U.S. patent application number 15/711576 was filed with the patent office on 2018-03-29 for peptidomimetic macrocycles.
The applicant listed for this patent is AILERON Therapeutics, Inc.. Invention is credited to Jia-Wen HAN, Rosana KAPELLER-LIBERMANN, Noriyuki KAWAHATA, Huw M. NASH, Justin NOEHRE, Tomi K. SAWYER.
Application Number | 20180085426 15/711576 |
Document ID | / |
Family ID | 43796193 |
Filed Date | 2018-03-29 |
United States Patent
Application |
20180085426 |
Kind Code |
A1 |
NASH; Huw M. ; et
al. |
March 29, 2018 |
PEPTIDOMIMETIC MACROCYCLES
Abstract
The present invention provides novel peptidomimetic macrocycles
and methods of using such macrocycles for the treatment of
disease.
Inventors: |
NASH; Huw M.; (Concord,
MA) ; KAPELLER-LIBERMANN; Rosana; (Chestnut Hill,
MA) ; HAN; Jia-Wen; (Newton, MA) ; SAWYER;
Tomi K.; (Southborough, MA) ; NOEHRE; Justin;
(Cambridge, MA) ; KAWAHATA; Noriyuki; (West
Roxbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AILERON Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
43796193 |
Appl. No.: |
15/711576 |
Filed: |
September 21, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15349478 |
Nov 11, 2016 |
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15711576 |
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13497522 |
Dec 5, 2012 |
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PCT/US10/49892 |
Sep 22, 2010 |
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15349478 |
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61244819 |
Sep 22, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/16 20130101;
A61K 38/12 20130101; A61P 35/04 20180101; A61K 38/00 20130101; C07K
7/50 20130101; C07K 14/00 20130101; C07K 7/64 20130101; A61P 35/00
20180101; A61P 35/02 20180101 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 7/64 20060101 C07K007/64 |
Claims
1.-21. (canceled)
22. A method of treating cancer in a subject in need thereof, the
method comprising administering to the subject a
therapeutically-effective amount of a peptidomimetic macrocycle,
wherein the cancer is associated with aberrant beta-catenin
activity; wherein the peptidomimetic macrocycle comprises: a) a
helical region; b) a cyclic amino acid; c) an alpha, alpha
disubstituted amino acid, wherein the cyclic amino acid and the
alpha, alpha disubstituted amino acid are at positions ranging from
i+4 to i+7; and d) a crosslinker linking the cyclic amino acid and
the alpha, alpha disubstituted amino acid within the peptidomimetic
macrocycle; wherein the crosslinker is of the formula: ##STR00112##
wherein X is CH.sub.2, Y is CH.sub.2, m is 0-10, n is 0-10, o is
0-10, and p is 0-10, and m, n, o, and p are chosen such that: 1)
the crosslinker has a length equal to 5 carbon-carbon bonds to 22
carbon-carbon bonds; 2) the crosslinker has 13 atoms to 33 atoms;
and 3) the peptidomimetic macrocycle has a ring that has 17 to 82
members.
23. The method of claim 22, wherein the helical region comprises an
alpha-helical region.
24. The method of claim 22, wherein the crosslinker links the
alpha-positions of the cyclic amino acid and the alpha, alpha
disubstituted amino acid.
25. The method of claim 22, wherein the peptidomimetic macrocycle
modulates an activity of beta-catenin in the subject.
26. The method of claim 22, wherein the peptidomimetic macrocycle
antagonizes an interaction between beta-catenin and TCF/LEF
proteins in the subject.
27. The method of claim 22, wherein the crosslinker has a length
equal to 8 carbon-carbon to 14 carbon-carbon atoms.
28. The method of claim 22, wherein the peptidomimetic macrocycle
has a ring that has 17 to 35 members.
29. The method of claim 22, wherein the peptidomimetic macrocycle
has a ring that has 17 to 44 members.
30. The method of claim 22, wherein one of the substituents on the
alpha, alpha disubstituted amino acid is a methyl group.
31. A method of treating cancer in a subject in need thereof, the
method comprising: administering to the subject a
therapeutically-effective amount of a peptidomimetic macrocycle,
wherein the cancer is associated with aberrant beta-catenin
activity; wherein the peptidomimetic macrocycle comprises a
crosslinker linking a first non-natural amino acid and a second
non-natural amino acid within the peptidomimetic macrocycle;
wherein the crosslinker has the formula -L.sub.1-L.sub.2-; wherein
each L.sub.1 and L.sub.2 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5; wherein each R.sub.4 is independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene; wherein each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;
wherein each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5; wherein each R.sub.5 is independently
halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6,
--SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent
moiety, a radioisotope or a therapeutic agent; wherein each R.sub.6
is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a
radioisotope or a therapeutic agent; wherein each n is
independently an integer from 1-5.
32. The method of claim 31, wherein the peptidomimetic macrocycle
comprises a cyclic amino acid.
33. The method of claim 31, wherein at least one of the two
non-natural amino acids is a cyclic amino acid.
34. The method of claim 31, wherein the first non-natural amino
acid is a cyclic amino acid.
35. The method of claim 34, wherein the second non-natural amino
acid is an alpha, alpha-disubstituted amino acid.
36. The method of claim 35, wherein one of the substituents on the
alpha, alpha disubstituted amino acid is a methyl group.
37. The method of claim 31, wherein the crosslinker links the
alpha-positions of the first non-natural amino acid and the second
non-natural amino acid.
38. The method of claim 31, wherein the peptidomimetic macrocycle
comprises a helix.
39. The method of claim 31, wherein the peptidomimetic macrocycle
comprises an alpha-helix.
40. The method of claim 31, wherein L.sub.1 and L.sub.2 are
independently alkylene, alkenylene, or alkynylene.
41. The method of claim 31, wherein L.sub.1 and L.sub.2 are
independently C.sub.3-C.sub.6 alkylene or C.sub.3-C.sub.6
alkenylene.
42. The method of claim 31, wherein the peptidomimetic macrocycle
modulates an activity of beta-catenin in the subject.
43. The method of claim 31, wherein the peptidomimetic macrocycle
antagonizes an interaction between beta-catenin and TCF/LEF
proteins in the subject.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 15/349,478, filed Nov. 11, 2016, which is a continuation of
U.S. application Ser. No. 13/497,522, filed Dec. 5, 2012, which is
a National Stage of International Application No. PCT/US10/49892,
filed Sep. 22, 2010, which claims the benefit of U.S. Provisional
Application No. 61/244,819, filed on Sep. 22, 2009, the content of
each of which is incorporated herein in its entirety by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 21, 2017, is named 35224-777.302_SL.txt and is 85,606 bytes
in size.
BACKGROUND OF THE INVENTION
[0003] .beta.-catenin is a subunit of the cadherin protein complex.
.beta.-catenin is critically required for cell adhesion and as an
intracellular mediator of the Wnt pathway. The Wnt signaling
pathway plays critical roles in embryonic development and
tumorigenesis. A smaller pool of .beta.-catenin in the nucleus and
cytoplasm is regulated by Wnt signals. The Wnt signaling activates
gene transcription through forming a complex between DNA-binding
high monility group (HMG)-box proteins of the Tcf/LEF family and
.beta.-catenin. In unstimulated cells, cytosolic .beta.-catenin is
constitutively degraded by a ubiquitin ligase-proteosome system.
Wnt signaling inhibits this process, allowing .beta.-catenin to
accumulate and subsequently translocate to the nucleus where it
forms a transcriptional activating complex with members of the
TCF/LEF-1 family of transcription factors. Tcf/LEF-1 proteins by
themselves have no innate transcriptional activity and they repress
transcription of Wnt target genes by recruiting corepressors to the
promoter. Transcriptional activation of target genes occurs when
.beta.-catenin binds the Tcf/LEF-1 factors and recruits
transcription factors, such as p300/CBP and the TATA binding
protein, to the promoter (Hecht, A. et. al. J. Biol. Chem. 274
(1999), pp. 18017-18025). Genetic and biochemical studies have
demonstrated that the Wnt signaling pathway controls many processes
in embryonic development in both vertebrates and invertebrates.
Inappropriate activation of the Wnt intracellular pathway is
associated with various human cancers, in particular colon cancer
(K. W. Kinzler and B. Vogelstein, Cell 87 (1996), pp. 159-170). Key
molecular lesions in colorectal, hepatocellular carcinoma (HCC),
and other cancers are caused by .beta.-catenin-dependent
transactivation of T cell factor (TCF)-dependent genes, for
example, c-myc, cyclin D1, VEGF, and others. For tumorigenesis,
formation of the complex between .beta.-catenin and TCF is the
critical step in the activation of Wnt target genes (M. Bienz and
H. Clevers Cell 103 (2000), pp. 311-320). Mutations in the
Adenomatous polyposis coli (APC) gene, a key regulator of cellular
.beta.-catenin levels, are found in most colorectal cancers.
Targeting elements downstream of APC in the Wnt pathway, such as
formation and activity of the Tcf4-.beta.-catenin protein complex
represents a potentially powerful means of treating common human
cancers, and there is a strong need for therapeutic approaches
targeting components of the Wnt signaling pathway such as the
Tcf4-.beta.-catenin complex.
SUMMARY OF THE INVENTION
[0004] In one aspect, the present invention provides a
peptidomimetic macrocycle comprising an amino acid sequence which
is at least about 60%, 80%, 90%, or 95% identical to an amino acid
sequence chosen from the group consisting of the amino acid
sequences in Table 1. Alternatively, an amino acid sequence of said
peptidomimetic macrocycle is chosen from the group consisting of
the amino acid sequences in Table 1. In some embodiments, the
peptidomimetic macrocycle comprises a helix, such as an
.alpha.-helix. In other embodiments, the peptidomimetic macrocycle
comprises an .alpha.,.alpha.-disubstituted amino acid. A
peptidomimetic macrocycle of the invention may comprise a
crosslinker linking the .alpha.-positions of at least two amino
acids. At least one of said two amino acids may be an
.alpha.,.alpha.-disubstituted amino acid.
[0005] In some embodiments, the peptidomimetic macrocycle has the
formula:
##STR00001##
[0006] wherein:
[0007] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0008] B is a natural or non-natural amino acid, amino acid
analog,
##STR00002##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0009] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0010] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0011] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0012] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0013] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0014] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0015] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0016] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0017] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0018] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0019] v and w are independently integers from 1-1000;
[0020] u, x, y and z are independently integers from 0-10; and
[0021] n is an integer from 1-5.
[0022] In other embodiments, the peptidomimetic macrocycle may
comprise a crosslinker linking a backbone amino group of a first
amino acid to a second amino acid within the peptidomimetic
macrocycle. For example, the invention provides peptidomimetic
macrocycles of the formula (IV) or (IVa):
##STR00003##
[0023] wherein:
[0024] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0025] B is a natural or non-natural amino acid, amino acid
analog,
##STR00004##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0026] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-, or part
of a cyclic structure with an E residue;
[0027] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0028] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0029] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0030] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0031] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0032] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0033] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0034] v and w are independently integers from 1-1000;
[0035] u, x, y and z are independently integers from 0-10; and
[0036] n is an integer from 1-5.
[0037] Additionally, the invention provides a method of treating
cancer in a subject comprising administering to the subject a
peptidomimetic macrocycle of the invention. Also provided is a
method of modulating the activity of .beta.-catenin in a subject
comprising administering to the subject a peptidomimetic macrocycle
of the invention, or a method of antagonizing the interaction
between .beta.-catenin and TCF/LEF proteins in a subject comprising
administering to the subject such a peptidomimetic macrocycle.
INCORPORATION BY REFERENCE
[0038] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0040] FIG. 1 illustrates a possible binding mode of a TCF
peptidomimetic macrocycle precursor of the invention to
.beta.-catenin.
[0041] FIG. 2 illustrates a possible binding mode of a TCF
peptidomimetic macrocycle precursor of the invention to
.beta.-catenin.
[0042] FIG. 3 illustrates a possible binding mode of a TCF
peptidomimetic macrocycle precursor of the invention to
.beta.-catenin.
[0043] FIGS. 4a, 4b, and 4c show exemplary peptidomimetic
macrocycles of the invention (SEQ ID NOS 66-116 & 115-129,
respectively, in order of appearance).
DETAILED DESCRIPTION OF THE INVENTION
[0044] As used herein, the term "macrocycle" refers to a molecule
having a chemical structure including a ring or cycle formed by at
least 9 covalently bonded atoms.
[0045] As used herein, the term "peptidomimetic macrocycle" or
"crosslinked polypeptide" refers to a compound comprising a
plurality of amino acid residues joined by a plurality of peptide
bonds and at least one macrocycle-forming linker which forms a
macrocycle between a first naturally-occurring or
non-naturally-occurring amino acid residue (or analog) and a second
naturally-occurring or non-naturally-occurring amino acid residue
(or analog) within the same molecule. Peptidomimetic macrocycle
include embodiments where the macrocycle-forming linker connects
the .alpha. carbon of the first amino acid residue (or analog) to
the .alpha. carbon of the second amino acid residue (or analog).
The peptidomimetic macrocycles optionally include one or more
non-peptide bonds between one or more amino acid residues and/or
amino acid analog residues, and optionally include one or more
non-naturally-occurring amino acid residues or amino acid analog
residues in addition to any which form the macrocycle. A
"corresponding uncrosslinked polypeptide" when referred to in the
context of a peptidomimetic macrocycle is understood to relate to a
polypeptide of the same length as the macrocycle and comprising the
equivalent natural amino acids of the wild-type sequence
corresponding to the macrocycle.
[0046] Unless otherwise stated, compounds and structures referred
to herein are also meant to include compounds which differ only in
the presence of one or more isotopically enriched atoms. For
example, compounds having the present structures wherein hydrogen
is replaced by deuterium or tritium, or wherein carbon atom is
replaced by .sup.13C- or .sup.14C-enriched carbon, or wherein
.alpha. carbon atom is replaced by silicon, are within the scope of
this invention. The compounds of the present invention may also
contain unnatural proportions of atomic isotopes at one or more of
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present invention,
whether radioactive or not, are encompassed within the scope of the
present invention.
[0047] As used herein, the term "stability" refers to the
maintenance of a defined secondary structure in solution by a
peptidomimetic macrocycle of the invention as measured by circular
dichroism, NMR or another biophysical measure, or resistance to
proteolytic degradation in vitro or in vivo. Non-limiting examples
of secondary structures contemplated in this invention are
.alpha.-helices, .beta.-turns, and .beta.-pleated sheets.
[0048] As used herein, the term "helical stability" refers to the
maintenance of .alpha. helical structure by a peptidomimetic
macrocycle of the invention as measured by circular dichroism or
NMR. For example, in some embodiments, the peptidomimetic
macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or
2-fold increase in .alpha.-helicity as determined by circular
dichroism compared to a corresponding uncrosslinked macrocycle.
[0049] The term ".alpha.-amino acid" or simply "amino acid" refers
to a molecule containing both an amino group and a carboxyl group
bound to a carbon which is designated the .alpha.-carbon. Suitable
amino acids include, without limitation, both the D- and L-isomers
of the naturally-occurring amino acids, as well as non-naturally
occurring amino acids prepared by organic synthesis or other
metabolic routes. Unless the context specifically indicates
otherwise, the term amino acid, as used herein, is intended to
include amino acid analogs.
[0050] The term "naturally occurring amino acid" refers to any one
of the twenty amino acids commonly found in peptides synthesized in
nature, and known by the one letter abbreviations A, R, N, C, D, Q,
E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0051] The term "amino acid analog" or "non-natural amino acid"
refers to a molecule which is structurally similar to an amino acid
and which can be substituted for an amino acid in the formation of
a peptidomimetic macrocycle. Amino acid analogs include, without
limitation, compounds which are structurally identical to an amino
acid, as defined herein, except for the inclusion of one or more
additional methylene groups between the amino and carboxyl group
(e.g., .alpha.-amino .beta.-carboxy acids), or for the substitution
of the amino or carboxy group by a similarly reactive group (e.g.,
substitution of the primary amine with a secondary or tertiary
amine, or substitution or the carboxy group with an ester).
[0052] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of a polypeptide without
abolishing or substantially altering its essential biological or
biochemical activity (e.g., receptor binding or activation). An
"essential" amino acid residue is a residue that, when altered from
the wild-type sequence of the polypeptide, results in abolishing or
substantially abolishing the polypeptide's essential biological or
biochemical activity.
[0053] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., K, R, H), acidic side
chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S,
T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W),
beta-branched side chains (e.g., T, V, I) and aromatic side chains
(e.g., Y, F, W, H). Thus, a predicted nonessential amino acid
residue in a polypeptide, for example, is preferably replaced with
another amino acid residue from the same side chain family. Other
examples of acceptable substitutions are substitutions based on
isosteric considerations (e.g. norleucine for methionine) or other
properties (e.g. 2-thienylalanine for phenylalanine).
[0054] The term "member" as used herein in conjunction with
macrocycles or macrocycle-forming linkers refers to the atoms that
form or can form the macrocycle, and excludes substituent or side
chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and
1,3-dimethyl cyclodecane are all considered ten-membered
macrocycles as the hydrogen or fluoro substituents or methyl side
chains do not participate in forming the macrocycle.
[0055] The symbol "" when used as part of a molecular structure
refers to a single bond or a trans or cis double bond.
[0056] The term "amino acid side chain" refers to a moiety attached
to the .alpha.-carbon in an amino acid. For example, the amino acid
side chain for alanine is methyl, the amino acid side chain for
phenylalanine is phenylmethyl, the amino acid side chain for
cysteine is thiomethyl, the amino acid side chain for aspartate is
carboxymethyl, the amino acid side chain for tyrosine is
4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino
acid side chains are also included, for example, those that occur
in nature (e.g., an amino acid metabolite) or those that are made
synthetically (e.g., an .alpha.,.quadrature..alpha. di-substituted
amino acid).
[0057] In addition to naturally occurring amino acid side chain
moieties, the amino acid side chain moieties of the present
invention also include various derivatives thereof. Derivatives of
amino acid side chain moieties include other straight chain or
branched, cyclic or noncyclic, substituted or unsubstituted,
saturated or unsaturated lower chain alkyl or aryl moieties.
Moreover, cyclic lower chain alkyl and aryl moieties of this
invention include naphthalene, as well as heterocyclic compounds
such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole,
pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine,
quinoline, isoquinoline and carbazole.
[0058] The term ".alpha.,.quadrature..alpha. di-substituted amino"
acid refers to a molecule or moiety containing both an amino group
and a carboxyl group bound to a carbon (the .alpha.-carbon) that is
attached to two natural or non-natural amino acid side chains.
[0059] The term "polypeptide" encompasses two or more naturally or
non-naturally-occurring amino acids joined by a covalent bond
(e.g., an amide bond). Polypeptides as described herein include
full length proteins (e.g., fully processed proteins) as well as
shorter amino acid sequences (e.g., fragments of
naturally-occurring proteins or synthetic polypeptide
fragments).
[0060] The term "macrocyclization reagent" or "macrocycle-forming
reagent" as used herein refers to any reagent which may be used to
prepare a peptidomimetic macrocycle of the invention by mediating
the reaction between two reactive groups. Reactive groups may be,
for example, an azide and alkyne, in which case macrocyclization
reagents include, without limitation, Cu reagents such as reagents
which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf,
as well as Cu(II) salts such as Cu(CO.sub.2CH.sub.3).sub.2,
CuSO.sub.4, and CuCl.sub.2 that can be converted in situ to an
active Cu(I) reagent by the addition of a reducing agent such as
ascorbic acid or sodium ascorbate. Macrocyclization reagents may
additionally include, for example, Ru reagents known in the art
such as Cp*RuCl(PPh.sub.3).sub.2, [Cp*RuCl].sub.4 or other Ru
reagents which may provide a reactive Ru(II) species. In other
cases, the reactive groups are terminal olefins. In such
embodiments, the macrocyclization reagents or macrocycle-forming
reagents are metathesis catalysts including, but not limited to,
stabilized, late transition metal carbene complex catalysts such as
Group VIII transition metal carbene catalysts. For example, such
catalysts are Ru and Os metal centers having a +2 oxidation state,
an electron count of 16 and pentacoordinated. Additional catalysts
are disclosed in Grubbs et al., "Ring Closing Metathesis and
Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28,
446-452, and U.S. Pat. No. 5,811,515. In yet other cases, the
reactive groups are thiol groups. In such embodiments, the
macrocyclization reagent is, for example, a linker functionalized
with two thiol-reactive groups such as halogen groups.
