa Lys Phe Xaa His
Ala Ala Lys Xaa Phe Ala Lys Ala Phe1 5 10 15Val Xaa Glu Ile Xaa Asn
Ser 2024523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(7)..(7)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(13)..(13)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(17)..(17)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 245Gly Ile Xaa Lys Phe Leu Xaa
Ala Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn
Ser 2024623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(8)..(8)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(13)..(13)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(17)..(17)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 246Gly Ile Gly Xaa Phe Leu His
Xaa Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn
Ser 2024723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(6)..(6)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(13)..(13)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 247Gly Xaa Gly Lys Phe Xaa His
Ala Ala Lys Lys Phe Xaa Lys Ala Phe1 5 10 15Val Ala Glu Xaa Xaa Asn
Ser 2024823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(23)..(23)Any amino acid
whose side chain has been replaced by an internal staple 248Gly Xaa
Gly Lys Phe Leu His Ala Xaa Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val
Ala Glu Ile Xaa Asn Xaa 2024923PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(3)..(3)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(10)..(10)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(15)..(15)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)NorleucineMOD_RES(22)..(22)Any amino acid
whose side chain has been replaced by an internal staple 249Gly Ile
Xaa Lys Phe Leu His Ala Ala Xaa Lys Phe Ala Lys Xaa Phe1 5 10 15Val
Ala Glu Ile Xaa Xaa Ser 2025023PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(8)..(8)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(17)..(17)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Any amino acid whose side chain has been
replaced by an internal staple 250Xaa Ile Gly Lys Phe Leu His Xaa
Ala Lys Lys Phe Ala Lys Ala Phe1 5 10 15Xaa Ala Glu Ile Xaa Asn Ser
2025123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(9)..(9)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(16)..(16)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(20)..(20)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 251Gly Xaa Gly Lys Phe Leu His
Ala Xaa Lys Lys Phe Ala Lys Ala Xaa1 5 10 15Val Ala Glu Xaa Xaa Asn
Ser 2025223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid whose side chain has
been replaced by an internal stapleMOD_RES(10)..(10)Any amino acid
whose side chain has been replaced by an internal
stapleMOD_RES(15)..(15)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(19)..(19)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 252Gly Ile Xaa Lys Phe Leu His
Ala Ala Xaa Lys Phe Ala Lys Xaa Phe1 5 10 15Val Ala Xaa Ile Xaa Asn
Ser 2025322PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(1)..(1)(S)-alpha-(4-Pentenyl)alanineMOD_RES(8)..(8)(S-
)-alpha-(4-Pentenyl)alanine 253Xaa Ile Gly Lys Phe Leu Lys Xaa Ala
Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Lys Ile Leu Lys Lys
2025422PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(1)..(1)(R)-alpha-(7-Octenyl)alanineMOD_RES(6)..(6)(S)-alph-
a-(4-Pentenyl)alanine 254Xaa Ile Gly Lys Phe Xaa Lys Lys Ala Lys
Lys Phe Gly Lys Ala Phe1 5 10 15Val Lys Ile Leu Lys Lys
2025522PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(1)..(1)(R)-alpha-(7-Octenyl)alanineMOD_RES(5)..(5)(R)-alph-
a-Allylalanine 255Xaa Ile Gly Lys Xaa Leu Lys Lys Ala Lys Lys Phe
Gly Lys Ala Phe1 5 10 15Val Lys Ile Leu Lys Lys
2025622PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(1)..(1)(S)-alpha-(4-Pentenyl)alanineMOD_RES(8)..(8)(S)-alp-
ha-(4-Pentenyl)alanineMOD_RES(15)..(15)(S)-alpha-(4-Pentenyl)alanineMOD_RE-
S(22)..(22)(S)-alpha-(4-Pentenyl)alanine 256Xaa Ile Gly Lys Phe Leu
Lys Xaa Ala Lys Lys Phe Gly Lys Xaa Phe1 5 10 15Val Lys Ile Leu Lys
Xaa 2025722PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptideMOD_RES(1)..(1)(R)-alpha-(7-Octenyl)alanineMOD_RES(6)..(6)(S)-
-alpha-(4-Pentenyl)alanineMOD_RES(17)..(17)(R)-alpha-(7-Octenyl)alanineMOD-
_RES(22)..(22)(S)-alpha-(4-Pentenyl)alanine 257Xaa Ile Gly Lys Phe
Xaa Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Xaa Lys Ile Leu
Lys Xaa 2025822PRTArtificial SequenceDescription of Artificial
Sequence Synthetic
peptideMOD_RES(1)..(1)(R)-alpha-(7-Octenyl)alanineMOD_RES(5)..(5)(R)-
-alpha-AllylalanineMOD_RES(18)..(18)(R)-alpha-(7-Octenyl)alanineMOD_RES(22-
)..(22)(R)-alpha-Allylalanine 258Xaa Ile Gly Lys Xaa Leu Lys Lys
Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Xaa Ile Leu Lys Xaa
2025922PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(3)Any naturally occurring amino
acidMOD_RES(5)..(6)Any naturally occurring amino
acidMOD_RES(9)..(9)Any naturally occurring amino
acidMOD_RES(12)..(13)Any naturally occurring amino
acidMOD_RES(15)..(17)Any naturally occurring amino
acidMOD_RES(19)..(20)Any naturally occurring amino acid 259Xaa Xaa
Xaa Lys Xaa Xaa Lys Lys Xaa Lys Lys Xaa Xaa Lys Xaa Xaa1 5 10 15Xaa
Lys Xaa Xaa Lys Lys 2026022PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(12)..(12)Phe or
SerMOD_RES(15)..(15)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(19)..(19)Any amino acid whose
side chain has been replaced by an internal
stapleMOD_RES(21)..(21)Norleucine 260Gly Ile Gly Lys Phe Leu His
Ser Ala Lys Lys Xaa Gly Lys Xaa Phe1 5 10 15Val Gly Xaa Ile Xaa Asn
2026122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(12)..(12)Phe or SerMOD_RES(16)..(16)Any
amino acid whose side chain has been replaced by an internal
stapleMOD_RES(20)..(20)Any amino acid whose side chain has been
replaced by an internal stapleMOD_RES(21)..(21)Norleucine 261Gly
Ile Gly Lys Phe Leu His Ser Ala Lys Lys Xaa Gly Lys Ala Xaa1 5 10
15Val Gly Glu Xaa Xaa Asn 2026222PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideMOD_RES(12)..(12)Phe or Ser
262Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Xaa Gly Lys Ala Phe1
5 10 15Val Gly Glu Ile Met Asn 20
* * * * *