U.S. patent application number 16/282908 was filed with the patent office on 2019-08-15 for novel a4 7 peptide dimer antagonists.
The applicant listed for this patent is Protagonist Therapeutics, Inc.. Invention is credited to Ashok Bhandari, Larry C. Mattheakis, Dinesh V. Patel.
Application Number | 20190248870 16/282908 |
Document ID | / |
Family ID | 50477907 |
Filed Date | 2019-08-15 |
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United States Patent
Application |
20190248870 |
Kind Code |
A1 |
Bhandari; Ashok ; et
al. |
August 15, 2019 |
NOVEL a4 7 PEPTIDE DIMER ANTAGONISTS
Abstract
The invention relates to disulfide-rich dimer molecules which
inhibit binding of .alpha.4.beta.7 to the mucosal addressin cell
adhesion molecule (MAdCAM) in vivo, and show high selectivity
against .alpha.4.beta.7 binding.
Inventors: |
Bhandari; Ashok;
(Pleasanton, CA) ; Patel; Dinesh V.; (Fremont,
CA) ; Mattheakis; Larry C.; (Cupertino, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Protagonist Therapeutics, Inc. |
Newark |
CA |
US |
|
|
Family ID: |
50477907 |
Appl. No.: |
16/282908 |
Filed: |
February 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16032400 |
Jul 11, 2018 |
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16282908 |
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15831120 |
Dec 4, 2017 |
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16032400 |
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15493471 |
Apr 21, 2017 |
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15831120 |
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15000923 |
Jan 19, 2016 |
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15493471 |
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14050349 |
Oct 10, 2013 |
9273093 |
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15000923 |
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61807714 |
Apr 2, 2013 |
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61712722 |
Oct 11, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
1/18 20180101; C07K 7/08 20130101; A61K 38/00 20130101; A61P 11/02
20180101; A61P 35/00 20180101; A61P 1/00 20180101; C07K 14/47
20130101; A61P 3/10 20180101; C07K 7/06 20130101; A61P 43/00
20180101; A61P 11/00 20180101; A61P 1/16 20180101; A61P 29/00
20180101; A61P 11/06 20180101; C07K 14/70546 20130101; A61P 37/06
20180101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C07K 7/06 20060101 C07K007/06; C07K 14/47 20060101
C07K014/47; C07K 7/08 20060101 C07K007/08 |
Claims
1. A peptide dimer compound comprising two peptide monomer subunits
of Formula (I)
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Xaa.sup.7-Xaa-
.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Xaa.sup.13-Xaa.sup.14-Xa-
a.sup.15 (I), or a pharmaceutically acceptable salt thereof,
wherein Xaa.sup.1 is selected from the group consisting of a
suitable linker moiety, absent, hydrogen, Ac-, Gln, Asn, Asp, Pro,
Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Leu, Val, Tye, Trp,
Met, Thr, a suitable isostere, a corresponding D-amino acid, and a
suitable linker moiety; Xaa.sup.2 is selected from the group
consisting of Ac-, NH.sub.2, a suitable linker moiety, absent, Gln,
Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Leu,
Val, Tye, Trp, Met, Thr, a suitable isostere, a suitable linker
moiety, and a corresponding D-amino acid; Xaa.sup.3 is selected
from the group consisting of Ac-, NH.sub.2, a suitable linker
moiety, Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn,
Glu, Leu, Val, Tye, Trp, Met, Thr, a suitable isostere, a suitable
linker moiety, and a corresponding D-amino acid; Xaa.sup.4 is
selected from the group consisting of Cys, Pen, Asp, Glu, hGlu,
Lys, homo-Lys, Orn, Dap, Dab, a suitable isostere, and a
corresponding D-amino acid; Xaa.sup.5 is selected from the group
consisting of Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,
Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, Thr, homo-Arg, Dap, Dab,
N-Me-Arg, Arg-(Me)sym, Arg-(me)asym, 4-Guan, Cit, Cav, a suitable
isostere, and a corresponding D-amino acid; Xaa.sup.6 is selected
from the group consisting of Ser, Gln, Asn, Asp, Pro, Gly, His,
Ala, Ile, Phe, Lys, Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, a
suitable isostere replacement and a corresponding D-amino acid;
Xaa.sup.7 is selected from the group consisting of Asp, N-Me-Asp, a
suitable isostere replacement for Asp, and a corresponding D-amino
acid; Xaa.sup.8 is selected from the group consisting of Thr, Gln,
Ser, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu,
Val, Tye, Trp, Met, an N-Methyl amino acid; a suitable isostere,
and a corresponding D-amino acid; Xaa.sup.9 is selected from the
group consisting of Gln, Asn, Asp, Pro, Gly, Ala, Phe, Leu, Asn,
Glu, Val, homo-Leu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala,
N-Me-Leu, a suitable isostere, and a corresponding D-amino acid;
Xaa.sup.10 is selected from the group consisting of Cys, Asp, Pen,
Lys, homo-Lys, Orn, GluDap, Dab, a suitable isostere, and a
corresponding D-amino acid; Xaa.sup.11 is selected from the group
consisting of Gly, Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr,
Lys, Trp, Tyr, CONH.sub.2, His, Glu, Ser, Arg, Pro, Phe, Sar, 1Nal,
2Nal, hPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac),
Orn, D-Orn, N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip,
Ala(3,3diphenyl), Biphenyl-Ala, an aromatic ring substituted Phe,
an aromatic ring substituted Trp, an aromatic ring substituted His,
a hetero aromatic amino acid, N-Me-Lys, N-Me-Lys(Ac), 4-Me-Phe, a
corresponding D-amino acid; a suitable isostere; and a suitable
linker moiety. Xaa.sup.12 is selected from the group consisting of
Glu, Amide, Lys, COOH, CONH.sub.2, Gln, Pro, Gly, His, Ala, Ile,
Phe, Lys, Arg, Leu, Val, Tye, Trp, Met, Gla, Ser, Asn, Dap, Dab,
Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab, a
suitable isostere, a suitable linker moiety, and a corresponding
D-amino acid; Xaa.sup.13 is selected from the group consisting of
Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tye, Trp,
Met, Glu, Gla, Ser, Asn, Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap,
N-Me-Dab, N-Me Lys, D-Dap, D-Dab, COOH, CONH.sub.2, NH.sub.2,
absent, a suitable linker moiety, a suitable isostere, and a
corresponding D-amino acid; Xaa.sup.14 is selected from the group
consisting of a natural amino acid, absent, COOH, CONH.sub.2,
NH.sub.2, a suitable isostere, a suitable linker, a corresponding
D-amino acid, and an N-Methyl amino acid; and Xaa.sup.15 is
selected from the group consisting of a suitable linker, and
absent.
2-7. (canceled)
8. A method for treating inflammatory bowel disease in a patient,
comprising administering to the patient an effective amount of a
peptide dimer compound of claim 1.
9-11. (canceled)
12. A method for treating a human having an inflammatory bowel
disease, comprising the steps of administering to the human an
effective amount of a peptide dimer according to the composition of
claim 1.
13-19. (canceled)
20. A method for treating a human afflicted with a condition that
is associated with a biological function of .alpha.4.beta.7, the
method comprising administering to the human a peptide dimer
according to the composition of claim 1.
21-22. (canceled)
23. A method for stabilizing a peptide dimer compound according to
claim 1, the method comprising a step for substituting Xaa.sup.4
and Xaa.sup.10 with an amino acid residue selected from the group
consisting of Cys and Pen, wherein Xaa.sup.4 and Xaa.sup.10 form a
cyclized structure through a disulfide bond.
24. A method for stabilizing a peptide dimer compound of Formula
(II)
Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Xaa.sup.7-Xaa.sup.8-Xaa.sup.9-Xaa.sup.10-Xa-
a.sup.11-Xaa.sup.12 (II), or a pharmaceutically acceptable salt
thereof, wherein the method comprises a step for substituting
Xaa.sup.4 and Xaa.sup.10 with compatible amino acid residues that
are capable of forming a cyclized structure through at least one of
an amide bond and a disulfide bond.
25-26. (canceled)
27. A pharmaceutical composition comprising a peptide dimer
compound according to at least one of Formula (I) and Formula
(II).
28-29. (canceled)
30. A method for treating a condition in a subject comprising
administering the pharmaceutical composition of claim 27 to the
subject, wherein the condition is treatable by reducing the
activity (partially or fully) of .alpha.4.beta.7 in the
subject.
31-22. (canceled)
33. A method for treating a human afflicted with a condition that
is associated with a biological function .alpha.4.beta.7 and
comprising administering to the individual a peptide dimer of
Formula (I) in an amount sufficient to inhibit (partially or fully)
the biological function of .alpha.4.beta.7 to tissues expressing
MAdCAM.
34-42. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 16/032,400, filed Jul. 11, 2018; which is a
Continuation of U.S. patent application Ser. No. 15/831,120, filed
Dec. 4, 2017; which is a Continuation of U.S. patent application
Ser. No. 15/493,471, filed Apr. 21, 2017, which is a Continuation
of U.S. patent application Ser. No. 15/000,923, filed Jan. 19,
2016; which is a Continuation of U.S. patent application Ser. No.
14/050,349, filed Oct. 10, 2013, now U.S. Pat. No. 9,273,093,
issued Mar. 1, 2016; which claims the benefit of U.S. Provisional
Application Nos. 61/807,714, filed on Apr. 2, 2013, and 61/712,722,
filed on Oct. 11, 2012, each of which is incorporated herein in its
entirety.
STATEMENT REGARDING SEQUENCE LINSTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and it hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is PRTH_007_06US_
ST25.txt. The text file is about 108 KB, was created on Feb. 22,
2019, and is being submitted electronically via EFS-Web.
FIELD OF THE INVENTION
[0003] The present invention relates to novel compounds having
activity useful for treating conditions which arise or are
exacerbated by integrin binding, pharmaceutical compositions
comprising the compounds, methods of treatment using the compounds,
and methods of blocking or disrupting integrin binding.
BACKGROUND OF THE INVENTION
[0004] Integrins are noncovalently associated .alpha./.beta.
heterodimeric cell surface receptors involved in numerous cellular
processes ranging from cell adhesion and migration to gene
regulation (Dubree, et al., Selective .alpha.4.beta.7 Integrin
Antagonist and Their Potential as Antiinflammatory Agents, J. Med.
Chem. 2002, 45, 3451-3457). Differential expression of integrins
can regulate a cell's adhesive properties, allowing different
leukocyte populations to be recruited to specific organs in
response to different inflammatory signals. If left unchecked,
integrins-mediated adhesion process can lead to chronic
inflammation and autoimmune disease.
[0005] The .alpha.4 integrins, .alpha.4.beta.1 and .alpha.4.beta.7,
play essential roles in lymphocyte migration throughout the
gastrointestinal tract. They are expressed on most leukocytes,
including B and T lymphocytes, where they mediate cell adhesion via
binding to their respective primary ligands, vascular cell adhesion
molecule (VCAM), and mucosal addressin cell adhesion molecule
(MAdCAM), respectively. The proteins differ in binding specificity
in that VCAM binds both .alpha.4.beta.1 and to a lesser extent
.alpha.4.beta.7, while MAdCAM is highly specific for
.alpha.4.beta.7. In addition to pairing with the .alpha.4 subunit,
the .beta.7 subunit also forms a heterodimeric complex with
.alpha.E subunit to form .alpha.E.beta.7, which is primarily
expressed on intraepithelial lymphocytes (IEL) in the intestine,
lung and genitourinary tract. .alpha.E.beta.7 is also expressed on
dendritic cells in the gut. The .alpha.E.beta.7 heterodimer binds
to E-cadherin on the epithelial cells. The IEL cells are thought to
provide a mechanism for immune surveillance within the epithelial
compartment. Therefore, blocking .alpha.E.beta.7 and
.alpha.4.beta.7 together may be a useful method for treating
inflammatory conditions of the intestine.
[0006] Inhibitors of specific integrins-ligand interactions have
been shown effective as anti-inflammatory agents for the treatment
of various autoimmune diseases. For example, monoclonal antibodies
displaying high binding affinity for .alpha.4.beta.7 have displayed
therapeutic benefits for gastrointestinal
auto-inflammatory/autoimmune diseases, such as Crohn's disease, and
ulcerative colitis. Id. However, these therapies interfered with
.alpha.4.beta.1 integrin-ligand interactions thereby resulting in
dangerous side effects to the patient. Therapies utilizing small
molecule antagonists have shown similar side effects in animal
models, thereby preventing further development of these
techniques.
[0007] Accordingly, there is a need in the art for an integrin
antagonist molecule having high affinity for the .alpha.4.beta.7
integrin and high selectivity against the .alpha.4.beta.1 integrin,
as a therapy for various gastrointestinal autoimmune diseases.
[0008] Such an integrin antagonist molecule is disclosed
herein.
SUMMARY OF THE INVENTION
[0009] The present invention has been developed in response to the
present state of the art, and in particular, in response to the
problems and needs in the art that have not yet been fully solved
by currently available integrin antagonists that are selective for
.alpha.4.beta.7. Thus, the present invention provides
.alpha.4.beta.7 antagonist dimer peptides for use as
anti-inflammatory and/or immunosuppressive agents. Further, the
present invention provides .alpha.4.beta.7 antagonist dimer peptide
for use in treating a condition that is associated with a
biological function of .alpha.4.beta.7 to tissues expressing
MAdCAM.
[0010] The invention relates to a novel class of peptidic compounds
exhibiting integrin antagonist activity. The present invention
further relates to a novel class of peptidic compounds exhibiting
high specificity for .alpha.4.beta.7 integrin. Compounds of the
present invention comprise two paired subunits that are linked
together by their C- or N-terminus via a linking moiety. Each
subunit of the present invention further comprises two natural or
unnatural amino acids that are capable of bridging to form a
cyclized structure. Thus, the compounds of the present invention
comprise dimerized peptides, each subunit of the dimer forming a
cyclized structure through at least one of a disulfide salt bridge,
an amide bond, or an equivalent connection. This feature provides
increased stability to the compound when administered orally as a
therapeutic agent. This feature further provides for increased
specificity and potency as compared to non-cyclized analogs.