[0061] The term "halo" or "halogen" refers to fluorine, chlorine,
bromine or iodine or a radical thereof.
[0062] The term "alkyl" refers to a hydrocarbon chain that is a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.10 indicates that the
group has from 1 to 10 (inclusive) carbon atoms in it. In the
absence of any numerical designation, "alkyl" is a chain (straight
or branched) having 1 to 20 (inclusive) carbon atoms in it.
[0063] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0064] The term "alkenyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
double bonds. The alkenyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkenyl" refers to a C.sub.2-C.sub.6 alkenyl chain. In the
absence of any numerical designation, "alkenyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0065] The term "alkynyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkynyl" refers to a C.sub.2-C.sub.6 alkynyl chain. In the
absence of any numerical designation, "alkynyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0066] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of
each ring are substituted by a substituent. Examples of aryl groups
include phenyl, naphthyl and the like. The term "arylalkyl" or the
term "aralkyl" refers to alkyl substituted with an aryl. The term
"arylalkoxy" refers to an alkoxy substituted with aryl.
[0067] "Arylalkyl" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with a C.sub.1-C.sub.5 alkyl group, as defined above.
Representative examples of an arylalkyl group include, but are not
limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl,
2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl,
3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl,
4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl,
2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,
2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl,
2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl,
2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.
[0068] "Arylamido" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with one or more --C(O)NH.sub.2 groups. Representative examples of
an arylamido group include 2-C(O)NH2-phenyl, 3-C(O)NH.sub.2-phenyl,
4-C(O)NH.sub.2-phenyl, 2-C(O)NH.sub.2-pyridyl,
3-C(O)NH.sub.2-pyridyl, and 4-C(O)NH.sub.2-pyridyl,
[0069] "Alkylheterocycle" refers to a C.sub.1-C.sub.5 alkyl group,
as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a heterocycle. Representative
examples of an alkylheterocycle group include, but are not limited
to, --CH.sub.2CH.sub.2-morpholine, --CH.sub.2CH.sub.2-piperidine,
--CH.sub.2CH.sub.2CH.sub.2-morpholine, and
--CH.sub.2CH.sub.2CH.sub.2-imidazole.
[0070] "Alkylamido" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --C(O)NH.sub.2 group.
Representative examples of an alkylamido group include, but are not
limited to, --CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.3,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--C(CH.sub.3).sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3--CH3, and
--CH.sub.2--CH.sub.2--NH--C(O)--CH.dbd.CH.sub.2.
[0071] "Alkanol" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a hydroxyl group.
Representative examples of an alkanol group include, but are not
limited to, --CH.sub.2OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2 CH.sub.2CH.sub.2OH,
--CH.sub.2CH(OH)CH.sub.3, --CH.sub.2CH(OH)CH.sub.2CH.sub.3,
--CH(OH)CH.sub.3 and --C(CH.sub.3).sub.2CH.sub.2OH.
[0072] "Alkylcarboxy" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --COOH group.
Representative examples of an alkylcarboxy group include, but are
not limited to, --CH.sub.2COOH, --CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH(COOH)CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH(COOH)CH.sub.2CH.sub.3, --CH(COOH)CH.sub.2CH.sub.3 and
--C(CH.sub.3).sub.2CH.sub.2COOH.
[0073] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, preferably 3 to 8 carbons, and more preferably 3 to 6
carbons, wherein the cycloalkyl group additionally is optionally
substituted. Some cycloalkyl groups include, without limitation,
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cycloheptyl, and cyclooctyl.
[0074] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are
substituted by a substituent. Examples of heteroaryl groups include
pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,
thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the
like.
[0075] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0076] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0077] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring are
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[0078] The term "substituent" refers to a group replacing a second
atom or group such as a hydrogen atom on any molecule, compound or
moiety. Suitable substituents include, without limitation, halo,
hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl,
aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido,
carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
[0079] In some embodiments, the compounds of this invention contain
one or more asymmetric centers and thus occur as racemates and
racemic mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds
are included in the present invention unless expressly provided
otherwise. In some embodiments, the compounds of this invention are
also represented in multiple tautomeric forms, in such instances,
the invention includes all tautomeric forms of the compounds
described herein (e.g., if alkylation of a ring system results in
alkylation at multiple sites, the invention includes all such
reaction products). All such isomeric forms of such compounds are
included in the present invention unless expressly provided
otherwise. All crystal forms of the compounds described herein are
included in the present invention unless expressly provided
otherwise.
[0080] As used herein, the terms "increase" and "decrease" mean,
respectively, to cause a statistically significantly (i.e.,
p<0.1) increase or decrease of at least 5%.
[0081] As used herein, the recitation of a numerical range for a
variable is intended to convey that the invention may be practiced
with the variable equal to any of the values within that range.
Thus, for a variable which is inherently discrete, the variable is
equal to any integer value within the numerical range, including
the end-points of the range. Similarly, for a variable which is
inherently continuous, the variable is equal to any real value
within the numerical range, including the end-points of the range.
As an example, and without limitation, a variable which is
described as having values between 0 and 2 takes the values 0, 1 or
2 if the variable is inherently discrete, and takes the values 0.0,
0.1, 0.01, 0.001, or any other real values .gtoreq.0 and .ltoreq.2
if the variable is inherently continuous.
[0082] As used herein, unless specifically indicated otherwise, the
word "or" is used in the inclusive sense of "and/or" and not the
exclusive sense of "either/or."
[0083] The term "on average" represents the mean value derived from
performing at least three independent replicates for each data
point.
[0084] The term "biological activity" encompasses structural and
functional properties of a macrocycle of the invention. Biological
activity is, for example, structural stability, alpha-helicity,
affinity for a target, resistance to proteolytic degradation, cell
penetrability, intracellular stability, in vivo stability, or any
combination thereof.
[0085] The details of one or more particular embodiments of the
invention are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages of the
invention will be apparent from the description and drawings, and
from the claims.
[0086] In some embodiments, the peptide sequence is derived from
proteins containing a .beta.-catenin binding domain (CBD).
[0087] The primary structure of .beta.-catenin includes an
N-terminal region of .about.130 residues that is required for its
phosphorylation-dependent degradation, a C-terminal
.about.100-residue segment that recruits transcriptional
coactivators and a central domain of 12 armadillo repeats spanning
residues 134-668 (Peifer M. et. al, Cell 76, 789-791, 1994). This
core armadillo repeat region interacts with cadherins, APC and Tcf
family transcription factors. The crystal structure of this region
reveals that each armadillo repeat consists of three helices; the
12 repeats stack to form a superhelix of helices. The resulting
rod-shaped structure has a long, positively charged surface that
was postulated to be the binding site for acidic sequences in
interacting proteins. These binding partners use both similar and
divergent sequences to bind distinct regions on the surface of
.beta.-catenin.
[0088] There are four members of the Tcf/LEF family. Tcf/LEF-1
family members bind .beta.-catenin in the large central core of the
protein, which contains 12 armadillo repeats. Each armadillo repeat
consists of three a helices, and together the 12 repeats form a
superhelix that features a long positively charged groove (A. H.
Huber, W. J. Nelson and W. I. Weis, Cell 90 (1997), pp. 871-882).
The CBD of Tcf/LEF-1 family members, which include TCF1, TCF3, and
TCF4, corresponds to approximately 60 amino acids at the very N
terminus of the protein (J. Behrens, et. al., Nature 382 (1996),
pp. 638-642). For example, the structure of the Tcf4/.beta.-catenin
complex reveals two distinct sites of interaction between the
proteins: an extended region (residues 13-25 of Tcf4) that binds
.beta.-catenin armadillo repeats 4-9 and a C-terminal helix
(residues 40-50) that binds armadillo repeats 3-5 (Poy F, et. al.
Nat Structural Biol., vol. 8 no 12, 2001). The extended portion of
the Tcf4 (residues 13-25) peptide binds in a positively charged
groove created by the twist between consecutive armadillo repeats.
Asp 16 and Glu 17 form salt bridge hydrogen bonds with
.beta.-catenin Lys 435 and Lys 508, respectively, which flank the
bound peptide on either side of the recognition groove. The
C-terminal end of the extended region is also anchored by an
electrostatic interaction, a salt bridge between Glu 24 in Tcf4 and
Lys 312 in .beta.-catenin. Many of the intervening contacts are
hydrophobic in nature; the side chains of Tcf4 residues Ile 19 and
Phe 21 pack together into a cleft lined by .beta.-catenin residues
Cys 466, Pro 463 and the aliphatic portion of Arg 386. Calorimetric
studies have identified Asp 16 in Tcf4 as a crucial residue for
high affinity binding. Mutation of Lys 435 or Lys 312 in
.beta.-catenin abolishes coprecipitation of Xenopus Tcf3 (XTcf3)
(Graham, T. A., Weaver, C., Mao, F., Kimelman, D & Xu, W. Cell
103, 885-896 2000). Residues 40-50 in Tcf4 form an amphipathic
helix that is cradled in a shallow groove formed by armadillo
repeats 3, 4 and 5 in .beta.-catenin. Hydrophobic residues on one
face of the Tcf4 helix intercalate in the bottom of the groove.
Tcf4 residues Asp 40, Lys 45 and Ser 47 form polar and
electrostatic interactions along the edge of the groove,
contributing to recognition of the helix. The side chain of Asp 40
extends between Arg 376 and Lys 335 in .beta.-catenin, Lys 45
hydrogen bonds with His 260 and Asn 261, and Ser 47 hydrogen bonds
with Lys 292. These interactions are also present in the
XTcf3-.beta.-catenin complex (Graham, T. A., Weaver, C., Mao, F.,
Kimelman, D. & Xu, W. Cell 103, 885-896 (2000)).
[0089] All Tcf/LEF-1 family members also have a highly conserved
HMG DNA binding domain, located within the C-terminal half of the
protein. In particular, XTcf3-CBD from Xenopus consists of, from N
terminus to C terminus, a .beta. hairpin module, an extended region
that contains a .beta. strand, and an .alpha. helix. XTcf3-CBD
wraps around the armadillo repeat region of .beta.-catenin in an
antiparallel fashion along the major axes of the superhelix (Graham
T A et. al. Cell, Vol 103 issue 6, 2000). The helical region of
XTcf3-CBD consists of residues Asp-40 to Glu-51. The helix of
XTcf3-CBD lies approximately antiparallel to the third helices of
armadillo repeats 3 and 4 in .beta.-catenin. The XTcf3-CBD helix
consists of the Tcf/LEF-1 family consensus sequence N'-DLAKSSLV-C'
(SEQ ID NO: 130). The side chains of Asp-40 and Lys-45 of the
XTcf3-CBD coordinate with the side chains of Lys-335 and Asn-261 of
.beta.-catenin, respectively. These two interactions may act as
tethers to stabilize the relative positioning of the helix. The
.alpha.-helical binding region of xTcf3 packs against the H3
helices of arm repeats 3 and 4 of .beta.-catenin. Four hydrophobic
residues in the XTcf3-CBD helix, Leu-41, Val-44, Leu-48, and
Val-49, form a surface complementary to the .beta.-catenin groove
(Graham T A et. al. Cell, Vol 103 issue 6, 2000). Since compounds
that block the interaction between .beta.-catenin and TCF could be
useful pharmacological agents for the treatment of cancers that
result from inappropriate activation of the Wnt pathway, any novel
structures of the CBD peptides generated by the method of the
present invention are useful in preventing and/or treating various
types of cancer in which Wnt pathway plays a role. Such cancers
include colorectal tumors, hepatocellular carcinoma, melanoma and
other tumors with mutations in Wnt pathway components.
[0090] Targeting .beta.-catenin/TCF interaction allows selective
targeting of many types of cancers including but not limited to
colorectal tumors, hepatocellular carcinoma, melanoma and other
tumors with mutations in Wnt pathway components by inhibiting
.beta.-catenin/TCF complex. Currently there is no small molecule
inhibitor of .beta.-catenin/TCF interaction. The substantial
overlap in the binding surfaces used by other Tcf family members,
cadherins and likely APC itself represents a complication in drug
development as inhibitors that disrupt .beta.-catenin/TCF
interaction might disrupt .beta.-catenin/cadherin interaction or
.beta.-catenin/APC or axin interactions. Destabilizing any of these
complexes could cause toxicity or, in the case of APC, provoke
malignant activation of Tcf4 in normal cells. Localized regions of
thermodynamic differences in binding among .beta.-catenin ligands
could make it possible to design specific inhibitors of the
.beta.-catenin-Tcf interaction despite the similarities seen in the
E-cadherin and xTcf3 binding mechanisms. Mutation studies suggest
that the .alpha.-helix region is less crucial for cadherin binding
to .beta.-catenin and therefore more specific for
.beta.-catenin/TCF interaction. Thus, the present invention also
provides a method of treating diseases including but not limited to
cancer and hyperproliferative diseases comprising administering a
TCF peptidomimetic macrocycle of the invention to
.beta.-catenin.
[0091] A non-limiting exemplary list of suitable .beta.-catenin/TCF
peptides for use in the present invention is given below:
TABLE-US-00001 TABLE 1 SEQ ID (X = cross-linked amino acid); Notes
NOS Ac-LGANDELISFKDEGEQEEKSSENSSAERDLADVKSSL V -NH2 WT TCF4 1 Ac-
NLXNVKXSL VNQS -NH2 i,i + 4 x + link 2 Ac- RDLXNVKXSL VNES -NH2 i,i
+ 4 x + link 3 Ac- RDLAXVVSXL VNES -NH2 i,i + 4 x + link 4 Ac-
RNLAXVKSXL VNES -NH2 i,i + 4 x + link 5 Ac- RDLAXVKSXL VNQS -NH2
i,i + 4 x + link 6 Ac- RDLADVKXSL VXES -NH2 i,i + 4 x + link 7 Ac-
RNLADVKXSL VXES -NH2 i,i + 4 x + link 8 Ac- RDLANVIXSL VXES -NH2
i,i + 4 x + link 9 Ac- RDLANVVXSL VXES -NH2 i,i + 4 x + link 10 Ac-
RDLANVKXSNleVXES -NH2 i,i + 4 x + link 11 Ac- RGLANVKXSL VXES -NH2
i,i + 4 x + link 12 Ac- VERGLANVKXSL VXES -NH2 i,i + 4 x + link 13
Ac- RNLADVKSXL VNXS -NH2 i,i + 4 x + link 14 Ac- RDLANVKSXL VNXS
-NH2 i,i + 4 x + link 15 Ac- RDLAXVKSSL VXQS -NH2 i,i + 4 x + link
16 Ac- SAXRDLXDVKSSL VNESE-NH2 i,i + 4 x + link 17 Ac-
SAEXDLAXVKSSL VNESE-NH2 i,i + 4 x + link 18 Ac- SAERDLXDVKXSL
VNESE-NH2 i,i + 4 x + link 19 Ac- SAERDLAXVKSXL VNESE-NH2 i,i + 4 x
+ link 20 Ac- SAERDLADVKXSL VXESE-NH2 i,i + 4 x + link 21 Ac-
SAERDLADVKSXL VNXSE-NH2 i,i + 4 x + link 22 Ac- SAXRNLXDVKSSL V
-NH2 i,i + 4 x + link 23 Ac- SAXRDLXNVKSSL V -NH2 i,i + 4 x + link
24 Ac- SAXRDLXNVKSSL V -NH2 i,i + 4 x + link 25 Ac- SAXRNLXDVKSSL V
-NH2 i,i + 4 x + link 26 Ac-LGANDELISFKDEGEQEEKSS -NH2 wt TCF4 27
Ac-LGANDELISFXDEGXQEEKSSN -NH2 i,i + 4 x + link 28
Ac-LGANDELISFXDQGXQEEKSSN -NH2 i,i + 4 x + link 29
Ac-LGANDELISFXDQGXQQEKSSN -NH2 i,i + 4 x + link 30
Ac-LGANDELISFXDQGXQEQKSSN -NH2 i,i + 4 x + link 31
Ac-LGANDELISFXDEGXQQQKSSN -NH2 i,i + 4 x + link 32
Ac-LGANDQLISFXDEGXQEEKSSN -NH2 i,i + 4 x + link 33
Ac-LGANDELISFXDEGXQQEKSSN -NH2 i,i + 4 x + link 34
Ac-LGANDELISFXDEGXQEQKSSN -NH2 i,i + 4 x + link 35
Ac-LGANDELISFKXEGEXEEKSSN -NH2 i,i + 4 x + link 36
Ac-LGANDELISFKDEGXQEEXSSN -NH2 i,i + 4 x + link 37
Ac-LGANDELISFKDEGEXEEKXSN -NH2 i,i + 4 x + link 38
Ac-LGANDELISFXDEGEQEXKSSN -NH2 i,i + 4 x + link 39
Ac-LGANDELISFKXEGEQEESXXN -NH2 i,i + 4 x + link 40
Ac-LGANDELISFKXEGEQEESXXN -NH2 i,i + 4 x + link 41
Ac-LGANDELISFKXEGEQEEXSSN -NH2 i,i + 4 x + link 42
Ac-LGANAELISFKDEGEQEEKSSENSSAERDLADVKSSLV -NH2 mutant TCF4 43
Ac-LGANAELISFKDEGEQEEKSSENSSAERDLADAKSSAV -NH2 mutant TCF4 44
Ac-LGANDELISFXDEGXQEEKSSNNSSAERDLADVKSSLV -NH2 i,i + 4 x + link 45
Ac-LGANDELISFXDEGXQEEKSSNNSSAXRDLXDVKSSLV -NH2 i,i + 4 x + link 46
(2) Ac-LGANDELISFXDEGXQEEKSSENSSAXRDLXNVKSSLV -NH2 i,i + 4 x + link
47 (2) Ac-LGANDELLSFXDEGXQQEKSSENSSAXRDLXNVKSSLV -NH2 i,i + 4 x +
link 48 (2) Ac-LGANDELISFXDEGXQEEKSSNNSSAXRDLXAVKSSLV -NH2 i,i + 4
x + link 49 (2) Ac-LGANDELISFXDQGXQEQKSSENSSAXRDLXNVKSSLV -NH2 i,i
+ 4 x + link 50 (2) Ac-LGANDELISWXDEGXQQEKSSENSSAXRDLXNVKSSLV -NH2
i,i + 4 x + link 51 (2) Ac-LGANDELISFXDEGXQQQKSSENSSAXRDLXNVKSSLV
-NH2 i,i + 4 x + link 52 (2)
Ac-LGANDELISFXNEGXQEEKSSNNSSAXRDLXDVKSSLV -NH2 i,i + 4 x + link 53
(2) Ac-LGANDELISFXNQGXQEEKSSNNSSAXRNLXDVKSSLV -NH2 i,i + 4 x + link
54 (2) Ac-LGANDELISFXNEGXQEEKSSNNSSAXRNLXDVKSSLV -NH2 i,i + 4 x +
link 55 (2) Ac-LGANDELISFXNEGXQAEKSSNNSSAXRNLXDVKSSLV -NH2 i,i + 4
x + link 56 (2) Ac-LGANDELISFXNQGXQAAKSSNNSSAXRNLXAVKSSLV -NH2 i,i
+ 4 x + link 57 (2) Ac-LGANDELISFXDEGXQEEKSSNNSSAEXDLAXVKSSLV -NH2
i,i + 4 x + link 58 (2) Ac-LGANDELISFXDEGXQEEKSSspacerAXRDLXNVKSSLV
-NH2 i,i + 4 x + link 59 (2) and 60
Ac-LGANDELISFXDEGXQEEKSSspacerAXRDLXNVKSSLV i,i + 4 x + link 59 (2)
and 60 Ac-LGANDELISFKXDGEXEEKSSNNSSAERDLADVKSSLV i,i + 4 x + link
61 Ac-LGANDELISFKDEGXQEEXSSNNSSAERDLADVKSSLV i,i + 4 x + link 62
Ac-LGANDELISFKDEGEXEEKXSNNSSAERDLADVKSSLV i,i + 4 x + link 63
Ac-LGANDELISFKDEGEQEEKSSENSSAXRDLXDVKSSLV i,i + 4 x + link 64
Ac-LGANDELISFKDEGEQEEKSSENSSAEXDLAXVKSSLV i,i + 4 x + link 65
Table 1 shows .beta.-catenin/TCF sequences suitable for synthesis
of peptidomimetic macrocycles. "Spacer" represents a non-peptide
linker chain such as PEGn.
