U.S. patent application number 17/155330 was filed with the patent office on 2022-01-06 for peptidomimetic macrocycles.
The applicant listed for this patent is Aileron Therapeutics, Inc.. Invention is credited to Carl ELKIN, Eric FEYFANT, Bradford J. GRAVES, Vincent GUERLAVAIS, Huw M. NASH, Tomi K. SAWYER.
Application Number | 20220000968 17/155330 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220000968 |
Kind Code |
A1 |
GUERLAVAIS; Vincent ; et
al. |
January 6, 2022 |
PEPTIDOMIMETIC MACROCYCLES
Abstract
Provided herein are peptidomimetic macrocycles containing amino
acid sequences with at least two modified amino acids that form an
intramolecular cross-link that can help to stabilize a secondary
structure of the amino acid sequence. Suitable sequences for
stabilization include those with homology to the p53 protein. These
sequences can bind to the MDM2 and/or MDMX proteins. Also provided
herein are methods of using such macrocycles for the treatment of
diseases and disorders, such as cancers or other disorders
characterized by a low level or low activity of a p53 protein or
high level of activity of a MDM2 and/or MDMX protein.
Inventors: |
GUERLAVAIS; Vincent;
(Arlington, MA) ; ELKIN; Carl; (Arlington, MA)
; NASH; Huw M.; (Lexington, MA) ; SAWYER; Tomi
K.; (Southborough, MA) ; GRAVES; Bradford J.;
(Nutley, NJ) ; FEYFANT; Eric; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aileron Therapeutics, Inc. |
Watertown |
MA |
US |
|
|
Appl. No.: |
17/155330 |
Filed: |
January 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16223473 |
Dec 18, 2018 |
10967042 |
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17155330 |
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15229517 |
Aug 5, 2016 |
10213477 |
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16223473 |
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14498063 |
Sep 26, 2014 |
9505804 |
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15229517 |
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13767852 |
Feb 14, 2013 |
8927500 |
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14498063 |
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61599328 |
Feb 15, 2012 |
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61656962 |
Jun 7, 2012 |
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61723770 |
Nov 7, 2012 |
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International
Class: |
A61K 38/10 20060101
A61K038/10; C07K 7/08 20060101 C07K007/08; A61K 38/03 20060101
A61K038/03; C07K 7/06 20060101 C07K007/06; A61K 38/08 20060101
A61K038/08; C07K 1/113 20060101 C07K001/113; C07K 14/47 20060101
C07K014/47; C07K 7/54 20060101 C07K007/54; A61K 45/06 20060101
A61K045/06 |
Claims
1.-102. (canceled)
103. A method of screening a peptidomimetic macrocycle by
affinity-selection mass spectrometry, comprising: (a) mixing the
peptidomimetic macrocycle and human homolog of mouse double minute
2 (hMDM2) protein in a solution; (b) clarifying the solution by
centrifugation; (c) incubating the solution at elevated
temperature; and (d) analyzed by size-exclusion
chromatography-liquid chromatograph mass spectrometry (LC-MS),
thereby determining the affinity of the peptidomimetic macrocycle
to the hMDM2 protein.
104. The method of claim 103, wherein the solution comprises
phosphate-buffered saline (PBS)
105. The method of claim 104, wherein the solution further
comprises DMSO.
106. The method of claim 103, wherein the hMDM3 protein is added in
two batches.
107. The method of claim 103, wherein the centrifugation is
conducted at 10,000 g for 10 minutes.
108. A method of screening a peptidomimetic macrocycle by
protein-ligand Kd titration, comprising: (a) preparing serially
diluted stock solutions of the peptidomimetic macrocycle; (b)
clarifying the stock solution by centrifugation to afford
supernatants; (c) adding human homolog of mouse double minute 2
(hMDM2) protein to the supernatants; and (d) analyzed by
size-exclusion chromatography-liquid chromatograph mass
spectrometry (LC-MS), thereby determining binding and affinity of
the peptidomimetic macrocycle to the hMDM2 protein.
109. The method of claim 108, wherein the solution comprises
phosphate-buffered saline (PBS)
110. The method of claim 109, wherein the solution further
comprises DMSO.
111. The method of claim 108, wherein the hMDM3 protein is added in
two batches.
112. The method of claim 108, wherein the centrifugation is
conducted at 10,000 g for 10 minutes.
113. A method of preparing a capsule, comprising: (a) preparing a
peptidomimetic macrocycle; (b) combining the peptidomimetic
macrocycle with a pharmaceutically acceptable carrier to afford a
mixture; and (c) encapsulating the mixture.
114. The method of claim 113, wherein the mixture further comprises
a diluent.
115. The method of claim 113, wherein the mixture further comprises
a flavoring agent.
116. The method of claim 113, wherein the mixture further comprises
a binder.
117. The method of claim 113, wherein the mixture further comprises
a disintegrating agent.
118. The method of claim 113, wherein the mixture further comprises
a preservative.
119. The method of claim 113, wherein the peptidomimetic macrocycle
comprising an amino acid sequence with at least about 90% sequence
identity to an amino acid sequence selected from the group
consisting of SEQ IP NOs: 10-457, or pharmaceutically acceptable
salt thereof; and wherein the peptidomimetic macrocycle has a
Formula: ##STR00068## wherein: each of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is
independently an amino acid, wherein at least three of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acids as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12(SEQ ID NO: 8) or
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12(SEQ ID NO: 9), wherein each
X.sub.4 and X.sub.11 is independently an amino acid: each D is
independently an amino acid; each E is independently an amino acid
selected from the group consisting of Ala (alanine), D-Ala
(D-alanine), Aib (.alpha.-aminoisobutyric acid), Sar (N-methyl
glycine), and Ser (serine); R.sub.1 and R.sub.2 are independently
--H, alkyl, alkenyl, alkyenyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-; or forms a macrocycle-forming linker L'
connected to the alpha position of one of said D or E amino acids;
L and L' are independently a macrocycle-forming linker; 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; each R.sub.6 is independently --H, alkyl,
alkenyl, alkenyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope or a therapeutic agent; R.sub.7
is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or
heteroaryl, optionally substituted with R.sub.5, or part of a
cyclic structure with a D residue; R.sub.8 is --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, aryl, or heteroaryl, optionally substituted with
R.sub.5, or part of a cyclic structure with an E residue; v is an
integer from 1-10; and w is an integer from 3-10.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 16/223,473 filed Dec. 18, 2018, which is a continuation
application of U.S. application Ser. No. 15/229,517, filed Aug. 5,
2016, now U.S. Pat. No. 10,213,477, which is a continuation
application of U.S. application Ser. No. 14/498,063, filed Sep. 26,
2014, now U.S. Pat. No. 9,505,804, issued Nov. 29, 2016, which is a
continuation application of U.S. application Ser. No. 13/767,852,
filed Feb. 14, 2013, now U.S. Pat. No. 8,927,500, issued Jan. 6,
2015, which claims the benefit of U.S. Provisional Application No.
61/723,770, filed Nov. 7, 2012, U.S. Provisional Application No.
61/656,962, filed Jun. 7, 2012, and U.S. Provisional Application
No. 61/599,328, filed Feb. 15, 2012; each of which is incorporated
herein by reference in their entirety.
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 Jan. 21, 2021, is named 35224-766.304_SL.txt and is 1,202,980
bytes in size.
BACKGROUND OF THE INVENTION
[0003] The human transcription factor protein p53 induces cell
cycle arrest and apoptosis in response to DNA damage and cellular
stress, and thereby plays a critical role in protecting cells from
malignant transformation. The E3 ubiquitin ligase MDM2 (also known
as HDM2) negatively regulates p53 function through a direct binding
interaction that neutralizes the p53 transactivation activity,
leads to export from the nucleus of p53 protein, and targets p53
for degradation via the ubiquitylation-proteasomal pathway. Loss of
p53 activity, either by deletion, mutation, or MDM2 overexpression,
is the most common defect in human cancers. Tumors that express
wild type p53 are vulnerable to pharmacologic agents that stabilize
or increase the concentration of active p53. In this context,
inhibition of the activities of MDM2 has emerged as a validated
approach to restore p53 activity and resensitize cancer cells to
apoptosis in vitro and in vivo. MDMX (MDM4) has more recently been
identified as a similar negative regulator of p53, and studies have
revealed significant structural homology between the p53 binding
interfaces of MDM2 and MDMX. The p53-MDM2 and p53-MDMX
protein-protein interactions are mediated by the same 15-residue
alpha-helical transactivation domain of p53, which inserts into
hydrophobic clefts on the surface of MDM2 and MDMX. Three residues
within this domain of p53 (F19, W23, and L.sub.26) are essential
for binding to MDM2 and MDMX.