[0011] In one aspect, the present invention provides a dimer
compound comprising two linked subunits of Formula (I):
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Xaa.sup.7-Xa-
a.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Xaa.sup.13-Xaa.sup.14-X-
aa.sup.15, (I)
(SEQ ID NO:1) or a pharmaceutically acceptable salt thereof,
wherein Formula (I) is a homo- or monomer that is linked to form a
dimer molecule in accordance with the present invention, and
wherein Xaa.sup.1 is absent, Xaa.sup.1 is a suitable linker moiety,
or Xaa.sup.1 is selected from the group consisting of hydrogen,
Ac-, Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn,
Glu, Leu, Val, Tye, Trp, Met, Thr, suitable isostere, and
corresponding D-amino acids; Xaa.sup.2 is absent, Xaa.sup.2 is Ac-,
Xaa.sup.2 is NH.sub.2, Xaa.sup.2 is a suitable linker moiety, or
Xaa.sup.2 is selected from the group consisting of Gln, Asn, Asp,
Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Leu, Val, Tye,
Trp, Met, Thr, a suitable isostere and corresponding D-amino acids;
Xaa.sup.3 is absent, Xaa.sup.3 is Ac-, Xaa.sup.3 is NH.sub.2, Xaa3
is a suitable linker moiety, or Xaa.sup.3 is selected from the
group consisting of an Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe,
Lys, Arg, Asn, Glu, Leu, Val, Tye, Trp, Met and Thr, a suitable
isostere and corresponding D-amino acids; Xaa.sup.4 is selected
from the group consisting of Cys, Pen, Asp, Glu, hGlu, .beta.-Asp,
.beta.-Glu, Lys, homo-Lys, Orn, Dap, Dab, a suitable isostere and
corresponding D-amino acids; Xaa.sup.5 is selected from the group
consisting of Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,
Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, Thr, homo-Arg, Dap, Dab,
N-Me-Arg, Arg-(Me)sym, Arg-(Me)asym, 4-Guan, Cit, Cav, and suitable
isostere replacements; Xaa.sup.6 is selected from the group
consisting of Ser, Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe,
Lys, Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, and suitable isostere
replacements; Xaa.sup.7 is selected from the group consisting of
Asp, N-Me-Asp and a suitable isostere replacement for Asp;
Xaa.sup.8 is selected from the group consisting of Thr, Gln, Ser,
Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val,
Tye, Trp, Met, and N-Methyl amino acids including N-Me-Thr;
Xaa.sup.9 is selected from the group consisting of Gln, Asn, Asp,
Pro, Gly, Ala, Phe, Leu, Asn, Glu, Val, homo-Leu, n-Butyl Ala,
n-Pentyl Ala, n-Hexyl Ala, N-Me-Leu, and suitable isostere
replacements; Xaa.sup.10 is selected from the group consisting of
Cys, Asp, Pen, Lys, homo-Lys, Orn, Glu, .beta.-Asp, .beta.-Glu,
Dap, and Dab; Xaa.sup.11 is selected from the group consisting of
Gly, Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr,
CONH.sub.2, COOH, His, Glu, Ser, Arg, Pro, Phe, Sar, 1Nal, 2Nal,
hPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac), Orn,
D-Orn, N-Me-Orn, N-Me-Dap, D-Dap, D-Dab Bip, Ala(3,3diphenyl),
Biphenyl-Ala, aromatic ring substituted Phe, aromatic ring
substituted Trp, aromatic ring substituted His, hetero aromatic
amino acids, N-Me-Lys, N-Me-Lys(Ac), 4-Me-Phe, and corresponding
D-amino acids and suitable isostere replacements; Xaa.sup.12 is
absent, Xaa.sup.12 is a suitable linker moiety, or Xaa.sup.12 is
selected from the group consisting of Glu, Amide, Lys, COOH,
CONH.sub.2, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val,
Tye, Trp, Met, Gla, Ser, Asn, Dap, Dab, Orn, D-Orn, N-Me-Orn,
N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab, suitable isosteres, and
corresponding D-amino acids; Xaa.sup.13 is absent, Xaa.sup.13 is
Ac, Xaa.sup.13 is a suitable linker moiety, or Xaa.sup.13 is
selected from the group consisting of Gln, Pro, Gly, His, Ala, Ile,
Phe, Lys, Arg, Leu, Val, Tye, Trp, Met, Glu, Gla, Ser, Asn, Dap,
Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap,
D-Dab, COOH, CONH.sub.2, suitable isosteres, and corresponding
D-amino acids; Xaa.sup.14 is absent, Xaa.sup.14 is a suitable
linker moiety, or Xaa.sup.14 is selected from the group consisting
of natural amino acids, COOH, CONH.sub.2, suitable isostere
replacements, corresponding D-amino acids, and corresponding
N-Methyl amino acids; Xaa.sup.15 is a suitable linker moiety, as
defined herein, wherein Xaa.sup.15 is selected from the group
consisting of DIG, DIG-OH, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K,
PEG4K, PEG5K, IDA, IDA-Palm, IDA-Boc, IDA-Isovaleric acid,
Triazine, Triazine-Boc, Trifluorobutyric acid,
2-Me-trifluorobutyric acid, Trifluoropentanoic acid, Isophthalic
acid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid,
glutaric acid, Azelaic acid, Pimelic acid, and Dodecanedioic acid;
wherein Formula (I) comprises a dimer formed from two subunits
joined by a suitable C- or N-terminal linker selected from the
group consisting of DIG, DIG-OH, PEG13, PEG25, PEG1K, PEG2K,
PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, IDA-Boc, IDA-Isovaleric acid,
Triazine, Triazine-Boc, Isophthalic acid, 1,3-phenylenediacetic
acid, 1,4-phenylenediacetic acid, glutaric acid, Azelaic acid,
Pimelic acid, Dodecanedioic acid, suitable aliphatics, suitable
aromatics, heteroaromatics, and polyethylene glycols having a
molecular weight from approximately 400 Da to approximately 40,000
Da. One having skill in the art will appreciate that the C- and
N-terminal linker moieties disclosed herein are non-limiting
examples of suitable, and that the present invention may include
any suitable linker moiety. Thus, some embodiments of the present
invention comprises a homo- or heterodimer molecule comprised of
two monomer subunits selected from the peptide molecules
represented by SEQ ID NOs: 1-136, wherein the C- or N-termini of
the respective monomers are linked by any suitable linker moiety to
provide a dimer molecule having integrin antagonist activity.
[0012] In another aspect, the present invention provides a
composition for treating a patient in need of integrin-antagonist
therapy comprising a compound of Formula (I) in combination with a
pharmaceutically acceptable carrier.
[0013] Yet another aspect of the present invention provides a
composition for treating a patient in need of
.alpha.4.beta.7-specific antagonist therapy comprising a compound
of Formula (I) having high selectivity for .alpha.4.beta.7 integrin
in combination with a pharmaceutically acceptable carrier.
[0014] Yet another aspect of the present invention provides a
composition for treating a patient in need of
.alpha.4.beta.7-specific antagonist therapy comprising a compound
of Formula (I) having high selectivity for .alpha.4.beta.7 against
.alpha.4.beta.1 integrins in combination with a pharmaceutically
acceptable carrier.
[0015] Yet another aspect of the present invention provides a
composition for treating a patient in need of
.alpha.4.beta.7-specific antagonist therapy comprising a compound
of Formula (I) having high selectivity for .alpha.4.beta.7 against
.alpha.E.beta.7 integrins in combination with a pharmaceutically
acceptable carrier.
[0016] Yet another aspect of the present invention provides a
composition for treating a patient in need of
.alpha.4.beta.7-specific antagonist therapy comprising a compound
of Formula (I) having low selectivity for .alpha.4.beta.7 against
.alpha.E.beta.7 integrins in combination with a pharmaceutically
acceptable carrier.
[0017] Yet another aspect of the present invention provides a
method for treating a patient in need of integrin-antagonist
therapy comprising administering to the patient a therapeutically
effective amount of a compound of Formula (I).
[0018] Still, yet another aspect of the present invention provides
a composition for the treatment of a disease from ulcerative
colitis, Crohn's disease, Celiac disease (nontropical Sprue),
enteropathy associated with seronegative arthropathies, microscopic
or collagenous colitis, eosinophilic gastroenteritis, radio- or
chemo-therapy, or pouchitis resulting after proctocolectomy and
ileoanal anastomosis, and various forms of gastrointestinal cancer.
In another embodiment, the condition is pancreatitis,
insulin-dependent diabetes mellitus, mastitis, cholecystitis,
cholangitis, pericholangitis, chronic bronchitis, chronic
sinusitis, asthma or graft versus host disease. In addition, these
compounds may be useful in the prevention or reversal of these
diseases when used in combination with currently available
therapies, medical procedures, and therapeutic agents.
[0019] In yet another aspect, the present invention provides a
diagnostic method for visualizing and diagnosing a disease
comprising administering an orally stable compound of Formula (I)
that is further labeled with at least one of a chelating group and
a detectable label for use as an in vivo imaging agent for
non-invasive diagnostic procedures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order that the manner in which the above-recited and
other features and advantages of the invention are obtained will be
readily understood, a more particular description of the invention
briefly described above will be rendered by reference to specific
embodiments thereof which are illustrated in the appended drawings.
Understanding that these drawings depict only typical embodiments
of the invention and are not therefore to be considered to be
limiting of its scope, the invention will be described and
explained with additional specificity and detail through the use of
the accompanying drawings in which:
[0021] FIG. 1 is a schematic showing C and N-terminal
dimerizations.
[0022] FIG. 2 is a schematic showing a pair of integrin antagonist
monomer subunits according to SEQ ID NO: 58, wherein the subunits
are aligned and linked at their respective C-termini by a DIG
linker in accordance with a representative embodiment of the
present invention.
[0023] FIG. 3 is a chart demonstrating stability data for integrin
antagonist homodimer molecules represented by SEQ ID NOs: 39, 57,
82, 102 and 121 in accordance with various representative
embodiment of the present invention.
[0024] FIG. 4 is a chart demonstrating potency and selectivity for
integrin antagonist monomer and homodimer molecules represented by
SEQ ID NOs: 71, 49. 63, 59, 61, 63, 65, 66, and 83 in accordance
with a representative selection of various embodiments of the
present invention.
SEQUENCE LISTING
[0025] The amino acid sequences listed in the accompanying sequence
listing are shown using three letter code for amino acids, as
defined in 37 C.F.R. 1.822. Only the monomer subunit sequences are
shown, however it is understood that the monomer subunits are
dimerized to form peptide dimer molecules, in accordance with the
present teaching and as shown generally in FIGS. 1 and 2. The
monomer subunits may be dimerized by a suitable linker moiety, as
defined herein. Some of the monomer subunits are shown having C-
and N-termini that both comprise free amine. Thus, a user must
modify the monomer subunit to eliminate either the C- or N-terminal
free amine, thereby permitting dimerization at the remaining free
amine. Thus, some of the monomer subunits comprise both a free
carboxy terminal and a free amino terminal, whereby a user may
selectively modify the subunit to achieve dimerization at a desired
terminus. Therefore, one having skill in the art will appreciate
that the monomer subunits of the instant invention may be
selectively modified to achieve a single, specific amine for a
desired dimerization.
[0026] It is further understood that the C-terminal residues of the
monomer subunits disclosed herein are amides, unless otherwise
indicated. Further, it is understood that dimerization at the
C-terminal is facilitated by using a suitable amino acid with a
side chain having amine functionality, as is generally understood
in the art. Regarding the N-terminal residues, it is generally
understood that dimerization may be achieved through the free amine
of the terminal residue, or may be achieved by using a suitable
amino acid side chain having a free amine, as is generally
understood in the art.
[0027] In the accompanying sequence listing:
[0028] SEQ ID NO: 1 shows a monomer subunit of a dimer compound of
Formula (I).
[0029] SEQ ID NO: 2 shows a monomer subunit of a dimer compound of
Formula (II).
[0030] SEQ ID NOs: 3-38, 49, 57-71, 76-117 and 124-136 show amino
acid sequences of monomer subunits that are dimerized to form
various dimer compounds in accordance with the present invention,
wherein these sequences have been substituted with an N-methylated
arginine.
[0031] SEQ ID NOs: 39-44, 58-65, 67-71, 74-76, 82, 83, 85, 86,
100-114, and 116-136 show amino acid sequences of monomer subunits
that are dimerized at their respective C-termini to form various
dimer compounds in accordance with the present invention.
[0032] SEQ ID NOs: 45-57, 66, 72-73, 77-81, 84, 87-99 and 115 show
amino acid sequences of monomer subunits that are dimerized at
their respective N-termini to form various dimer compounds in
accordance with the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] As used herein, the singular forms "a," "and" and "the"
include plural references unless the context clearly dictates
otherwise.
[0034] As used in the present specification the following terms
have the meanings indicated:
[0035] The term "peptide," as used herein, refers broadly to a
sequence of two or more amino acids joined together by peptide
bonds. It should be understood that this term does not connote a
specific length of a polymer of amino acids, nor is it intended to
imply or distinguish whether the polypeptide is produced using
recombinant techniques, chemical or enzymatic synthesis, or is
naturally occurring.
[0036] The term "DRP," as used herein, refers to disulfide rich
peptides.
[0037] The term "dimer," as used herein, refers broadly to a
peptide comprising two or more subunits, wherein the subunits are
DRPs that are linked at their C- or N-termini. Dimers of the
present invention may include homodimers and heterodimers and
function as integrin antagonists.
[0038] The term "L-amino acid," as used herein, refers to the "L"
isomeric form of a peptide, and conversely the term "D-amino acid"
refers to the "D" isomeric form of a peptide. The amino acid
residues described herein are preferred to be in the "L" isomeric
form, however, residues in the "D" isomeric form can be substituted
for any L-amino acid residue, as long as the desired functional is
retained by the peptide.
[0039] The term "NH2," as used herein, refers to the free amino
group present at the amino terminus of a polypeptide. The term
"OH," as used herein, refers to the free carboxy group present at
the carboxy terminus of a peptide. Further, the term "Ac," as used
herein, refers to Acetyl protection through acylation of the C- or
N-terminus of a polypeptide.
[0040] The term "carboxy," as used herein, refers to
--CO.sub.2H.
[0041] The term "isostere replacement," as used herein, refers to
any amino acid or other analog moiety having chemical and/or
structural properties similar to a specified amino acid.
[0042] The term "cyclized," as used herein, refers to a reaction in
which one part of a polypeptide molecule becomes linked to another
part of the polypeptide molecule to form a closed ring, such as by
forming a disulfide bridge or other similar bond.
[0043] The term "subunit," as used herein, refers to one of a pair
of polypeptides monomers that are joined at the C- or N- terminus
to form a dimer peptide composition.
[0044] The term "dimer," as used herein, refers to a chemical
entity consisting of two structurally similar monomers joined by
terminus bonds and/or a terminus linker.
[0045] The term "linker," as used herein, refers broadly to a
chemical structure that is capable of linking together a plurality
of DRP monomer subunits to form a dimer.
[0046] The term "receptor," as used herein, refers to chemical
groups of molecules on the cell surface or in the cell interior
that have an affinity for a specific chemical group or molecule.
Binding between dimer peptides and targeted integrins can provide
useful diagnostic tools.
[0047] The term "integrin-related diseases," as used herein, refer
to indications that manifest as a result of integrin binding, and
which may be treated through the administration of an integrin
antagonist.
[0048] The term "pharmaceutically acceptable salt," as used herein,
represents salts or zwitterionic forms of the compounds of the
present invention which are water or oil-soluble or dispersible,
which are suitable for treatment of diseases without undue
toxicity, irritation, and allergic response; which are commensurate
with a reasonable benefit/risk ratio, and which are effective for
their intended use. The salts can be prepared during the final
isolation and purification of the compounds or separately by
reacting an amino group with a suitable acid. Representative acid
addition salts include acetate, adipate, alginate, citrate,
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
camphorate, camphorsulfonate, digluconate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, formate, fumarate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate
(isethionate), lactate, maleate, mesitylenesulfonate,
methanesulfonate, naphthylenesulfonate, nicotinate,
2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,
3-phenylproprionate, picrate, pivalate, propionate, succinate,
tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate,
bicarbonate, para-toluenesulfonate, and undecanoate. Also, amino
groups in the compounds of the present invention can be quaternized
with methyl, ethyl, propyl, and butyl chlorides, bromides, and
iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl,
lauryl, myristyl, and steryl chlorides, bromides, and iodides; and
benzyl and phenethyl bromides. Examples of acids which can be
employed to form therapeutically acceptable addition salts include
inorganic acids such as hydrochloric, hydrobromic, sulfuric, and
phosphoric, and organic acids such as oxalic, maleic, succinic, and
citric.
[0049] All peptide sequences are written according to the generally
accepted convention whereby the .alpha.-N-terminal amino acid
residue is on the left and the .alpha.-C-terminal is on the right.
As used herein, the term ".alpha.-N-terminal" refers to the free
.alpha.-amino group of an amino acid in a peptide, and the term
".alpha.-C-terminal" refers to the free .alpha.-carboxylic acid
terminus of an amino acid in a peptide.
[0050] For the most part, the names on naturally occurring and
non-naturally occurring aminoacyl residues used herein follow the
naming conventions suggested by the IUPAC Commission on the
Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on
Biochemical Nomenclature as set out in "Nomenclature of
.alpha.-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2),
(1975). To the extent that the names and abbreviations of amino
acids and aminoacyl residues employed in this specification and
appended claims differ from those suggestions, they will be made
clear to the reader. Some abbreviations useful in describing the
invention are defined below in the following Table 1.
TABLE-US-00001 TABLE 1 Abbreviation Definition DIG Diglycolic acid
(Linker) Dap Diaminopropionic acid Dab Diaminobutyric acid Pen
Penicillamine Sar Sarcosine Cit Citroline Cav Cavanine 4-Guan
4-Guanidine-Phenylalanine N--Me-Arg N-Methyl-Arginine Ac-- Acetyle
2-Nal 2-Napthylalanine 1-Nal 1-Napthylalanine Bip Biphenylalanine
O--Me-Tyr Tyrisine (O-Methocy) N--Me-Lys N-Methyl-Lysine N--Me-Lys
(Ac) N-e-Acetyl-D-lysine Ala (3,3 diphenyle) 3,3 diphenyl alanine
NH2 Free Amine CONH2 Amide COOH Acid Phe (4-F) 4-Fluoro-Phenylanine
PEG13 Bifunctional PEG linker with 13 PolyEthylene Glycol units
PEG25 Bifunctional PEG linker with 25 PolyEthylene Glycol units
PEG1K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of
1000 Da PEG2K Bifunctional PEG linker with PolyEthylene Glycol Mol
wt of 2000 Da PEG3.4K Bifunctional PEG linker with PolyEthylene
Glycol Mol wt of 3400 Da PEG5K Bifunctional PEG linker with
PolyEthylene Glycol Mol wt of 5000 Da IDA (Affymax)
.beta.-Ala-Iminodiacetic acid (Linker) IDA-Palm .beta.-Ala
(Palityl)-Iminodiacetic acid hPhe Homo Phenylalanine Ahx
Aminohexanoic acid DIG-OH Glycolic monoacid Triazine Amino propyl
Triazine di-acid (Linker) Boc-Triazine Boc-Triazine di-acid
(Linker) Trifluorobutyric acid Acylated with 4,4,4-Trifluorobutyric
acid 2-Methly-trifluorobutyric acid Acylated with
2-methy-4,4,4-Butyric acid Trifluorpentanoic acid Acylated with
5,5,5-Trifluoropentnoic acid 1,4-Phenylenediacetic acid Para -
Phenylenediacetic acid (Linker) 1,3-Phenylenediacetic acid meta -
Phenylenediacetic acid (Linker)
[0051] The present invention relates generally to DRPs that have
been shown to have integrin antagonist activity. In particular, the
present invention relates to various peptide dimers comprising
hetero- or homo-monomer subunits that each form cyclized structures
through disulfide bonds. The monomer subunits are linked at either
their C- or N-termini, as shown in FIG. 1. The cyclized structure
of each subunit has been shown to increase potency and selectivity
of the dimer molecules, as discussed below. A non-limiting,
representative illustration of the cyclized structure is shown in
FIG. 2.