[0092] Peptidomimetic Macrocycles of the Invention
[0093] In some embodiments, a peptidomimetic macrocycle of the
invention has the Formula (I):
##STR00005##
[0094] wherein:
[0095] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0096] B is a natural or non-natural amino acid, amino acid
analog,
##STR00006##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0097] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0098] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0099] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0100] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0101] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0102] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0103] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0104] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0105] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0106] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0107] v and w are independently integers from 1-1000;
[0108] u, x, y and z are independently integers from 0-10; and
[0109] n is an integer from 1-5.
[0110] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0111] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle
or macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges. Similarly, when u is greater
than 1, each compound of the invention may encompass peptidomimetic
macrocycles which are the same or different. For example, a
compound of the invention may comprise peptidomimetic macrocycles
comprising different linker lengths or chemical compositions.
[0112] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00007##
[0113] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0114] In one embodiment, the peptidomimetic macrocycle of Formula
(I) is:
##STR00008##
[0115] wherein each R.sub.1 and R.sub.2 is independently
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-.
[0116] In related embodiments, the peptidomimetic macrocycle of
Formula (I) is:
##STR00009##
[0117] In other embodiments, the peptidomimetic macrocycle of
Formula (I) is a compound of any of the formulas shown below:
##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014##
wherein "AA" represents any natural or non-natural amino acid side
chain and "" is [D].sub.v, [E].sub.w as defined above, and n is an
integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some
embodiments, n is 0. In other embodiments, n is less than 50.
[0118] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00015##
[0119] In some embodiments, the peptidomimetic macrocycles of the
invention have the Formula (II):
##STR00016##
[0120] wherein:
[0121] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0122] B is a natural or non-natural amino acid, amino acid
analog,
##STR00017##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0123] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0124] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0125] L is a macrocycle-forming linker of the formula
##STR00018##
[0126] L.sub.1, L.sub.2 and L.sub.3 are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0127] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0128] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0129] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0130] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0131] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0132] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0133] v and w are independently integers from 1-1000;
[0134] u, x, y and z are independently integers from 0-10; and
[0135] n is an integer from 1-5.
[0136] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0137] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle
or macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0138] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00019##
[0139] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0140] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029##
##STR00030##
[0141] In other embodiments, the invention provides peptidomimetic
macrocycles of Formula (III):
##STR00031##
[0142] wherein:
[0143] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0144] B is a natural or non-natural amino acid, amino acid
analog
##STR00032##
[--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-];
[0145] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0146] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, unsubstituted or substituted with
R.sub.5;
[0147] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4--]n, each being unsubstituted or substituted
with R.sub.5;
[0148] K is O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;
[0149] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0150] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0151] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0152] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, unsubstituted or substituted with
R.sub.5, or part of a cyclic structure with a D residue;
[0153] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, unsubstituted or substituted with
R.sub.5, or part of a cyclic structure with an E residue;
[0154] v and w are independently integers from 1-1000;
[0155] u, x, y and z are independently integers from 0-10; and
[0156] n is an integer from 1-5.
[0157] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0158] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9
or 10. Each occurrence of A, B, C, D or E in a macrocycle or
macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0159] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00033##
[0160] In other embodiments, the length of the macrocycle-forming
linker [-L.sub.1-S-L.sub.2-S-L.sub.3-] as measured from a first
C.alpha. to a second C.alpha. is selected to stabilize a desired
secondary peptide structure, such as an .alpha.-helix formed by
residues of the peptidomimetic macrocycle including, but not
necessarily limited to, those between the first C.alpha. to a
second C.alpha..
[0161] Macrocycles or macrocycle precursors are synthesized, for
example, by solution phase or solid-phase methods, and can contain
both naturally-occurring and non-naturally-occurring amino acids.
See, for example, Hunt, "The Non-Protein Amino Acids" in Chemistry
and Biochemistry of the Amino Acids, edited by G. C. Barrett,
Chapman and Hall, 1985. In some embodiments, the thiol moieties are
the side chains of the amino acid residues L-cysteine, D-cysteine,
.alpha.-methyl-L cysteine, .alpha.-methyl-D-cysteine,
L-homocysteine, D-homocysteine, .alpha.-methyl-L-homocysteine or
.alpha.-methyl-D-homocysteine. A bis-alkylating reagent is of the
general formula X-L.sub.2-Y wherein L.sub.2 is a linker moiety and
X and Y are leaving groups that are displaced by --SH moieties to
form bonds with L.sub.2. In some embodiments, X and Y are halogens
such as I, Br, or Cl.
[0162] In other embodiments, D and/or E in the compound of Formula
I, II or III are further modified in order to facilitate cellular
uptake. In some embodiments, lipidating or PEGylating a
peptidomimetic macrocycle facilitates cellular uptake, increases
bioavailability, increases blood circulation, alters
pharmacokinetics, decreases immunogenicity and/or decreases the
needed frequency of administration.
[0163] In other embodiments, at least one of [D] and [E] in the
compound of Formula I, II or III represents a moiety comprising an
additional macrocycle-forming linker such that the peptidomimetic
macrocycle comprises at least two macrocycle-forming linkers. In a
specific embodiment, a peptidomimetic macrocycle comprises two
macrocycle-forming linkers.
[0164] In the peptidomimetic macrocycles of the invention, any of
the macrocycle-forming linkers described herein may be used in any
combination with any of the sequences shown in Tables 1-4 and also
with any of the R-- substituents indicated herein.
[0165] In some embodiments, the peptidomimetic macrocycle comprises
at least one .alpha.-helix motif. For example, A, B and/or C in the
compound of Formula I, II or III include one or more
.alpha.-helices. As a general matter, .alpha.-helices include
between 3 and 4 amino acid residues per turn. In some embodiments,
the .alpha.-helix of the peptidomimetic macrocycle includes 1 to 5
turns and, therefore, 3 to 20 amino acid residues. In specific
embodiments, the .alpha.-helix includes 1 turn, 2 turns, 3 turns, 4
turns, or 5 turns. In some embodiments, the macrocycle-forming
linker stabilizes an .alpha.-helix motif included within the
peptidomimetic macrocycle. Thus, in some embodiments, the length of
the macrocycle-forming linker L from a first C.alpha. to a second
C.alpha. is selected to increase the stability of an .alpha.-helix.
In some embodiments, the macrocycle-forming linker spans from 1
turn to 5 turns of the .alpha.-helix. In some embodiments, the
macrocycle-forming linker spans approximately 1 turn, 2 turns, 3
turns, 4 turns, or 5 turns of the .alpha.-helix. In some
embodiments, the length of the macrocycle-forming linker is
approximately 5 .ANG. to 9 .ANG. per turn of the .alpha.-helix, or
approximately 6 .ANG. to 8 .ANG. per turn of the .alpha.-helix.
Where the macrocycle-forming linker spans approximately 1 turn of
an .alpha.-helix, the length is equal to approximately 5
carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7
carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 2 turns of an .alpha.-helix, the length is equal to
approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds,
approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or
approximately 12 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 3 turns of an .alpha.-helix, the length
is equal to approximately 14 carbon-carbon bonds to 22
carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20
carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where
the macrocycle-forming linker spans approximately 4 turns of an
.alpha.-helix, the length is equal to approximately 20
carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22
carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 5 turns of an .alpha.-helix, the length is equal to
approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds,
approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or
approximately 30 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 1 turn of an .alpha.-helix, the linkage
contains approximately 4 atoms to 12 atoms, approximately 6 atoms
to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming
linker spans approximately 2 turns of the .alpha.-helix, the
linkage contains approximately 7 atoms to 15 atoms, approximately 9
atoms to 13 atoms, or approximately 11 atoms. Where the
macrocycle-forming linker spans approximately 3 turns of the
.alpha.-helix, the linkage contains approximately 13 atoms to 21
atoms, approximately 15 atoms to 19 atoms, or approximately 17
atoms. Where the macrocycle-forming linker spans approximately 4
turns of the .alpha.-helix, the linkage contains approximately 19
atoms to 27 atoms, approximately 21 atoms to 25 atoms, or
approximately 23 atoms. Where the macrocycle-forming linker spans
approximately 5 turns of the .alpha.-helix, the linkage contains
approximately 25 atoms to 33 atoms, approximately 27 atoms to 31
atoms, or approximately 29 atoms. Where the macrocycle-forming
linker spans approximately 1 turn of the .alpha.-helix, the
resulting macrocycle forms a ring containing approximately 17
members to 25 members, approximately 19 members to 23 members, or
approximately 21 members. Where the macrocycle-forming linker spans
approximately 2 turns of the .alpha.-helix, the resulting
macrocycle forms a ring containing approximately 29 members to 37
members, approximately 31 members to 35 members, or approximately
33 members. Where the macrocycle-forming linker spans approximately
3 turns of the .alpha.-helix, the resulting macrocycle forms a ring
containing approximately 44 members to 52 members, approximately 46
members to 50 members, or approximately 48 members. Where the
macrocycle-forming linker spans approximately 4 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 59 members to 67 members, approximately 61 members to
65 members, or approximately 63 members. Where the
macrocycle-forming linker spans approximately 5 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 74 members to 82 members, approximately 76 members to
80 members, or approximately 78 members.
[0166] In other embodiments, the invention provides peptidomimetic
macrocycles of Formula (IV) or (IVa):
##STR00034##
[0167] wherein:
[0168] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0169] B is a natural or non-natural amino acid, amino acid
analog,
##STR00035##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0170] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-, or part
of a cyclic structure with an E residue;
[0171] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0172] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0173] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0174] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0175] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0176] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0177] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0178] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0179] v and w are independently integers from 1-1000;
[0180] u, x, y and z are independently integers from 0-10; and
[0181] n is an integer from 1-5.
[0182] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0183] In some embodiments of the invention, x+y+z is at least 1.
In other embodiments of the invention, x+y+z is at least 2. In
other embodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9 or
10. Each occurrence of A, B, C, D or E in a macrocycle or
macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0184] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00036##
[0185] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0186] Exemplary embodiments of the macrocycle-forming linker
-L.sub.1-L.sub.2- are shown below.
##STR00037##
Preparation of Pentidomimetic Macrocycles
[0187] Peptidomimetic macrocycles of the invention may be prepared
by any of a variety of methods known in the art. For example, any
of the residues indicated by "X" in Tables 1, 2, 3 or 4 may be
substituted with a residue capable of forming a crosslinker with a
second residue in the same molecule or a precursor of such a
residue.
[0188] Various methods to effect formation of peptidomimetic
macrocycles are known in the art. For example, the preparation of
peptidomimetic macrocycles of Formula I is described in
Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000);
Schafmeister & Verdin, J. Am. Chem. Soc. 122:5891 (2005);
Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No.
7,192,713. The .alpha.,.alpha.-disubstituted amino acids and amino
acid precursors disclosed in the cited references may be employed
in synthesis of the peptidomimetic macrocycle precursor
polypeptides. For example, the "S5 olefin amino acid" is
(S)-.alpha.-(2'-pentenyl) alanine and the "R8 olefin amino acid" is
(R)-.alpha.-(2'-octenyl) alanine. Following incorporation of such
amino acids into precursor polypeptides, the terminal olefins are
reacted with a metathesis catalyst, leading to the formation of the
peptidomimetic macrocycle.
[0189] In other embodiments, the peptidomimetic macrocyles of the
invention are of Formula IV or IVa. Methods for the preparation of
such macrocycles are described, for example, in U.S. Pat. No.
7,202,332.
[0190] In some embodiments, the synthesis of these peptidomimetic
macrocycles involves a multi-step process that features the
synthesis of a peptidomimetic precursor containing an azide moiety
and an alkyne moiety; followed by contacting the peptidomimetic
precursor with a macrocyclization reagent to generate a
triazole-linked peptidomimetic macrocycle. Such a process is
described, for example, in U.S. application Ser. No. 12/037,041,
filed on Feb. 25, 2008. Macrocycles or macrocycle precursors are
synthesized, for example, by solution phase or solid-phase methods,
and can contain both naturally-occurring and
non-naturally-occurring amino acids. See, for example, Hunt, "The
Non-Protein Amino Acids" in Chemistry and Biochemistry of the Amino
Acids, edited by G. C. Barrett, Chapman and Hall, 1985.
[0191] In some embodiments, an azide is linked to the
.alpha.-carbon of a residue and an alkyne is attached to the
.alpha.-carbon of another residue. In some embodiments, the azide
moieties are azido-analogs of amino acids L-lysine, D-lysine,
alpha-methyl-L-lysine, alpha-methyl-D-lysine, L-ornithine,
D-ornithine, alpha-methyl-L-ornithine or alpha-methyl-D-ornithine.
In another embodiment, the alkyne moiety is L-propargylglycine. In
yet other embodiments, the alkyne moiety is an amino acid selected
from the group consisting of L-propargylglycine,
D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
(R)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-2-methyl-5-hexynoic acid,
(R)-2-amino-2-methyl-5-hexynoic acid,
(S)-2-amino-2-methyl-6-heptynoic acid,
(R)-2-amino-2-methyl-6-heptynoic acid,
(S)-2-amino-2-methyl-7-octynoic acid,
(R)-2-amino-2-methyl-7-octynoic acid,
(S)-2-amino-2-methyl-8-nonynoic acid and
(R)-2-amino-2-methyl-8-nonynoic acid.
[0192] In some embodiments, the invention provides a method for
synthesizing a peptidomimetic macrocycle, the method comprising the
steps of contacting a peptidomimetic precursor of Formula V or
Formula VI:
##STR00038##
[0193] with a macrocyclization reagent;
[0194] wherein v, w, x, y, z, A, B, C, D, E, R.sub.1, R.sub.2,
R.sub.7, R.sub.8, L.sub.1 and L.sub.2 are as defined for Formula
(II); R.sub.12 is --H when the macrocyclization reagent is a Cu
reagent and R.sub.12 is --H or alkyl when the macrocyclization
reagent is a Ru reagent; and further wherein said contacting step
results in a covalent linkage being formed between the alkyne and
azide moiety in Formula III or Formula IV. For example, R.sub.12
may be methyl when the macrocyclization reagent is a Ru
reagent.
[0195] In the peptidomimetic macrocycles of the invention, at least
one of R.sub.1 and R.sub.2 is alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-. In some embodiments, both
R.sub.1 and R.sub.2 are independently alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid.
[0196] For example, at least one of R.sub.1 and R.sub.2 is alkyl,
unsubstituted or substituted with halo-. In another example, both
R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl. The macrocyclization reagent may be a Cu
reagent or a Ru reagent.
[0197] In some embodiments, the peptidomimetic precursor is
purified prior to the contacting step. In other embodiments, the
peptidomimetic macrocycle is purified after the contacting step. In
still other embodiments, the peptidomimetic macrocycle is refolded
after the contacting step. The method may be performed in solution,
or, alternatively, the method may be performed on a solid
support.
[0198] Also envisioned herein is performing the method of the
invention in the presence of a target macromolecule that binds to
the peptidomimetic precursor or peptidomimetic macrocycle under
conditions that favor said binding. In some embodiments, the method
is performed in the presence of a target macromolecule that binds
preferentially to the peptidomimetic precursor or peptidomimetic
macrocycle under conditions that favor said binding. The method may
also be applied to synthesize a library of peptidomimetic
macrocycles.
[0199] In some embodiments, the alkyne moiety of the peptidomimetic
precursor of Formula V or Formula VI is a sidechain of an amino
acid selected from the group consisting of L-propargylglycine,
D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
(R)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-2-methyl-5-hexynoic acid,
(R)-2-amino-2-methyl-5-hexynoic acid,
(S)-2-amino-2-methyl-6-heptynoic acid,
(R)-2-amino-2-methyl-6-heptynoic acid,
(S)-2-amino-2-methyl-7-octynoic acid,
(R)-2-amino-2-methyl-7-octynoic acid,
(S)-2-amino-2-methyl-8-nonynoic acid, and
(R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, the
azide moiety of the peptidomimetic precursor of Formula V or
Formula VI is a sidechain of an amino acid selected from the group
consisting of -azido-L-lysine, -azido-D-lysine,
-azido-.alpha.-methyl-L-lysine, -azido-.alpha.-methyl-D-lysine,
.delta.-azido-.alpha.-methyl-L-ornithine, and
.delta.-azido-.alpha.-methyl-D-ornithine.
[0200] In some embodiments, x+y+z is 3, and and A, B and C are
independently natural or non-natural amino acids. In other
embodiments, x+y+z is 6, and and A, B and C are independently
natural or non-natural amino acids.
[0201] In some embodiments, the contacting step is performed in a
solvent selected from the group consisting of protic solvent,
aqueous solvent, organic solvent, and mixtures thereof. For
example, the solvent may be chosen from the group consisting of
H.sub.2O, THF, THF/H.sub.2O, tBuOH/H.sub.2O, DMF, DIPEA, CH.sub.3CN
or CH.sub.2Cl.sub.2, ClCH.sub.2CH.sub.2Cl or a mixture thereof. The
solvent may be a solvent which favors helix formation.
[0202] Alternative but equivalent protecting groups, leaving groups
or reagents are substituted, and certain of the synthetic steps are
performed in alternative sequences or orders to produce the desired
compounds. Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein include, for example, those such as
described in Larock, Comprehensive Organic Transformations, VCH
Publishers (1989); Greene and Wuts, Protective Groups in Organic
Synthesis, 2d. Ed., John Wiley and Sons (1991); Fieser and Fieser,
Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and
Sons (1994); and Paquette, ed., Encyclopedia of Reagents for
Organic Synthesis, John Wiley and Sons (1995), and subsequent
editions thereof.
[0203] The peptidomimetic macrocycles of the invention are made,
for example, by chemical synthesis methods, such as described in
Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed.
Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence,
for example, peptides are synthesized using the automated
Merrifield techniques of solid phase synthesis with the amine
protected by either tBoc or Fmoc chemistry using side chain
protected amino acids on, for example, an automated peptide
synthesizer (e.g., Applied Biosystems (Foster City, Calif.), Model
430A, 431, or 433).
[0204] One manner of producing the peptidomimetic precursors and
peptidomimetic macrocycles described herein uses solid phase
peptide synthesis (SPPS). The C-terminal amino acid is attached to
a cross-linked polystyrene resin via an acid labile bond with a
linker molecule. This resin is insoluble in the solvents used for
synthesis, making it relatively simple and fast to wash away excess
reagents and by-products. The N-terminus is protected with the Fmoc
group, which is stable in acid, but removable by base. Side chain
functional groups are protected as necessary with base stable, acid
labile groups.