[0004] There remains a considerable need for compounds capable of
binding to and modulating the activity of p53, MDM2 and/or MDMX.
Provided herein are p53-based peptidomimetic macrocycles that
modulate an activity of p53. Also provided herein are p53-based
peptidomimetic macrocycles that inhibit the interactions between
p53, MDM2 and/or MDMX proteins. Further, provided herein are
p53-based peptidomimetic macrocycles that can be used for treating
diseases including but not limited to cancer and other
hyperproliferative diseases.
SUMMARY OF THE INVENTION
[0005] Described herein are stably cross-linked peptides related to
a portion of human p53 ("p53 peptidomimetic macrocycles"). These
cross-linked peptides contain at least two modified amino acids
that together form an intramolecular cross-link that can help to
stabilize the alpha-helical secondary structure of a portion of p53
that is thought to be important for binding of p53 to MDM2 and for
binding of p53 to MDMX. Accordingly, a cross-linked polypeptide
described herein can have improved biological activity relative to
a corresponding polypeptide that is not cross-linked. The p53
peptidomimetic macrocycles are thought to interfere with binding of
p53 to MDM2 and/or of p53 to MDMX, thereby liberating functional
p53 and inhibiting its destruction. The p53 peptidomimetic
macrocycles described herein can be used therapeutically, for
example to treat cancers and other disorders characterized by an
undesirably low level or a low activity of p53, and/or to treat
cancers and other disorders characterized by an undesirably high
level of activity of MDM2 or MDMX. The p53 peptidomimetic
macrocycles can also be useful for treatment of any disorder
associated with disrupted regulation of the p53 transcriptional
pathway, leading to conditions of excess cell survival and
proliferation such as cancer and autoimmunity, in addition to
conditions of inappropriate cell cycle arrest and apoptosis such as
neurodegeneration and immune deficiencies. In some embodiments, the
p53 peptidomimetic macrocycles bind to MDM2 (e.g., GenBank.RTM.
Accession No.: 228952; GI:228952) and/or MDMX (also referred to as
MDM4; GenBank.RTM. Accession No.: 88702791; GI:88702791).
[0006] In one aspect, provided herein is 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, Table 1a, Table 1b, or Table 1c. Alternatively, an amino
acid sequence of said peptidomimetic macrocycle is chosen from the
group consisting of the amino acid sequences in Table 4. In some
embodiments, the peptidomimetic macrocycle is not a peptide as
shown in Table 2a or 2b. In other cases, the peptidomimetic
macrocycle does not comprise a structure as shown in Table 2a or
2b. In some embodiments, the peptidomimetic macrocycle has an amino
acid sequence chosen from Table 1. In some embodiments, the
peptidomimetic macrocycle has an amino acid sequence chosen from
Table 1a. In some embodiments, the peptidomimetic macrocycle has an
amino acid sequence chosen from Table 1b. In some embodiments, the
peptidomimetic macrocycle has an amino acid sequence chosen from
Table 1c.
[0007] Alternatively, an amino acid sequence of said peptidomimetic
macrocycle is chosen as above, and further wherein the macrocycle
does not include a thioether or a triazole. 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 can comprise a crosslinker linking the
.alpha.-positions of at least two amino acids. At least one of said
two amino acids can be an .alpha.,.alpha.-disubstituted amino
acid.
[0008] In some embodiments, provided are peptidomimetic macrocycle
of the formula:
##STR00001##
[0009] wherein:
[0010] each A, C, D, and E is independently an amino acid;
[0011] B is an amino acid,
##STR00002##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0012] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0013] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0014] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-; L.sub.1 and 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;
[0015] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0016] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0017] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0018] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0019] 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;
[0020] 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;
[0021] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10;
[0022] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0023] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0024] n is an integer from 1-5.
[0025] In some embodiments, w>2 and each of the first two amino
acid represented by E comprises an uncharged side chain or a
negatively charged side chain.
[0026] some embodiments, the first C-terminal amino acid and/or the
second C-terminal amino acid represented by E comprise a
hydrophobic side chain. For example, the first C-terminal amino
acid and/or the second C-terminal amino acid represented by E
comprises a hydrophobic side chain, for example a large hydrophobic
side chain.
[0027] In some embodiments, w is between 3 and 1000. For example,
the third amino acid represented by E comprises a large hydrophobic
side chain.
[0028] In other embodiments, the peptidomimetic macrocycle as
claimed excludes the sequence of: [0029]
Ac-RTQATF$r8NQWAibAN1e$TNAibTR-NH.sub.2 (SEQ ID NO: 1),
Ac-RTQATF$r8NQWAibAN1e$TNAibTR-NH.sub.2 (SEQ ID NO: 2), [0030]
Ac-$r8SQQTFS$LWRLLAibQN-NH2 (SEQ ID NO: 3),
Ac-QSQ$r8TFSNLW$LLAibQN-NH2 (SEQ ID NO: 4), [0031]
Ac-QS$r5QTFStNLW$LLAibQN-NH2 (SEQ ID NO: 5), or
Ac-QSQQ$r8FSNLWR$LAibQN-NH2 (SEQ ID NO: 6).
[0032] In other embodiments, the peptidomimetic macrocycle as
claimed excludes the sequence of:
[0033] Ac-Q$r8QQTFSN$WRLLAibQN-NH2 (SEQ ID NO: 7).
[0034] Peptidomimetic macrocycles are also provided of the
formula:
##STR00003##
[0035] wherein:
[0036] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8), where each X is an amino
acid;
[0037] each D and E is independently an amino acid;
[0038] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0039] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-;
[0040] 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;
[0041] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0042] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0043] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0044] 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;
[0045] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0046] 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;
[0047] 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;
[0048] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0049] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0050] n is an integer from 1-5.
[0051] In some embodiments, a peptidomimetic macrocycle has the
Formula:
##STR00004##
[0052] wherein:
[0053] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9), where each X
is an amino acid;
[0054] each D is independently an amino acid;
[0055] each E is independently an amino acid, for example an amino
acid selected from Ala (alanine), D-Ala (D-alanine), Aib
(.alpha.-aminoisobutyric acid), Sar (N-methyl glycine), and Ser
(serine);
[0056] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0057] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-;
[0058] 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;
[0059] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0060] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0061] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0062] 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;
[0063] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0064] 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;
[0065] 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;
[0066] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0067] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0068] n is an integer from 1-5.
[0069] In some embodiments, a peptidomimetic macrocycle has the
Formula:
##STR00005##
[0070] wherein:
[0071] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least two of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.5,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9), where each X
is an amino acid;
[0072] each D and E is independently an amino acid;
[0073] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0074] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-, wherein L comprises at least one
double bond in the E configuration;
[0075] 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;
[0076] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0077] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0078] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0079] 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;
[0080] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0081] 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;
[0082] 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;
[0083] v is an integer from 1-1000;
[0084] w is an integer from 3-1000;
[0085] n is an integer from 1-5; and
[0086] Xaa.sub.7 is Boc-protected tryptophan.
[0087] In some embodiments of any of the Formulas described herein,
[D].sub.v is -Leu.sub.1-Thr.sub.2. In other embodiments of the
Formulas described herein, each E other than the third amino acid
represented by E is an amino acid selected from Ala (alanine),
D-Ala (D-alanine), Aib (.alpha.-aminoisobutyric acid), Sar
(N-methyl glycine), and Ser (serine).
[0088] In some embodiments, w is an integer from 3-10, for example
3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other
embodiments, w is 6. In some embodiments, v is an integer from
1-10, for example 2-5. In some embodiments, v is 2.