[0052] The linker moieties of the present invention may include any
structure, length, and/or size that is compatible with the
teachings herein. In at least one embodiment, a linker moiety is
selected from the non-limiting group consisting of DIG, PEG4,
PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, Boc-IDA,
Glutaric acid, Isophthalic acid, 1,3-phenylenediacetic acid,
1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, Triazine,
Boc-Triazine, suitable aliphatics, aromatics, heteroaromatics, and
polyethylene glycol based linkers having a molecular weight from
approximately 400 Da to approximately 40,000 Da. Non-limiting
examples of suitable linker moieties are provided in Table 2.
TABLE-US-00002 TABLE 2 Abbreviation Description Structure DIG
DIGlycolic acid, ##STR00001## PEG4 Bifunctional PEG linker with 4
PolyEthylene Glycol units ##STR00002## PEG13 Bifunctional PEG
linker with 13 PolyEthylene Glycol units ##STR00003## PEG25
Bifunctional PEG linker with 25 PolyEthylene Glycol units
##STR00004## PEG1K Bifunctional PEG linker with PolyEthylene Glycol
Mol wt of 1000Da PEG2K Bifunctional PEG linker with PolyEthylene
Glycol Mol wt of 2000Da PEG3.4K Bifunctional PEG linker with
PolyEthylene Glycol Mol wt of 3400Da PEG5K Bifunctional PEG linker
with PolyEthylene Glycol Mol wt of 5000Da DIG DIGlycolic acid,
##STR00005## IDA (Affymax) N-.beta.-Ala-Iminodiacetic acid
##STR00006## Boc-IDA Boc-.beta.-Ala-Iminodiacetic acid ##STR00007##
GTA Glutaric acid ##STR00008## PMA Pemilic acid ##STR00009## AZA
Azelaic acid ##STR00010## DDA Dodecanedioic acid ##STR00011## IPA
Isopthalic acid ##STR00012## 1,3-PDA 1,3-Phenylenediacetic acid
##STR00013## 1,4-PDA 1,4-Phenylenediacetic acid ##STR00014##
1,2-PDA 1,2-Phenylenediacetic acid ##STR00015## Triazine
N-[4,6-bis(.beta.-ala)-1,3,5-triazin-2-yl]-N-(n-propyl)amine
##STR00016## Boc-Triazine
N[4,6-bis(.beta.-ala)-1,3,5-triazin-2-yl]-N-(Boc-n-propyl) amine
##STR00017##
[0053] The present invention further includes various DRP that have
been substituted with various amino acids. For example, some
peptides include Dab, Dap, Pen, Sar, Cit, Cav, 4-guan, and various
N-methylated amino acids. One having skill in the art will
appreciate that additional substitutions may be made to achieve
similar desired results, and that such substitutions are within the
teaching and spirit of the present invention.
[0054] In one aspect, the present invention relates to dimer
compounds, each subunit of the dimer compound comprising the
structure
Xaa.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Xaa.sup.7-Xa-
a.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Xaa.sup.13-Xaa.sup.14-X-
aa.sup.15 (I),
wherein Formula (II)
Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Xaa.sup.7-Xaa.sup.8-Xaa.sup.9-Xaa.sup.10-Xa-
a.sup.11-Xaa.sup.12, (II) (SEQ ID NO: 2) or a pharmaceutically
acceptable salt thereof, further represent a subunit of a homo- or
heterodimer molecule, wherein each subunit comprises 9 amino acids.
The N-terminus of the nonapeptide can be modified by one to three
suitable groups, as represented by Xaa.sup.1, Xaa.sup.2, and
Xaa.sup.3 of Formula (I). The groups Xaa.sup.13, Xaa.sup.14, and
Xaa.sup.15 of Formula (I) represent one to three groups suitable
for modifying the C-terminus of the peptide.
[0055] In some embodiments, Xaa.sup.1, Xaa.sup.2, and Xaa.sup.3 are
absent. In other embodiments, Xaa.sup.1 is absent, and Xaa.sup.2
and Xaa.sup.3 represent suitable groups for modifying the
N-terminus of the nonapeptide. Further, in some embodiments
Xaa.sup.1 and Xaa.sup.2 are absent, and Xaa.sup.3 represents a
single suitable group for modifying the N-terminus of the
nonapeptide subunit.
[0056] Xaa.sup.1 is an amino acyl residue selected from the group
consisting of Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,
Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, Thr, suitable isosteres,
and corresponding D-amino acids. In some embodiments, Xaa.sup.1 is
the N-terminus and is therefore either Ac or free NH.sub.2. In at
least one embodiment, Xaa.sup.1 is Ser. In other embodiments,
Xaa.sup.1 is absent. Further, in at least one embodiment Xaa.sup.1
is an N-terminal linker moiety selected from the group consisting
of DIG, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA,
IDA-Palm, IDA-Boc, IDA-Isovaleric acid, Triazine, Triazine-Boc,
Isophthalic acid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic
acid, glutaric acid, Azelaic acid, Pimelic acid, Dodecanedioic
acid, suitable aliphatics, aromatics, heteroaromatics, and
polyethylene glycol based linkers having a molecular weight from
approximately 400 Da to approximately 20,000 kDa. Preferred
Xaa.sup.1 groups for modifying the N-terminus of the compounds in
the scope of the invention are free NH.sub.2, Ac, Lys, dLys.
[0057] Xaa.sup.2 is an amino acyl residue selected from the group
consisting of Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,
Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, and Thr. In some
embodiments, Xaa.sup.2 is Thr or a corresponding D-amino acid. When
Xaa.sup.1 is absent, Xaa.sup.2 is the N-terminus and is therefore
either Ac, free NH.sub.2, or a suitable linker moiety. Further, in
at least one embodiment Xaa.sup.2 is an N-terminal linker moiety
selected from the group consisting of DIG, PEG13, PEG25, PEG1K,
PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, IDA-Boc,
IDA-Isovaleric acid, Triazine, Triazine-Boc, Isophthalic acid,
1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid, glutaric
acid, Azelaic acid, Pimelic acid, Dodecanedioic acid, suitable
aliphatics, aromatics, heteroaromatics, and polyethylene glycol
based linkers having a molecular weight from approximately 400 Da
to approximately 40,000 Da. In other embodiments, Xaa.sup.2 is
absent. Preferred Xaa.sup.2 groups for modifying the N-terminus of
the compounds in the scope of the invention are Ac, NH.sub.2, Lys,
dLys and a suitable linker moiety.
[0058] Xaa.sup.3 is an amino acyl residue selected from the group
consisting of Gln, Asn, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,
Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, Thr, and corresponding
D-amino acids. When Xaa.sup.1 and Xaa.sup.2 are absent, Xaa.sup.3
is the N-terminus and is therefore either Ac or free NH2. Further,
in at least one embodiment Xaa.sup.3 is an N-terminal linker moiety
selected from the group consisting of DIG, PEG13, PEG25, PEG1K,
PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, IDA-Boc,
IDA-Isovaleric acid, Triazine, Triazine-Boc, Isophthalic acid,
1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid, glutaric
acid, Azelaic acid, Pimelic acid, Dodecanedioic acid, suitable
aliphatics, aromatics, heteroaromatics, and polyethylene glycol
based linkers having a molecular weight from approximately 400 Da
to approximately 20,000 kDa. In other embodiments Xaa3 is absent.
Preferred Xaa.sup.3 groups for modifying the N-terminus of the
compounds in the scope of the invention are Ac, Lys, dLys, NH2, and
a suitable linker moiety.
[0059] In some embodiments, Xaa.sup.4 is an amino acyl residue or
analog selected from the group consisting of Cys, Pen, Asp, Glu,
hGlu, .beta.-Asp, .beta.-Glu, Lys, homo-Lys, Orn, Dap, and Dab.
When Xaa.sup.10 is Lys, homo-Lys, Orn, Dap or Dab, suitable groups
for Xaa.sup.4 are Asp, Glu, hGlu. When Xaa.sup.10 is Asp, Glu,
hGlu, suitable groups for Xaa.sup.4 are Lys, homo-Lys, Orn, Dap,
and Dab. When Xaa.sup.4 and Xaa.sup.10 are either Cys or Pen, each
subunit of the dimer is cyclized though a disulfide bond between
Xaa.sup.4 and Xaa.sup.10. When Xaa.sup.4 is Lys, homo-Lys, Orn,
Dap, or Dab, and when Xaa.sup.10 is Asp, Glu, hGlu, each subunit of
the dimer is cyclized through an amide bond between Xaa.sup.4 and
Xaa.sup.10. Preferably, in one embodiment Xaa.sup.4 is Cys. In
another embodiment, preferably Xaa.sup.4 is Pen.
[0060] Xaa.sup.5 is an amino acyl residue or analog selected from
the group consisting of Gln, Asn, Asp, Pro, Gly, His, Ala, Ile,
Phe, Lys, Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, Thr, homo-Arg,
Dap, Dab, N-Me-Arg, Arg-(Me)sym, Arg-(me)asym, 4-Guan, Cit, Cav,
and suitable isostere replacements. Preferably, Xaa.sup.5 is
N-Me-Arg. In another embodiment, preferably Xaa.sup.5 is Arg.
[0061] Xaa.sup.6 is an amino acyl residue or analog selected from
the group consisting of Ser, Gln, Asn, Asp, Pro, Gly, His, Ala,
Ile, Phe, Lys, Arg, Asn, Glu, Leu, Val, Tye, Trp, Met, and suitable
isostere replacements. Preferably, Xaa.sup.6 is Ser, Gly.
[0062] Xaa.sup.7 is an amino acyl residue or analog selected from
the group consisting of Asp, N-Me-Asp, and a suitable isostere
replacement for Asp. Preferably, Xaa.sup.7 is Asp.
[0063] Xaa.sup.8 is an amino acyl residue or analog selected from
the group consisting of Thr, Gln, Ser, Asn, Asp, Pro, Gly, His,
Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tye, Trp, Met, and N-Methyl
amino acids including N-Me-Thr and a suitable isostere replacement
for Thr. Preferably, Xaa.sup.8 is Thr.
[0064] Xaa.sup.9 is an amino acyl residue or analog selected from
the group consisting of Gln, Asn, Asp, Pro, Gly, Ala, Phe, Leu,
Asn, Glu, Val, homo-Leu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala,
N-Me-Leu, amino acids with hydrophobic side chains, and suitable
isostere replacements. Preferably, Xaa.sup.9 is Leu.
[0065] Xaa.sup.10 is an amino acyl residue selected from the group
consisting of Cys, Asp, Pen, Lys, homo-Lys, Orn, Glu, Dap, and Dab.
In some embodiments, Xaa.sup.10 is selected from the group
consisting of Asp, Glu, and hGlu, when Xaa.sup.4 is Lys, Dap, Dab,
homo-Lys, or Orn. In other embodiments, Xaa.sup.10 selected from
the group consisting of Lys, homo-Lys, Orn, Dap, or Dab when
Xaa.sup.4 is Asp, Glu, or hGlu. In at least one embodiment,
Xaa.sup.10 is Pen. When Xaa.sup.10 and Xaa.sup.4 are both either
Cys or Pen, each subunit of the dimer is cyclized through a
disulfide bond between Xaa.sup.4 and Xaa.sup.10. When Xaa.sup.10 is
Asp, Glu, or hGlu, and when Xaa.sup.4 is Lys, homo-Lys, Orn, Dap,
or Dab, each subunit of the dimer is cyclized through an amide bond
between Xaa.sup.4 and Xaa.sup.10. When Xaa.sup.11 is absent and
Xaa.sup.10 is the C-terminus of the subunit, Xaa.sup.10 is either
COOH or amide CONH2. Preferably, in one embodiment Xaa.sup.10 is
Pen. In another embodiment, Xaa.sup.10 is preferably Cys.
[0066] Xaa.sup.11 is an amino acyl residue selected from the group
consisting of Gly, Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr,
Lys, Trp, Tyr, CONH.sub.2, COOH, His, Glu, Ser, Arg, Pro, Phe, Sar,
1Nal, 2Nal, hPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab,
Dab(Ac), Orn, D-Orn, N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip,
Ala(3,3diphenyl), Biphenyl-Ala, aromatic ring substituted Phe,
aromatic ring substituted Trp, aromatic ring substituted His,
hetero aromatic amino acids, N-Me-Lys, N-Me-Lys(Ac), 4-Me-Phe, and
corresponding D-amino acids and suitable isostere replacements.
When Xaa.sup.12 and Xaa.sup.13 are absent, and Xaa.sup.11 is the
C-terminus of the subunit, Xaa.sup.11 is either COOH or CONH.sub.2.
In at least one embodiment, Xaa.sup.11 and Xaa.sup.12 are absent.
When Xaa.sup.12 and Xaa.sup.13 are absent, Xaa.sup.11 is a linker
moiety selected from the group consisting of DIG, PEG13, PEG25,
PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, IDA-Boc,
IDA-Isovaleric acid, Triazine, Triazine-Boc, Isophthalic acid,
1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid, glutaric
acid, Azelaic acid, Pimelic acid, Dodecanedioic acid, suitable
aliphatics, aromatics, heteroaromatics, and polyethylene glycol
based linkers having a molecular weight from approximately 400 Da
to approximately 40,000 Da. Preferably, Xaa.sup.11 is Trp. In other
embodiments Xaa.sup.11 is selected from group consisting of Lys,
dLys, and N-Me-Lys.
[0067] Xaa.sup.12 is an amino acyl residue selected from the group
consisting of Glu, Lys, COOH, CONH.sub.2, Gln, Pro, Gly, His, Ala,
Ile, Phe, Lys, Arg, Leu, Val, Tye, Trp, Met, Gla, Ser, Asn, Dap,
Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap,
D-Dab, suitable isosters, and corresponding D-amino acids. When
Xaa.sup.13 to Xaa.sup.15 are absent, and Xaa.sup.12 is the
C-terminus of the subunit, Xaa.sup.12 is either COOH or CONH.sub.2.
In some embodiments Xaa.sup.12 is absent. Preferably, Xaa.sup.12 is
selected from the group consisting of Lys, dLys, and N-Me-Lys.
[0068] Xaa.sup.13 is an amino acyl residue selected from the group
consisting of Gln, Pro, Gly, His, Ala, Be, Phe, Lys, Arg, Leu, Val,
Tye, Trp, Met, Glu, Gla, Ser, Asn, Dap, Dab, Orn, D-Orn, N-Me-Orn,
N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab, COOH, CONH.sub.2,
suitable isosteres, and corresponding D-amino acids. In some
embodiments, when Xaa.sup.14 and Xaa.sup.15 are absent, Xaa.sup.12
is the C-terminus and Xaa.sup.13 comprises a linker moiety selected
from the group consisting of DIG, DIG-OH, PEG13, PEG25, PEG1K,
PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, IDA-Boc,
IDA-Isovaleric acid, Triazine, Triazine-Boc, Isophthalic acid,
1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid, glutaric
acid, Azelaic acid, Pimelic acid, Dodecanedioic acid, suitable
aliphatics, aromatics, heteroaromatics, and polyethylene glycol
based linkers having a molecular weight from approximately 400 Da
to approximately 40,000 kDa. In other embodiments, the dimer
molecule comprises an N-terminal linker, and therefore when
Xaa.sup.14 and Xaa.sup.15 are absent, Xaa.sup.13 is the C-terminus
and is therefore either COOH, or CONH.sub.2. In at least one
embodiment, Xaa.sup.13 is Lys. In other embodiments, Xaa.sup.13 is
absent. In at least one embodiment, Xaa.sup.14 is a C-terminal
linker. Preferred Xaa.sup.13 groups for modifying the C-terminus
are free NH.sub.2, COOH, CONH.sub.2 and a suitable linker
moiety.