[0205] Longer peptidomimetic precursors are produced, for example,
by conjoining individual synthetic peptides using native chemical
ligation. Alternatively, the longer synthetic peptides are
biosynthesized by well known recombinant DNA and protein expression
techniques. Such techniques are provided in well-known standard
manuals with detailed protocols. To construct a gene encoding a
peptidomimetic precursor of this invention, the amino acid sequence
is reverse translated to obtain a nucleic acid sequence encoding
the amino acid sequence, preferably with codons that are optimum
for the organism in which the gene is to be expressed. Next, a
synthetic gene is made, typically by synthesizing oligonucleotides
which encode the peptide and any regulatory elements, if necessary.
The synthetic gene is inserted in a suitable cloning vector and
transfected into a host cell. The peptide is then expressed under
suitable conditions appropriate for the selected expression system
and host. The peptide is purified and characterized by standard
methods.
[0206] The peptidomimetic precursors are made, for example, in a
high-throughput, combinatorial fashion using, for example, a
high-throughput polychannel combinatorial synthesizer (e.g.,
Thuramed TETRAS multichannel peptide synthesizer from CreoSalus,
Louisville, Ky. or Model Apex 396 multichannel peptide synthesizer
from AAPPTEC, Inc., Louisville, Ky.).
[0207] The following synthetic schemes are provided solely to
illustrate the present invention and are not intended to limit the
scope of the invention, as described herein. To simplify the
drawings, the illustrative schemes depict azido amino acid analogs
-azido-.alpha.-methyl-L-lysine and -azido-.alpha.-methyl-D-lysine,
and alkyne amino acid analogs L-propargylglycine,
(S)-2-amino-2-methyl-4-pentynoic acid, and
(S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the following
synthetic schemes, each R.sub.1, R.sub.2, R.sub.7 and R.sub.8 is
--H; each L.sub.1 is --(CH.sub.2).sub.4--; and each L.sub.2 is
--(CH.sub.2)--. However, as noted throughout the detailed
description above, many other amino acid analogs can be employed in
which R.sub.1, R.sub.2, R.sub.7, R.sub.8, L.sub.1 and L.sub.2 can
be independently selected from the various structures disclosed
herein.
##STR00039## ##STR00040##
[0208] Synthetic Scheme 1 describes the preparation of several
compounds of the invention. Ni(II) complexes of Schiff bases
derived from the chiral auxiliary
(S)-2-[N--(N'-benzylprolyl)amino]benzophenone (BPB) and amino acids
such as glycine or alanine are prepared as described in Belokon et
al. (1998), Tetrahedron Asymm. 9:4249-4252. The resulting complexes
are subsequently reacted with alkylating reagents comprising an
azido or alkynyl moiety to yield enantiomerically enriched
compounds of the invention. If desired, the resulting compounds can
be protected for use in peptide synthesis.
##STR00041##
[0209] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 2, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solution-phase or solid-phase peptide synthesis
(SPPS) using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl- -azido-L-lysine, and N-methyl- -azido-D-lysine. The
peptidomimetic precursor is then deprotected and cleaved from the
solid-phase resin by standard conditions (e.g., strong acid such as
95% TFA). The peptidomimetic precursor is reacted as a crude
mixture or is purified prior to reaction with a macrocyclization
reagent such as a Cu(I) in organic or aqueous solutions (Rostovtsev
et al. (2002), Angew. Chem. Int. Ed. 41:2596-2599; Tornoe et al.
(2002), J. Org. Chem. 67:3057-3064; Deiters et al. (2003), J. Am.
Chem. Soc. 125:11782-11783; Punna et al. (2005), Angew. Chem. Int.
Ed. 44:2215-2220). In one embodiment, the triazole forming reaction
is performed under conditions that favor .alpha.-helix formation.
In one embodiment, the macrocyclization step is performed in a
solvent chosen from the group consisting of H.sub.2O, THF,
CH.sub.3CN, DMF, DIPEA, tBuOH or a mixture thereof. In another
embodiment, the macrocyclization step is performed in DMF. In some
embodiments, the macrocyclization step is performed in a buffered
aqueous or partially aqueous solvent.
##STR00042##
[0210] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 3, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solid-phase peptide synthesis (SPPS) using the
commercially available amino acid N-.alpha.-Fmoc-L-propargylglycine
and the N-.alpha.-Fmoc-protected forms of the amino acids
(S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic
acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl-
-azido-L-lysine, and N-methyl- -azido-D-lysine. The peptidomimetic
precursor is reacted with a macrocyclization reagent such as a
Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al.
(2002), Angew. Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002),
J. Org. Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem.
Soc. 125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed.
44:2215-2220). The resultant triazole-containing peptidomimetic
macrocycle is then deprotected and cleaved from the solid-phase
resin by standard conditions (e.g., strong acid such as 95% TFA).
In some embodiments, the macrocyclization step is performed in a
solvent chosen from the group consisting of CH.sub.2Cl.sub.2,
ClCH.sub.2CH.sub.2Cl, DMF, THF, NMP, DIPEA, 2,6-lutidine, pyridine,
DMSO, H.sub.2O or a mixture thereof. In some embodiments, the
macrocyclization step is performed in a buffered aqueous or
partially aqueous solvent.
##STR00043##
[0211] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 4, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solution-phase or solid-phase peptide synthesis
(SPPS) using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl- -azido-L-lysine, and N-methyl- -azido-D-lysine. The
peptidomimetic precursor is then deprotected and cleaved from the
solid-phase resin by standard conditions (e.g., strong acid such as
95% TFA). The peptidomimetic precursor is reacted as a crude
mixture or is purified prior to reaction with a macrocyclization
reagent such as a Ru(II) reagents, for example
Cp*RuCl(PPh.sub.3).sub.2 or [Cp*RuCl].sub.4 (Rasmussen et al.
(2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem.
Soc. 127:15998-15999). In some embodiments, the macrocyclization
step is performed in a solvent chosen from the group consisting of
DMF, CH.sub.3CN and THF.
##STR00044##
[0212] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 5, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solid-phase peptide synthesis (SPPS) using the
commercially available amino acid N-.alpha.-Fmoc-L-propargylglycine
and the N-.alpha.-Fmoc-protected forms of the amino acids
(S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic
acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl-
-azido-L-lysine, and N-methyl- -azido-D-lysine. The peptidomimetic
precursor is reacted with a macrocyclization reagent such as a
Ru(II) reagent on the resin as a crude mixture. For example, the
reagent can be Cp*RuCl(PPh.sub.3).sub.2 or [Cp*RuCl].sub.4
(Rasmussen et al. (2007), Org. Lett. 9:5337-5339; Zhang et al.
(2005), J. Am. Chem. Soc. 127:15998-15999). In some embodiments,
the macrocyclization step is performed in a solvent chosen from the
group consisting of CH.sub.2Cl.sub.2, ClCH.sub.2CH.sub.2Cl,
CH.sub.3CN, DMF, and THF.
[0213] The present invention contemplates the use of
non-naturally-occurring amino acids and amino acid analogs in the
synthesis of the peptidomimetic macrocycles described herein. Any
amino acid or amino acid analog amenable to the synthetic methods
employed for the synthesis of stable triazole containing
peptidomimetic macrocycles can be used in the present invention.
For example, L-propargylglycine is contemplated as a useful amino
acid in the present invention. However, other alkyne-containing
amino acids that contain a different amino acid side chain are also
useful in the invention. For example, L-propargylglycine contains
one methylene unit between the .alpha.-carbon of the amino acid and
the alkyne of the amino acid side chain. The invention also
contemplates the use of amino acids with multiple methylene units
between the .alpha.-carbon and the alkyne. Also, the azido-analogs
of amino acids L-lysine, D-lysine, alpha-methyl-L-lysine, and
alpha-methyl-D-lysine are contemplated as useful amino acids in the
present invention. However, other terminal azide amino acids that
contain a different amino acid side chain are also useful in the
invention. For example, the azido-analog of L-lysine contains four
methylene units between the .alpha.-carbon of the amino acid and
the terminal azide of the amino acid side chain. The invention also
contemplates the use of amino acids with fewer than or greater than
four methylene units between the .alpha.-carbon and the terminal
azide. Table 2 shows some amino acids useful in the preparation of
peptidomimetic macrocycles of the invention.
TABLE-US-00002 TABLE 2 ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064##
[0214] Table 2 shows exemplary amino acids useful in the
preparation of peptidomimetic macrocycles of the invention.
[0215] In some embodiments the amino acids and amino acid analogs
are of the D-configuration. In other embodiments they are of the
L-configuration. In some embodiments, some of the amino acids and
amino acid analogs contained in the peptidomimetic are of the
D-configuration while some of the amino acids and amino acid
analogs are of the L-configuration. In some embodiments the amino
acid analogs are .alpha.,.alpha.-disubstituted, such as
.alpha.-methyl-L-propargylglycine,
.alpha.-methyl-D-propargylglycine, -azido-alpha-methyl-L-lysine,
and -azido-alpha-methyl-D-lysine. In some embodiments the amino
acid analogs are N-alkylated, e.g., N-methyl-L-propargylglycine,
N-methyl-D-propargylglycine, N-methyl- -azido-L-lysine, and
N-methyl- -azido-D-lysine.
[0216] In some embodiments, the --NH moiety of the amino acid is
protected using a protecting group, including without
limitation-Fmoc and -Boc. In other embodiments, the amino acid is
not protected prior to synthesis of the peptidomimetic
macrocycle.
[0217] In other embodiments, peptidomimetic macrocycles of Formula
III are synthesized. The preparation of such macrocycles is
described, for example, in U.S. application Ser. No. 11/957,325,
filed on Dec. 17, 2007. The following synthetic schemes describe
the preparation of such compounds. To simplify the drawings, the
illustrative schemes depict amino acid analogs derived from L- or
D-cysteine, in which L.sub.1 and L.sub.3 are both --(CH.sub.2)--.
However, as noted throughout the detailed description above, many
other amino acid analogs can be employed in which L.sub.1 and
L.sub.3 can be independently selected from the various structures
disclosed herein. The symbols "[AA].sub.m", "[AA].sub.n",
"[AA].sub.o" represent a sequence of amide bond-linked moieties
such as natural or unnatural amino acids. As described previously,
each occurrence of "AA" is independent of any other occurrence of
"AA", and a formula such as "[AA].sub.m" encompasses, for example,
sequences of non-identical amino acids as well as sequences of
identical amino acids.
##STR00065##
[0218] In Scheme 6, the peptidomimetic precursor contains two --SH
moieties and is synthesized by solid-phase peptide synthesis (SPPS)
using commercially available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-trityl-L-cysteine or
N-.alpha.-Fmoc-S-trityl-D-cysteine. Alpha-methylated versions of
D-cysteine or L-cysteine are generated by known methods (Seebach et
al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and
references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-trityl monomers by known methods
("Bioorganic Chemistry: Peptides and Proteins", Oxford University
Press, New York: 1998, the entire contents of which are
incorporated herein by reference). The precursor peptidomimetic is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The precursor
peptidomimetic is reacted as a crude mixture or is purified prior
to reaction with X-L.sub.2-Y in organic or aqueous solutions. In
some embodiments the alkylation reaction is performed under dilute
conditions (i.e. 0.15 mmol/L) to favor macrocyclization and to
avoid polymerization. In some embodiments, the alkylation reaction
is performed in organic solutions such as liquid NH.sub.3 (Mosberg
et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al.
(1992), Int. J. Peptide Protein Res. 40:233-242), NH.sub.3/MeOH, or
NH.sub.3/DMF (Or et al. (1991), J. Org. Chem. 56:3146-3149). In
other embodiments, the alkylation is performed in an aqueous
solution such as 6M guanidinium HCL, pH 8 (Brunel et al. (2005),
Chem. Commun. (20):2552-2554). In other embodiments, the solvent
used for the alkylation reaction is DMF or dichloroethane.
##STR00066##
[0219] In Scheme 7, the precursor peptidomimetic contains two or
more --SH moieties, of which two are specially protected to allow
their selective deprotection and subsequent alkylation for
macrocycle formation. The precursor peptidomimetic is synthesized
by solid-phase peptide synthesis (SPPS) using commercially
available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-p-methoxytrityl-L-cysteine or
N-.alpha.-Fmoc-S-p-methoxytrityl-D-cysteine. Alpha-methylated
versions of D-cysteine or L-cysteine are generated by known methods
(Seebach et al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748,
and references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-p-methoxytrityl monomers by known
methods (Bioorganic Chemistry: Peptides and Proteins, Oxford
University Press, New York: 1998, the entire contents of which are
incorporated herein by reference). The Mmt protecting groups of the
peptidomimetic precursor are then selectively cleaved by standard
conditions (e.g., mild acid such as 1% TFA in DCM). The precursor
peptidomimetic is then reacted on the resin with X-L.sub.2-Y in an
organic solution. For example, the reaction takes place in the
presence of a hindered base such as diisopropylethylamine. In some
embodiments, the alkylation reaction is performed in organic
solutions such as liquid NH.sub.3 (Mosberg et al. (1985), J. Am.
Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J. Peptide
Protein Res. 40:233-242), NH.sub.3/MeOH or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). In other embodiments, the
alkylation reaction is performed in DMF or dichloroethane. The
peptidomimetic macrocycle is then deprotected and cleaved from the
solid-phase resin by standard conditions (e.g., strong acid such as
95% TFA).
##STR00067##
[0220] In Scheme 8, the peptidomimetic precursor contains two or
more --SH moieties, of which two are specially protected to allow
their selective deprotection and subsequent alkylation for
macrocycle formation. The peptidomimetic precursor is synthesized
by solid-phase peptide synthesis (SPPS) using commercially
available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-p-methoxytrityl-L-cysteine,
N-.alpha.-Fmoc-S-p-methoxytrityl-D-cysteine,
N-.alpha.-Fmoc-S--S-t-butyl-L-cysteine, and
N-.alpha.-Fmoc-S--S-t-butyl-D-cysteine. Alpha-methylated versions
of D-cysteine or L-cysteine are generated by known methods (Seebach
et al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and
references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-p-methoxytrityl or
N-.alpha.-Fmoc-S--S-t-butyl monomers by known methods (Bioorganic
Chemistry: Peptides and Proteins, Oxford University Press, New
York: 1998, the entire contents of which are incorporated herein by
reference). The S--S-tButyl protecting group of the peptidomimetic
precursor is selectively cleaved by known conditions (e.g., 20%
2-mercaptoethanol in DMF, reference: Galande et al. (2005), J.
Comb. Chem. 7:174-177). The precursor peptidomimetic is then
reacted on the resin with a molar excess of X-L.sub.2-Y in an
organic solution. For example, the reaction takes place in the
presence of a hindered base such as diisopropylethylamine. The Mmt
protecting group of the peptidomimetic precursor is then
selectively cleaved by standard conditions (e.g., mild acid such as
1% TFA in DCM). The peptidomimetic precursor is then cyclized on
the resin by treatment with a hindered base in organic solutions.
In some embodiments, the alkylation reaction is performed in
organic solutions such as NH.sub.3/MeOH or NH.sub.3/DMF (Or et al.
(1991), J Org. Chem. 56:3146-3149). The peptidomimetic macrocycle
is then deprotected and cleaved from the solid-phase resin by
standard conditions (e.g., strong acid such as 95% TFA).
##STR00068##
[0221] In Scheme 9, the peptidomimetic precursor contains two
L-cysteine moieties. The peptidomimetic precursor is synthesized by
known biological expression systems in living cells or by known in
vitro, cell-free, expression methods. The precursor peptidomimetic
is reacted as a crude mixture or is purified prior to reaction with
X-L2-Y in organic or aqueous solutions. In some embodiments the
alkylation reaction is performed under dilute conditions (i.e. 0.15
mmol/L) to favor macrocyclization and to avoid polymerization. In
some embodiments, the alkylation reaction is performed in organic
solutions such as liquid NH.sub.3 (Mosberg et al. (1985), J. Am.
Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J. Peptide
Protein Res. 40:233-242), NH.sub.3/MeOH, or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). In other embodiments, the
alkylation is performed in an aqueous solution such as 6M
guanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.
(20):2552-2554). In other embodiments, the alkylation is performed
in DMF or dichloroethane. In another embodiment, the alkylation is
performed in non-denaturing aqueous solutions, and in yet another
embodiment the alkylation is performed under conditions that favor
.alpha.-helical structure formation. In yet another embodiment, the
alkylation is performed under conditions that favor the binding of
the precursor peptidomimetic to another protein, so as to induce
the formation of the bound .alpha.-helical conformation during the
alkylation.
[0222] Various embodiments for X and Y are envisioned which are
suitable for reacting with thiol groups. In general, each X or Y is
independently be selected from the general category shown in Table
5. For example, X and Y are halides such as --Cl, --Br or --I. Any
of the macrocycle-forming linkers described herein may be used in
any combination with any of the sequences shown in Tables 1-4 and
also with any of the R-- substituents indicated herein.
TABLE-US-00003 TABLE 3 Examples of Reactive Groups Capable of
Reacting with Thiol Groups and Resulting Linkages X or Y Resulting
Covalent Linkage acrylamide Thioether halide (e.g. alkyl or aryl
halide) Thioether sulfonate Thioether aziridine Thioether epoxide
Thioether haloacetamide Thioether maleimide Thioether sulfonate
ester Thioether
[0223] The present invention contemplates the use of both
naturally-occurring and non-naturally-occurring amino acids and
amino acid analogs in the synthesis of the peptidomimetic
macrocycles of Formula (III). Any amino acid or amino acid analog
amenable to the synthetic methods employed for the synthesis of
stable bis-sulfhydryl containing peptidomimetic macrocycles can be
used in the present invention. For example, cysteine is
contemplated as a useful amino acid in the present invention.
However, sulfur containing amino acids other than cysteine that
contain a different amino acid side chain are also useful. For
example, cysteine contains one methylene unit between the
.alpha.-carbon of the amino acid and the terminal --SH of the amino
acid side chain. The invention also contemplates the use of amino
acids with multiple methylene units between the .alpha.-carbon and
the terminal --SH. Non-limiting examples include
.alpha.-methyl-L-homocysteine and .alpha.-methyl-D-homocysteine. In
some embodiments the amino acids and amino acid analogs are of the
D-configuration. In other embodiments they are of the
L-configuration. In some embodiments, some of the amino acids and
amino acid analogs contained in the peptidomimetic are of the
D-configuration while some of the amino acids and amino acid
analogs are of the L-configuration. In some embodiments the amino
acid analogs are .alpha.,.alpha.-disubstituted, such as
.alpha.-methyl-L-cysteine and .alpha.-methyl-D-cysteine.
[0224] The invention includes macrocycles in which
macrocycle-forming linkers are used to link two or more --SH
moieties in the peptidomimetic precursors to form the
peptidomimetic macrocycles of the invention. As described above,
the macrocycle-forming linkers impart conformational rigidity,
increased metabolic stability and/or increased cell penetrability.
Furthermore, in some embodiments, the macrocycle-forming linkages
stabilize the .alpha.-helical secondary structure of the
peptidomimetic macrocyles. The macrocycle-forming linkers are of
the formula X-L.sub.2-Y, wherein both X and Y are the same or
different moieties, as defined above. Both X and Y have the
chemical characteristics that allow one macrocycle-forming linker
-L.sub.2- to bis alkylate the bis-sulfhydryl containing
peptidomimetic precursor. As defined above, the linker -L.sub.2-
includes alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene, or
heterocycloarylene, or --R.sub.4--K--R.sub.4--, all of which can be
optionally substituted with an R.sub.5 group, as defined above.
Furthermore, one to three carbon atoms within the
macrocycle-forming linkers -L.sub.2-, other than the carbons
attached to the --SH of the sulfhydryl containing amino acid, are
optionally substituted with a heteroatom such as N, S or O.