[0089] In some embodiments, peptides disclosed herein bind a
binding site defined at least in part by the MDMX amino acid side
chains of L.sub.17, V46, M50, Y96 (forming the rim of the pocket)
and L.sub.99. Without being bound by theory, binding to such a
binding site improves one or more properties such as binding
affinity, induction of apoptosis, in vitro or in vivo anti-tumor
efficacy, or reduced ratio of binding affinities to MDMX versus
MDM2.
[0090] In some embodiments, the peptidomimetic macrocycle has
improved binding affinity to MDM2 or MDMX relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
other instances, the peptidomimetic macrocycle has a reduced ratio
of binding affinities to MDMX versus MDM2 relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
still other instances, the peptidomimetic macrocycle has improved
in vitro anti-tumor efficacy against p53 positive tumor cell lines
relative to a corresponding peptidomimetic macrocycle where w is 0,
1 or 2. In some embodiments, the peptidomimetic macrocycle shows
improved in vitro induction of apoptosis in p53 positive tumor cell
lines relative to a corresponding peptidomimetic macrocycle where w
is 0, 1 or 2. In other instances, the peptidomimetic macrocycle of
claim 1, wherein the peptidomimetic macrocycle has an improved in
vitro anti-tumor efficacy ratio for p53 positive versus p53
negative or mutant tumor cell lines relative to a corresponding
peptidomimetic macrocycle where w is 0, 1 or 2. In some instances
the improved efficacy ratio in vitro, is 1-29, .gtoreq.30-49, or
.gtoreq.50. In still other instances, the peptidomimetic macrocycle
has improved in vivo anti-tumor efficacy against p53 positive
tumors relative to a corresponding peptidomimetic macrocycle where
w is 0, 1 or 2. In some instances the improved efficacy ratio in
vivo is -29, .gtoreq.30-49, or .gtoreq.50. In yet other instances,
the peptidomimetic macrocycle has improved in vivo induction of
apoptosis in p53 positive tumors relative to a corresponding
peptidomimetic macrocycle where w is 0, 1 or 2. In some
embodiments, the peptidomimetic macrocycle has improved cell
permeability relative to a corresponding peptidomimetic macrocycle
where w is 0, 1 or 2. In other cases, the peptidomimetic macrocycle
has improved solubility relative to a corresponding peptidomimetic
macrocycle where w is 0, 1 or 2.
[0091] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof. In some embodiments, Xaa.sub.5 is Glu or an amino
acid analog thereof and wherein the peptidomimetic macrocycle has
an improved property, such as improved binding affinity, improved
solubility, improved cellular efficacy, improved cell permeability,
improved in vivo or in vitro anti-tumor efficacy, or improved
induction of apoptosis relative to a corresponding peptidomimetic
macrocycle where Xaa.sub.5 is Ala.
[0092] In some embodiments, the peptidomimetic macrocycle has
improved binding affinity to MDM2 or MDMX relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala. In
other embodiments, the peptidomimetic macrocycle has a reduced
ratio of binding affinities to MDMX vs MDM2 relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala. In
some embodiments, the peptidomimetic macrocycle has improved
solubility relative to a corresponding peptidomimetic macrocycle
where Xaa.sub.5 is Ala, or the peptidomimetic macrocycle has
improved cellular efficacy relative to a corresponding
peptidomimetic macrocycle where Xaa.sub.5 is Ala.
[0093] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof and wherein the peptidomimetic macrocycle has
improved biological activity, such as improved binding affinity,
improved solubility, improved cellular efficacy, improved helicity,
improved cell permeability, improved in vivo or in vitro anti-tumor
efficacy, or improved induction of apoptosis relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala.
[0094] In some embodiments, the peptidomimetic macrocycle has an
activity against a p53+/+ cell line which is at least 2-fold,
3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 70-fold, or
100-fold greater than its binding affinity against a p53-/- cell
line. In some embodiments, the peptidomimetic macrocycle has an
activity against a p53+/+ cell line which is between 1 and 29-fold,
between 30 and 49-fold, or .gtoreq.50-fold greater than its binding
affinity against a p53-/- cell line. Activity can be measured, for
example, as an IC50 value. For example, the p53+/+ cell line is
SJSA-1, RKO, HCT-116, or MCF-7 and the p53-/- cell line is RKO-E6
or SW-480. In some embodiments, the peptide has an IC50 against the
p53+/+ cell line of less than 1 .mu.M.
[0095] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof and the peptidomimetic macrocycle has an activity
against a p53+/+ cell line which is at least 10-fold greater than
its binding affinity against a p53-/- cell line.
[0096] Additionally, a method is provided of treating cancer in a
subject comprising administering to the subject a peptidomimetic
macrocycle. In some embodiments, the cancer is head and neck
cancer, melanoma, lung cancer, breast cancer, or glioma.
[0097] Also provided is a method of modulating the activity of p53
or MDM2 or MDMX in a subject comprising administering to the
subject a peptidomimetic macrocycle, or a method of antagonizing
the interaction between p53 and MDM2 and/or MDMX proteins in a
subject comprising administering to the subject such a
peptidomimetic macrocycle.
[0098] Provided herein is a method of preparing a composition
comprising a peptidomimetic macrocycle of Formula (I):
##STR00006##
comprising an amino acid sequence which is about 60% to about 100%
identical to an amino acid sequence selected from the group
consisting of the amino acid sequences in Table 1, Table 1a, Table
1b, or Table 1c, the method comprising treating a compound of
Formula (II)
##STR00007##
with a catalyst to result in the compound of Formula I
[0099] wherein in the compound(s) of Formulae (I) and (II)
[0100] each A, C, D, and E is independently an amino acid;
[0101] each B is independently an amino acid,
##STR00008##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0102] each R.sub.1 and R.sub.2 are independently hydrogen, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with
halogen; or at least one of R.sub.1 and R.sub.2 forms a
macrocycle-forming linker L' connected to the alpha position of one
of the D or E amino acids;
[0103] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0104] each L' is independently a macrocycle-forming linker of the
formula -L.sub.1-L.sub.2-;
[0105] each 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;
[0106] each R.sub.4 and R.sub.4' is independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene;
[0107] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0108] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0109] each R.sub.6 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope or a therapeutic agent;
[0110] each R.sub.7 is independently hydrogen, 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;
[0111] each R.sub.8 is independently hydrogen, 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;
[0112] each v and w are independently integers from 1-1000;
[0113] u is an integer from 1-10;
[0114] each x, y and z are independently integers from 0-10;
[0115] each n is independently an integer from 1-5;
[0116] each o is independently an integer from 1 to 15;
[0117] each p is independently an integer from 1 to 15;
[0118] "(E)" indicates a trans double bond; and
[0119] one or more of the amino acids A, C and/or B when B is an
amino acid, present in the compounds of Formulae (I) and (II), has
a side chain bearing a protecting group.
[0120] In some embodiments, the protecting group is a nitrogen atom
protecting group.
[0121] In some embodiments, the protecting group is a Boc
group.
[0122] In some embodiments, the side chain of the amino acid
bearing the protecting group comprises a protected indole.
[0123] In some embodiments, the amino acid bearing the protecting
group on its side chain is tryptophan (W) that is protected by the
protecting group on its indole nitrogen. For example, the
protecting group is a Boc group.
[0124] In some embodiments, after the step of contacting the
compound of Formula II with catalyst the compound of Formula (I) is
obtained in equal or higher amounts than a corresponding compound
which is a Z isomer. For example, after the step of contacting the
compound of Formula II with catalyst the compound of Formula (I) is
obtained in a 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher amount than
the corresponding compound which is a Z isomer.
[0125] In some embodiments, the catalyst is a ruthenium
catalyst.
[0126] In some embodiments, the method further comprises the step
of treating the compounds of Formula (I) with a reducing agent or
an oxidizing agent.
[0127] In some embodiments, the compound of Formula (II) is
attached to a solid support. In other embodiments, the compound of
Formula (II) is not attached to a solid support.