[0069] Xaa.sup.14 is an amino acyl residue selected from the group
consisting of natural amino acids, COOH, CONH.sub.2, suitable
isostere replacements, corresponding D-amino acids, and
corresponding N-Methyl amino acids. In some embodiments, when
Xaa.sup.15 is absent, Xaa.sup.13 is the C-terminus and Xaa.sup.14
comprises a linker moiety selected from the group consisting of
DIG, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA,
IDA-Palm, IDA-Boc, IDA-Isovaleric acid, Triazine, Triazine-Boc,
Isophthalic acid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic
acid, glutaric acid, Azelaic acid, Pimelic acid, Dodecanedioic
acid, suitable aliphatics, aromatics, heteroaromatics, and
polyethylene glycol based linkers having a molecular weight from
approximately 400 Da to approximately 40,000 Da. In other
embodiments, the dimer molecule comprises an N-terminal linker, and
therefore when Xaa.sup.15 is absent Xaa.sup.14 is the C-terminus
and is therefore either COOH, or CONH.sub.2. In at least one
embodiment, Xaa.sup.14 is absent. In at least one embodiment,
Xaa.sup.14 is a C-terminal linker. Preferred Xaa.sup.14 groups for
modifying the C-terminus are COOH, CONH.sub.2 or a suitable linker
moiety.
[0070] Xaa.sup.15 is a linker moiety selected from the group
consisting of DIG, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K,
PEG5K, IDA, IDA-Palm, IDA-Boc, IDA-Isovaleric acid, Triazine,
Triazine-Boc, Isophthalic acid, 1,3-phenylenediacetic acid,
1,4-phenylenediacetic acid, glutaric acid, Azelaic acid, Pimelic
acid, Dodecanedioic acid, suitable aliphatics, aromatics,
heteroaromatics, and polyethylene glycol based linkers having a
molecular weight from approximately 400 Da to approximately 40,000
Da. In at least one embodiment, Xaa.sup.15 is absent. Preferably
Xaa.sup.15 is DIG.
[0071] Some embodiments of the present invention further include a
DRP homodimer or heterodimer molecule, wherein each subunit of the
dimer molecule comprises an amino acid sequence represented by at
least one of SEQ ID NOs: 1-136. Other embodiments comprise a DRP
homodimer or heterodimer molecule, wherein each subunit of the
dimer molecule comprises an amino acid sequence comprising an
N-methylated arginine residue, as represented by at least one of
SEQ ID NOs: 1-38, 49, 57-71, 75-117 and 124-136. Further, some
embodiments of the present invention comprise a DRP homodimer or
hetereodimer molecule, wherein each subunit of the dimer molecule
is cyclized through a disulfide bond, as represented by at least
one of SEQ ID NOs: 1-136. In other embodiments, a DRP homo- or
heterodimer molecule is provided, wherein each subunit of the dimer
molecule is cyclized through an amide bond, as represented by at
least one of SEQ ID NOs: 1 and 2, wherein Xaa.sup.4 and Xaa.sup.10
are selected from the group consisting of Lys, homo-Lys, Orn Dap or
Dab, Asp, Glu and hGlu.
Dimer Structure and Biological Activity
[0072] The present invention provides various novel antagonist
disulfide peptide dimers. These compounds have been tested to more
clearly characterize the increased affinity for .alpha.4.beta.7
binding, increased selectivity against .alpha.4.beta.1, and
increased stability in simulated intestinal fluid (SIF). These
novel antagonist molecules demonstrate high binding affinity with
.alpha.4.beta.7, thereby preventing binding between .alpha.4.beta.7
and the MAdCAM ligand. Accordingly, these antagonist peptides have
shown to be effective in eliminating and/or reducing the
inflammation process in various experiments.
[0073] The present invention thus provides various dimer peptide
compounds which bind or associate with the .alpha.4.beta.7
integrin, in serum and SIF, to disrupt or block binding between
.alpha.4.beta.7 and the MAdCAM ligand. The various peptide
compounds of the invention may be constructed solely of natural
amino acids. Alternatively, the peptide compounds may include
non-natural amino acids including, but not limited to, modified
amino acids. Modified amino acids include natural amino acids which
have been chemically modified to include a group, groups, or
chemical moiety not naturally present on the amino acid. The
peptide compounds of the invention may additionally include D-amino
acids. Still further, the peptide compounds of the invention may
include amino acid analogs.
[0074] Some antagonist disulfide dimers have been shown to be
gastrointestinal stable and provide high levels of specificity and
affinity for the .alpha.4.beta.7 integrin. Some implementations of
the present invention provide a disulfide dimer comprising a
half-life of greater than 60 minutes when exposed to simulated
intestinal fluids (SIF). Some implementations further provide a DRP
comprising a half-life from approximately 1 minute to approximately
63 minutes.
[0075] The compounds of the present invention are homo- or
heterodimers formed by linking two subunit monomers at their C- or
N-termini. Dimerization of the monomer subunits represented by SEQ
ID NOs: 1-136 demonstrate increased potency over their
non-dimerized, monomer analogs. Some dimer compounds of the present
invention demonstrated further increased potency as a result of
substituting various natural amino acyl residues with N-methylated
analog residues. For example, SEQ ID NOs.: 1-38, 49, 57-71, 75-117
and 124-136 represent subunit monomers sequences that were
substituted with N-Me-Arg. Further still, some dimer compounds of
the present invention comprise monomer subunits that undergo
independent cyclization, whereby the cyclized structures
demonstrate increased stability over their non-cyclized monomer and
dimer analogs. Specific examples and data illustrating these
improvements is provided in FIGS. 3 and 4.
[0076] Referring now to FIG. 3, a chart is provided which includes
various data illustrating increased stability for various
non-limiting sample homodimer molecules in accordance with the
instant invention. Simulated Intestinal Fluid (SIF) Stability
assays were performed for all of the monomer peptides, and their
respective homodimer molecules, represented by SEQ ID NOs: 39-136.
A selective sampling of these results is provided in FIG. 3.
[0077] According to the protocols discussed herein, applicant
successfully synthesized, purified and dimerized all of the
integrin antagonist dimer molecules represented by SEQ ID NOs:
39-139 to form homodimers.
[0078] Dimerization of the monomer disulfide peptide subunits
generally demonstrated increase stability, as compared to the
monomer disulfide subunit peptides. Further, substitutions at
arginine with N-Me-Arg increased half-life substantially in SIF, as
demonstrated by SEQ ID NOs: 57 when compared to SEQ ID NO: 39 with
Arg. In some embodiments, substitution of Cys with Penicillamine
(Pen) increased stability significantly in simulated intestinal
fluids (SIF), as demonstrated by SEQ ID NOs: 82, 102 and 121 when
compared to SEQ ID NO: 39 with Cys. The substitution of Cys with
Pen also increased stability under reduced conditions (DTT)
indication improved gastric stability.
[0079] Referring now to FIG. 4, a chart is provided which includes
various data illustrating increased potency and selectivity for
various non-limiting sample homodimer molecules in accordance with
the instant invention. Potency and selectivity assays were
performed for all of the monomer peptides, and their respective
homodimer molecules, represented by SEQ ID NOs: 39-136. A selective
sampling of these results is provided in FIG. 4 wherein the
homodimer peptides are represented by Samples 2, 4, 5, 7, 9, 11,
13, 15, 17-19 and 21, and the respective monomer subunits molecules
are represented by Samples 1, 3, 6, 8, 10, 12, 14, 16 and 20.
Through dimerization, significant improvement in potency achieved
for .alpha.4.beta.7 in ELISA as well as cell adhesion assay. In
addition, dimerization lead to significant improvement achieved in
selectivity against .alpha.4.beta.1 through improved potency for
.alpha.4.beta.7. The peptides also have low efficacy for
.alpha.4.beta.1 when compared to .alpha.4.beta.7 indicating
selectivity against .alpha.4.beta.7.
[0080] According to the protocols discussed herein, applicant
successfully synthesized, purified and dimerized all of the
integrin antagonist dimer molecules represented by SEQ ID NOs:
39-136 to form homodimers. Each of these molecules were subjected
to an .alpha.4.beta.7-MAdCAM Competition ELISA assay, an
.alpha.4.beta.1-VCAM Competition ELISA assay, an
.alpha.4.beta.7-MadCAM cell adhesion assay. For many sequences,
these assays were also performed on both the monomer subunit and
dimer molecules. A small sampling of these results is provided in
FIG. 4.
[0081] Dimerization of the monomer disulfide peptides subunits
generally demonstrated increased affinity for .alpha.4.beta.7
and/or decreased affinity for .alpha.4.beta.1 leading to increased
selectivity against .alpha.4.beta.1, as compared to the monomer
disulfide subunit peptides.
[0082] Upon C and N-terminal dimerization a significant improvement
in potency for .alpha.4.beta.7 was also observed. In addition
dimerization also lead to loss of potency for .alpha.4.beta.1
leading to increased selectivity for .alpha.4.beta.7 in ELISA and
cell adhesion assays. When Arg is replaced with N-Me-Arg, a
significant improvement in potency for .alpha.4.beta.7 in both
ELISA as well as in cell adhesion assays.
Compositions
[0083] As discussed above, integrins are heterodimers that function
as cell adhesion molecules. The .alpha.4 integrins, .alpha.4.beta.1
and .alpha.4.beta.7, play essential roles in lymphocyte migration
throughout the gastrointestinal tract. They are expressed on most
leukocytes, including B and T lymphocytes, monocytes, and dendritic
cells, where they mediate cell adhesion via binding to their
respective primary ligands, namely vascular cell adhesion molecule
(VCAM) and mucosal addressin cell adhesion molecule (MAdCAM). VCAM
and MAdCAM differ in binding specificity, in that VCAM binds both
.alpha.4.beta.1 and .alpha.4.beta.7, while MAdCAM is highly
specific for .alpha.4.beta.7.
[0084] Differences in the expression profiles of VCAM and MAdCAM
provide the most convincing evidence of their role in inflammatory
diseases. Both are constitutively expressed in the gut; however,
VCAM expression extends into peripheral organs, while MAdCAM
expression is confined to organs of the gastrointestinal tract. In
addition, elevated MAdCAM expression in the gut has now been
correlated with several gut-associated inflammatory diseases,
including Crohn's disease, ulcerative colitis, and hepatitis C.
[0085] The compounds of the invention, including but not limited to
those specified in the examples, possess integrin-antagonist
activity. In one embodiment, the condition or medical indication
comprises at least one of Inflammatory Bowel Disease (IBD),
ulcerative colitis, Crohn's disease, Celiac disease (nontropical
Sprue), enteropathy associated with seronegative arthropathies,
microscopic or collagenous colitis, eosinophilic gastroenteritis,
radio- and chemotherapy, or pouchitis resulting after
proctocolectomy and ileoanal anastomosis and various forms of
gastrointestinal cancer, osteoporosis, arthritis, multiple
sclerosis, chronic pain, weight gain, and depression. In another
embodiment, the condition is pancreatitis, insulin-dependent
diabetes mellitus, mastitis, cholecystitis, cholangitis,
pericholangitis, chronic bronchitis, chronic sinusitis, asthma or
graft versus host disease. In addition, these compounds may be
useful in the prevention or reversal of these diseases when used in
combination with currently available therapies, medical procedures,
and therapeutic agents.
[0086] The compounds of the invention may be used in combination
with other compositions and procedures for the treatment of
disease. Additionally, the compounds of the present invention may
be combined with pharmaceutically acceptable excipients, and
optionally sustained-release matrices, such as biodegradable
polymers, to form therapeutic compositions.
Methods of Treatment
[0087] In some embodiments, the present invention provides a method
for treating an individual afflicted with a condition or indication
characterized by integrin binding, wherein the method comprises
administering to the individual an integrin antagonist dimer
molecule according to Formulas (I) or (II). In one embodiment, a
method is provided for treating an individual afflicted with a
condition or indication characterized by inappropriate trafficking
of cells expressing .alpha.4.beta.7 to tissues comprising cells
expressing MAdCAM, comprising administering to the individual an
.alpha.4.beta.7-antagonist dimer molecule according to at least one
of Formula (I) and Formula (II) in an amount sufficient to inhibit
(partially or fully) the trafficking of cells expressing
.alpha.4.beta.7 to tissues comprising cells expressing MAdCAM.
[0088] In some embodiments, the present invention provides a method
whereby a pharmaceutical composition comprising an integrin
antagonist dimer molecule according to Formula (I) is administered
to a patient as a first treatment. In another embodiment, the
method further comprises administering to the subject a second
treatment. In another embodiment, the second treatment is
administered to the subject before and/or simultaneously with
and/or after the pharmaceutical composition is administered to the
subject. In other embodiment, the second treatment comprises an
anti-inflammatory agent. In another embodiment, the second
pharmaceutical composition comprises an agent selected from the
group consisting of non-steroidal anti-inflammatory drugs,
steroids, and immune modulating agents. In another embodiment, the
method comprises administering to the subject a third
treatment.
[0089] In one embodiment, a method is provided for treating an
individual afflicted with a condition or indication characterized
by .alpha.4.beta.7 integrin binding, wherein the method comprises
administering to the individual an effective amount of an
.alpha.4.beta.7 integrin antagonist dimer molecule according to at
least one of Formula (I) and Formula (II). In some instances, an
.alpha.4.beta.7 integrin antagonist dimer molecule according to at
least one of Formula (I) and Formula (II) having high specificity
for .alpha.4.beta.7 is administered to an individual as part of a
therapeutic treatment for a condition or indication characterized
by .alpha.4.beta.7 integrin binding. Some embodiments of the
present invention further provide a method for treating an
individual with an .alpha.4.beta.7 integrin antagonist dimer
molecule that is suspended in a sustained-release matrix. A
sustained-release matrix, as used herein, is a matrix made of
materials, usually polymers, which are degradable by enzymatic or
acid-base hydrolysis or by dissolution. Once inserted into the
body, the matrix is acted upon by enzymes and body fluids. A
sustained-release matrix desirably is chosen from biocompatible
materials such as liposomes, polylactides (polylactic acid),
polyglycolide (polymer of glycolic acid), polylactide co-glycolide
(copolymers of lactic acid and glycolic acid) polyanhydrides,
poly(ortho)esters, polypeptides, hyaluronic acid, collagen,
chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,
polysaccharides, nucleic acids, polyamino acids, amino acids such
as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene, polyvinylpyrrolidone and silicone. A preferred
biodegradable matrix is a matrix of one of either polylactide,
polyglycolide, or polylactide co-glycolide (co-polymers of lactic
acid and glycolic acid).
[0090] In some aspects, the invention provides a pharmaceutical
composition for oral delivery. The various embodiments and dimer
compositions of the instant invention may be prepared for oral
administration according to any of the methods, techniques, and/or
delivery vehicles described herein. Further, one having skill in
the art will appreciate that the dimer compositions of the instant
invention may be modified or integrated into a system or delivery
vehicle that is not disclosed herein, yet is well known in the art
and compatible for use in oral delivery of small dimer peptide
molecules.
[0091] Oral dosage forms or unit doses compatible for use with the
dimer peptides of the present invention may include a mixture of
dimer peptide active drug components, and nondrug components or
excipients, as well as other non-reusable materials that may be
considered either as an ingredient or packaging. Oral compositions
may include at least one of a liquid, a solid, and a semi-solid
dosage forms. In some embodiments, an oral dosage form is provided
comprising an effective amount of dimer peptide according to
Formula (I), wherein the dosage form comprises at least one of a
pill, a tablet, a capsule, a gel, a paste, a drink, and a syrup. In
some instances, an oral dosage form is provided that is designed
and configured to achieve delayed release of the peptide dimer in
the subjects small intestine.
[0092] In one embodiment, an oral pharmaceutical composition
according to Formula (I) comprises an enteric coating that is
designed to delay release of the peptide dimer in the small
intestine. In at least some embodiments, a pharmaceutical
composition is provided which comprises a peptide dimer compound
according to Formula (I) and a protease inhibitor, such as
aprotinin, in a delayed release pharmaceutical formulation. In some
instances it is preferred that a pharmaceutical composition of the
instant invention comprise an enteric coat that is soluble in
gastric juice at a pH of about 5.0 or higher. In at least one
embodiment, a pharmaceutical composition is provided comprising an
enteric coating comprising a polymer having dissociable carboxylic
groups, such as derivatives of cellulose, including
hydroxypropylmethyl cellulose phthalate, cellulose acetate
phthalate and cellulose acetate trimellitate and similar
derivatives of cellulose and other carbohydrate polymers.