[0225] The L.sub.2 component of the macrocycle-forming linker
X-L.sub.2-Y may be varied in length depending on, among other
things, the distance between the positions of the two amino acid
analogs used to form the peptidomimetic macrocycle. Furthermore, as
the lengths of L.sub.1 and/or L.sub.3 components of the
macrocycle-forming linker are varied, the length of L.sub.2 can
also be varied in order to create a linker of appropriate overall
length for forming a stable peptidomimetic macrocycle. For example,
if the amino acid analogs used are varied by adding an additional
methylene unit to each of L.sub.1 and L.sub.3, the length of
L.sub.2 are decreased in length by the equivalent of approximately
two methylene units to compensate for the increased lengths of
L.sub.1 and L.sub.3.
[0226] In some embodiments, L.sub.2 is an alkylene group of the
formula --(CH.sub.2).sub.n--, where n is an integer between about 1
and about 15. For example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In
other embodiments, L.sub.2 is an alkenylene group. In still other
embodiments, L.sub.2 is an aryl group.
[0227] Table 4 shows additional embodiments of X-L.sub.2-Y
groups.
TABLE-US-00004 TABLE 4 Exemplary X--L.sub.2--Y groups of the
invention. ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087##
##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##
##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102##
##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107##
##STR00108## ##STR00109## ##STR00110## ##STR00111## Each X and Y in
this table, is for example, independently Cl--, Br-- or I--.
[0228] Additional methods of forming peptidomimetic macrocycles
which are envisioned as suitable to perform the present invention
include those disclosed by Mustapa, M. Firouz Mohd et al., J. Org.
Chem (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem.
Lett. (2004), 14, pp. 1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat.
No. 5,446,128; U.S. Pat. No. 5,824,483; U.S. Pat. No. 6,713,280;
and U.S. Pat. No. 7,202,332. In such embodiments, aminoacid
precursors are used containing an additional substituent R-- at the
alpha position. Such aminoacids are incorporated into the
macrocycle precursor at the desired positions, which may be at the
positions where the crosslinker is substituted or, alternatively,
elsewhere in the sequence of the macrocycle precursor. Cyclization
of the precursor is then effected according to the indicated
method.
Assays
[0229] The properties of the peptidomimetic macrocycles of the
invention are assayed, for example, by using the methods described
below. In some embodiments, a peptidomimetic macrocycle of the
invention has improved biological properties relative to a
corresponding polypeptide lacking the substituents described
herein.
Assay to Determine .alpha.-Helicity.
[0230] In solution, the secondary structure of polypeptides with
.alpha.-helical domains will reach a dynamic equilibrium between
random coil structures and .alpha.-helical structures, often
expressed as a "percent helicity". Thus, for example, unmodified
alpha-helical domains are predominantly random coils in solution,
with .alpha.-helical content usually under 25%. Peptidomimetic
macrocycles with optimized linkers, on the other hand, possess, for
example, an alpha-helicity that is at least two-fold greater than
that of a corresponding uncrosslinked polypeptide. In some
embodiments, macrocycles of the invention will possess an
alpha-helicity of greater than 50%. To assay the helicity of
peptidomimetic macrocyles of the invention, the compounds are
dissolved in an aqueous solution (e.g. 50 mM potassium phosphate
solution at pH 7, or distilled H.sub.2O, to concentrations of 25-50
.mu.M). Circular dichroism (CD) spectra are obtained on a
spectropolarimeter (e.g., Jasco J-710) using standard measurement
parameters (e.g. temperature, 20.degree. C.; wavelength, 190-260
nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;
response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The
.alpha.-helical content of each peptide is calculated by dividing
the mean residue ellipticity (e.g. [.PHI.]222obs) by the reported
value for a model helical decapeptide (Yang et al. (1986), Methods
Enzymol. 130:208)).
Assay to Determine Melting Temperature (Tm).
[0231] A peptidomimetic macrocycle of the invention comprising a
secondary structure such as an .alpha.-helix exhibits, for example,
a higher melting temperature than a corresponding uncrosslinked
polypeptide. Typically peptidomimetic macrocycles of the invention
exhibit Tm of >60.degree. C. representing a highly stable
structure in aqueous solutions. To assay the effect of macrocycle
formation on meltine temperature, peptidomimetic macrocycles or
unmodified peptides are dissolved in distilled H.sub.2O (e.g. at a
final concentration of 50 .mu.M) and the Tm is determined by
measuring the change in ellipticity over a temperature range (e.g.
4 to 95.degree. C.) on a spectropolarimeter (e.g., Jasco J-710)
using standard parameters (e.g. wavelength 222 nm; step resolution,
0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec;
bandwidth, 1 nm; temperature increase rate: 1.degree. C./min; path
length, 0.1 cm).
Protease Resistance Assay.
[0232] The amide bond of the peptide backbone is susceptible to
hydrolysis by proteases, thereby rendering peptidic compounds
vulnerable to rapid degradation in vivo. Peptide helix formation,
however, typically buries the amide backbone and therefore may
shield it from proteolytic cleavage. The peptidomimetic macrocycles
of the present invention may be subjected to in vitro trypsin
proteolysis to assess for any change in degradation rate compared
to a corresponding uncrosslinked polypeptide. For example, the
peptidomimetic macrocycle and a corresponding uncrosslinked
polypeptide are incubated with trypsin agarose and the reactions
quenched at various time points by centrifugation and subsequent
HPLC injection to quantitate the residual substrate by ultraviolet
absorption at 280 nm. Briefly, the peptidomimetic macrocycle and
peptidomimetic precursor (5 mcg) are incubated with trypsin agarose
(Pierce) (S/E.about.125) for 0, 10, 20, 90, and 180 minutes.
Reactions are quenched by tabletop centrifugation at high speed;
remaining substrate in the isolated supernatant is quantified by
HPLC based peak detection at 280 nm. The proteolytic reaction
displays first order kinetics and the rate constant, k, is
determined from a plot of ln[S] versus time (k=-1.times.
slope).
Ex Vivo Stability Assay.
[0233] Peptidomimetic macrocycles with optimized linkers possess,
for example, an ex vivo half-life that is at least two-fold greater
than that of a corresponding uncrosslinked polypeptide, and possess
an ex vivo half-life of 12 hours or more. For ex vivo serum
stability studies, a variety of assays may be used. For example, a
peptidomimetic macrocycle and a corresponding uncrosslinked
polypeptide (2 mcg) are incubated with fresh mouse, rat and/or
human serum (2 mL) at 37.degree. C. for 0, 1, 2, 4, 8, and 24
hours. To determine the level of intact compound, the following
procedure may be used: The samples are extracted by transferring
100 .mu.l of sera to 2 ml centrifuge tubes followed by the addition
of 10 .mu.L of 50% formic acid and 500 .mu.L acetonitrile and
centrifugation at 14,000 RPM for 10 min at 4.+-.2.degree. C. The
supernatants are then transferred to fresh 2 ml tubes and
evaporated on Turbovap under N.sub.2<10 psi, 37.degree. C. The
samples are reconstituted in 100 .mu.L of 50:50 acetonitrile:water
and submitted to LC-MS/MS analysis.
In Vitro Binding Assays.
[0234] To assess the binding and affinity of peptidomimetic
macrocycles and peptidomimetic precursors to acceptor proteins, a
fluorescence polarization assay (FPA) issued, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution).
[0235] For example, fluoresceinated peptidomimetic macrocycles (25
nM) are incubated with the acceptor protein (25-1000 nM) in binding
buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Binding activity is measured, for example, by
fluorescence polarization on a luminescence spectrophotometer (e.g.
Perkin-Elmer LS50B). Kd values may be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic
macrocycle of the invention shows, in some instances, similar or
lower Kd than a corresponding uncrosslinked polypeptide.
In Vitro Displacement Assays to Characterize Antagonists of
Peptide-Protein Interactions.
[0236] To assess the binding and affinity of compounds that
antagonize the interaction between a peptide and an acceptor
protein, a fluorescence polarization assay (FPA) utilizing a
fluoresceinated peptidomimetic macrocycle derived from a
peptidomimetic precursor sequence is used, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution). A compound that antagonizes the
interaction between the fluoresceinated peptidomimetic macrocycle
and an acceptor protein will be detected in a competitive binding
FPA experiment.
[0237] For example, putative antagonist compounds (1 nM to 1 mM)
and a fluoresceinated peptidomimetic macrocycle (25 nM) are
incubated with the acceptor protein (50 nM) in binding buffer (140
mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature
Antagonist binding activity is measured, for example, by
fluorescence polarization on a luminescence spectrophotometer (e.g.
Perkin-Elmer LS50B). Kd values may be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.).
[0238] Any class of molecule, such as small organic molecules,
peptides, oligonucleotides or proteins can be examined as putative
antagonists in this assay.
Assay for Protein-Ligand Binding by Affinity Selection-Mass
Spectrometry
[0239] To assess the binding and affinity of test compounds for
proteins, an affinity-selection mass spectrometry assay is used,
for example. Protein-ligand binding experiments are conducted
according to the following representative procedure outlined for a
system-wide control experiment using 1 .mu.M peptidomimetic
macrocycle plus 5 .mu.M target protein. A 1 .mu.L DMSO aliquot of a
40 .mu.M stock solution of peptidomimetic macrocycle is dissolved
in 19 .mu.L of PBS (Phosphate-buffered saline: 50 mM, pH 7.5
Phosphate buffer containing 150 mM NaCl). The resulting solution is
mixed by repeated pipetting and clarified by centrifugation at 10
000 g for 10 min. To a 4 .mu.L aliquot of the resulting supernatant
is added 4 .mu.L of 10 .mu.M target protein in PBS. Each 8.0 .mu.L
experimental sample thus contains 40 pmol (1.5 .mu.g) of protein at
5.0 .mu.M concentration in PBS plus 1 .mu.M peptidomimetic
macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each
concentration point are incubated for 60 min at room temperature,
and then chilled to 4.degree. C. prior to size-exclusion
chromatography-LC-MS analysis of 5.0 .mu.L injections. Samples
containing a target protein, protein-ligand complexes, and unbound
compounds are injected onto an SEC column, where the complexes are
separated from non-binding component by a rapid SEC step. The SEC
column eluate is monitored using UV detectors to confirm that the
early-eluting protein fraction, which elutes in the void volume of
the SEC column, is well resolved from unbound components that are
retained on the column. After the peak containing the protein and
protein-ligand complexes elutes from the primary UV detector, it
enters a sample loop where it is excised from the flow stream of
the SEC stage and transferred directly to the LC-MS via a valving
mechanism. The (M+3H).sup.3+ ion of the peptidomimetic macrocycle
is observed by ESI-MS at the expected m/z, confirming the detection
of the protein-ligand complex.
Assay for Protein-Ligand Kd Titration Experiments.
[0240] To assess the binding and affinity of test compounds for
proteins, a protein-ligand Kd titration experiment is performed,
for example. Protein-ligand K.sub.d titrations experiments are
conducted as follows: 2 .mu.L DMSO aliquots of a serially diluted
stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . .
, 0.098 mM) are prepared then dissolved in 38 .mu.L of PBS. The
resulting solutions are mixed by repeated pipetting and clarified
by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of
the resulting supernatants is added 4.0 .mu.L of 10 .mu.M target
protein in PBS. Each 8.0 .mu.L experimental sample thus contains 40
pmol (1.5 .mu.g) of protein at 5.0 .mu.M concentration in PBS,
varying concentrations (125, 62.5, . . . , 0.24 .mu.M) of the
titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for
each concentration point are incubated at room temperature for 30
min, then chilled to 4.degree. C. prior to SEC-LC-MS analysis of
2.0 .mu.L injections. The (M+H).sup.1+, (M+2H).sup.2+,
(M+3H).sup.3+, and/or (M+Na).sup.1+ ion is observed by ESI-MS;
extracted ion chromatograms are quantified, then fit to equations
to derive the binding affinity K.sub.d as described in "A General
Technique to Rank Protein-Ligand Binding Affinities and Determine
Allosteric vs. Direct Binding Site Competition in Compound
Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.;
Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in
"ALIS: An Affinity Selection-Mass Spectrometry System for the
Discovery and Characterization of Protein-Ligand Interactions" D.
A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in
Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH;
2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors):
Methods and Principles in Medicinal Chemistry.
Assay for Competitive Binding Experiments by Affinity
Selection-Mass Spectrometry
[0241] To determine the ability of test compounds to bind
competitively to proteins, an affinity selection mass spectrometry
assay is performed, for example. A mixture of ligands at 40 .mu.M
per component is prepared by combining 2 .mu.L aliquots of 400
.mu.M stocks of each of the three compounds with 14 .mu.L of DMSO.
Then, 1 .mu.L aliquots of this 40 .mu.M per component mixture are
combined with 1 .mu.L DMSO aliquots of a serially diluted stock
solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . ,
0.078 mM). These 2 .mu.L samples are dissolved in 38 .mu.L of PBS.
The resulting solutions were mixed by repeated pipetting and
clarified by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L
aliquots of the resulting supernatants is added 4.0 .mu.L of 10
.mu.M target protein in PBS. Each 8.0 .mu.L experimental sample
thus contains 40 pmol (1.5 .mu.g) of protein at 5.0 .mu.M
concentration in PBS plus 0.5 .mu.M ligand, 2.5% DMSO, and varying
concentrations (125, 62.5, . . . , 0.98 .mu.M) of the titrant
peptidomimetic macrocycle. Duplicate samples thus prepared for each
concentration point are incubated at room temperature for 60 min,
then chilled to 4.degree. C. prior to SEC-LC-MS analysis of 2.0
.mu.L injections. Additional details on these and other methods are
provided in "A General Technique to Rank Protein-Ligand Binding
Affinities and Determine Allosteric vs. Direct Binding Site
Competition in Compound Mixtures." Annis, D. A.; Nazef, N.; Chuang,
C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126,
15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry
System for the Discovery and Characterization of Protein-Ligand
Interactions" D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass
Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G:
Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series
Editors): Methods and Principles in Medicinal Chemistry.
Binding Assays in Intact Cells.
[0242] It is possible to measure binding of peptides or
peptidomimetic macrocycles to their natural acceptors in intact
cells by immunoprecipitation experiments. For example, intact cells
are incubated with fluoresceinated (FITC-labeled) compounds for 4
hrs in the absence of serum, followed by serum replacement and
further incubation that ranges from 4-18 hrs. Cells are then
pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM
NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at
4.degree. C. Extracts are centrifuged at 14,000 rpm for 15 minutes
and supernatants collected and incubated with 10 .mu.l goat
anti-FITC antibody for 2 hrs, rotating at 4.degree. C. followed by
further 2 hrs incubation at 4.degree. C. with protein A/G Sepharose
(50 .mu.l of 50% bead slurry). After quick centrifugation, the
pellets are washed in lysis buffer containing increasing salt
concentration (e.g., 150, 300, 500 mM). The beads are then
re-equilibrated at 150 mM NaCl before addition of SDS-containing
sample buffer and boiling. After centrifugation, the supernatants
are optionally electrophoresed using 4%-12% gradient Bis-Tris gels
followed by transfer into Immobilon-P membranes. After blocking,
blots are optionally incubated with an antibody that detects FITC
and also with one or more antibodies that detect proteins that bind
to the peptidomimetic macrocycle.
Cellular Penetrability Assays.
[0243] A peptidomimetic macrocycle is, for example, more cell
penetrable compared to a corresponding uncrosslinked macrocycle.
Peptidomimetic macrocycles with optimized linkers possess, for
example, cell penetrability that is at least two-fold greater than
a corresponding uncrosslinked macrocycle, and often 20% or more of
the applied peptidomimetic macrocycle will be observed to have
penetrated the cell after 4 hours. To measure the cell
penetrability of peptidomimetic macrocycles and corresponding
uncrosslinked macrocycle, intact cells are incubated with
fluoresceinated peptidomimetic macrocycles or corresponding
uncrosslinked macrocycle (10 .mu.M) for 4 hrs in serum free media
at 37.degree. C., washed twice with media and incubated with
trypsin (0.25%) for 10 min at 37.degree. C. The cells are washed
again and resuspended in PBS. Cellular fluorescence is analyzed,
for example, by using either a FACSCalibur flow cytometer or
Cellomics' Kinetic Scan.RTM. HCS Reader.
Cellular Efficacy Assays.
[0244] The efficacy of certain peptidomimetic macrocycles is
determined, for example, in cell-based killing assays using a
variety of tumorigenic and non-tumorigenic cell lines and primary
cells derived from human or mouse cell populations. Cell viability
is monitored, for example, over 24-96 hrs of incubation with
peptidomimetic macrocycles (0.5 to 50 .mu.M) to identify those that
kill at EC50<10 .mu.M. Several standard assays that measure cell
viability are commercially available and are optionally used to
assess the efficacy of the peptidomimetic macrocycles. In addition,
assays that measure Annexin V and caspase activation are optionally
used to assess whether the peptidomimetic macrocycles kill cells by
activating the apoptotic machinery. For example, the Cell Titer-glo
assay is used which determines cell viability as a function of
intracellular ATP concentration.
In Vivo Stability Assay.
[0245] To investigate the in vivo stability of the peptidomimetic
macrocycles, the compounds are, for example, administered to mice
and/or rats by IV, IP, PO or inhalation routes at concentrations
ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0',
5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection.
Levels of intact compound in 25 .mu.L of fresh serum are then
measured by LC-MS/MS as above.
In Vivo Efficacy in Animal Models.
[0246] To determine the anti-oncogenic activity of peptidomimetic
macrocycles of the invention in vivo, the compounds are, for
example, given alone (IP, IV, PO, by inhalation or nasal routes) or
in combination with sub-optimal doses of relevant chemotherapy
(e.g., cyclophosphamide, doxorubicin, etoposide). In one example,
5.times.10.sup.6 RS4; 11 cells (established from the bone marrow of
a patient with acute lymphoblastic leukemia) that stably express
luciferase are injected by tail vein in NOD-SCID mice 3 hrs after
they have been subjected to total body irradiation. If left
untreated, this form of leukemia is fatal in 3 weeks in this model.
The leukemia is readily monitored, for example, by injecting the
mice with D-luciferin (60 mg/kg) and imaging the anesthetized
animals (e.g., Xenogen In Vivo Imaging System, Caliper Life
Sciences, Hopkinton, Mass.). Total body bioluminescence is
quantified by integration of photonic flux (photons/sec) by Living
Image Software (Caliper Life Sciences, Hopkinton, Mass.).
Peptidomimetic macrocycles alone or in combination with sub-optimal
doses of relevant chemotherapeutics agents are, for example,
administered to leukemic mice (10 days after injection/day 1 of
experiment, in bioluminescence range of 14-16) by tail vein or IP
routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21
days. Optionally, the mice are imaged throughout the experiment
every other day and survival monitored daily for the duration of
the experiment. Expired mice are optionally subjected to necropsy
at the end of the experiment. Another animal model is implantation
into NOD-SCID mice of DoHH2, a cell line derived from human
follicular lymphoma, that stably expresses luciferase. These in
vivo tests optionally generate preliminary pharmacokinetic,
pharmacodynamic and toxicology data.
Clinical Trials.
[0247] To determine the suitability of the peptidomimetic
macrocycles of the invention for treatment of humans, clinical
trials are performed. For example, patients diagnosed with cancer
and in need of treatment are selected and separated in treatment
and one or more control groups, wherein the treatment group is
administered a peptidomimetic macrocycle of the invention, while
the control groups receive a placebo or a known anti-cancer drug.
The treatment safety and efficacy of the peptidomimetic macrocycles
of the invention can thus be evaluated by performing comparisons of
the patient groups with respect to factors such as survival and
quality-of-life. In this example, the patient group treated with a
peptidomimetic macrocyle show improved long-term survival compared
to a patient control group treated with a placebo.