[0128] In some embodiments, the method further comprises removing
the protecting group(s) from the compounds of Formula (I).
[0129] In some embodiments, the ring closing metathesis is
conducted at a temperature ranging from about 20.degree. C. to
about 80.degree. C.
[0130] In some embodiments, the peptidomimetic macrocycle of
Formula (I) has the Formula:
##STR00009##
[0131] wherein:
[0132] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least two of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.5,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8), where each X is an amino
acid;
[0133] each D and E is independently an amino acid;
[0134] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0135] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-, wherein L comprises at least one
double bond in the E configuration;
[0136] 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;
[0137] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0138] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0139] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0140] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0141] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0142] 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;
[0143] 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;
[0144] v is an integer from 1-1000;
[0145] w is an integer from 3-1000;
[0146] n is an integer from 1-5; and
[0147] Xaa.sub.7 is Boc-protected tryptophan.
[0148] In some embodiments, the peptidomimetic macrocycle of
Formula (I) comprises an .alpha.-helix.
INCORPORATION BY REFERENCE
[0149] 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
[0150] 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:
[0151] FIG. 1 shows a structure of peptidomimetic macrocycle 46
(Table 2b), a p53 peptidomimetic macrocycle, complexed with MDMX
(Primary SwissProt accession number Q7ZUW7; Entry MDM4_DANRE).
[0152] FIG. 2 shows overlaid structures of p53 peptidomimetic
macrocycles 142 (Table 2b) and SP43 bound to MDMX (Primary
SwissProt accession number Q7ZUW7; Entry MDM4_DANRE).
[0153] FIG. 3 shows the effect of SP154, a peptidomimetic
macrocycle, on tumor growth in a mouse MCF-7 xenograft model.
[0154] FIG. 4 shows the effect of SP249, a peptidomimetic
macrocycle, on tumor growth in a mouse MCF-7 xenograft model.
[0155] FIG. 5 shows the effect of SP315, a peptidomimetic
macrocycle, on tumor growth in a mouse MCF-7 xenograft model.
[0156] FIG. 6 shows the effect of SP252, a point mutation of SP154,
on tumor growth in a mouse MCF-7 xenograft model.
[0157] FIG. 7 shows a plot of solubility for peptidomimetic
macrocycles with varying C-terminal extensions.
DETAILED DESCRIPTION OF THE INVENTION
[0158] 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.
[0159] 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 a carbon of the first amino acid residue (or analog) to the a
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.
[0160] As used herein, the term "stability" refers to the
maintenance of a defined secondary structure in solution by a
peptidomimetic macrocycle 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 herein are .alpha.-helices, 3.sub.10
helices, .beta.-turns, and .beta.-pleated sheets.
[0161] As used herein, the term "helical stability" refers to the
maintenance of a helical structure by a peptidomimetic macrocycle
as measured by circular dichroism or NMR. For example, in some
embodiments, a peptidomimetic macrocycle exhibits at least a 1.25,
1.5, 1.75 or 2-fold increase in .alpha.-helicity as determined by
circular dichroism compared to a corresponding uncrosslinked
macrocycle.
[0162] The term "amino acid" refers to a molecule containing both
an amino group and a carboxyl group. 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. The term amino acid, as used herein, includes, without
limitation, .alpha.-amino acids, natural amino acids, non-natural
amino acids, and amino acid analogs.
[0163] The term ".alpha.-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.
[0164] The term ".beta.-amino acid" refers to a molecule containing
both an amino group and a carboxyl group in a 13 configuration.
[0165] 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.
[0166] The following table shows a summary of the properties of
natural amino acids:
TABLE-US-00001 3- 1- Side-chain Amino Letter Letter Side-chain
charge Hydropathy Acid Code Code Polarity (pH 7.4) Index Alanine
Ala A nonpolar neutral 1.8 Arginine Arg R polar positive -4.5
Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar
negative -3.5 Cysteine Cys C polar neutral 2.5 Glutamic acid Glu E
polar negative -3.5 Glutamine Gln Q polar neutral -3.5 Glycine Gly
G nonpolar neutral -0.4 Histidine His H polar positive(10%) -3.2
neutral(90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L
nonpolar neutral 3.8 Lysine Lys K polar positive -3.9 Methionine
Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8
Proline Pro P nonpolar neutral -1.6 Serine Ser S polar neutral -0.8
Threonine Thr T polar neutral -0.7 Tryptophan Trp W nonpolar
neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Val V
nonpolar neutral 4.2
[0167] "Hydrophobic amino acids" include small hydrophobic amino
acids and large hydrophobic amino acids. "Small hydrophobic amino
acid" are glycine, alanine, proline, and analogs thereof "Large
hydrophobic amino acids" are valine, leucine, isoleucine,
phenylalanine, methionine, tryptophan, and analogs thereof "Polar
amino acids" are serine, threonine, asparagine, glutamine,
cysteine, tyrosine, and analogs thereof "Charged amino acids" are
lysine, arginine, histidine, aspartate, glutamate, and analogs
thereof.
[0168] The term "amino acid analog" 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, .beta.-amino acids
and amino acids where the amino or carboxy group is substituted by
a similarly reactive group (e.g., substitution of the primary amine
with a secondary or tertiary amine, or substitution of the carboxy
group with an ester).
[0169] The term "non-natural amino acid" refers to an amino acid
which is not 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. Non-natural amino acids or amino acid analogs include,
without limitation, structures according to the following:
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015##
[0170] Amino acid analogs include .beta.-amino acid analogs.
Examples of .beta.-amino acid analogs include, but are not limited
to, the following: cyclic .beta.-amino acid analogs;
.beta.-alanine; (R)-.beta.-phenylalanine;
(R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;
(R)-3-amino-4-(1-naphthyl)-butyric acid;
(R)-3-amino-4-(2,4-dichlorophenyl)butyric acid;
(R)-3-amino-4-(2-chlorophenyl)-butyric acid;
(R)-3-amino-4-(2-cyanophenyl)-butyric acid;
(R)-3-amino-4-(2-fluorophenyl)-butyric acid;
(R)-3-amino-4-(2-furyl)-butyric acid;
(R)-3-amino-4-(2-methylphenyl)-butyric acid;
(R)-3-amino-4-(2-naphthyl)-butyric acid;
(R)-3-amino-4-(2-thienyl)-butyric acid;
(R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-(3,4-dichlorophenyl)butyric acid;
(R)-3-amino-4-(3,4-difluorophenyl)butyric acid;
(R)-3-amino-4-(3-benzothienyl)-butyric acid;
(R)-3-amino-4-(3-chlorophenyl)-butyric acid;
(R)-3-amino-4-.beta.-cyanophenyl)-butyric acid;
(R)-3-amino-4-(3-fluorophenyl)-butyric acid;
(R)-3-amino-4-.beta.-methylphenyl)-butyric acid;
(R)-3-amino-4-(3-pyridyl)-butyric acid;
(R)-3-amino-4-(3-thienyl)-butyric acid;
(R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-(4-bromophenyl)-butyric acid;
(R)-3-amino-4-(4-chlorophenyl)-butyric acid;
(R)-3-amino-4-(4-cyanophenyl)-butyric acid;
(R)-3-amino-4-(4-fluorophenyl)-butyric acid;
(R)-3-amino-4-(4-iodophenyl)-butyric acid;