[0093] In one embodiment, a pharmaceutical composition according to
Formula (I) is provided in an enteric coating, the enteric coating
being designed to protect and release the pharmaceutical
composition in a controlled manner within the subjects lower
gastrointestinal system, and to avoid systemic side effects. In
addition to enteric coatings, the dimer peptides of the instant
invention may be encapsulated, coated, engaged or otherwise
associated within any compatible oral drug delivery system or
component. For example, in some embodiments a dimer peptide of the
present invention is provided in a lipid carrier system comprising
at least one of polymeric hydrogels, nanoparticles, microspheres,
micelles, and other lipid systems.
[0094] To overcome peptide degradation in the small intestine, some
implementations of the present invention comprise a hydrogel
polymer carrier system in which a peptide dimer in accordance with
the present invention is contained, whereby the hydrogel polymer
protect the peptide dimer from proteolysis in the small intestine.
The peptide dimers of the present invention may further be
formulated for compatible use with a carrier system that is
designed to increase the dissolution kinetics and enhance
intestinal absorption of the dimer peptides. These methods include
the use of liposomes, micelles and nanoparticles to increase GI
tract permeation of peptides.
[0095] Various bioresponsive systems may also be combined with one
or more peptide dimers of the present invention to provide a
pharmaceutical agent for oral delivery. In some embodiments, a
peptide dimer of the instant invention is used in combination with
a bioresponsive system, such as hydrogels and mucoadhesive polymers
with hydrogen bonding groups (e.g., PEG, poly(methacrylic) acid
[PMAA], cellulose, Eudragit.RTM., chitosan and alginate) to provide
a therapeutic agent for oral administration. Other embodiments
include a method for optimizing or prolonging drug residence time
for a peptide dimer disclosed herein, wherein the surface of the
peptide dimer surface is modified to comprise mucoadhesive
properties through hydrogen bonds, polymers with linked mucins
or/and hydrophobic interactions. These modified dimer molecules may
demonstrate increase drug residence time within the subject, in
accordance with a desired feature of the invention. Moreover,
targeted mucoadhesive systems may specifically bind to receptors at
the enterocytes and M-cell surfaces, thereby further increasing the
uptake of particles containing the dimer peptide.
[0096] Other embodiments comprise a method for oral delivery of a
dimer peptide according to Formula (I), wherein the dimer peptide
is used in combination with permeation enhancers that promote the
transport of the dimer peptides across the intestinal mucosa by
increasing paracellular or transcellular permeation. For example,
in one embodiment a permeation enhancer is combined with a dimer
peptide according to Formula (I), wherein the permeation enhancer
comprises at least one of a long-chain fatty acid, a bile salt, an
amphiphilic surfactant, and a chelating agent. In one embodiment, a
permeation enhancer comprising sodium N-[hydroxybenzoyl)amino]
caprylate is used to form a weak noncovalent association with the
dimer peptide of the instant invention, wherein the permeation
enhancer favors membrane transport and further dissociation once
reaching the blood circulation. In another embodiment, a peptide
dimer of the present invention is conjugated to oligoarginine,
thereby increasing cellular penetration of the dimer peptides into
various cell types. Further, in at least one embodiment a
noncovalent bond is provided between a dimer peptide according to
Formula (I) and a permeation enhancer selected from the group
consisting of a cyclodextrin (CD) and a dendrimers, wherein the
permeation enhancer reduces peptide aggregation and increasing
stability and solubility for the peptide dimer molecule.
[0097] Other embodiments of the invention provide a method for
treating an individual with an .alpha.4.beta.7 integrin antagonist
dimer molecule having an increased half-life. In one aspect, the
present invention provides an integrin antagonist dimer molecule
having a half-life of at least several hours to one day in vitro or
in vivo (e.g., when administered to a human subject) sufficient for
daily (q.d.) or twice daily (b.i.d.) dosing of a therapeutically
effective amount. In another embodiment, the dimer molecule has a
half-life of three days or longer sufficient for weekly (q.w.)
dosing of a therapeutically effective amount. Further, in another
embodiment the dimer molecule has a half-life of eight days or
longer sufficient for bi-weekly (b.i.w.) or monthly dosing of a
therapeutically effective amount. In another embodiment, the dimer
molecule is derivatized or modified such that is has a longer
half-life as compared to the underivatized or unmodified dimer
molecule. In another embodiment, the dimer molecule contains one or
more chemical modifications to increase serum half-life.
[0098] When used in at least one of the treatments or delivery
systems described herein, a therapeutically effective amount of one
of the compounds of the present invention may be employed in pure
form or, where such forms exist, in pharmaceutically acceptable
salt form. As used herein, a "therapeutically effective amount" of
the compound of the invention is meant to describe a sufficient
amount of the peptide dimer compound to treat an integrin-related
disease, (for example, to reduce inflammation associated with IBD)
at a desired benefit/risk ratio applicable to any medical
treatment. It will be understood, however, that the total daily
usage of the compounds and compositions of the present invention
will be decided by the attending physician within the scope of
sound medical judgment. The specific therapeutically effective dose
level for any particular patient will depend upon a variety of
factors including: a) the disorder being treated and the severity
of the disorder; b) activity of the specific compound employed; c)
the specific composition employed, the age, body weight, general
health, sex and diet of the patient; d) the time of administration,
route of administration, and rate of excretion of the specific
compound employed; e) the duration of the treatment; f) drugs used
in combination or coincidental with the specific compound employed,
and like factors well known in the medical arts. For example, it is
well within the skill of the art to start doses of the compound at
levels lower than those required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved.
[0099] Alternatively, a compound of the present invention may be
administered as pharmaceutical compositions containing the compound
of interest in combination with one or more pharmaceutically
acceptable excipients. A pharmaceutically acceptable carrier or
excipient refers to a non-toxic solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type. The compositions may be administered parenterally,
intracisternally, intravaginally, intraperitoneally, intrarectally,
topically (as by powders, ointments, drops, suppository, or
transdermal patch), rectally, or buccally. The term "parenteral" as
used herein refers to modes of administration which include
intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous, intradermal and intraarticular injection and
infusion.
[0100] Pharmaceutical compositions for parenteral injection
comprise pharmaceutically acceptable sterile aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, as well as
sterile powders for reconstitution into sterile injectable
solutions or dispersions just prior to use. Examples of suitable
aqueous and nonaqueous carriers, diluents, solvents or vehicles
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), carboxymethylcellulose
and suitable mixtures thereof, vegetable oils (such as olive oil),
and injectable organic esters such as ethyl oleate. Proper fluidity
may be maintained, for example, by the use of coating materials
such as lecithin, by the maintenance of the required particle size
in the case of dispersions, and by the use of surfactants.
[0101] These compositions may also contain adjuvants such as
preservative, wetting agents, emulsifying agents, and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents such as
sugars, sodium chloride, and the like. Prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption, such as aluminum
monostearate and gelatin.
[0102] Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide, poly(orthoesters), poly(anhydrides), and
(poly)glycols, such as PEG. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Depot injectable formulations
are also prepared by entrapping the drug in liposomes or
microemulsions which are compatible with body tissues.
[0103] The injectable formulations may be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium just prior to use.
[0104] Topical administration includes administration to the skin
or mucosa, including surfaces of the lung and eye. Compositions for
topical lung administration, including those for inhalation and
intranasal, may involve solutions and suspensions in aqueous and
non-aqueous formulations and can be prepared as a dry powder which
may be pressurized or non-pressurized. In non-pressurized powder
compositions, the active ingredient in finely divided form may be
used in admixture with a larger-sized pharmaceutically acceptable
inert carrier comprising particles having a size, for example, of
up to 100 micrometers in diameter. Suitable inert carriers include
sugars such as lactose.
[0105] Alternatively, the composition may be pressurized and
contain a compressed gas, such as nitrogen or a liquified gas
propellant. The liquified propellant medium and indeed the total
composition is preferably such that the active ingredient does not
dissolve therein to any substantial extent. The pressurized
composition may also contain a surface active agent, such as a
liquid or solid non-ionic surface active agent or may be a solid
anionic surface active agent. It is preferred to use the solid
anionic surface active agent in the form of a sodium salt.
[0106] A further form of topical administration is to the eye. A
compound of the invention is delivered in a pharmaceutically
acceptable ophthalmic vehicle, such that the compound is maintained
in contact with the ocular surface for a sufficient time period to
allow the compound to penetrate the corneal and internal regions of
the eye, as for example the anterior chamber, posterior chamber,
vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary,
lens, choroid/retina and sclera. The pharmaceutically acceptable
ophthalmic vehicle may, for example, be an ointment, vegetable oil
or an encapsulating material. Alternatively, the compounds of the
invention may be injected directly into the vitreous and aqueous
humour.
[0107] Compositions for rectal or vaginal administration are
preferably suppositories which may be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at room temperature but liquid at
body temperature and therefore melt in the rectum or vaginal cavity
and release the active compound.
[0108] Compounds of the present invention may also be administered
in the form of liposomes. As is known in the art, liposomes are
generally derived from phospholipids or other lipid substances.
Liposomes are formed by mono- or multi-lamellar hydrated liquid
crystals that are dispersed in an aqueous medium. Any non-toxic,
physiologically acceptable and metabolizable lipid capable of
forming liposomes can be used. The present compositions in liposome
form can contain, in addition to a compound of the present
invention, stabilizers, preservatives, excipients, and the like.
The preferred lipids are the phospholipids and the phosphatidyl
cholines (lecithins), both natural and synthetic. Methods to form
liposomes are known in the art.
[0109] Total daily dose of the compositions of the invention to be
administered to a human or other mammal host in single or divided
doses may be in amounts, for example, from 0.0001 to 300 mg/kg body
weight daily and more usually 1 to 300 mg/kg body weight.
Non-Invasive Detection of Intestinal Inflammation
[0110] The peptides of the invention may be used for detection,
assessment and diagnosis of intestinal inflammation by microPET
imaging using an orally stable compound of Formula (I) that is
further labeled with at least one of a chelating group and a
detectable label as part of a non-invasive diagnostic procedure. In
one embodiment, an integrin antagonist dimer molecule is conjugated
with a bifunctional chelator to provide an orally stable dimer
molecule. In another embodiment, an integrin antagonist dimer
molecule is radiolabeled to provide an orally stable dimer
molecule. The orally stable, chelated or radiolabeled dimer
molecule is then administered to a subject orally or rectally. In
one embodiment, the orally stable dimer molecule is included in
drinking water. Following uptake of the dimer molecules, microPET
imaging may be used to visualize inflammation throughout the
subject's bowels and digestive track.
Synthesis of Peptide Subunits
[0111] The monomer peptide subunits of the present invention may be
synthesized by many techniques that are known to those skilled in
the art. novel and unique monomer subunits were synthesized,
purified, and dimerized using the techniques provided herein.
[0112] The peptides of the present invention were synthesized using
the Merrifield solid phase synthesis techniques on Protein
Technology's Symphony multiple channel synthesizer. The peptides
were assembled using HBTU
(O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate)-
, Diisopropylethylamine (DIEA) coupling conditions. Rink Amide MBHA
resin (100-200 mesh, 0.57 mmol/g) is used for peptide with
C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected
amino acid is used for peptide with C-terminal acids. The coupling
reagents (HBTU and DIEA premixed) were prepared at 100 mmol
concentration. Similarly amino acids solutions were prepared at 100
mmol concentration. The peptides were assembled using standard
Symphony protocols.
Assembly
[0113] The peptide sequences were assembled as follows: Resin (250
mg, 0.14 mmol) in each reaction vial was washed twice with 4 ml of
DMF followed by treatment with 2.5 ml of 20% 4-methyl piperidine
(Fmoc de-protection) for 10 min. The resin was then filtered and
washed two times with DMF (4 ml) and re-treated with N-methyl
piperifine for additional 30 minutes. The resin was again washed
three times with DMF (4 ml) followed by addition 2.5 ml of amino
acid and 2.5 ml of HBTU-DIEA mixture. After 45 min of frequent
agitations, the resin was filtered and washed three times with DMF
(4 ml each). For a typical peptide of the present invention, double
couplings were performed for first 25 amino acid, and triple
couplings were performed for the remaining residues. After
completing the coupling reaction, the resin was washed three times
with DMF (4 ml each) before proceeding to the next amino acid
coupling.
Cleavage
[0114] Following completion of the peptide assembly, the peptide
was cleaved from the resin by treatment with cleavage reagent, such
as reagent K (82.5% trigluoroacetic acid, 5% water, 5% thioanisole,
5% phenol, 2.5% 1,2-ethanedithiol). The cleavage reagent was able
to successfully cleave the peptide from the resin, as well as all
remaining side chain protecting groups.
[0115] The cleaved were precipitated in cold diethyl ether followed
by two washings with ethyl ether. The filtrate was poured off and a
second aliquot of cold ether was added, and the procedure repeated.
The crude peptide was dissolved in a solution of acetonitrile:water
(7:3 with 1% TFA) and filtered. The quality of linear peptide was
then verified using electrospray ionization mass spectrometry
(ESI-MS) (Micromass/Waters ZQ) before being purified.
Disulfide Bond Formation via Oxidation
[0116] 50 mg of crude, cleaved peptide was dissolved in 20 ml of
water:acetonitrile. Saturated Iodine in acetic acid was then added
drop wise with stifling until yellow color persisted. The solution
was stirred for 15 minutes and the reaction was monitored with
analytic HPLC and LCMS. When the reaction was completed, solid
ascorbic acid was added until the solution became clear. The
solvent mixture was then purified by first being diluted with water
and then loaded onto a reverse phase HPLC machine (Luna C18
support, 10 u, 100 A, Mobile phase A: water containing 0.1% TFA,
mobile phase B: Acetonitrile (ACN) containing 0.1% TFA, gradient
began with 5% B, and changed to 50% B over 60 minutes at a flow
rate of 15 ml/min). Fractions containing pure product were then
freeze-dried on a lyophilyzer.
Lactam Bond Formation
[0117] 100 mg of crude, cleaved peptide (approx. 0.12 mmol) was
dissolved in 100 ml of anhydrous dichloromethane. HOBt
(1-Hydroxybenzotriazole hydrate) (0.24 mmol, 2 equivalents) was
added followed by DIEA (N,N-Diisopropylethylamine) (1.2 mmol, 10
equivalents) and TBTU
(O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate) (0.24 mmol, 2 equivalents). The mixture was
stirred overnight and followed the reaction by HPLC. When the
reaction was completed, dichloromethane was evaporated and diluted
with water and Acetonitrile and then loaded onto a reverse phase
HPLC machine (Luna C18 support, 10 u, 100 A, Mobile phase A: water
containing 0.1% TFA, mobile phase B: Acetonitrile (ACN) containing
0.1% TFA, gradient began with 5% B, and changed to 50% B over 60
minutes at a flow rate of 15 ml/min). Fractions containing pure
product were then freeze-dried on a lyophilyzer.
Purification
[0118] Analytical reverse-phase, high performance liquid
chromatography (HPLC) was performed on a Gemini C18 column (4.6
mm.times.250 mm) (Phenomenex). Semi-Preparative reverse phase HPLC
was performed on a Gemini 10 .mu.m C18 column (22 mm.times.250 mm)
(Phenomenex) or Jupiter 10 .mu.m, 300 A .degree. C18 column (21.2
mm.times.250 mm) (Phenomenex). Separations were achieved using
linear gradients of buffer B in A (Mobile phase A: water containing
0.15% TFA, mobile phase B: Acetonitrile (ACN) containing 0.1% TFA),
at a flow rate of 1 mL/min (analytical) and 15 mL/min
(preparative). Separations were achieved using linear gradients of
buffer B in A (Mobile phase A: water containing 0.15% TFA, mobile
phase B: Acetonitrile (ACN) containing 0.1% TFA), at a flow rate of
1 mL/min (analytical) and 15 mL/min (preparative).
Linker Activation and Dimerization
[0119] Small Scale DIG Linker Activation Procedure: 5 mL of NMP was
added to a glass vial containing IDA diacid (304.2 mg, 1 mmol),
N-hydroxysuccinimide (NHS, 253.2 mg, 2.2 eq. 2.2 mmol) and a
stirring bar. The mixture was stirred at room temperature to
completely dissolve the solid starting materials.
N,N'-Dicyclohexylcarbodiimide (DCC, 453.9 mg, 2.2 eq., 2.2 mmol)
was then added to the mixture. Precipitation appeared within 10 min
and the reaction mixture was further stirred at room temperature
overnight. The reaction mixture was then filtered to remove the
precipitated dicyclohexylurea (DCU). The activated linker was kept
in a closed vial prior to use for dimerization. The nominal
concentration of the activated linker was approximately 0.20 M.