Pharmaceutical Compositions and Routes of Administration
[0248] The peptidomimetic macrocycles of the invention also include
pharmaceutically acceptable derivatives or prodrugs thereof. A
"pharmaceutically acceptable derivative" means any pharmaceutically
acceptable salt, ester, salt of an ester, pro-drug or other
derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
pharmaceutically acceptable derivatives are those that increase the
bioavailability of the compounds of the invention when administered
to a mammal (e g., by increasing absorption into the blood of an
orally administered compound) or which increases delivery of the
active compound to a biological compartment (e.g., the brain or
lymphatic system) relative to the parent species. Some
pharmaceutically acceptable derivatives include a chemical group
which increases aqueous solubility or active transport across the
gastrointestinal mucosa.
[0249] In some embodiments, the peptidomimetic macrocycles of the
invention are modified by covalently or non-covalently joining
appropriate functional groups to enhance selective biological
properties. Such modifications include those which increase
biological penetration into a given biological compartment (e.g.,
blood, lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism, and alter rate of excretion.
[0250] Pharmaceutically acceptable salts of the compounds of this
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, benzoate, benzenesulfonate,
butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate,
glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,
hydrobromide, hydroiodide, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
palmoate, phosphate, picrate, pivalate, propionate, salicylate,
succinate, sulfate, tartrate, tosylate and undecanoate. Salts
derived from appropriate bases include alkali metal (e.g., sodium),
alkaline earth metal (e.g., magnesium), ammonium and
N-(alkyl).sub.4.sup.+ salts.
[0251] For preparing pharmaceutical compositions from the compounds
of the present invention, pharmaceutically acceptable carriers
include either solid or liquid carriers. Solid form preparations
include powders, tablets, pills, capsules, cachets, suppositories,
and dispersible granules. A solid carrier can be one or more
substances, which also acts as diluents, flavoring agents, binders,
preservatives, tablet disintegrating agents, or an encapsulating
material. Details on techniques for formulation and administration
are well described in the scientific and patent literature, see,
e.g., the latest edition of Remington's Pharmaceutical Sciences,
Maack Publishing Co, Easton Pa.
[0252] In powders, the carrier is a finely divided solid, which is
in a mixture with the finely divided active component. In tablets,
the active component is mixed with the carrier having the necessary
binding properties in suitable proportions and compacted in the
shape and size desired.
[0253] Suitable solid excipients are carbohydrate or protein
fillers include, but are not limited to sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
and gums including arabic and tragacanth; as well as proteins such
as gelatin and collagen. If desired, disintegrating or solubilizing
agents are added, such as the cross-linked polyvinyl pyrrolidone,
agar, alginic acid, or a salt thereof, such as sodium alginate.
[0254] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water/propylene glycol solutions.
For parenteral injection, liquid preparations can be formulated in
solution in aqueous polyethylene glycol solution.
[0255] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form.
[0256] When the compositions of this invention comprise a
combination of a peptidomimetic macrocycle and one or more
additional therapeutic or prophylactic agents, both the compound
and the additional agent should be present at dosage levels of
between about 1 to 100%, and more preferably between about 5 to 95%
of the dosage normally administered in a monotherapy regimen. In
some embodiments, the additional agents are administered
separately, as part of a multiple dose regimen, from the compounds
of this invention. Alternatively, those agents are part of a single
dosage form, mixed together with the compounds of this invention in
a single composition.
Methods of Use
[0257] In one aspect, the present invention provides novel
peptidomimetic macrocycles that are useful in competitive binding
assays to identify agents which bind to the natural ligand(s) of
the proteins or peptides upon which the peptidomimetic macrocycles
are modeled. For example, in the TCF/.beta.-catenin system, labeled
peptidomimetic macrocycles based on CBD peptides of TCF can be used
in a .beta.-catenin binding assay along with small molecules that
competitively bind to .beta.-catenin. Competitive binding studies
allow for rapid in vitro evaluation and determination of drug
candidates specific for the TCF/.beta.-catenin system. Such binding
studies may be performed with any of the peptidomimetic macrocycles
disclosed herein and their binding partners.
[0258] The invention further provides for the generation of
antibodies against the peptidomimetic macrocycles. In some
embodiments, these antibodies specifically bind both the
peptidomimetic macrocycle and the precursor peptides, such as
TCF-CBD, to which the peptidomimetic macrocycles are related. Such
antibodies, for example, disrupt the native protein-protein
interaction, for example, binding between TCF and
.beta.-catenin.
[0259] In other aspects, the present invention provides for both
prophylactic and therapeutic methods of treating a subject at risk
of (or susceptible to) a disorder or having a disorder associated
with aberrant (e.g., insufficient or excessive) expression or
activity of the molecules including .beta.-catenin.
[0260] In another embodiment, a disorder is caused, at least in
part, by an abnormal level of .beta.-catenin, (e.g., over or under
expression), or by the presence of .beta.-catenin exhibiting
abnormal activity. As such, the reduction in the level and/or
activity of the .beta.-catenin, or the enhancement of the level
and/or activity of .beta.-catenin, by peptidomimetic macrocycles
derived from a CBD-containing protein such as TCF, is used, for
example, to ameliorate or reduce the adverse symptoms of the
disorder.
[0261] In another aspect, the present invention provides methods
for treating or preventing a disease including hyperproliferative
disease and inflammatory disorder by interfering with the
interaction or binding between binding partners, for example,
between TCF and .beta.-catenin. These methods comprise
administering an effective amount of a compound of the invention to
a warm blooded animal, including a human. In some embodiments, the
administration of the compounds of the present invention induces
cell growth arrest or apoptosis.
[0262] As used herein, the term "treatment" is defined as the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disease, a
symptom of disease or a predisposition toward a disease, with the
purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve or affect the disease, the symptoms of disease
or the predisposition toward disease.
[0263] In some embodiments, the peptidomimetics macrocycles of the
invention is used to treat, prevent, and/or diagnose cancers and
neoplastic conditions. As used herein, the terms "cancer",
"hyperproliferative" and "neoplastic" refer to cells having the
capacity for autonomous growth, i.e., an abnormal state or
condition characterized by rapidly proliferating cell growth.
Hyperproliferative and neoplastic disease states may be categorized
as pathologic, i.e., characterizing or constituting a disease
state, or may be categorized as non-pathologic, i.e., a deviation
from normal but not associated with a disease state. The term is
meant to include all types of cancerous growths or oncogenic
processes, metastatic tissues or malignantly transformed cells,
tissues, or organs, irrespective of histopathologic type or stage
of invasiveness. A metastatic tumor can arise from a multitude of
primary tumor types, including but not limited to those of breast,
lung, liver, colon and ovarian origin. "Pathologic
hyperproliferative" cells occur in disease states characterized by
malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair. Examples of cellular proliferative and/or
differentiative disorders include cancer, e.g., carcinoma, sarcoma,
or metastatic disorders. In some embodiments, the peptidomimetics
macrocycles are novel therapeutic agents for controlling breast
cancer, ovarian cancer, colon cancer, lung cancer, metastasis of
such cancers and the like.
[0264] Examples of cancers or neoplastic conditions include, but
are not limited to, a fibrosarcoma, myosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,
pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,
cancer of the head and neck, skin cancer, brain cancer, squamous
cell carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular cancer, small cell lung
carcinoma, non-small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi
sarcoma.
[0265] Examples of proliferative disorders include hematopoietic
neoplastic disorders. As used herein, the term "hematopoietic
neoplastic disorders" includes diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof. Preferably, the diseases arise from poorly
differentiated acute leukemias, e.g., erythroblastic leukemia and
acute megakaryoblastic leukemia. Additional exemplary myeloid
disorders include, but are not limited to, acute promyeloid
leukemia (APML), acute myelogenous leukemia (AML) and chronic
myelogenous leukemia (CIVIL) (reviewed in Vaickus (1991), Crit Rev.
Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are
not limited to acute lymphoblastic leukemia (ALL) which includes
B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia
(CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and
Waldenstrom's macroglobulinemia (WM). Additional forms of malignant
lymphomas include, but are not limited to non-Hodgkin lymphoma and
variants thereof, peripheral T cell lymphomas, adult T cell
leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large
granular lymphocytic leukemia (LGF), Hodgkin's disease and
Reed-Stemberg disease.
[0266] Examples of cellular proliferative and/or differentiative
disorders of the breast include, but are not limited to,
proliferative breast disease including, e.g., epithelial
hyperplasia, sclerosing adenosis, and small duct papillomas;
tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor,
and sarcomas, and epithelial tumors such as large duct papilloma;
carcinoma of the breast including in situ (noninvasive) carcinoma
that includes ductal carcinoma in situ (including Paget's disease)
and lobular carcinoma in situ, and invasive (infiltrating)
carcinoma including, but not limited to, invasive ductal carcinoma,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0267] Examples of cellular proliferative and/or differentiative
disorders of the lung include, but are not limited to, bronchogenic
carcinoma, including paraneoplastic syndromes, bronchioloalveolar
carcinoma, neuroendocrine tumors, such as bronchial carcinoid,
miscellaneous tumors, and metastatic tumors; pathologies of the
pleura, including inflammatory pleural effusions, noninflammatory
pleural effusions, pneumothorax, and pleural tumors, including
solitary fibrous tumors (pleural fibroma) and malignant
mesothelioma.
[0268] Examples of cellular proliferative and/or differentiative
disorders of the colon include, but are not limited to,
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0269] Examples of cellular proliferative and/or differentiative
disorders of the liver include, but are not limited to, nodular
hyperplasias, adenomas, and malignant tumors, including primary
carcinoma of the liver and metastatic tumors.
[0270] Examples of cellular proliferative and/or differentiative
disorders of the ovary include, but are not limited to, ovarian
tumors such as, tumors of coelomic epithelium, serous tumors,
mucinous tumors, endometrioid tumors, clear cell adenocarcinoma,
cystadenofibroma, Brenner tumor, surface epithelial tumors; germ
cell tumors such as mature (benign) teratomas, monodermal
teratomas, immature malignant teratomas, dysgerminoma, endodermal
sinus tumor, choriocarcinoma; sex cord-stomal tumors such as,
granulosa-theca cell tumors, thecomafibromas, androblastomas, hill
cell tumors, and gonadoblastoma; and metastatic tumors such as
Krukenberg tumors.
[0271] In other or further embodiments, the peptidomimetics
macrocycles described herein are used to treat, prevent or diagnose
conditions characterized by overactive cell death or cellular death
due to physiologic insult, etc. Some examples of conditions
characterized by premature or unwanted cell death are or
alternatively unwanted or excessive cellular proliferation include,
but are not limited to hypocellular/hypoplastic,
acellular/aplastic, or hypercellular/hyperplastic conditions. Some
examples include hematologic disorders including but not limited to
fanconi anemia, aplastic anemia, thalaessemia, congenital
neutropenia, and myelodysplasia.
[0272] In other or further embodiments, the peptidomimetics
macrocycles of the invention that act to decrease apoptosis are
used to treat disorders associated with an undesirable level of
cell death. Thus, in some embodiments, the anti-apoptotic
peptidomimetics macrocycles of the invention are used to treat
disorders such as those that lead to cell death associated with
viral infection, e.g., infection associated with infection with
human immunodeficiency virus (HIV). A wide variety of neurological
diseases are characterized by the gradual loss of specific sets of
neurons. One example is Alzheimer's disease (AD). Alzheimer's
disease is characterized by loss of neurons and synapses in the
cerebral cortex and certain subcortical regions. This loss results
in gross atrophy of the affected regions. Both amyloid plaques and
neurofibrillary tangles are visible in brains of those afflicted by
AD. Alzheimer's disease has been identified as a protein misfolding
disease, due to the accumulation of abnormally folded A-beta and
tau proteins in the brain. Plaques are made up of .beta.-amyloid.
.beta.-amyloid is a fragment from a larger protein called amyloid
precursor protein (APP). APP is critical to neuron growth, survival
and post-injury repair. In AD, an unknown process causes APP to be
cleaved into smaller fragments by enzymes through proteolysis. One
of these fragments is fibrils of .beta.-amyloid, which form clumps
that deposit outside neurons in dense formations known as senile
plaques. Plaques continue to grow into insoluble twisted fibers
within the nerve cell, often called tangles. Disruption of the
interaction between .beta.-amyloid and its native receptor is
therefore important in the treatment of AD. The anti-apoptotic
peptidomimetics macrocycles of the invention are used, in some
embodiments, in the treatment of AD and other neurological
disorders associated with cell apoptosis. Such neurological
disorders include Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal
muscular atrophy, and various forms of cerebellar degeneration. The
cell loss in these diseases does not induce an inflammatory
response, and apoptosis appears to be the mechanism of cell
death.
[0273] In addition, a number of hematologic diseases are associated
with a decreased production of blood cells. These disorders include
anemia associated with chronic disease, aplastic anemia, chronic
neutropenia, and the myelodysplastic syndromes. Disorders of blood
cell production, such as myelodysplastic syndrome and some forms of
aplastic anemia, are associated with increased apoptotic cell death
within the bone marrow. These disorders could result from the
activation of genes that promote apoptosis, acquired deficiencies
in stromal cells or hematopoietic survival factors, or the direct
effects of toxins and mediators of immune responses. Two common
disorders associated with cell death are myocardial infarctions and
stroke. In both disorders, cells within the central area of
ischemia, which is produced in the event of acute loss of blood
flow, appear to die rapidly as a result of necrosis. However,
outside the central ischemic zone, cells die over a more protracted
time period and morphologically appear to die by apoptosis. In
other or further embodiments, the anti-apoptotic peptidomimetics
macrocycles of the invention are used to treat all such disorders
associated with undesirable cell death.
[0274] Some examples of neurologic disorders that are treated with
the peptidomimetics macrocycles described herein include but are
not limited to Alzheimer's Disease, Down's Syndrome, Dutch Type
Hereditary Cerebral Hemorrhage Amyloidosis, Reactive Amyloidosis,
Familial Amyloid Nephropathy with Urticaria and Deafness,
Muckle-Wells Syndrome, Idiopathic Myeloma;
Macroglobulinemia-Associated Myeloma, Familial Amyloid
Polyneuropathy, Familial Amyloid Cardiomyopathy, Isolated Cardiac
Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,
Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the
Thyroid, Familial Amyloidosis, Hereditary Cerebral Hemorrhage With
Amyloidosis, Familial Amyloidotic Polyneuropathy, Scrapie,
Creutzfeldt-Jacob Disease, Gerstmann Straussler-Scheinker Syndrome,
Bovine Spongiform Encephalitis, a prion-mediated disease, and
Huntington's Disease.
[0275] In another embodiment, the peptidomimetics macrocycles
described herein are used to treat, prevent or diagnose
inflammatory disorders. Numerous types of inflammatory disorders
exist. Certain inflammatory diseases are associated with the immune
system, for example, autoimmune diseases. Autoimmune diseases arise
from an overactive immune response of the body against substances
and tissues normally present in the body, i.e. self antigens. In
other words, the immune system attacks its own cells. Autoimmune
diseases are a major cause of immune-mediated diseases. Rheumatoid
arthritis is an example of an autoimmune disease, in which the
immune system attacks the joints, where it causes inflammation
(i.e. arthritis) and destruction. It can also damage some organs,
such as the lungs and skin. Rheumatoid arthritis can lead to
substantial loss of functioning and mobility. Rheumatoid arthritis
is diagnosed with blood tests especially the rheumatoid factor
test. Some examples of autoimmune diseases that are treated with
the peptidomimetics macrocycles described herein include, but are
not limited to, acute disseminated encephalomyelitis (ADEM),
Addison's disease, ankylosing spondylitis, antiphospholipid
antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune inner ear disease, Bechets disease, bullous
pemphigoid, coeliac disease, Chagas disease, Churg-Strauss
syndrome, chronic obstructive pulmonary disease (COPD), Crohn's
disease, dermatomyositis, diabetes mellitus type 1, endometriosis,
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome
(GBS), Hashimoto's disease, Hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, inflammatory bowl disease (IBD),
interstitial cystitis, lupus erythematosus, morphea, multiple
sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus
vulgaris, pernicious anaemia, Polymyositis, polymyalgia rheumatica,
primary biliary cirrhosis, psoriasis, rheumatoid arthritis,
schizophrenia, scleroderma, Sjogren's syndrome, temporal arteritis
(also known as "giant cell arteritis"), Takayasu's arteritis,
Vasculitis, Vitiligo, and Wegener's granulomatosis.
[0276] Some examples of other types of inflammatory disorders that
are treated with the peptidomimetics macrocycles described herein
include, but are not limited to, allergy including allergic
rhinitis/sinusitis, skin allergies (urticaria/hives, angioedema,
atopic dermatitis), food allergies, drug allergies, insect
allergies, and rare allergic disorders such as mastocytosis,
asthma, arthritis including osteoarthritis, rheumatoid arthritis,
and spondyloarthropathies, primary angitis of the CNS, sarcoidosis,
organ transplant rejection, fibromyalgia, fibrosis, pancreatitis,
and pelvic inflammatory disease.
[0277] Examples of cardiovascular disorders (e g., inflammatory
disorders) that are treated or prevented with the peptidomimetics
macrocycles of the invention include, but are not limited to,
aortic valve stenosis, atherosclerosis, myocardial infarction,
stroke, thrombosis, aneurism, heart failure, ischemic heart
disease, angina pectoris, sudden cardiac death, hypertensive heart
disease; non-coronary vessel disease, such as arteriolosclerosis,
small vessel disease, nephropathy, hypertriglyceridemia,
hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma,
hypertension, emphysema and chronic pulmonary disease; or a
cardiovascular condition associated with interventional procedures
("procedural vascular trauma"), such as restenosis following
angioplasty, placement of a shunt, stent, synthetic or natural
excision grafts, indwelling catheter, valve or other implantable
devices. Preferred cardiovascular disorders include
atherosclerosis, myocardial infarction, aneurism, and stroke.
EXAMPLES
Example 1
[0278] A peptidomimetic macrocycle of the invention is prepared,
for example, starting with the sequence RDLADVKSSLVNES (SEQ ID NO:
131) by replacing the 4.sup.th and 8.sup.th amino acids with an
alpha, alpha-disubstituted amino acid (e.g. the S5 olefin amino
acid). An olefin metathesis reaction is performed resulting in a
peptidomimetic macrocycle comprising an i to i+4 crosslink.
.alpha.-helical crosslinked polypeptides are synthesized, purified
and analyzed as previously described (Schafmeister et al. (2000),
J. Am. Chem. Soc. 122:5891-5892; Walensky et al (2004) Science
305:1466-70; Walensky et al (2006) Mol Cell 24:199-210). The
.alpha.,.alpha.-disubstituted amino acids and amino acid precursors
disclosed in the cited references may be employed in synthesis of
the peptidomimetic macrocycle precursor polypeptides.
Alpha,alpha-disubstituted non-natural amino acids containing
olefinic side chains are synthesized according to Williams et al.
(1991) J. Am. Chem. Soc. 113:9276; and Schafmeister et al. (2000)
J. Am. Chem Soc. 122:5891. Crosslinked polypeptides are designed by
replacing two naturally occurring amino acids (see above) with the
corresponding synthetic amino acids. Substitutions are made at i
and i+4 positions and at i and i+7 positions. Additional
peptidomimetic macrocycles are synthesized as shown in FIGS. 4a,
4b, and 4c.
[0279] In the sequences shown in FIGS. 4a, 4b, and 4c and
elsewhere, Nle represents norleucine, Ac represents N-terminal
acetyl, NH2 represents C-terminal amide, PEG3 represents a
NH-(PEG).sub.3-COOH (16 atoms) linker (Novabiochem cat#01-63-0199),
PEG4 represents a NH-(PEG).sub.4-COOH (19 atoms) linker
(Novabiochem cat#01-63-0200), and PEG5 represents a
NH-(PEG).sub.5-COOH (22 atoms) linker (Novabiochem cat#01-63-0204).