(R)-3-amino-4-(4-methylphenyl)-butyric acid;
(R)-3-amino-4-(4-nitrophenyl)-butyric acid;
(R)-3-amino-4-(4-pyridyl)-butyric acid;
(R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-pentafluoro-phenylbutyric acid;
(R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid;
(R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic
acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;
(S)-3-amino-4-(1-naphthyl)-butyric acid;
(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;
(S)-3-amino-4-(2-chlorophenyl)-butyric acid;
(S)-3-amino-4-(2-cyanophenyl)-butyric acid;
(S)-3-amino-4-(2-fluorophenyl)-butyric acid;
(S)-3-amino-4-(2-furyl)-butyric acid;
(S)-3-amino-4-(2-methylphenyl)-butyric acid;
(S)-3-amino-4-(2-naphthyl)-butyric acid;
(S)-3-amino-4-(2-thienyl)-butyric acid;
(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-(3,4-dichlorophenyl)butyric acid;
(S)-3-amino-4-(3,4-difluorophenyl)butyric acid;
(S)-3-amino-4.beta.-(3)-benzothienyl)-butyric acid;
(S)-3-amino-4-(3-chlorophenyl)-butyric acid;
(S)-3-amino-4-(3-cyanophenyl)-butyric acid;
(S)-3-amino-4-(3-fluorophenyl)-butyric acid;
(S)-3-amino-4-(3-methylphenyl)-butyric acid;
(S)-3-amino-4-(3-pyridyl)-butyric acid;
(S)-3-amino-4-(3-thienyl)-butyric acid;
(S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-(4-bromophenyl)-butyric acid;
(S)-3-amino-4-(4-chlorophenyl)-butyric acid;
(S)-3-amino-4-(4-cyanophenyl)-butyric acid;
(S)-3-amino-4-(4-fluorophenyl)-butyric acid;
(S)-3-amino-4-(4-iodophenyl)-butyric acid;
(S)-3-amino-4-(4-methylphenyl)-butyric acid;
(S)-3-amino-4-(4-nitrophenyl)-butyric acid;
(S)-3-amino-4-(4-pyridyl)-butyric acid;
(S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-pentafluoro-phenylbutyric acid;
(S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid;
(S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic
acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid;
1,2,5,6-tetrahydropyridine-4-carboxylic acid;
3-amino-3-(2-chlorophenyl)-propionic acid;
3-amino-3-(2-thienyl)-propionic acid;
3-amino-3-.beta.-bromophenyl)-propionic acid;
3-amino-3-(4-chlorophenyl)-propionic acid;
3-amino-3-(4-methoxyphenyl)-propionic acid;
3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid;
D-.beta.-phenylalanine; .beta.-leucine; L-.beta.-homoalanine;
L-.beta.-homoaspartic acid .gamma.-benzyl ester;
L-.beta.-homoglutamic acid .delta.-benzyl ester;
L-.beta.-homoisoleucine; L-.beta.-homoleucine;
L-.beta.-homomethionine; L-.beta.-homophenylalanine;
L-.beta.-homoproline; L-.beta.-homotryptophan; L-.beta.-homovaline;
L-N.omega.-benzyloxycarbonyl-.beta.-homolysine;
N.omega.-L-.beta.-homoarginine;
O-benzyl-L-.sym.-homohydroxyproline; O-benzyl-L-.beta.-homoserine;
O-benzyl-L-.beta.-homothreonine; O-benzyl-L-.beta.-homotyrosine;
.gamma.-trityl-L-.beta.-homoasparagine; (R)-.beta.-phenylalanine;
L-.beta.-homoaspartic acid .gamma.-t-butyl ester;
L-.beta.-homoglutamic acid .delta.-t-butyl ester;
L-N.omega.-.beta.-homolysine;
N.delta.-trityl-L-.beta.-homoglutamine;
N.omega.-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-.beta.-homo-
arginine; O-t-butyl-L-.beta.-homohydroxy-proline;
O-t-butyl-L-.beta.-homoserine; O-t-butyl-L-.beta.-homothreonine;
O-t-butyl-L-.beta.-homotyrosine; 2-aminocyclopentane carboxylic
acid; and 2-aminocyclohexane carboxylic acid.
[0171] Amino acid analogs include analogs of alanine, valine,
glycine or leucine. Examples of amino acid analogs of alanine,
valine, glycine, and leucine include, but are not limited to, the
following: .alpha.-methoxyglycine; .alpha.-allyl-L-alanine;
.alpha.-aminoisobutyric acid; .alpha.-methyl-leucine;
.beta.-(1-naphthyl)-D-alanine; .beta.-(1-naphthyl)-L-alanine;
.beta.-(2-naphthyl)-D-alanine; .beta.-(2-naphthyl)-L-alanine;
.beta.-(2-pyridyl)-D-alanine; .beta.-(2-pyridyl)-L-alanine;
.beta.-(2-thienyl)-D-alanine; .beta.-(2-thienyl)-L-alanine;
.beta..beta.-(3)-benzothienyl)-D-alanine;
.beta..beta.-(3)-benzothienyl)-L-alanine;
.beta.-.beta.-pyridyl)-D-alanine; .beta.-.beta.-pyridyl)-L-alanine;
.beta.-(4-pyridyl)-D-alanine; .beta.-(4-pyridyl)-L-alanine;
.beta.-chloro-L-alanine; .beta.-cyano-L-alanine;
.beta.-cyclohexyl-D-alanine; .beta.-cyclohexyl-L-alanine;
.beta.-cyclopentene-1-yl-alanine; .beta.-cyclopentyl-alanine;
.beta.-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt;
.beta.-t-butyl-D-alanine; .beta.-t-butyl-L-alanine;
.gamma.-aminobutyric acid; L-.alpha.,.beta.-diaminopropionic acid;
2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine;
2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine;
3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine;
4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium
salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine;
4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic
acid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt;
cyclopentyl-Gly-OH.dicyclohexylammonium salt;
D-.alpha.,.beta.-diaminopropionic acid; D-.alpha.-aminobutyric
acid; D-.alpha.-t-butylglycine; D-(2-thienyl)glycine;
D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine;
D-allylglycine-dicyclohexylammonium salt; D-cyclohexylglycine;
D-norvaline; D-phenylglycine; .beta.-aminobutyric acid;
.beta.-aminoisobutyric acid; (2-bromophenyl)glycine;
(2-methoxyphenyl)glycine; (2-methylphenyl)glycine;
(2-thiazoyl)glycine; (2-thienyl)glycine;
2-amino-3-(dimethylamino)-propionic acid;
L-.alpha.,.beta.-diaminopropionic acid; L-.alpha.-aminobutyric
acid; L-.alpha.-t-butylglycine; L-.beta.-thienyl)glycine;
L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid
dicyclohexyl-ammonium salt; L-2-indanylglycine;
L-allylglycine-dicyclohexyl ammonium salt; L-cyclohexylglycine;
L-phenylglycine; L-propargylglycine; L-norvaline;
N-.alpha.-aminomethyl-L-alanine; D-.alpha.,.gamma.-diaminobutyric
acid; L-.alpha.,.gamma.-diaminobutyric acid;
.beta.-cyclopropyl-L-alanine;
(N-.beta.-(2,4-dinitrophenyl))-L-.alpha.,.beta.-diaminopropionic
acid;
(N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.b-
eta.-diaminopropionic acid;
(N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.b-
eta.-diaminopropionic acid;
(N-.beta.-4-methyltrityl)-L-.alpha.,.beta.-diaminopropionic acid;
(N-.beta.-allyloxycarbonyl)-L-.alpha.,.beta.-diaminopropionic acid;
(N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.-
gamma.-diaminobutyric acid;
(N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.-
gamma.-diaminobutyric acid;
(N-.gamma.-4-methyltrityl)-D-.alpha.,.gamma.-diaminobutyric acid;
(N-.gamma.-4-methyltrityl)-L-.alpha.,.gamma.-diaminobutyric acid;
(N-.gamma.-allyloxycarbonyl)-L-.alpha.,.gamma.-diaminobutyric acid;
D-.alpha.,.gamma.-diaminobutyric acid; 4,5-dehydro-L-leucine;
cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine;
D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid;
L-allylglycine; L-homocyclohexylalanine; and
N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.
[0172] Amino acid analogs include analogs of arginine or lysine.
Examples of amino acid analogs of arginine and lysine include, but
are not limited to, the following: citrulline;
L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic
acid; L-citrulline; Lys(Me).sub.2-OH; Lys(N.sub.3)--OH;
N.delta.-benzyloxycarbonyl-L-ornithine; N.omega.-nitro-D-arginine;
N.omega.-nitro-L-arginine; .alpha.-methyl-ornithine;
2,6-diaminoheptanedioic acid; L-ornithine;
(N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine-
;
(N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithin-
e; (N.delta.-4-methyltrityl)-D-ornithine;
(N.delta.-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine;
Arg(Me)(Pbf)-OH; Arg(Me).sub.2-OH (asymmetrical); Arg(Me).sub.2-OH
(symmetrical); Lys(ivDde)-OH; Lys(Me)2-OH.HCl; Lys(Me3)-OH
chloride; N.omega.-nitro-D-arginine; and
N.omega.-nitro-L-arginine.