[0120] For dimerization using PEG linkers, there is no
pre-activation step involved. Commercially available pre-activated
bi-functional PEG linkers were used.
[0121] Dimerization Procedure: 2 mL of anhydrous DMF was added to a
vial containing peptide monomer (0.1 mmol). The pH of the peptide
was the adjusted to 8-9 with DIEA. Activated linker (IDA or PEG13,
PEG 25) (0.48 eq relative to monomer, 0.048 mmol) was then added to
the monomer solution. The reaction mixture was stirred at room
temperature for one hour. Completion of the dimerization reaction
was monitored using analytical HPLC. The time for completion of
dimerization reaction varied depending upon the linker. After
completion of reaction, the peptide was precipitated in cold ether
and centrifuged. The supernatant ether layer was discarded. The
precipitation step was repeated twice. The crude dimer was then
purified using reverse phase HPLC (Luna C18 support, 10 u, 100 A,
Mobile phase A: water containing 0.1% TFA, mobile phase B:
Acetonitrile (ACN) containing 0.1% TFA, gradient of 15% B and
change to 45% B over 60 min, flow rate 15 ml/min). Fractions
containing pure product were then freeze-dried on a
lyophilyzer.
Simulated Intestinal Fluid (SIF) Stability Assay
[0122] Studies were carried out in simulated intestinal fluid (SIF)
to evaluate gastric stability of the dimer molecules of the instant
invention. SIF was prepared by adding 6.8 g of monobasic potassium
phosphate and 10.0 g of pancreatin to 1.0 L of water. After
dissolution, the pH was adjusted to 6.8 using NaOH. DMSO stocks (2
mM) were first prepared for the test compounds. Aliquots of the
DMSO solutions were dosed into 6 individual tubes, each containing
0.5 mL of SIF, which had been pre-warmed to 37.degree. C.
[0123] The final test compound concentration was 20 .mu.M. The
vials were kept in a benchtop Thermomixer.RTM. for the duration of
the experiment. At each timepoint (0, 5, 10, 20, 40, and 60
minutes), 1.0 mL of acetonitrile containing 1% formic acid was
added to one vial to terminate the reaction. Samples were stored at
4.degree. C. until the end of the experiment. After the final
timepoint was sampled, the tubes were mixed and then centrifuged at
3,000 rpm for 10 minutes. Aliquots of the supernatant were removed,
diluted 1:1 into distilled water containing internal standard, and
analyzed by LCMS/MS. Percent remaining at each timepoint was
calculated based on the peak area response ratio of test to
compound to internal standard. Time 0 was set to 100 %, and all
later timepoints were calculated relative to time 0. Half-lives
were calculated by fitting to a first-order exponential decay
equation using GraphPad. A small sampling of the results of these
studies is provided and discussed in connection FIG. 3, above.
EXAMPLES
.alpha.4.beta.7-MAdCAM Competition ELISA
[0124] A nickel coated plate (Pierce #15442) was coated with
recombinant human integrin .alpha.4.beta.7 (R&D Systems
#5397-A30) at 800 ng/well and incubated at room temperature with
shaking for 1 hr. The solution was then remove by shaking and
blocked with assay buffer (50 Mm Tris-HCl pH 7.6, 150 mM NaCl, 1 mM
MnCl.sub.2, 0.05% Tween-20 and 0.5% BSA) at 250 ul/well. The plate
was then incubated at room temperature for 1 hr. Each well was
washed 3 times with wash buffer (50 mM Tris-HCl pH 7.6, 100 mM
NaCl, 1 mM MnCl.sub.2, 0.05% Tween-20). To each well was added 25
ul of a serial dilution (3-fold dilutions in assay buffer) of
peptides starting at 20 .mu.M. 25 ul of recombinant human MAdCAM-1
(R&D Systems #6056-MC) was then added to each well at a fixed
concentration 20 nM. The final starting peptide concentration was
10 .mu.M, and the final MAdCAM-1 concentration was 10 nM. The
plates were then incubated at room temperature for 1 hr to reach
binding equilibrium. The wells were then washed three times with
wash buffer. 50 ul of mouse anti-human IgG1-HRP (Invitrogen
#A10648) diluted in 1:2000 in assay buffer was then added to each
well. The wells were incubated at room temperature for 45 min with
shaking. The wells were then washed 3 times with wash buffer. 100
ul of TMB were then added to each well and closely observe during
development time. The reaction was stopped with 2N H.sub.2SO.sub.4
and absorbance was read at 450 nm.
.alpha.4.beta.1-VCAM Competition ELISA
[0125] A Nunc MaxiSorp plate was coated with rh VCAM-1/CD106 Fc
chimera (R&D #862-VC) at 400 ng/well in 50 ul per well in 1XPBS
and incubated overnight at 4.degree. C. The solution was removed by
shaking and then blocked with 250 ul of 1% BSA in 1XPBS per well.
The wells were then incubated at room temperature for 1 hr with
shaking. Each well was then washed once with wash buffer (50 mM
Tris-HCl pH 7.6, 100 mM NaCL, 1 mM MnCl.sub.2, 0.05% Tween-20). 25
ul of serial dilutions of peptides starting at 200 .mu.M in assay
buffer (Assay buffer: 50 mM Tris-HCl pH 7.6, 100 mM NaCl, 1 mM
MnCl.sub.2, 0.05% Tween-20) was added to each well. Additionally,
25 ul of .alpha.4.beta.1 (R&D Systems #5668-A4 ) was added to
each well at a fixed concentration of 120 nM. The final peptide and
.alpha.4.beta.1 concentrations were 100 .mu.M and 60 nM,
respectively. The plates were then incubated at 37.degree. C. for 2
hr. The solution was then removed by shaking and each well was
washed three times with wash buffer. 50 ul of 9F10 antibody at 4
ug/ml (purified mouse anti-human CD49d, BD Bioscience Cat #555502)
was then added to each well, and the plate was incubated at room
temperature for 1 hr with shaking. The solution was again removed
by shaking, and each well was washed three times with wash buffer.
50 ul of peroxidase-conjugated AffiniPure Goat anti-mouse IgG
(Jackson immune research cat #115-035-003) diluted in 1:5000 in
assay buffer was added to each well. The plate was incubated at
room temperature for 30 min with shaking. Each well was then washed
3 times with wash buffer. 100 ul of TMB was then added to each well
and closely observe during developing time. The reaction was
stepped with 2N H.sub.2SO.sub.4 and absorbance was read at 450
nm.
Example 3: .alpha.4.beta.7-MAdCAM Cell Adhesion Assay
[0126] RPMI 8866 cells (Sigma #95041316) are cultured in RPMI 1640
HEPES medium (Invitrogen #22400-089) supplemented with 10% serum
(Fetal Bovine Serum, Invitrogen #16140-071), 1 mM sodium pyruvate
(Invitrogen #11360-070), 2 mM L-glutamine (Invitrogen #25030-081)
and Penicillin-Streptomycin (Invitrogen #15140-122) at 100 units of
penicillin and 100 lug of streptomycin per ml. The cells are washed
two times in DMEM medium (ATCC #30-2002) supplemented with 0.1%
BSA, 10 mM HEPES pH 7 and 1 mM MnCl.sub.2. The cells are
re-suspended in supplemented DMEM medium at a density of
4.times.10.sup.6 cells/ml.
[0127] A Nunc MaxiSorp plate was coated with rh MAdCAM-1/Fc Chimera
(R&D #6065-MC) at 200 ng per well in 50 ul per well in 1XPBS
and incubated at 4.degree. C. overnight. The solution was then
removed by shaking, blocked with 250 ul per well PBS containing 1%
BSA, and incubated at 37.degree. C. for 1 hr. The solution was
removed by shaking. Peptides are diluted by serial dilution in a
final volume of 50 ul per well (2.times. concentration). To each
well, 50 ul of cells (200,000 cells) are added and the plate is
incubated at 37.degree. C., 5% CO.sub.2 for 30-45 min to allow cell
adhesion. The wells are washed manually three times (100 ul per
wash) with supplemented DMEM. After the final wash, 100 ul/well of
supplemented DMEM and 10 ul/well of MTT reagent (ATTC cat
#30-1010K) are added. The plate is incubated at 37.degree. C., 5%
CO2 for 2-3hrs until a purple precipitate is visible. 100 ul of
Detergent Reagent (ATTC cat #30-1010K) is added to each well. The
plate is covered from the light, wrapped in Parafilm to prevent
evaporation, and left overnight at room temperature in the dark.
The plate is shaken for 5 min and the absorbance at 570 nm is
measured. To calculate the dose response, the absorbance value of
control wells not containing cells is subtracted from each test
well.
Example 4: .alpha.4.beta.1-VCAM Cell Adhesion Assay
[0128] Jurkat E6.1 cells (Sigma #88042803) are cultured in RPMI
1640 HEPES medium (Invitrogen #22400-089) supplemented with 10 %
serum (Fetal Bovine Serum, Invitrogen #16140-071), 1 mM sodium
pyruvate (Invitrogen #11360-070), 2 mM L-glutamine (Invitrogen
#25030-081) and Penicillin-Streptomycin (Invitrogen #15140-122) at
100 units of penicillin and 100 ug of streptomycin per ml. The
cells are washed two times in DMEM medium (ATCC #30-2002)
supplemented with 0.1% BSA, 10 mM HEPES pH 7 and 1 mM MnCl.sub.2.
The cells are re-suspended in supplemented DMEM medium at a density
of 4.times.10.sup.6 cells/ml.
[0129] A Nunc MaxiSorp plate was coated with rh VCAM-1/CD106 Fc
chimera (R&D #862-VC) at 400 ng per well in 50 ul per well in
1XPBS and incubated at 4.degree. C. overnight. The solution was
then removed by shaking, blocked with 250 ul per well PBS
containing 1% BSA, and incubated at 37.degree. C. for 1 hr. The
solution was removed by shaking. peptides are diluted by serial
dilution in a final volume of 50 ul per well (2.times.
concentration). To each well, 50 ul of cells (200,000 cells) are
added and the plate is incubated at 37.degree. C., 5% CO.sub.2 for
30-45 min to allow cell adhesion. The wells are washed manually
three times (100 ul per wash) with supplemented DMEM. After the
final wash, 100 ul/well of supplemented DMEM and 10 ul/well of MTT
reagent (ATTC cat #30-1010K) are added. The plate is incubated at
37.degree. C., 5% CO2 for 2-3 hrs until a purple precipitate is
visible. 100 ul of Detergent Reagent (ATTC cat #30-1010K) is added
to each well. The plate is covered from the light, wrapped in
Parafilm to prevent evaporation, and left overnight at room
temperature in the dark. The plate is shaken for 5 min and the
absorbance at 570 nm is measured. To calculate the dose response,
the absorbance value of control wells not containing cells is
subtracted from each test well.
[0130] The present invention may be embodied in other specific
forms without departing from its structures, methods, or other
essential characteristics as broadly described herein and claimed
hereinafter. The described embodiments are to be considered in all
respects only as illustrative, and not restrictive. The scope of
the invention is, therefore, indicated by the appended claims,
rather than by the foregoing description. All changes that come
within the meaning and range of equivalency of the claims are to be
embraced within their scope.
Sequence CWU 1
1
146114PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(2)absent, Gln, Asn, Asp, Pro, Gly, His, Ala,
Ile, Phe, Lys, Arg, Glu, Leu, Val, Tyr, Trp, Ser, Met,
ThrMOD_RES(1)..(4)Can be ACETYLATEDVARIANT(3)..(3)absent, Gln, Asn,
Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Glu, Leu, Val, Tyr,
Trp, Ser, Met, Thr, D-LysMOD_RES(3)..(3)Can be
N(alpha)METHYLATEDLACTAM(4)..(10)VARIANT(4)..(4)Cys, Asp, Glu, Lys,
Pen, HGlu, HLys, Orn, Dap, Dab, Beta-Asp,
Beta-GluDISULFID(4)..(10)Disulfide bond between Xaa at position 4
and Xaa at position 10VARIANT(5)..(5)Gln, Asn, Asp, Pro, Gly, His,
Ala, Ile, Phe, Lys, Arg, Glu, Leu, Val, Tyr, Trp, Met, Thr, HArg,
4-Guan, Cit, Cav, Dap, DabMOD_RES(5)..(5)Can be N(alpha)METHYLATED,
Arg-Me-sym, Arg-Me- asymVARIANT(6)..(6)Ser, Gln, Asn, Asp, Pro,
Gly, His, Ala, Ile, Phe, Lys, Arg, Glu, Leu, Val, Thr, Trp, Tyr,
MetMOD_RES(7)..(7)Can be N(alpha)METHYLATEDVARIANT(7)..(7)Asp,
D-AspVARIANT(8)..(8)Thr, Gln, Ser, Asn, Asp, Pro, Gly, His, Ala,
Ile, Phe, Lys, Arg, Glu, Val, Tyr, Trp, Leu, MetMOD_RES(8)..(8)Can
be N(alpha)METHYLATEDMOD_RES(9)..(9)Can be
N(alpha)METHYLATEDVARIANT(9)..(9)Gln, Asn, Asp, Pro, Gly, Ala, Phe,
Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala,
Nle, cyclobutyl-Ala, HChaVARIANT(10)..(10)Cys, Asp, Lys, Glu, Pen,
HAsp, HGlu, HLys, Orn, Dap, DabMOD_RES(11)..(11)absent, Gly, Gln,
Asn, Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu,
Ser, Arg, Pro, Phe, 1,1-Indane, 2,2-Indane, D-HPhe, Sar, 1-Nal,
2-Nal, HPhe, Phe(4-F), dihydro- Trp, Dap, Dab, Orn, D-Orn, D-Dap,
D-Dab, Bip, Ala(3,3 diphenyl),
Biphenyl-Ala,MOD_RES(11)..(11)(Continued) D-Phe, D-Trp, D-Tyr,
D-Glu, D-His, D-Lys, 3,3-diPhe, Beta-HTrp, F(4-CF3), O-Me-Tyr,
4-Me-Phe, Phe(2,4-Cl2), Phe(3,4-Cl2), D-1-Nal,
D-2-NalMOD_RES(11)..(11)Can be ACETYLATEDMOD_RES(11)..(11)Can be
N(alpha)METHYLATEDVARIANT(12)..(12)absent, Glu, Lys, Gln, Pro, Gly,
His, Ala, Ile, Phe, Arg, Leu, Val, Tyr, Trp, Met, Ser, Asn, Asp,
Dap, Dab, Orn, D-Orn, D-Dap, D-Dab, D-Lys, D-Glu, Beta-HGlu, 2-Nal,
1-Nal, D-Asp, Bip, Beta-HPhe, Beta-Glu, D-Tyr, Beta-HGlu,
GlaMOD_RES(12)..(12)Can be ACETYLATEDMOD_RES(12)..(12)Can be
N(alpha)METHYLATEDVARIANT(13)..(13)absent, Gln, Pro, Gly, His, Ala,
Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Dap,
Dab, Orn, D-Orn, D-Dap, D-Dab, D-Lys, GlaMOD_RES(13)..(13)Can be
ACETYLATEDMOD_RES(13)..(13)Can be
N(alpha)METHYLATEDMOD_RES(14)..(14)Can be
ACETYLATEDVARIANT(14)..(14)absent, can be any naturally occuring
amino acidMOD_RES(14)..(14)Can be N(alpha)METHYLATED 1Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10210PRTArtificial
SequenceSynthetic ConstructVARIANT(1)..(1)Cys, Asp, Glu, Lys, Pen,
HGlu, HLys, Orn, Dap, DabMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Xaa at position 7LACTAM(1)..(7)Lactam bond between Xaa at
position 1 and Xaa at position 7VARIANT(2)..(2)Gln, Asn, Asp, Pro,
Gly, His, Ala, Ile, Phe, Lys, Arg, Glu, Leu, Val, Tye, Trp, Met,
Thr, HArg, Dap, Dab, 4-Guan, Cit,
CavMOD_RES(2)..(2)N(alpha)METHYLATION; N-Me-Arg; Arg-Me-sym;
Arg-Me-asymVARIANT(3)..(3)Ser, Thr, Gln, Asn, Asp, Pro, Gly, His,
Ala, Ile, Phe, Lys, Arg, Glu, Leu, Val, Tyr, Trp,
MetMOD_RES(4)..(4)Can be
N(alpha)METHYLATEDVARIANT(4)..(4)AspMOD_RES(5)..(5)Can be
N(alpha)METHYLATEDVARIANT(5)..(5)Thr, Gln, Ser, Asn, Asp, Pro, Gly,
His, Ala, Ile, Phe, Lys, Arg, Glu, Val, Tyr, Trp,
MetMOD_RES(6)..(6)Can be N(alpha)METHYLATEDVARIANT(6)..(6)Gln, Asn,
Asp, Pro, Gly, Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala,
n-Pentyl AlaVARIANT(7)..(7)Cys, Asp, Lys, Glu, Pen, HLys, Orn,
HGlu, Dap, DabVARIANT(8)..(8)Gly, Gln, Asn, Asp, Ala, Ile, Leu,
Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg, Pro, Phe,
1,1-Indane, 2,2-Indane, D-HPhe, Sar, 1-Nal, 2-Nal, HPhe, Phe(4-F),
dihydro- Trp, Dap, Dab, Orn, D-Orn, D-Dap, D-Dab, Bip, Ala(3,3
diphenyl), Biphenyl-Ala,MOD_RES(8)..(8)(Continued) D-Phe, D-Trp,
D-Tyr, D-Glu, D-His, D-Lys, 3,3-diPhe, Beta-HTrp, F(4-CF3),
O-Me-Tyr, 4-Me-Phe, Phe(2,4-Cl2), Phe(3,4-Cl2), D-1-Nal,
D-2-NalMOD_RES(8)..(8)Can be N(alpha)METHYLATEDMOD_RES(8)..(10)Can
be ACETYLATEDMOD_RES(9)..(9)Can be
N(alpha)METHYLATEDVARIANT(9)..(9)absent, Glu, Lys, Gln, Pro, Gly,
His, Ala, Asp, Ile, Phe, Arg, Leu, Val, Tyr, Trp, Met, Ser, Asn,
Dap, Dab, Orn, D-Orn, D-Dap, D-Dab, D-Lys, D-Glu, Beta-HGlu, 2-Nal,
1-Nal, D-Asp, Bip, Beta-HPhe, Beta-Glu, D-Tyr,
Beta-HGluVARIANT(10)..(10)absent, Gln, Pro, Gly, His, Ala, Ile,
Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Dap, Dab,
Orn, D-Orn, D-Dap, D-Dab, D-Lys, GlaMOD_RES(10)..(10)Can be
N(alpha)METHYLATED 2Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
1039PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can be any
naturally occurring amino acidVARIANT(5)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(8)Xaa can be any
naturally occurring amino acidMOD_RES(9)..(9)Can be
ACETYLATEDMOD_RES(9)..(9)Can be
N(alpha)METHYLATEDVARIANT(9)..(9)Xaa can be any naturally occurring
amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-Dap 3Cys Arg Xaa
Asp Xaa Xaa Cys Xaa Xaa1 549PRTArtificial SequenceSynthetic
ConstructDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position 7MOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can
be any naturally occurring amino acidVARIANT(5)..(6)Xaa can be any
naturally occurring amino acidMOD_RES(7)..(7)PenVARIANT(8)..(8)Can