The amino acid represented as $ is (S)-.alpha.-(2'-pentenyl)
alanine ("S5-olefin amino acid") connected by an all-carbon
crosslinker comprising one double bond. The amino acids represented
as $r8 is (R)-.alpha.-(2'-octenyl) alanine ("R8 olefin amino
acid"), connected by an all-carbon crosslinker comprising one
double bond.
[0280] The non-natural amino acids (R and S enantiomers of the
5-carbon olefinic amino acid and the S enantiomer of the 8-carbon
olefinic amino acid) are characterized by nuclear magnetic
resonance (NMR) spectroscopy (Varian Mercury 400) and mass
spectrometry (Micromass LCT). Peptide synthesis is performed either
manually or on an automated peptide synthesizer (Applied
Biosystems, model 433A), using solid phase conditions, rink amide
AM resin (Novabiochem), and Fmoc main-chain protecting group
chemistry. For the coupling of natural Fmoc-protected amino acids
(Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio
of coupling reagents HBTU/HOBt (Novabiochem)/DIEA are employed.
Non-natural amino acids (4 equiv) are coupled with a 1:1:2 molar
ratio of HATU (Applied Biosystems)/HOBt/DIEA. Olefin metathesis is
performed in the solid phase using 10 mM Grubbs catalyst
(Blackewell et al. 1994 supra) (Materia) dissolved in degassed
dichloromethane and reacted for 2 hours at room temperature.
Isolation of metathesized compounds is achieved by trifluoroacetic
acid-mediated deprotection and cleavage, ether precipitation to
yield the crude product, and high performance liquid chromatography
(HPLC) (Varian ProStar) on a reverse phase C18 column (Varian) to
yield the pure compounds. Chemical composition of the pure products
is confirmed by LC/MS mass spectrometry (Micromass LCT interfaced
with Agilent 1100 HPLC system) and amino acid analysis (Applied
Biosystems, model 420A).
Example 2. TCF/Beta-Catenin Competitive Fluorescence Polarization
Assay
[0281] The following experiments are performed at room temperature
unless otherwise noted. First, an assay buffer is prepared composed
of 25 mM Tris-Hcl pH 7.5, 200 mM Sodium Chloride, and 5 mM CHAPS.
Next, 1M DTT is added to assay buffer to a final concentration of 2
mM. An aliquot of C-terminally, 6.times. Histidine-tagged (SEQ ID
NO: 132) Beta-Catenin (aa 134-668, 59 KD, see Poy F. et al, Nature
Structure Bio., 8, 1053 (2001)) (50.8 .mu.M stock) is thawed on ice
and diluted to a final concentration of 125 nM in assay buffer.
Forty .mu.l of this protein stock is added to all but six wells of
a 96 well plate (which will serve as the free peptide and Blank).
Test competitor peptides are diluted from 1 mM DMSO stocks to
2.times. working stocks in assay buffer. Further dilutions are made
in assay buffer-DMSO as to maintain a constant DMSO concentration
in all wells. The dilution is carried out such that the peptides
have a working concentration range of 40 .mu.M-0.7 .mu.M
(2.times.). As a benchmark, a linear, non-crosslinked peptide
competitor is prepared as above but with a working concentration
range of 13-0.7 .mu.M. Fifty .mu.l of the competitor 2.times.
stocks are added to the 40 .mu.l of protein solution previously
transferred into the assay 96 well plate. Next, a 10.times. stock
of the fluorescent probe is prepared. The probe is identical to the
linear benchmark peptide except for the n-terminal conjugation of a
FAM flourophore with Beta-Alanine spacer. A 1 mM DMSO stock of the
probe is serially diluted to a final concentration of 25 nM
(10.times.) and 10 .mu.l of this stock is added to all wells except
for those that will serve as the blank. The assay plate is stored
in the dark and the reaction is allowed to run at room temperature
for three hours. The reaction is then read on a Biotek Synergy 2
with the following settings: 100 ms delay, 40 measurements per
well, with excitation filters of 485/20 nM, and emission filters of
528/20 nM. Data is then analyzed in Graphpad Prism.
[0282] Peptidomimetic macrocycles were tested for binding ability
to beta-catenin as shown in Table 5.
TABLE-US-00005 TABLE 5 Binding Activity: SEQ ID SP# IC50 (nM) NO:
SP1 Ac-NL$NVK$SLVNQS-NH2 100000 66 SP2 Ac-RDLADVK$SLV$ES-NH2 100000
67 SP3 Ac-RNLA$VKS$LVNES-NH2 100000 68 SP4 Ac-RNLADVK$SLV$ES-NH2
100000 69 SP5 Ac-RNLADVKS$LVN$S-NH2 100000 70 SP6
Ac-RDLA$VKS$LVNQS-NH2 100000 71 SP7 Ac-RDLA$r8VKSSLV$QS-NH2 100000
72 SP8 Ac-RDLANVKS$LVN$S-NH2 100000 73 SP9 Ac-RDL$NVK$SLVNES-NH2
100000 74 SP10 Ac-SAERDL$DVK$SLVNESEKR-NH2 100000 75 SP11
Ac-SAERDLA$VKS$LVNESEKR-NH2 100000 76 SP12
Ac-SAERDLADVK$SLV$ESEKR-NH2 84950 77 SP13
Ac-SAERDLADVKS$LVN$SEKR-NH2 100000 78 SP14
Ac-LGANDELISFKDEGEQEEKSSENSSAERDLADVKSSLV-NH2 95 79 SP15
Ac-LGANDELISFKDEGEQEEKSS-NH2 6078 80 SP16
Ac-LGANDELISF$DEG$QEEKSSN-NH2 100000 81 SP17
Ac-LGANDELISFK$EGE$EEKSSN-NH2 52879 82 SP18
Ac-LGANDELISFKDEG$QEE$SSN-NH2 60133 83 SP19
Ac-LGANDELISFKDEGE$EEK$SN-NH2 100000 84 SP20
Ac-LGANDELISF$r8DEGEQE$KSSN-NH2 100000 85 SP21
Ac-LGANDELISFK$r8EGEQEE$SSN-NH2 23878 86 SP22
Ac-SA$RDL$DVKSSLVNESEKR-NH2 100000 87 SP23
Ac-SAE$DLA$VKSSLVNESEKR-NH2 100000 88 SP24
Ac-LGANAELISFKDEGEQEEKSSENSSAERDLADVKSSLV-NH2 100000 89 SP25
Ac-LGANAELISFKDEGEQEEKSSENSSAERDLADAKSSAV-NH2 100000 90 SP26
Ac-LGANDELISF$DEG$QEEKSSNNSSAERDLADVKSSLV-NH2 42 91 SP27
Ac-LGANDELISFK$EGE$EEKSSNNSSAERDLADVKSSLV-NH2 158 92 SP28
Ac-LGANDELISFKDEG$QEE$SSNNSSAERDLADVKSSLV-NH2 158 93 SP29
Ac-LGANDELISFKDEGE$EEK$SNNSSAERDLADVKSSLV-NH2 223 94 SP30
Ac-LGANDELISFKDEGEQEEKSSENSSA$RDL$DVKSSLV-NH2 98 95 SP31
Ac-LGANDELISFKDEGEQEEKSSENSSAE$DLA$VKSSLV-NH2 248 96 SP32
Ac-RDLANVI$SLV$ES-NH2 100000 97 SP33 Ac-RDLANVV$SLV$ES-NH2 100000
98 SP34 Ac-RDLANVK$SNleV$ES-NH2 100000 99 SP35
Ac-RGLANVK$SLV$ES-NH2 100000 100 SP36 Ac-VERGLANVK$SLV$ES-NH2
100000 101 SP37 Ac-LGANDELISF$DEG$QEEKSSNNSSA$RDL$DVKSSLV-NH2 56
102 SP38 Ac-LGANDELISF$DEG$QEEKSSNNSSAE$DLA$VKSSLV-NH2 83 103 SP39
Ac-LGANDELISF$DQG$QEEKSSN-NH2 3081 104 SP40
Ac-LGANDELISF$DEG$QQEKSSN-NH2 6627 105 SP41
Ac-LGANDELISF$DEG$QEQKSSN-NH2 19546 106 SP42 Ac-SA$RNL$DVKSSLV-NH2
100000 107 SP43 Ac-SA$RDL$NVKSSLV-NH2 100000 108 SP44
Ac-LGANDELISF$DQG$QQEKSSN-NH2 3752 109 SP45
Ac-LGANDELISF$DQG$QEQKSSN-NH2 10935 110 SP46
Ac-LGANDELISF$DEG$QQQKSSN-NH2 23724 111 SP47
Ac-LGANDELISF$DEG$QEEKSSENSSA$RDL$NVKSSLV-NH2 176 112 SP48
Ac-LGANDELISF$DQG$QEQKSSENSSA$RDL$NVKSSLV-NH2 461 113 SP49
Ac-LGANDELISF$DEG$QQQKSSENSSA$RDL$NVKSSLV-NH2 1647 114 SP50
Ac-LGANDELISF$DEG$QEEKSS(PEG3)A$RDL$NVKSSLV-NH2 1104 115 & 116
SP51 Ac-LGANDELISF$DEG$QEEKSS(PEG4)A$RDL$NVKSSLV-NH2 603 115 &
116 SP52 Ac-LGANDELISFM8EGEQEE$SSN-NH2 25981 117 SP53
Ac-LGANDQLISF$DEG$QEEKSSN-NH2 24418 118 SP54 Ac-SA$RDL$NVKSSLV-NH2
100000 119 SP55 Ac-SA$RNL$DVKSSLV-NH2 100000 120 SP56
Ac-RDLA$VVS$LVNES-NH2 100000 121 SP57
Ac-LGANDELISF$NQG$QEEKSSNNSSA$RNL$DVKSSLV-NH2 407 122 SP58
Ac-LGANDELLSF$DEG$QQEKSSENSSA$RDL$NVKSSLV-NH2 324 123 SP59
Ac-LGANDELISF$DEG$QEEKSSNNSSA$RDL$AVKSSLV-NH2 300 124 SP60
Ac-LGANDELISF$NQG$QAAKSSNNSSA$RNL$AVKSSLV-NH2 100000 125 SP61
Ac-LGANDELISW$DEG$QQEKSSENSSA$RDL$NVKSSLV-NH2 1002 126 SP62
Ac-LGANDELISF$NEG$QEEKSSNNSSA$RDL$DVKSSLV-NH2 70 127 SP63
Ac-LGANDELISF$NEG$QAEKSSNNSSA$RNL$DVKSSLV-NH2 1186 128 SP64
Ac-LGANDELISF$NEG$QEEKSSNNSSA$RNL$DVKSSLV-NH2 541 129
Example 3. HEK293T-3XTCF Transient Reporter Assay of TCf4/b-Catenin
Activity
[0283] HEK-293T cells cultured in DMEM/10% FBS media supplemented
with 1% antimycotic-antibiotic suspension are seeded at the density
of 4 million cells per 100 mm dish a day prior to transfections.
Cells are allowed to attain about 60% confluency overnight in
incubators at 37.degree. C., 5% CO.sub.2 On the day of
transfections, cells are washed and transiently transfected with a
combination of 3XTCF-luc reporter (Millipore, catalog#21-170)
(14.25 .mu.g) and pRL-TK (renilla-Luc) reporter (Promega Inc,
Catalog# E224A) (0.75 .mu.g) using Fugene 6 transfection reagent
(Roche catalog #11814443001) at 3:1 (Fugene:DNA) ratio following
manufacturer's instructions for transient transfections. Briefly,
45 .mu.l of Fugene is diluted in OptiMem and a total of 15 .mu.g
DNA is added. The complex is formed at room temperature for 30 min
under serum-free conditions and added to cells in DMEM-10% FBS
media (without antibiotics or selection agents). Plates are
returned to incubators for 24 h. A mock transfection plate, in
which no DNA is used for transfection, is kept as negative control.
At 24 h post transfection, cells are harvested, washed and counted.
Cells are then seeded in 96-well plates at the density of 20,000
cells/60 .mu.l per well in either OptiMem (serum-free) or a
specified % of FBS added to OptiMem. Peptidomimetic macrocycles are
diluted from 10 mM DMSO stocks to 8.times. working stock in sterile
water. Further dilutions are made in DMSO-water solvent so as to
keep final concentration of DMSO constant in all wells. 10 .mu.l
diluted macrocycle (10.times. desired concentration) is added to
each well. As a benchmark molecule NO-Aspirin is used at a
concentration range of 20 .mu.M-0.4 .mu.M range. The beta-catenin
signaling pathway is activated using GSK-3b-inhibitor IV (BIO,
CalBiochem Catalog#. 361550)) at 2 .mu.M final concentration. The
TCF activity reporter is measured on Synergy Multiplate reader at
24 h &/or 48 h post macrocycle treatment using Dual-Glo
Luciferase assay system (Promega, catalog # E2940) as per
manufacturer's instructions Inhibition of the reporter activity is
calculated against the DMSO treated cells stimulated with Bio.
[0284] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
132138PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term amidated 1Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15
Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(3)..(3)Any amino acid
available for cross-linkingMOD_RES(7)..(7)Any amino acid available
for cross-linkingmisc_feature(3)..(7)Cross-link between residues
2Asn Leu Xaa Asn Val Lys Xaa Ser Leu Val Asn Gln Ser 1 5 10
314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(4)..(4)Any amino acid available for
cross-linkingMOD_RES(8)..(8)Any amino acid available for
cross-linkingmisc_feature(4)..(8)Cross-link between residues 3Arg
Asp Leu Xaa Asn Val Lys Xaa Ser Leu Val Asn Glu Ser 1 5 10
414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)Any amino acid available for
cross-linkingMOD_RES(9)..(9)Any amino acid available for
cross-linkingmisc_feature(5)..(9)Cross-link between residues 4Arg
Asp Leu Ala Xaa Val Val Ser Xaa Leu Val Asn Glu Ser 1 5 10
514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)Any amino acid available for
cross-linkingMOD_RES(9)..(9)Any amino acid available for
cross-linkingmisc_feature(5)..(9)Cross-link between residues 5Arg
Asn Leu Ala Xaa Val Lys Ser Xaa Leu Val Asn Glu Ser 1 5 10
614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)Any amino acid available for
cross-linkingMOD_RES(9)..(9)Any amino acid available for
cross-linkingmisc_feature(5)..(9)Cross-link between residues 6Arg
Asp Leu Ala Xaa Val Lys Ser Xaa Leu Val Asn Gln Ser 1 5 10
714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(8)..(12)Cross-link between residues 7Arg
Asp Leu Ala Asp Val Lys Xaa Ser Leu Val Xaa Glu Ser 1 5 10
814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(8)..(12)Cross-link between residues 8Arg
Asn Leu Ala Asp Val Lys Xaa Ser Leu Val Xaa Glu Ser 1 5 10
914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(8)..(12)Cross-link between residues 9Arg
Asp Leu Ala Asn Val Ile Xaa Ser Leu Val Xaa Glu Ser 1 5 10
1014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(8)..(12)Cross-link between residues 10Arg
Asp Leu Ala Asn Val Val Xaa Ser Leu Val Xaa Glu Ser 1 5 10
1114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(10)..(10)NorleucineMOD_RES(12)..(12)Any amino
acid available for cross-linkingmisc_feature(8)..(12)Cross-link
between residues 11Arg Asp Leu Ala Asn Val Lys Xaa Ser Leu Val Xaa
Glu Ser 1 5 10 1214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(8)..(12)Cross-link between residues 12Arg
Gly Leu Ala Asn Val Lys Xaa Ser Leu Val Xaa Glu Ser 1 5 10
1316PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(10)..(10)Any amino acid available for
cross-linkingMOD_RES(14)..(14)Any amino acid available for
cross-linkingmisc_feature(10)..(14)Cross-link between residues
13Val Glu Arg Gly Leu Ala Asn Val Lys Xaa Ser Leu Val Xaa Glu Ser 1
5 10 15 1414PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(9)..(9)Any amino acid available for
cross-linkingMOD_RES(13)..(13)Any amino acid available for
cross-linkingmisc_feature(9)..(13)Cross-link between residues 14Arg
Asn Leu Ala Asp Val Lys Ser Xaa Leu Val Asn Xaa Ser 1 5 10
1514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(9)..(9)Any amino acid available for
cross-linkingMOD_RES(13)..(13)Any amino acid available for
cross-linkingmisc_feature(9)..(13)Cross-link between residues 15Arg
Asp Leu Ala Asn Val Lys Ser Xaa Leu Val Asn Xaa Ser 1 5 10
1614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(5)..(12)Cross-link between residues 16Arg
Asp Leu Ala Xaa Val Lys Ser Ser Leu Val Xaa Gln Ser 1 5 10
1718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)Any amino acid available for
cross-linkingMOD_RES(7)..(7)Any amino acid available for
cross-linkingmisc_feature(3)..(7)Cross-link between residues 17Ser
Ala Xaa Arg Asp Leu Xaa Asp Val Lys Ser Ser Leu Val Asn Glu 1 5 10
15 Ser Glu 1818PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(4)..(4)Any amino acid available for
cross-linkingMOD_RES(8)..(8)Any amino acid available for
cross-linkingmisc_feature(4)..(8)Cross-link between residues 18Ser
Ala Glu Xaa Asp Leu Ala Xaa Val Lys Ser Ser Leu Val Asn Glu 1 5 10
15 Ser Glu 1918PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(7)..(7)Any amino acid available for
cross-linkingMOD_RES(11)..(11)Any amino acid available for
cross-linkingmisc_feature(7)..(11)Cross-link between residues 19Ser
Ala Glu Arg Asp Leu Xaa Asp Val Lys Xaa Ser Leu Val Asn Glu 1 5 10
15 Ser Glu 2018PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)Any amino acid available for
cross-linkingMOD_RES(12)..(12)Any amino acid available for
cross-linkingmisc_feature(8)..(12)Cross-link between residues 20Ser
Ala Glu Arg Asp Leu Ala Xaa Val Lys Ser Xaa Leu Val Asn Glu 1 5 10
15 Ser Glu 2118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingmisc_feature(11)..(15)Cross-link between residues
21Ser Ala Glu Arg Asp Leu Ala Asp Val Lys Xaa Ser Leu Val Xaa Glu 1
5 10 15 Ser Glu 2218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)Any amino acid available for
cross-linkingMOD_RES(16)..(16)Any amino acid available for
cross-linkingmisc_feature(12)..(16)Cross-link between residues
22Ser Ala Glu Arg Asp Leu Ala Asp Val Lys Ser Xaa Leu Val Asn Xaa 1
5 10 15 Ser Glu 2314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)Any amino acid available for
cross-linkingMOD_RES(7)..(7)Any amino acid available for
cross-linkingmisc_feature(3)..(7)Cross-link between residues 23Ser
Ala Xaa Arg Asn Leu Xaa Asp Val Lys Ser Ser Leu Val 1 5 10
2414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)Any amino acid available for
cross-linkingMOD_RES(7)..(7)Any amino acid available for
cross-linkingmisc_feature(3)..(7)Cross-link between residues 24Ser
Ala Xaa Arg Asp Leu Xaa Asn Val Lys Ser Ser Leu Val 1 5 10
2514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)Any amino acid available for
cross-linkingMOD_RES(7)..(7)Any amino acid available for
cross-linkingmisc_feature(3)..(7)Cross-link between residues 25Ser
Ala Xaa Arg Asp Leu Xaa Asn Val Lys Ser Ser Leu Val 1 5 10
2614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)Any amino acid available for
cross-linkingMOD_RES(7)..(7)Any amino acid available for
cross-linkingmisc_feature(3)..(7)Cross-link between residues 26Ser
Ala Xaa Arg Asn Leu Xaa Asp Val Lys Ser Ser Leu Val 1 5 10
2721PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term amidated 27Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15
Glu Glu Lys Ser Ser 20 2822PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingmisc_feature(11)..(15)Cross-link between residues
28Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly Xaa Gln 1
5 10 15 Glu Glu Lys Ser Ser Asn 20 2922PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 29Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Gln Gly
Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn 20 3022PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 30Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Gln Gly
Xaa Gln 1 5 10 15 Gln Glu Lys Ser Ser Asn 20 3122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 31Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Gln Gly
Xaa Gln 1 5 10 15 Glu Gln Lys Ser Ser Asn 20 3222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 32Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly
Xaa Gln 1 5 10 15 Gln Gln Lys Ser Ser Asn 20 3322PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 33Leu Gly Ala Asn Asp Gln Leu Ile Ser Phe Xaa Asp Glu Gly
Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn 20 3422PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 34Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly
Xaa Gln 1 5 10 15 Gln Glu Lys Ser Ser Asn 20 3522PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 35Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly
Xaa Gln 1 5 10 15 Glu Gln Lys Ser Ser Asn 20 3622PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(12)..(12)Any amino acid
available for cross-linkingMOD_RES(16)..(16)Any amino acid
available for cross-linkingmisc_feature(12)..(16)Cross-link between
residues 36Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Xaa Glu Gly
Glu Xaa 1 5 10 15 Glu Glu Lys Ser Ser Asn 20 3722PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(15)..(15)Any amino acid
available for cross-linkingMOD_RES(19)..(19)Any amino acid
available for cross-linkingmisc_feature(15)..(19)Cross-link between
residues 37Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly
Xaa Gln 1 5 10 15 Glu Glu Xaa Ser Ser Asn 20 3822PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(16)..(16)Any amino acid
available for cross-linkingMOD_RES(20)..(20)Any amino acid
available for cross-linkingmisc_feature(16)..(20)Cross-link between
residues 38Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly
Glu Xaa 1 5 10 15 Glu Glu Lys Xaa Ser Asn 20 3922PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(18)..(18)Any amino acid
available for cross-linkingmisc_feature(11)..(18)Cross-link between
residues 39Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly
Glu Gln 1 5 10 15 Glu Xaa Lys Ser Ser Asn 20 4022PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(12)..(12)Any amino acid
available for cross-linkingMOD_RES(19)..(19)Any amino acid
available for cross-linkingmisc_feature(12)..(19)Cross-link between
residues 40Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Xaa Glu Gly
Glu Gln 1 5 10 15 Glu Glu Xaa Ser Ser Asn 20 4122PRTArtificial
SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)Any amino acid available for
cross-linkingMOD_RES(19)..(19)Any amino acid available for
cross-linkingmisc_feature(12)..(19)Cross-link between residues
41Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Xaa Glu Gly Glu Gln 1
5 10 15 Glu Glu Xaa Ser Ser Asn 20 4222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term amidatedMOD_RES(12)..(12)Any amino acid
available for cross-linkingMOD_RES(19)..(19)Any amino acid
available for cross-linkingmisc_feature(12)..(19)Cross-link between
residues 42Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Xaa Glu Gly
Glu Gln 1 5 10 15 Glu Glu Xaa Ser Ser Asn 20 4338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term amidated 43Leu Gly Ala Asn
Ala Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15 Glu Glu
Lys Ser Ser Glu Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20 25 30
Val Lys Ser Ser Leu Val 35 4438PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideN-term acetylated and
C-term amidated 44Leu Gly Ala Asn Ala Glu Leu Ile Ser Phe Lys Asp
Glu Gly Glu Gln 1 5 10 15 Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala
Glu Arg Asp Leu Ala Asp 20 25 30 Ala Lys Ser Ser Ala Val 35
4538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingmisc_feature(11)..(15)Cross-link between residues
45Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly Xaa Gln 1
5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala
Asp 20 25 30 Val Lys Ser Ser Leu Val 35 4638PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 46Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asp 20 25 30 Val Lys Ser Ser Leu Val 35
4738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 47Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asn 20 25 30 Val Lys Ser Ser Leu Val 35
4838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 48Leu Gly Ala Asn Asp Glu Leu Leu Ser Phe Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Gln Glu Lys Ser Ser Glu Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asn 20 25 30 Val Lys Ser Ser Leu Val 35
4938PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 49Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Ala 20 25 30 Val Lys Ser Ser Leu Val 35
5038PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 50Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp
Gln Gly Xaa Gln 1 5 10 15 Glu Gln Lys Ser Ser Glu Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asn 20 25 30 Val Lys Ser Ser Leu Val 35
5138PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 51Leu Gly Ala Asn Asp Glu Leu Ile Ser Trp Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Gln Glu Lys Ser Ser Glu Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asn 20 25 30 Val Lys Ser Ser Leu Val 35
5238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 52Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Gln Gln Lys Ser Ser Glu Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asn 20 25 30 Val Lys Ser Ser Leu Val 35
5338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 53Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asn
Glu Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asp Leu Xaa Asp 20 25 30 Val Lys Ser Ser Leu Val 35
5438PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 54Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asn
Gln Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asn Leu Xaa Asp 20 25 30 Val Lys Ser Ser Leu Val 35
5538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 55Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asn
Glu Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asn Leu Xaa Asp 20 25 30 Val Lys Ser Ser Leu Val 35
5638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 56Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asn
Glu Gly Xaa Gln 1 5 10 15 Ala Glu Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asn Leu Xaa Asp 20 25 30 Val Lys Ser Ser Leu Val 35
5738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(11)..(31)Cross-link between residues
where indicated 57Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asn
Gln Gly Xaa Gln 1 5 10 15 Ala Ala Lys Ser Ser Asn Asn Ser Ser Ala
Xaa Arg Asn Leu Xaa Ala 20 25 30 Val Lys Ser Ser Leu Val 35
5838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)Any amino acid available for
cross-linkingMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(28)..(28)Any amino acid available for
cross-linkingMOD_RES(32)..(32)Any amino acid available for
cross-linkingmisc_feature(11)..(32)Cross-link between residues
where indicated 58Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp
Glu Gly Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala
Glu Xaa Asp Leu Ala Xaa 20 25 30 Val Lys Ser Ser Leu Val 35
5921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylatedMOD_RES(11)..(11)Any amino acid
available for cross-linkingMOD_RES(15)..(15)Any amino acid
available for cross-linkingmisc_feature(11)..(15)Cross-link between
residues 59Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Xaa Asp Glu Gly
Xaa Gln 1 5 10 15 Glu Glu Lys Ser Ser 20 6013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Any amino acid available for
cross-linkingMOD_RES(6)..(6)Any amino acid available for
cross-linkingmisc_feature(2)..(6)Cross-link between residuesC-term
amidated 60Ala Xaa Arg Asp Leu Xaa Asn Val Lys Ser Ser Leu Val 1 5
10 6138PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)Any amino acid available for
cross-linkingMOD_RES(16)..(16)Any amino acid available for
cross-linkingmisc_feature(12)..(16)Cross-link between residues
61Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Xaa Glu Gly Glu Xaa 1
5 10 15 Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala
Asp 20 25 30 Val Lys Ser Ser Leu Val 35 6238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(15)..(15)Any amino acid available for
cross-linkingMOD_RES(19)..(19)Any amino acid available for
cross-linkingmisc_feature(15)..(19)Cross-link between residues
62Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Xaa Gln 1
5 10 15 Glu Glu Xaa Ser Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala
Asp 20 25 30 Val Lys Ser Ser Leu Val 35 6338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(16)..(16)Any amino acid available for
cross-linkingMOD_RES(20)..(20)Any amino acid available for
cross-linkingmisc_feature(16)..(20)Cross-link between residues
63Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Xaa 1
5 10 15 Glu Glu Lys Xaa Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala
Asp 20 25 30 Val Lys Ser Ser Leu Val 35 6438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(27)..(27)Any amino acid available for
cross-linkingMOD_RES(31)..(31)Any amino acid available for
cross-linkingmisc_feature(27)..(31)Cross-link between residues
64Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1
5 10 15 Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Xaa Arg Asp Leu Xaa
Asp 20 25 30 Val Lys Ser Ser Leu Val 35 6538PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(28)..(28)Any amino acid available for
cross-linkingMOD_RES(32)..(32)Any amino acid available for
cross-linkingmisc_feature(28)..(32)Cross-link between residues
65Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1
5 10 15 Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Glu Xaa Asp Leu Ala
Xaa 20 25 30 Val Lys Ser Ser Leu Val 35 6613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term
amidatedMOD_RES(3)..(3)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl) alanine 66Asn Leu Ala
Asn Val Lys Ala Ser Leu Val Asn Gln Ser 1 5 10 6714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 67Arg Asp
Leu Ala Asp Val Lys Ala Ser Leu Val Ala Glu Ser 1 5 10
6814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(9)..(9)(S)-alpha-(2'-pentenyl) alanine 68Arg Asn Leu
Ala Ala Val Lys Ser Ala Leu Val Asn Glu Ser 1 5 10
6914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 69Arg Asn
Leu Ala Asp Val Lys Ala Ser Leu Val Ala Glu Ser 1 5
10 7014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(9)..(9)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(13)..(13)(S)-alpha-(2'-pentenyl) alanine 70Arg Asn
Leu Ala Asp Val Lys Ser Ala Leu Val Asn Ala Ser 1 5 10
7114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(9)..(9)(S)-alpha-(2'-pentenyl) alanine 71Arg Asp Leu
Ala Ala Val Lys Ser Ala Leu Val Asn Gln Ser 1 5 10
7214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)(R)-alpha-(2'-octenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 72Arg Asp
Leu Ala Ala Val Lys Ser Ser Leu Val Ala Gln Ser 1 5 10
7314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(9)..(9)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(13)..(13)(S)-alpha-(2'-pentenyl) alanine 73Arg Asp
Leu Ala Asn Val Lys Ser Ala Leu Val Asn Ala Ser 1 5 10
7414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(4)..(4)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl) alanine 74Arg Asp Leu
Ala Asn Val Lys Ala Ser Leu Val Asn Glu Ser 1 5 10
7520PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl) alanine 75Ser Ala
Glu Arg Asp Leu Ala Asp Val Lys Ala Ser Leu Val Asn Glu 1 5 10 15
Ser Glu Lys Arg 20 7620PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 76Ser Ala
Glu Arg Asp Leu Ala Ala Val Lys Ser Ala Leu Val Asn Glu 1 5 10 15
Ser Glu Lys Arg 20 7720PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 77Ser Ala
Glu Arg Asp Leu Ala Asp Val Lys Ala Ser Leu Val Ala Glu 1 5 10 15
Ser Glu Lys Arg 20 7820PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(16)..(16)(S)-alpha-(2'-pentenyl) alanine 78Ser Ala
Glu Arg Asp Leu Ala Asp Val Lys Ser Ala Leu Val Asn Ala 1 5 10 15
Ser Glu Lys Arg 20 7938PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideN-term acetylated and
C-term amidated 79Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp
Glu Gly Glu Gln 1 5 10 15 Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala
Glu Arg Asp Leu Ala Asp 20 25 30 Val Lys Ser Ser Leu Val 35
8021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term amidated 80Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15
Glu Glu Lys Ser Ser 20 8122PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 81Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn 20 8222PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(16)..(16)(S)-alpha-(2'-pentenyl) alanine 82Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Ala Glu Gly Glu Ala 1 5 10 15
Glu Glu Lys Ser Ser Asn 20 8322PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(19)..(19)(S)-alpha-(2'-pentenyl) alanine 83Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Ala Ser Ser Asn 20 8422PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(16)..(16)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(20)..(20)(S)-alpha-(2'-pentenyl) alanine 84Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Ala 1 5 10 15
Glu Glu Lys Ala Ser Asn 20 8522PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(R)-alpha-(2'-octenyl)
alanineMOD_RES(18)..(18)(S)-alpha-(2'-pentenyl) alanine 85Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Glu Gln 1 5 10 15
Glu Ala Lys Ser Ser Asn 20 8622PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)(R)-alpha-(2'-octenyl)
alanineMOD_RES(19)..(19)(S)-alpha-(2'-pentenyl) alanine 86Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Ala Glu Gly Glu Gln 1 5 10 15
Glu Glu Ala Ser Ser Asn 20 8720PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl) alanine 87Ser Ala Ala
Arg Asp Leu Ala Asp Val Lys Ser Ser Leu Val Asn Glu 1 5 10 15 Ser
Glu Lys Arg 20 8820PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(4)..(4)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl) alanine 88Ser Ala Glu
Ala Asp Leu Ala Ala Val Lys Ser Ser Leu Val Asn Glu 1 5 10 15 Ser
Glu Lys Arg 20 8938PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideN-term acetylated and C-term amidated
89Leu Gly Ala Asn Ala Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1
5 10 15 Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Glu Arg Asp Leu Ala
Asp 20 25 30 Val Lys Ser Ser Leu Val 35 9038PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term amidated 90Leu Gly Ala Asn
Ala Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15 Glu Glu
Lys Ser Ser Glu Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20 25 30
Ala Lys Ser Ser Ala Val 35 9138PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideN-term acetylated and
C-term amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 91Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 9238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(16)..(16)(S)-alpha-(2'-pentenyl) alanine 92Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Ala Glu Gly Glu Ala 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 9338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(19)..(19)(S)-alpha-(2'-pentenyl) alanine 93Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Ala Ser Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 9438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(16)..(16)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(20)..(20)(S)-alpha-(2'-pentenyl) alanine 94Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Ala 1 5 10 15
Glu Glu Lys Ala Ser Asn Asn Ser Ser Ala Glu Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 9538PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 95Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15
Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Ala Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 9638PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(28)..(28)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(32)..(32)(S)-alpha-(2'-pentenyl) alanine 96Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Asp Glu Gly Glu Gln 1 5 10 15
Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Glu Ala Asp Leu Ala Ala 20
25 30 Val Lys Ser Ser Leu Val 35 9714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 97Arg Asp
Leu Ala Asn Val Ile Ala Ser Leu Val Ala Glu Ser 1 5 10
9814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 98Arg Asp
Leu Ala Asn Val Val Ala Ser Leu Val Ala Glu Ser 1 5 10
9914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(10)..(10)NorleucineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl-
) alanine 99Arg Asp Leu Ala Asn Val Lys Ala Ser Leu Val Ala Glu Ser
1 5 10 10014PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(8)..(8)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(12)..(12)(S)-alpha-(2'-pentenyl) alanine 100Arg Gly
Leu Ala Asn Val Lys Ala Ser Leu Val Ala Glu Ser 1 5 10
10116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(10)..(10)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(14)..(14)(S)-alpha-(2'-pentenyl) alanine 101Val Glu
Arg Gly Leu Ala Asn Val Lys Ala Ser Leu Val Ala Glu Ser 1 5 10 15
10238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 102Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 10338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(28)..(28)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(32)..(32)(S)-alpha-(2'-pentenyl) alanine 103Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Glu Ala Asp Leu Ala Ala 20
25 30 Val Lys Ser Ser Leu Val 35 10422PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 104Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Gln Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn 20 10522PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideN-term acetylated and
C-term amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 105Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Gln Glu Lys Ser Ser Asn 20 10622PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideN-term acetylated and
C-term amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 106Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Gln Lys Ser Ser Asn 20 10714PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideN-term acetylated and
C-term amidatedMOD_RES(3)..(3)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl) alanine 107Ser Ala
Ala Arg Asn Leu Ala Asp Val Lys Ser Ser Leu Val 1 5 10
10814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl) alanine 108Ser Ala
Ala Arg Asp Leu Ala Asn Val Lys Ser Ser Leu Val 1 5 10
10922PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 109Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Gln Gly Ala Gln 1 5 10 15
Gln Glu Lys Ser Ser Asn 20 11022PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideN-term acetylated and
C-term amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 110Leu Gly
Ala
Asn Asp Glu Leu Ile Ser Phe Ala Asp Gln Gly Ala Gln 1 5 10 15 Glu
Gln Lys Ser Ser Asn 20 11122PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 111Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Gln Gln Lys Ser Ser Asn 20 11238PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptideN-term acetylated and
C-term amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 112Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Glu Asn Ser Ser Ala Ala Arg Asp Leu Ala Asn 20
25 30 Val Lys Ser Ser Leu Val 35 11338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 113Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Gln Gly Ala Gln 1 5 10 15
Glu Gln Lys Ser Ser Glu Asn Ser Ser Ala Ala Arg Asp Leu Ala Asn 20
25 30 Val Lys Ser Ser Leu Val 35 11438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 114Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Gln Gln Lys Ser Ser Glu Asn Ser Ser Ala Ala Arg Asp Leu Ala Asn 20
25 30 Val Lys Ser Ser Leu Val 35 11521PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
acetylatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 115Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser 20 11613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(6)..(6)(S)-alpha-(2'-pentenyl) alanineC-term
amidated 116Ala Ala Arg Asp Leu Ala Asn Val Lys Ser Ser Leu Val 1 5
10 11722PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(12)..(12)(R)-alpha-(2'-octenyl)
alanineMOD_RES(19)..(19)(S)-alpha-(2'-pentenyl) alanine 117Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Lys Ala Glu Gly Glu Gln 1 5 10 15
Glu Glu Ala Ser Ser Asn 20 11822PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideN-term acetylated and
C-term amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl) alanine 118Leu Gly
Ala Asn Asp Gln Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn 20 11914PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideN-term acetylated and
C-term amidatedMOD_RES(3)..(3)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl) alanine 119Ser Ala
Ala Arg Asp Leu Ala Asn Val Lys Ser Ser Leu Val 1 5 10
12014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(3)..(3)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(7)..(7)(S)-alpha-(2'-pentenyl) alanine 120Ser Ala
Ala Arg Asn Leu Ala Asp Val Lys Ser Ser Leu Val 1 5 10
12114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideN-term acetylated and C-term
amidatedMOD_RES(5)..(5)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(9)..(9)(S)-alpha-(2'-pentenyl) alanine 121Arg Asp
Leu Ala Ala Val Val Ser Ala Leu Val Asn Glu Ser 1 5 10
12238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 122Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asn Gln Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asn Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 12338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 123Leu Gly
Ala Asn Asp Glu Leu Leu Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Gln Glu Lys Ser Ser Glu Asn Ser Ser Ala Ala Arg Asp Leu Ala Asn 20
25 30 Val Lys Ser Ser Leu Val 35 12438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 124Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asp Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asp Leu Ala Ala 20
25 30 Val Lys Ser Ser Leu Val 35 12538PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 125Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asn Gln Gly Ala Gln 1 5 10 15
Ala Ala Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asn Leu Ala Ala 20
25 30 Val Lys Ser Ser Leu Val 35 12638PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 126Leu Gly
Ala Asn Asp Glu Leu Ile Ser Trp Ala Asp Glu Gly Ala Gln 1 5 10 15
Gln Glu Lys Ser Ser Glu Asn Ser Ser Ala Ala Arg Asp Leu Ala Asn 20
25 30 Val Lys Ser Ser Leu Val 35 12738PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 127Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asn Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asp Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 12838PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 128Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asn Glu Gly Ala Gln 1 5 10 15
Ala Glu Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asn Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 12938PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideN-term acetylated and C-term
amidatedMOD_RES(11)..(11)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(15)..(15)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(27)..(27)(S)-alpha-(2'-pentenyl)
alanineMOD_RES(31)..(31)(S)-alpha-(2'-pentenyl) alanine 129Leu Gly
Ala Asn Asp Glu Leu Ile Ser Phe Ala Asn Glu Gly Ala Gln 1 5 10 15
Glu Glu Lys Ser Ser Asn Asn Ser Ser Ala Ala Arg Asn Leu Ala Asp 20
25 30 Val Lys Ser Ser Leu Val 35 1308PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 130Asp
Leu Ala Lys Ser Ser Leu Val 1 5 13114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 131Arg
Asp Leu Ala Asp Val Lys Ser Ser Leu Val Asn Glu Ser 1 5 10
1326PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 132His His His His His His 1 5
* * * * *