[0173] Amino acid analogs include analogs of aspartic or glutamic
acids. Examples of amino acid analogs of aspartic and glutamic
acids include, but are not limited to, the following:
.alpha.-methyl-D-aspartic acid; .alpha.-methyl-glutamic acid;
.alpha.-methyl-L-aspartic acid; .gamma.-methylene-glutamic acid;
(N-.gamma.-ethyl)-L-glutamine;
[N-.alpha.-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic
acid; L-.alpha.-aminosuberic acid; D-2-aminoadipic acid;
D-.alpha.-aminosuberic acid; .alpha.-aminopimelic acid;
iminodiacetic acid; L-2-aminoadipic acid;
threo-.beta.-methyl-aspartic acid; .gamma.-carboxy-D-glutamic acid
.gamma.,.gamma.-di-t-butyl ester; .gamma.-carboxy-L-glutamic acid
.gamma.,.gamma.-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and
pyroglutamic acid.
[0174] Amino acid analogs include analogs of cysteine and
methionine. Examples of amino acid analogs of cysteine and
methionine include, but are not limited to, Cys(farnesyl)-OH,
Cys(farnesyl)-OMe, .alpha.-methyl-methionine,
Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH,
2-amino-4-(ethylthio)butyric acid, buthionine,
buthioninesulfoximine, ethionine, methionine methylsulfonium
chloride, selenomethionine, cysteic acid,
[2-(4-pyridyl)ethyl]-DL-penicillamine,
[2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine,
4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine,
4-methylbenzyl-L-penicillamine, benzyl-D-cysteine,
benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine,
carboxyethyl-L-cysteine, carboxymethyl-L-cysteine,
diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine,
t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine,
cystathionine, homocystine, L-homocystine,
(2-aminoethyl)-L-cysteine, seleno-L-cysteine, cystathionine,
Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.
[0175] Amino acid analogs include analogs of phenylalanine and
tyrosine. Examples of amino acid analogs of phenylalanine and
tyrosine include .beta.-methyl-phenylalanine,
.beta.-hydroxyphenylalanine,
.alpha.-methyl-3-methoxy-DL-phenylalanine,
.alpha.-methyl-D-phenylalanine, .alpha.-methyl-L-phenylalanine,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,
2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine,
2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,
2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,
2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,
2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,
2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,
2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,
2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine,
3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,
3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,
3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,
3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,
3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine,
3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine,
3-(trifluoromethyl)-D-phenylalanine,
3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,
3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,
3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine,
3-chloro-L-tyrosine, 3-cyano-D-phenylalanine,
3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine,
3-fluoro-L-phenylalanine, 3-fluoro-tyrosine,
3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine,
3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine,
3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine,
3-nitro-L-phenylalanine, 3-nitro-L-tyrosine,
4-(trifluoromethyl)-D-phenylalanine,
4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,
4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,
4-benzoyl-L-phenylalanine,
4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine,
4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine,
4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine,
4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine,
4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine,
4-iodo-L-phenylalanine, homophenylalanine, thyroxine,
3,3-diphenylalanine, thyronine, ethyl-tyrosine, and
methyl-tyrosine.
[0176] Amino acid analogs include analogs of proline. Examples of
amino acid analogs of proline include, but are not limited to,
3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline,
thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.
[0177] Amino acid analogs include analogs of serine and threonine.
Examples of amino acid analogs of serine and threonine include, but
are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid,
2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic
acid, 2-amino-3-methoxybutanoic acid,
4-amino-3-hydroxy-6-methylheptanoic acid,
2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic
acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic
acid, and .alpha.-methylserine.
[0178] Amino acid analogs include analogs of tryptophan. Examples
of amino acid analogs of tryptophan include, but are not limited
to, the following: .alpha.-methyl-tryptophan;
.beta.-(3)-benzothienyl)-D-alanine;
.beta.-(3)-benzothienyl)-L-alanine; 1-methyl-tryptophan;
4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan;
5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan;
5-hydroxy-L-tryptophan; 5-methoxy-tryptophan;
5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan;
6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan;
6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan;
7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic
acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;
7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;
5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.
[0179] In some embodiments, amino acid analogs are racemic. In some
embodiments, the D isomer of the amino acid analog is used. In some
embodiments, the L isomer of the amino acid analog is used. In
other embodiments, the amino acid analog comprises chiral centers
that are in the R or S configuration. In still other embodiments,
the amino group(s) of a .beta.-amino acid analog is substituted
with a protecting group, e.g., tert-butyloxycarbonyl (BOC group),
9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. In yet
other embodiments, the carboxylic acid functional group of a
.beta.-amino acid analog is protected, e.g., as its ester
derivative. In some embodiments the salt of the amino acid analog
is used.
[0180] 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.
[0181] 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 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, or 6-Cl-tryptophan for
tryptophan).
[0182] The term "capping group" refers to the chemical moiety
occurring at either the carboxy or amino terminus of the
polypeptide chain of the subject peptidomimetic macrocycle. The
capping group of a carboxy terminus includes an unmodified
carboxylic acid (ie --COOH) or a carboxylic acid with a
substituent. For example, the carboxy terminus can be substituted
with an amino group to yield a carboxamide at the C-terminus.
Various substituents include but are not limited to primary and
secondary amines, including pegylated secondary amines.
Representative secondary amine capping groups for the C-terminus
include:
##STR00016## ##STR00017##
[0183] The capping group of an amino terminus includes an
unmodified amine (ie --NH.sub.2) or an amine with a substituent.
For example, the amino terminus can be substituted with an acyl
group to yield a carboxamide at the N-terminus. Various
substituents include but are not limited to substituted acyl
groups, including C.sub.1-C.sub.6 carbonyls, C.sub.7-C.sub.30
carbonyls, and pegylated carbamates. Representative capping groups
for the N-terminus include, but are not limited to, 4-FBzl
(4-fluoro-benzyl) and the following:
##STR00018##
[0184] 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.
[0185] The symbol "" when used as part of a molecular structure
refers to a single bond or a trans or cis double bond.
[0186] The term "amino acid side chain" refers to a moiety attached
to the .alpha.-carbon (or another backbone atom) 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.,.alpha.
di-substituted amino acid).
[0187] The term ".alpha.,.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.
[0188] 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).
[0189] The term "first C-terminal amino acid" refers to the amino
acid which is closest to the C-terminus. The term "second
C-terminal amino acid" refers to the amino acid attached at the
N-terminus of the first C-terminal amino acid.
[0190] The term "macrocyclization reagent" or "macrocycle-forming
reagent" as used herein refers to any reagent which can be used to
prepare a peptidomimetic macrocycle by mediating the reaction
between two reactive groups. Reactive groups can 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 can
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 can 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. In other examples,
catalysts have W or Mo centers. Various catalysts are disclosed in
Grubbs et al., "Ring Closing Metathesis and Related Processes in
Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452, U.S. Pat.
Nos. 5,811,515; 7,932,397; U.S. Application No. 2011/0065915; U.S.
Application No. 2011/0245477; Yu et al., "Synthesis of Macrocyclic
Natural Products by Catalyst-Controlled Stereoselective
Ring-Closing Metathesis," Nature 2011, 479, 88; and Peryshkov et
al., "Z-Selective Olefin Metathesis Reactions Promoted by Tungsten
Oxo Alkylidene Complexes," J. Am. Chem. Soc. 2011, 133, 20754. 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.
[0191] The term "halo" or "halogen" refers to fluorine, chlorine,
bromine or iodine or a radical thereof.
[0192] 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.
[0193] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0194] 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.
[0195] 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.
[0196] 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 "arylalkoxy" refers
to an alkoxy substituted with aryl.