be any naturally occurring amino acidVARIANT(9)..(9)Can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(9)..(9)Can be N(alpha)METHYLATEDMOD_RES(9)..(9)Can be
ACETYLATED 4Cys Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa1 559PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Cys at position 7MOD_RES(2)..(2)Can be
N(alpha)METHYLATEDVARIANT(3)..(3)Xaa can be any naturally occurring
amino acidVARIANT(5)..(6)Xaa can be any naturally occurring amino
acidVARIANT(8)..(8)Xaa can be any naturally occurring amino
acidVARIANT(9)..(9)Xaa can be any naturally occurring amino acid,
D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(9)..(9)Can be
N(alpha)METHYLATEDMOD_RES(9)..(9)Can be ACETYLATED 5Xaa Arg Xaa Asp
Xaa Xaa Cys Xaa Xaa1 569PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can be any
naturally occurring amino acidVARIANT(5)..(6)Xaa can be any
naturally occurring amino acidMOD_RES(7)..(7)PenVARIANT(8)..(8)Xaa
can be any naturally occurring amino acidMOD_RES(9)..(9)Can be
ACETYLATEDVARIANT(9)..(9)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(9)..(9)Can
be N(alpha)METHYLATED 6Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa1
5710PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa can
be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Cys at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidVARIANT(9)..(9)Xaa can be any
naturally occurring amino acidMOD_RES(10)..(10)Can be
ACETYLATEDVARIANT(10)..(10)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 7Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa Xaa1 5
10810PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(8)..(8)PenVARIANT(9)..(9)Xaa
can be any naturally occurring amino acidMOD_RES(10)..(10)Can be
ACETYLATEDVARIANT(10)..(10)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 8Xaa Cys Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa1 5
10910PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Cys at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidVARIANT(9)..(9)Xaa can be any
naturally occurring amino acidMOD_RES(10)..(10)Can be
ACETYLATEDMOD_RES(10)..(10)Can be
N(alpha)METHYLATEDVARIANT(10)..(10)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-Dap
9Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa Xaa1 5 101010PRTArtificial
SequenceSynthetic ConstructVARIANT(1)..(1)Xaa can be any naturally
occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(8)..(8)PenVARIANT(9)..(9)Xaa
can be any naturally occurring amino acidMOD_RES(10)..(10)Can be
ACETYLATEDVARIANT(10)..(10)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 10Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa1 5
101110PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position
1 and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can be any
naturally occurring amino acidVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino acidVARIANT(10)..(10)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(10)..(10)Can be N(alpha)METHYLATEDMOD_RES(10)..(10)Can
be ACETYLATED 11Cys Arg Xaa Asp Xaa Xaa Cys Xaa Xaa Xaa1 5
101210PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position
1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can be any
naturally occurring amino acidVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidMOD_RES(7)..(7)PenVARIANT(8)..(9)Xaa
can be any naturally occurring amino acidVARIANT(10)..(10)Xaa can
be any naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn,
D-Dab, D-DapMOD_RES(10)..(10)Can be ACETYLATEDMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 12Cys Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa1 5
101310PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can be any
naturally occurring amino acidVARIANT(5)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino acidMOD_RES(10)..(10)Can be
ACETYLATEDVARIANT(10)..(10)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 13Xaa Arg Xaa Asp Xaa Xaa Cys Xaa Xaa Xaa1 5
101410PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(3)..(3)Xaa can be any
naturally occurring amino acidVARIANT(5)..(6)Xaa can be any
naturally occurring amino acidMOD_RES(7)..(7)PenVARIANT(8)..(9)Xaa
can be any naturally occurring amino acidMOD_RES(10)..(10)Can be
ACETYLATEDVARIANT(10)..(10)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 14Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa1 5
101511PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Cys at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidVARIANT(9)..(10)Xaa can be any
naturally occurring amino acidMOD_RES(11)..(11)Can be
ACETYLATEDVARIANT(11)..(11)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(11)..(11)Can
be N(alpha)METHYLATED 15Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa Xaa
Xaa1 5 101611PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(1)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(2)..(8)Disulfide bond
between Cys at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(8)..(8)PenVARIANT(9)..(10)Xaa
can be any naturally occurring amino acidMOD_RES(11)..(11)Can be
ACETYLATEDVARIANT(11)..(11)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(11)..(11)Can
be N(alpha)METHYLATED 16Xaa Cys Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa
Xaa1 5 101711PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(1)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(2)..(8)Disulfide bond
between Xaa at position 2 and Cys at position
8MOD_RES(2)..(2)PenMOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa
can be any naturally occurring amino acidVARIANT(6)..(7)Xaa can be
any naturally occurring amino acidVARIANT(9)..(10)Xaa can be any
naturally occurring amino acidMOD_RES(11)..(11)Can be
ACETYLATEDVARIANT(11)..(11)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(11)..(11)Can
be N(alpha)METHYLATED 17Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa Xaa
Xaa1 5 101811PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(1)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(2)..(8)Disulfide bond
between Xaa at position 2 and Xaa at position
8MOD_RES(2)..(2)PenMOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa
can be any naturally occurring amino acidVARIANT(6)..(7)Xaa can be
any naturally occurring amino
acidMOD_RES(8)..(8)PenVARIANT(9)..(10)Xaa can be any naturally
occurring amino acidMOD_RES(11)..(11)Can be
ACETYLATEDMOD_RES(11)..(11)Can be
N(alpha)METHYLATEDVARIANT(11)..(11)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-Dap
18Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa1 5
101912PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(3)..(9)Disulfide bond between Cys at position 3
and Cys at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino acidVARIANT(10)..(11)Xaa can be any
naturally occurring amino acidVARIANT(12)..(12)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(12)..(12)Can be N(alpha)METHYLATEDMOD_RES(12)..(12)Can
be ACETYLATED 19Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa Xaa Xaa1 5
102012PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(3)..(9)Disulfide bond between Cys at position 3
and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino
acidMOD_RES(9)..(9)PenVARIANT(10)..(11)Xaa can be any naturally
occurring amino acidVARIANT(12)..(12)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(12)..(12)Can be N(alpha)METHYLATEDMOD_RES(12)..(12)Can
be ACETYLATED 20Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa1 5
102112PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(3)..(3)PenDISULFID(3)..(9)Disulfide bond between
Xaa at position 3 and Cys at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino acidVARIANT(10)..(11)Xaa can be any
naturally occurring amino acidVARIANT(12)..(12)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(12)..(12)Can be N(alpha)METHYLATEDMOD_RES(12)..(12)Can
be ACETYLATED 21Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa Xaa Xaa1 5
102212PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(3)..(3)PenDISULFID(3)..(9)Disulfide bond between
Xaa at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino
acidMOD_RES(9)..(9)PenVARIANT(10)..(11)Xaa can be any naturally
occurring amino acidVARIANT(12)..(12)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(12)..(12)Can be N(alpha)METHYLATEDMOD_RES(12)..(12)Can
be ACETYLATED 22Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa1 5
102313PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(3)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(4)..(10)Disulfide bond between Cys at position 4
and Cys at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino acidVARIANT(11)..(12)Xaa can be any
naturally occurring amino acidVARIANT(13)..(13)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(13)..(13)Can be N(alpha)METHYLATEDMOD_RES(13)..(13)Can
be ACETYLATED 23Xaa Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa Xaa
Xaa1 5 102413PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(3)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(4)..(10)Disulfide bond
between Cys at position 4 and Xaa at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino
acidMOD_RES(10)..(10)PenVARIANT(11)..(12)Xaa can be any naturally
occurring amino acidVARIANT(13)..(13)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(13)..(13)Can be N(alpha)METHYLATEDMOD_RES(13)..(13)Can
be ACETYLATED 24Xaa Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa
Xaa1 5 102513PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(3)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(4)..(4)PenDISULFID(4)..(10)Disulfide bond between
Xaa at position 4 and Cys at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino acidVARIANT(11)..(12)Xaa can be any
naturally occurring amino acidVARIANT(13)..(13)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(13)..(13)Can be ACETYLATEDMOD_RES(13)..(13)Can be
N(alpha)METHYLATED 25Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa
Xaa Xaa1 5 102613PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(3)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(4)..(4)PenDISULFID(4)..(10)Disulfide bond between
Xaa at position 4 and Xaa at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino
acidMOD_RES(10)..(10)PenVARIANT(11)..(12)Xaa can be any naturally
occurring amino acidVARIANT(13)..(13)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(13)..(13)Can be ACETYLATEDMOD_RES(13)..(13)Can be
N(alpha)METHYLATED 26Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10279PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(1)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(2)..(8)Disulfide bond
between Cys at position 2 and Cys at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(9)..(9)Can be
ACETYLATEDMOD_RES(9)..(9)Can be
N(alpha)METHYLATEDMOD_RES(9)..(9)D-Lys, Dap, Dab, Orn, D-Orn,
D-Dab, D-Dap 27Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa1
5289PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa can
be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(8)..(8)PenMOD_RES(9)..(9)Can
be ACETYLATEDVARIANT(9)..(9)Xaa can be any naturally occurring
amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(9)..(9)Can be N(alpha)METHYLATED 28Xaa Cys Arg Xaa Asp
Xaa Xaa Xaa Xaa1 5299PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(1)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Cys at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(9)..(9)Can be
ACETYLATEDVARIANT(9)..(9)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(9)..(9)Can
be N(alpha)METHYLATED 29Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa1
5309PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa can
be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONVARIANT(4)..(4)Xaa can be any
naturally occurring amino acidVARIANT(6)..(7)Xaa can be any
naturally occurring amino acidMOD_RES(8)..(8)PenMOD_RES(9)..(9)Can
be ACETYLATEDVARIANT(9)..(9)Xaa can be any naturally occurring
amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(9)..(9)Can be N(alpha)METHYLATED 30Xaa Xaa Arg Xaa Asp
Xaa Xaa Xaa Xaa1 53110PRTArtificial SequenceSynthetic
ConstructVARIANT(1)..(2)Xaa can be any naturally occurring amino
acidMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide bond
between Cys at position 3 and Cys at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino acidVARIANT(10)..(10)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(10)..(10)Can be N(alpha)METHYLATEDMOD_RES(10)..(10)Can
be ACETYLATED 31Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa1 5
103210PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(3)..(9)Disulfide bond between Cys at position 3
and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino
acidMOD_RES(9)..(9)PenVARIANT(10)..(10)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(10)..(10)Can be ACETYLATEDMOD_RES(10)..(10)Can be
N(alpha)METHYLATED 32Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Xaa Xaa1 5
103310PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(3)..(3)PenDISULFID(3)..(9)Disulfide bond between
Xaa at position 3 and Cys at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino acidVARIANT(10)..(10)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(10)..(10)Can be ACETYLATEDMOD_RES(10)..(10)Can be
N(alpha)METHYLATED 33Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa1 5
103410PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(2)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)May be
ACETYLATEDMOD_RES(3)..(3)PenDISULFID(3)..(9)Disulfide bond between
Xaa at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONVARIANT(5)..(5)Xaa can be any
naturally occurring amino acidVARIANT(7)..(8)Xaa can be any
naturally occurring amino
acidMOD_RES(9)..(9)PenVARIANT(10)..(10)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(10)..(10)May be ACETYLATEDMOD_RES(10)..(10)Can be
N(alpha)METHYLATED 34Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa1 5
103511PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(3)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(4)..(10)Disulfide bond between Cys at position 4
and Cys at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino acidVARIANT(11)..(11)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(11)..(11)Can be N(alpha)METHYLATEDMOD_RES(11)..(11)Can
be ACETYLATED 35Xaa Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Cys Xaa1 5
103611PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(3)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(4)..(10)Disulfide bond between Cys at position 4
and Xaa at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino
acidMOD_RES(10)..(10)PenVARIANT(11)..(11)Xaa can be any naturally
occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(11)..(11)Can be ACETYLATEDMOD_RES(11)..(11)Can be
N(alpha)METHYLATED 36Xaa Xaa Xaa Cys Arg Xaa Asp Xaa Xaa Xaa Xaa1 5
103711PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(3)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(4)..(4)PenDISULFID(4)..(10)Disulfide bond between
Xaa at position 4 and Cys at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino acidVARIANT(11)..(11)Xaa can be any
naturally occurring amino acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab,
D-DapMOD_RES(11)..(11)Can be ACETYLATEDMOD_RES(11)..(11)Can be
N(alpha)METHYLATED 37Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Cys Xaa1 5
103811PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(3)Xaa
can be any naturally occurring amino acidMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(4)..(4)PenDISULFID(4)..(10)Disulfide bond between
Xaa at position 4 and Xaa at position
10MOD_RES(5)..(5)N(alpha)METHYLATIONVARIANT(6)..(6)Xaa can be any
naturally occurring amino acidVARIANT(8)..(9)Xaa can be any
naturally occurring amino
acidMOD_RES(10)..(10)PenMOD_RES(11)..(11)Can be
ACETYLATEDVARIANT(11)..(11)Xaa can be any naturally occurring amino
acid, D-Lys, Dap, Dab, Orn, D-Orn, D-Dab, D-DapMOD_RES(11)..(11)Can