[0197] "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.
[0198] "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)NH.sub.2-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,
[0199] "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.
[0200] "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--CH.sub.3, and
--CH.sub.2--CH.sub.2--NH--C(O)--CH.dbd.CH.sub.2.
[0201] "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.2CH.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.
[0202] "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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] In some embodiments, the compounds disclosed herein 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 unless expressly provided otherwise. In some
embodiments, the compounds disclosed herein are also represented in
multiple tautomeric forms, in such instances, the compounds include
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 unless expressly
provided otherwise. All crystal forms of the compounds described
herein are included unless expressly provided otherwise.
[0210] 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%.
[0211] As used herein, the recitation of a numerical range for a
variable is intended to convey that the variable is 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.
[0212] 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."
[0213] The term "on average" represents the mean value derived from
performing at least three independent replicates for each data
point.
[0214] The term "biological activity" encompasses structural and
functional properties of a macrocycle. 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.
[0215] The term "binding affinity" refers to the strength of a
binding interaction, for example between a peptidomimetic
macrocycle and a target. Binding affinity can be expressed, for
example, as an equilibrium dissociation constant ("K.sub.D"), which
is expressed in units which are a measure of concentration (e.g. M,
mM, .mu.M, nM etc). Numerically, binding affinity and K.sub.D
values vary inversely, such that a lower binding affinity
corresponds to a higher K.sub.D value, while a higher binding
affinity corresponds to a lower K.sub.D value. Where high binding
affinity is desirable, "improved" binding affinity refers to higher
binding affinity and therefore lower K.sub.D values.
[0216] The term "in vitro efficacy" refers to the extent to which a
test compound, such as a peptidomimetic macrocycle, produces a
beneficial result in an in vitro test system or assay. In vitro
efficacy can be measured, for example, as an "IC.sub.50" or
"EC.sub.50" value, which represents the concentration of the test
compound which produces 50% of the maximal effect in the test
system.
[0217] The term "ratio of in vitro efficacies" or "in vitro
efficacy ratio" refers to the ratio of IC.sub.50 or EC.sub.50
values from a first assay (the numerator) versus a second assay
(the denominator). Consequently, an improved in vitro efficacy
ratio for Assay 1 versus Assay 2 refers to a lower value for the
ratio expressed as IC.sub.50(Assay 1)/IC.sub.50(Assay 2) or
alternatively as EC.sub.50(Assay 1)/EC.sub.50(Assay 2). This
concept can also be characterized as "improved selectivity" in
Assay 1 versus Assay 2, which can be due either to a decrease in
the IC.sub.50 or EC.sub.50 value for Target 1 or an increase in the
value for the IC.sub.50 or EC.sub.50 value for Target 2.
[0218] 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.
[0219] Peptidomimetic Macrocycles
[0220] In some embodiments, a peptidomimetic macrocycle has the
Formula (I):
##STR00019##
[0221] wherein:
[0222] each A, C, D, and E is independently an amino acid
(including natural or non-natural amino acids, and amino acid
analogs) and the terminal D and E independently optionally include
a capping group;
[0223] B is an amino acid (including natural or non-natural amino
acids, and amino acid analogs),
##STR00020##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0224] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0225] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0226] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0227] 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;
[0228] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0229] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0230] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0231] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0232] 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;
[0233] 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;
[0234] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10;
[0235] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0236] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0237] n is an integer from 1-5.
[0238] In some embodiments, v and w are integers between 1-30. In
some embodiments, w is an integer from 3-1000, for example 3-500,
3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, the
sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3.
In other embodiments, the sum of x+y+z is 6.
[0239] In some embodiments, peptidomimetic macrocycles are also
provided of the formula:
##STR00021##
[0240] wherein:
[0241] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8), where each X is an amino
acid;
[0242] each D and E is independently an amino acid;
[0243] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0244] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-;
[0245] 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;
[0246] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0247] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0248] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0249] 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;
[0250] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0251] 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;
[0252] 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;
[0253] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20 or 1-10;
[0254] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0255] n is an integer from 1-5.
[0256] In some embodiments, v and w are integers between 1-30. In
some embodiments, w is an integer from 3-1000, for example 3-500,
3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, the
sum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3.
In other embodiments, the sum of x+y+z is 6.
[0257] In some embodiments of any of the Formulas described herein,
at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the
amino acid at the corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-Xu-Sera (SEQ ID NO: 8). In other embodiments, at least four
of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at
least five of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the
amino acid at the corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at
least six of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at
least seven of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the
amino acid at the corresponding position of the sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 8).
[0258] In some embodiments, a peptidomimetic macrocycle has the
Formula:
##STR00022##
[0259] wherein:
[0260] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Alain (SEQ ID NO: 9), where each X is an
amino acid;
[0261] each D is independently an amino acid;
[0262] each E is independently an amino acid, for example an amino
acid selected from Ala (alanine), D-Ala (D-alanine), Aib
(.alpha.-aminoisobutyric acid), Sar (N-methyl glycine), and Ser
(serine);
[0263] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0264] each L or L' is independently a macrocycle-forming linker of
the formula -L.sub.1-L.sub.2-;
[0265] 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;
[0266] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0267] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0268] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0269] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0270] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0271] 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;
[0272] 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;
[0273] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0274] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0275] n is an integer from 1-5.
[0276] In some embodiments of the above Formula, at least three of
Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9,
and Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least four of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least five of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least six of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Alas-Gln.sub.9-Leu.sub.10-
/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least seven of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9).
[0277] In some embodiments, w is an integer from 3-10, for example
3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other
embodiments, w is 6. In some embodiments, v is an integer from
1-10, for example 2-5. In some embodiments, v is 2.
[0278] In an embodiment of any of the Formulas described herein,
L.sub.1 and L.sub.2, either alone or in combination, do not form a
triazole or a thioether.
[0279] 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.
[0280] In some embodiments, x+y+z is at least 3. In other
embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some
embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the
sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.
Each occurrence of A, B, C, D or E in a macrocycle or macrocycle
precursor is independently selected. For example, a sequence
represented by the formula [A].sub.x, when x is 3, encompasses
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 can encompass peptidomimetic macrocycles which are
the same or different. For example, a compound can comprise
peptidomimetic macrocycles comprising different linker lengths or
chemical compositions.
[0281] In some embodiments, the peptidomimetic macrocycle 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
##STR00023##
[0282] 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..
[0283] In one embodiment, the peptidomimetic macrocycle of Formula
(I) is:
##STR00024## [0284] wherein each R.sub.1 and R.sub.2 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-.
[0285] In related embodiments, the peptidomimetic macrocycle of
Formula (I) is:
##STR00025##
[0286] wherein each R.sub.1' and R.sub.2' is independently an amino
acid.
[0287] In other embodiments, the peptidomimetic macrocycle of
Formula (I) is a compound of any of the formulas shown below:
##STR00026## ##STR00027##
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.
[0288] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00028##
[0289] In other embodiments, D and/or E in the compound of Formula
I 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.
[0290] In other embodiments, at least one of [D] and [E] in the
compound of Formula I 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. In an embodiment, u is 2.
[0291] In some embodiments, any of the macrocycle-forming linkers
described herein can be used in any combination with any of the
sequences shown in Table 1, Table 1a, Table 1b, or Table 1c and
also with any of the R-- substituents indicated herein.
[0292] 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 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.
[0293] In other embodiments, provided are peptidomimetic
macrocycles of Formula (IV) or (IVa):
##STR00029##
[0294] wherein:
[0295] each A, C, D, and E is independently a natural or
non-natural amino acid, and the terminal D and E independently
optionally include a capping group;
[0296] B is a natural or non-natural amino acid, amino acid
analog,
##STR00030##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0297] 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 at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0298] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0299] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0300] 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;
[0301] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0302] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0303] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0304] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0305] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0306] v and w are independently integers from 1-1000;
[0307] u is an integer from 1-10;
[0308] x, y and z are independently integers from 0-10; and
[0309] n is an integer from 1-5.
[0310] In one example, L.sub.1 and L.sub.2, either alone or in
combination, do not form a triazole or a thioether.