be N(alpha)METHYLATED 38Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa
Xaa1 5 10398PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position
7VARIANT(2)..(2)Arg, Tyr 39Cys Xaa Ser Asp Thr Leu Cys Lys1
54010PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position 7 40Cys Arg Ser Asp Thr Leu Cys Gly Glu Lys1 5
10418PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Cys at position 8 41Lys Cys Arg Ser Asp Thr Leu Cys1
54210PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Cys at position 8 42Lys Cys Arg Ser Asp Thr Leu Cys Gly Glu1 5
10439PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position 7MOD_RES(2)..(2)METHYLATION Arg-Me-sym;
Arg-Me-asym 43Cys Arg Ser Asp Thr Leu Cys Gly Glu1
5449PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position 7MOD_RES(2)..(2)Dab, Cit, Cav, Dap 44Cys Xaa
Ser Asp Thr Leu Cys Gly Glu1 5459PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position 7 45Cys His Ser
Asp Thr Leu Cys Gly Glu1 5467PRTArtificial SequenceSynthetic
ConstructDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position 7MOD_RES(2)..(2)N(alpha)METHYLATION 46Cys Arg
Ser Asp Thr Leu Cys1 5478PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATION, Arg-Me-sym,
Arg-Me-asymMOD_RES(8)..(8)Dap, Dab, D-LysMOD_RES(8)..(8)Can be
ACETYLATED 47Cys Arg Ser Asp Thr Leu Cys Xaa1 5488PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(8)..(8)Can be
N(alpha)METHYLATED 48Cys Arg Ser Asp Thr Leu Cys Lys1
5498PRTArtificial SequenceSynthetic
ConstructDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(2)..(2)Guan, 4-Guan,
Dap, Dab, Phe(4-NH2)MOD_RES(8)..(8)Dap, Dab,
D-LysMOD_RES(8)..(8)Can be ACETYLATED 49Cys Xaa Ser Asp Thr Leu Cys
Xaa1 5507PRTArtificial SequenceSynthetic
ConstructDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position 7MOD_RES(2)..(2)N(alpha)METHYLATION 50Cys Arg
Ser Asp Thr Leu Cys1 55110PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONVARIANT(2)..(2)Arg,
4-GuanMOD_RES(10)..(10)Can be N(alpha)METHYLATED 51Cys Xaa Ser Asp
Thr Leu Cys Gly Glu Lys1 5 10529PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position
7MOD_RES(5)..(6)Can be N(alpha)METHYLATED 52Cys His Ser Asp Thr Leu
Cys Gly Glu1 55310PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position 7MOD_RES(8)..(8)Sar 53Cys Arg Ser Asp Thr Leu
Cys Xaa Glu Lys1 5 105410PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(10)..(10)D-Lys, Dab, Dab
(2-Me-trifluorobutyl), Dab (trifluorpentyl), Dab (Acetyl), Dab
(Octonyl) 54Cys Arg Ser Asp Thr Leu Cys Gly Glu Xaa1 5
10558PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Lys
55Cys Arg Ser Asp Thr Leu Xaa Xaa1 5568PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)D-LysDISULFID(2)..(8)Disulfide bond
between Cys at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 56Xaa Cys Arg
Ser Asp Thr Leu Xaa1 5579PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
57Cys Arg Ser Asp Thr Leu Xaa Trp Xaa1 5589PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
58Cys Arg Ser Asp Thr Leu Xaa Tyr Xaa1 5598PRTArtificial
SequenceSynthetic ConstructDISULFID(2)..(8)Disulfide bond between
Cys at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 59Arg Cys Arg
Ser Asp Thr Leu Xaa1 5608PRTArtificial SequenceSynthetic
ConstructDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 60Leu Cys Arg
Ser Asp Thr Leu Xaa1 5619PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 61Lys Leu Cys
Arg Ser Asp Thr Leu Xaa1 5628PRTArtificial SequenceSynthetic
ConstructDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 62His Cys Arg
Ser Asp Thr Leu Xaa1 5639PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 63Lys His Cys
Arg Ser Asp Thr Leu Xaa1 5648PRTArtificial SequenceSynthetic
ConstructDISULFID(2)..(8)Disulfide bond between Cys at position 3
and Xaa at position
9MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 64Glu Cys Arg
Ser Asp Thr Leu Xaa1 5658PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(8)Disulfide
bond between Cys at position 3 and Xaa at position
8MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 65Lys Glu Cys
Arg Ser Asp Thr Xaa1 5669PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 66Lys Trp Cys
Arg Ser Asp Thr Leu Xaa1 5678PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(2)..(8)Disulfide
bond between Cys at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 67Pro Cys Arg
Ser Asp Thr Leu Xaa1 5689PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 68Lys Pro Cys
Arg Ser Asp Thr Leu Xaa1 5699PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 69Lys Ser Cys
Arg Ser Asp Thr Leu Xaa1 5709PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 70Lys Asn Cys
Arg Ser Asp Thr Leu Xaa1 5719PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(3)..(9)Disulfide
bond between Cys at position 3 and Xaa at position
9MOD_RES(4)..(4)N(alpha)METHYLATIONMOD_RES(9)..(9)Pen 71Lys Tyr Cys
Arg Ser Asp Thr Leu Xaa1 5729PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
72Cys Arg Ser Asp Thr Leu Xaa Leu Xaa1 5739PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
73Cys Arg Ser Asp Thr Leu Xaa His Xaa1 5749PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
74Cys Arg Ser Asp Thr Leu Xaa Glu Xaa1 5759PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)Pen 75Cys Arg Ser
Asp Thr Leu Xaa Tyr Lys1 5769PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)Pen 76Cys Arg Ser
Asp Thr Leu Xaa Trp Lys1 5779PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
77Cys Arg Ser Asp Thr Leu Xaa Arg Xaa1 5789PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
78Cys Arg Ser Asp Thr Leu Xaa Pro Xaa1 5799PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
79Cys Arg Ser Asp Thr Leu Xaa Ser Xaa1 5809PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
80Cys Arg Ser Asp Thr Leu Xaa Asn Xaa1 58110PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)PenMOD_RES(9)..(9)1-Nal
81Arg Cys Arg Ser Asp Thr Leu Xaa Xaa Lys1 5 108210PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)PenMOD_RES(9)..(9)1-Nal
82Glu Cys Arg Ser Asp Thr Leu Xaa Xaa Lys1 5 108310PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 83Arg Cys Arg
Ser Asp Thr Leu Xaa Trp Lys1 5 108410PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 84Glu Cys Arg
Ser Asp Thr Leu Xaa Trp Lys1 5 108510PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 85Arg Cys Arg
Ser Asp Thr Leu Xaa Tyr Lys1 5 108610PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 86Glu Cys Arg
Ser Asp Thr Leu Xaa Tyr Lys1 5 10878PRTArtificial SequenceSynthetic
ConstructDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 87Tyr Cys Arg
Ser Asp Thr Leu Xaa1 5889PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)1-Nal-
MOD_RES(9)..(9)D-Lys 88Cys Arg Ser Asp Thr Leu Xaa Xaa Xaa1
5899PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)Pen 89Cys Arg Ser
Asp Thr Leu Xaa His Lys1 5909PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
90Xaa Arg Ser Asp Thr Leu Xaa His Xaa1 5919PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
91Xaa Arg Ser Asp Thr Leu Xaa Tyr Xaa1 5929PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
92Xaa Arg Ser Asp Thr Leu Xaa Trp Xaa1 5938PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Lys-
, D-Phe 93Xaa Arg Ser Asp Thr Leu Xaa Xaa1 5948PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Lys
94Cys Arg Ser Asp Thr Leu Xaa Xaa1 5958PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)PenMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Xaa at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(8)..(8)D-Lys 95Xaa Arg
Ser Asp Thr Leu Cys Xaa1 5969PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
96Cys Arg Ser Asp Thr Leu Xaa Phe Xaa1 5979PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Phe-
, 2-Nal, HPhe, Bip, Phe(4-F), Tyr(OMe), Ala(3,3 biphenyl),
Trp(di-hydro), dTrp (di-hydro), Phe(4-Me)MOD_RES(9)..(9)D-Lys 97Cys
Arg Ser Asp Thr Leu Xaa Xaa Xaa1 5988PRTArtificial
SequenceSynthetic ConstructDISULFID(2)..(8)Disulfide bond between
Cys at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 98Ser Cys Arg
Ser Asp Thr Leu Xaa1 5998PRTArtificial SequenceSynthetic
ConstructDISULFID(2)..(8)Disulfide bond between Cys at position 2
and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 99Asn Cys Arg
Ser Asp Thr Leu Xaa1 51008PRTArtificial SequenceSynthetic
ConstructDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)Pen 100Cys Arg
Ser Asp Thr Leu Xaa Trp1 51018PRTArtificial SequenceSynthetic
ConstructDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Trp-
, D-Phe 101Cys Arg Ser Asp Thr Leu Xaa Xaa1 51029PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)Can be
N(alpha)METHYLATEDMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond
between Xaa at position 2 and Xaa at position 8MOD_RES(2)..(2)Can
be
N(alpha)METHYLATEDMOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen
102Lys Xaa Arg Ser Asp Thr Leu Xaa Trp1 510310PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)Can be
N(alpha)METHYLATEDDISULFID(2)..(8)Disulfide bond between Xaa at
position 2 and Xaa at position
8MOD_RES(2)..(2)PenMOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)PenMO-
D_RES(10)..(10)Can be N(alpha)METHYLATED 103Lys Xaa Arg Ser Asp Thr
Leu Xaa Trp Lys1 5 1010410PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDMOD_RES(1)..(1)Can be
N(alpha)METHYLATEDMOD_RES(1)..(1)D-LysMOD_RES(2)..(2)PenDISULFID(2)..(8)D-
isulfide bond between Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)PenMOD_RES(9)..(9)Can
be N(alpha)METHYLATEDMOD_RES(10)..(10)D-Lys 104Xaa Xaa Arg Ser Asp
Thr Leu Xaa Trp Xaa1 5 1010510PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 105Xaa Arg Ser Asp Thr Leu Xaa Trp Phe Xaa1 5
1010610PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)ACETYLATIONMOD_RES(1)..(1)PenDISULFID(1)..(7)Disu-
lfide bond between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)Can
be N(alpha)METHYLATEDMOD_RES(10)..(10)D-LysMOD_RES(10)..(10)Can be
N(alpha)METHYLATED 106Xaa Arg Ser Asp Thr Leu Xaa Trp Glu Xaa1 5
1010710PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at postion 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 107Xaa Arg Ser Asp Thr Leu Xaa Trp Glu Lys1 5
1010810PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 108Xaa Arg Ser Asp Thr Leu Xaa Trp Ser Xaa1 5
1010910PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 109Xaa Arg Ser Asp Thr Leu Xaa Trp Tyr Xaa1 5
101109PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Trp-
, D-PheMOD_RES(9)..(9)D-Lys 110Xaa Arg Ser Asp Thr Leu Xaa Xaa Xaa1
51119PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)Can
be N(alpha)METHYLATED 111Xaa Arg Ser Asp Thr Leu Xaa Trp Lys1
51129PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(4)..(4)D-AspMOD_RES(7)..(7)Pen-
MOD_RES(9)..(9)D-Lys 112Xaa Arg Ser Xaa Thr Leu Xaa Trp Xaa1
51139PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 113Pro Xaa
Arg Ser Asp Thr Leu Xaa Trp1 51149PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 114Leu Xaa
Arg Ser Asp Thr Leu Xaa Trp1 51159PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 115His Xaa
Arg Ser Asp Thr Leu Xaa Trp1 51169PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 116Glu Xaa
Arg Ser Asp Thr Leu Xaa Trp1 51179PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 117Arg Xaa
Arg Ser Asp Thr Leu Xaa Trp1 51189PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 118Ser Xaa
Arg Ser Asp Thr Leu Xaa Trp1 51199PRTArtificial SequenceSynthetic
ConstructMOD_RES(2)..(2)PenDISULFID(2)..(8)Disulfide bond between
Xaa at position 2 and Xaa at position
8MOD_RES(3)..(3)N(alpha)METHYLATIONMOD_RES(8)..(8)Pen 119Phe Xaa
Arg Ser Asp Thr Leu Xaa Trp1 512010PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Phe-
, D-Trp, 2-Nal, 3-3-diPhe, Beta-HTrp, Phe(4-CF3), 1-Nal, 2-Nal,
Phe(2,4-Cl2), Phe(3,4-Cl2), D-1-Nal, D-2-Nal, HPhe, D-HPhe,
2,2-Indane, 1,1-IndaneMOD_RES(10)..(10)D-LysMOD_RES(10)..(10)Can be
N(alpha)METHYLATED 120Xaa Arg Ser Asp Thr Leu Xaa Xaa Glu Xaa1 5
1012110PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 121Xaa Arg Ser Asp Thr Leu Xaa Trp Leu Xaa1 5
1012210PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 122Xaa Arg Ser Asp Thr Leu Xaa Trp His Xaa1 5
1012310PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 123Xaa Arg Ser Asp Thr Leu Xaa Trp Arg Xaa1 5
1012410PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 124Xaa Arg Ser Asp Thr Leu Xaa Trp Trp Xaa1 5
1012510PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 125Xaa Arg Ser Asp Thr Leu Xaa Trp Pro Xaa1 5
1012610PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 126Xaa Arg Ser Asp Thr Leu Xaa Trp Asn Xaa1 5
101279PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Phe-
MOD_RES(9)..(9)D-Lys 127Xaa Arg Thr Asp Thr Leu Xaa Xaa Xaa1
51289PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Phe-
MOD_RES(9)..(9)D-Lys 128Xaa Arg Ile Asp Thr Leu Xaa Xaa Xaa1
51299PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(8)..(8)D-PheMOD_RES(9)..(9)D-L-
ys 129Cys Arg Ser Asp Ile Leu Cys Xaa Xaa1 51309PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(8)..(8)D-PheMOD_RES(9)..(9)D-L-
ys 130Cys Arg Ser Asp Val Leu Cys Xaa Xaa1 51319PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDDISULFID(1)..(7)Disulfide bond between Cys at position 1
and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(6)..(6)HLeu,
cyclobutyl-Ala, HCha, NleMOD_RES(9)..(9)D-Lys 131Cys Arg Ser Asp
Thr Xaa Cys Trp Xaa1 51329PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be ACETYLATEDDISULFID(1)..(7)Disulfide
bond between Cys at position 1 and Cys at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(9)..(9)D-Lys 132Cys Arg
Ser Asp Thr Leu Cys Trp Xaa1 513310PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Asp-
, D-Glu, Beta-HGlu, 1-Nal, 2-Nal, Bip, Beta-HPhe,
Beta-GluMOD_RES(10)..(10)D-Lys 133Xaa Arg Ser Asp Thr Leu Xaa Trp
Xaa Xaa1 5 1013410PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Phe-
, D-Trp, 1-Nal, 2-Nal, Phe(4-CF3), HPheMOD_RES(9)..(9)D-Glu,
D-TyrMOD_RES(10)..(10)D-Lys 134Xaa Arg Ser Asp Thr Leu Xaa Xaa Xaa
Xaa1 5 1013510PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Glu-
MOD_RES(10)..(10)D-Lys 135Xaa Arg Ser Asp Thr Leu Xaa Phe Xaa Xaa1
5 101369PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(9)..(9)D-Lys
136Xaa Lys Ser Asp Thr Leu Xaa Trp Xaa1 513710PRTArtificial
SequenceSynthetic ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)Can
be N(alpha)METHYLATED 137Xaa Arg Ser Asp Thr Leu Xaa Trp Tyr Lys1 5
101389PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(6)..(6)NleMOD_RES(7)..(7)PenMO-
D_RES(9)..(9)D-Lys 138Xaa Arg Ser Asp Thr Xaa Xaa Trp Xaa1
513910PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(6)..(6)NleMOD_RES(7)..(7)PenMO-
D_RES(10)..(10)D-Lys 139Xaa Arg Ser Asp Thr Xaa Xaa Trp Glu Xaa1 5
101408PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)D-Phe
140Xaa Arg Ser Asp Thr Leu Xaa Xaa1 51418PRTArtificial
SequenceSynthetic
ConstructMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)Pen 141Xaa Arg
Ser Asp Thr Leu Xaa Trp1 514210PRTArtificial SequenceSynthetic
ConstructMOD_RES(1)..(1)Can be
ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond between
Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 142Xaa Arg Ser Asp Thr Leu Xaa Trp Asp Xaa1 5
1014310PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 143Xaa Arg Ser Asp Thr Leu Xaa His Glu Xaa1 5
1014410PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 144Xaa Arg Ser Asp Thr Leu Xaa His Tyr Xaa1 5
1014510PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(10)..(10)D-L-
ys 145Xaa Arg Ser Asp Thr Leu Xaa Tyr Glu Xaa1 5
1014610PRTArtificial SequenceSynthetic ConstructMOD_RES(1)..(1)Can
be ACETYLATEDMOD_RES(1)..(1)PenDISULFID(1)..(7)Disulfide bond
between Xaa at position 1 and Xaa at position
7MOD_RES(2)..(2)N(alpha)METHYLATIONMOD_RES(7)..(7)PenMOD_RES(8)..(8)2-Nal-
MOD_RES(10)..(10)D-Lys 146Xaa Arg Ser Asp Thr Leu Xaa Xaa Tyr Xaa1
5 10
* * * * *