[0311] 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.
[0312] In some embodiments, x+y+z is at least 1. In other
embodiments, x+y+z is at least 2. In other embodiments, x+y+z is 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in
a macrocycle or macrocycle precursor is independently selected. For
example, a sequence represented by the formula [A].sub.x, when x is
3, encompasses 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.
[0313] In some embodiments, the peptidomimetic macrocycle 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
##STR00031##
[0314] 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..
[0315] Exemplary embodiments of the macrocycle-forming linker
-L.sub.1-L.sub.2- are shown below.
##STR00032##
[0316] Unless otherwise stated, any compounds (including
peptidomimetic macrocycles, macrocycle precursors, and other
compositions) are also meant to encompass compounds which differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the described structures except for
the replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by .sup.13C- or .sup.14C-enriched carbon
are within the scope of this invention.
[0317] In some embodiments, the compounds disclosed herein can
contain unnatural proportions of atomic isotopes at one or more of
atoms that constitute such compounds. For example, the compounds
can be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
In other embodiments, one or more carbon atoms is replaced with a
silicon atom. All isotopic variations of the compounds disclosed
herein, whether radioactive or not, are contemplated herein.
[0318] Preparation of Peptidomimetic Macrocycles
[0319] Peptidomimetic macrocycles can be prepared by any of a
variety of methods known in the art. For example, any of the
residues indicated by "$" or "$r8" in Table 1, Table 1a, Table 1b,
or Table 1c can 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.
[0320] 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 & Verdine, J. Am. Chem. Soc. 122:5891 (2005);
Walensky et al., Science 305:1466-1470 (2004); U.S. Pat. No.
7,192,713 and PCT application WO 2008/121767. The
.alpha.,.alpha.-disubstituted amino acids and amino acid precursors
disclosed in the cited references can 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. In various embodiments, the following amino acids can
be employed in the synthesis of the peptidomimetic macrocycle:
##STR00033##
[0321] In other embodiments, the peptidomimetic macrocycles 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.
[0322] Additional methods of forming peptidomimetic macrocycles
which are envisioned as suitable 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. Nos. 5,364,851; 5,446,128; 5,824,483;
6,713,280; and 7,202,332. In such embodiments, amino acid
precursors are used containing an additional substituent R-- at the
alpha position. Such amino acids are incorporated into the
macrocycle precursor at the desired positions, which can 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.
[0323] Assays
[0324] The properties of peptidomimetic macrocycles are assayed,
for example, by using the methods described below. In some
embodiments, a peptidomimetic macrocycle has improved biological
properties relative to a corresponding polypeptide lacking the
substituents described herein.
[0325] Assay to Determine .alpha.-Helicity
[0326] 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, 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 will possess an alpha-helicity of greater
than 50%. To assay the helicity of peptidomimetic macrocycles, 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)).
[0327] Assay to Determine Melting Temperature (Tm).
[0328] A peptidomimetic macrocycle comprising a secondary structure
such as an .alpha.-helix exhibits, for example, a higher melting
temperature than a corresponding uncrosslinked polypeptide.
Typically peptidomimetic macrocycles exhibit Tm of >60.degree.
C. representing a highly stable structure in aqueous solutions. To
assay the effect of macrocycle formation on melting 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).
[0329] Protease Resistance Assay.
[0330] 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 can
shield it from proteolytic cleavage. The peptidomimetic macrocycles
can 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).
[0331] Ex Vivo Stability Assay.
[0332] 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 can 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 can 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 5004 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 1004 of 50:50 acetonitrile:water and
submitted to LC-MS/MS analysis.
[0333] In vitro Binding Assays.
[0334] To assess the binding and affinity of peptidomimetic
macrocycles and peptidomimetic precursors to acceptor proteins, a
fluorescence polarization assay (FPA) 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).
[0335] 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 can be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic
macrocycle shows, In some embodiments, similar or lower Kd than a
corresponding uncrosslinked polypeptide.
[0336] In Vitro Displacement Assays to Characterize Antagonists of
Peptide-Protein Interactions.
[0337] 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.
[0338] 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 can be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.).
[0339] Any class of molecule, such as small organic molecules,
peptides, oligonucleotides or proteins can be examined as putative
antagonists in this assay.
[0340] Assay for Protein-Ligand Binding by Affinity Selection-Mass
Spectrometry
[0341] 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 hMDM2. 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 hMDM2 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.
[0342] 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.
[0343] Assay for Protein-Ligand Kd Titration Experiments.
[0344] 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 hMDM2 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.
[0345] Assay for Competitive Binding Experiments by Affinity
Selection-Mass Spectrometry
[0346] 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 hMDM2 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.
[0347] Binding Assays in Intact Cells.
[0348] 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.
[0349] Cellular Penetrability Assays.
[0350] 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
fluorescently-labeled (e.g. 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' KineticScan.RTM. HCS
Reader.
[0351] Cellular Efficacy Assays.
[0352] 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 EC.sub.50<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.
[0353] In Vivo Stability Assay.
[0354] 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.
[0355] In Vivo Efficacy in Animal Models.
[0356] To determine the anti-oncogenic activity of peptidomimetic
macrocycles in vivo, the compounds are, for example, given alone
(IP, W, 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.
[0357] Clinical Trials.
[0358] To determine the suitability of the peptidomimetic
macrocycles for treatment of humans, clinical trials are performed.
For example, patients diagnosed with cancer and in need of
treatment can be selected and separated in treatment and one or
more control groups, wherein the treatment group is administered a
peptidomimetic macrocycle, while the control groups receive a
placebo or a known anti-cancer drug. The treatment safety and
efficacy of the peptidomimetic macrocycles 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 macrocycle can show
improved long-term survival compared to a patient control group
treated with a placebo.
[0359] Pharmaceutical Compositions and Routes of Administration
[0360] Pharmaceutical compositions disclosed herein include
peptidomimetic macrocycles and 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 disclosed
herein which, upon administration to a recipient, is capable of
providing (directly or indirectly) a compound disclosed herein.
Particularly favored pharmaceutically acceptable derivatives are
those that increase the bioavailability of the compounds 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.
[0361] In some embodiments, peptidomimetic macrocycles 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.
[0362] Pharmaceutically acceptable salts of the compounds disclosed
herein 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.
[0363] For preparing pharmaceutical compositions from the compounds
disclosed herein, 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.
[0364] 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.
[0365] 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.
[0366] 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.
[0367] The pharmaceutical preparation can be 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.
[0368] When one or more compositions disclosed herein 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 one or more
compounds disclosed herein. Alternatively, those agents are part of
a single dosage form, mixed together with the compounds disclosed
herein in a single composition.
[0369] Methods of Use
[0370] In one aspect, provided herein are 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 p53/MDMX system, labeled peptidomimetic
macrocycles based on p53 can be used in a MDMX binding assay along
with small molecules that competitively bind to MDMX. Competitive
binding studies allow for rapid in vitro evaluation and
determination of drug candidates specific for the p53/MDMX system.
Such binding studies can be performed with any of the
peptidomimetic macrocycles disclosed herein and their binding
partners.
[0371] Further provided are methods 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 p53,
to which the peptidomimetic macrocycles are related. Such
antibodies, for example, disrupt the native protein-protein
interaction, for example, binding between p53 and MDMX.
[0372] In other aspects, provided herein are 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 p53, MDM2 or MDMX.
[0373] In another embodiment, a disorder is caused, at least in
part, by an abnormal level of p53 or MDM2 or MDMX, (e.g., over or
under expression), or by the presence of p53 or MDM2 or MDMX
exhibiting abnormal activity. As such, the reduction in the level
and/or activity of p53 or MDM2 or MDMX, or the enhancement of the
level and/or activity of p53 or MDM2 or MDMX, by peptidomimetic
macrocycles derived from p53, is used, for example, to ameliorate
or reduce the adverse symptoms of the disorder.
[0374] In another aspect, provided herein are 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 p53 and MDM2
or p53 and MDMX. These methods comprise administering an effective
amount of a compound to a warm blooded animal, including a human.
In some embodiments, the administration of one or more compounds
disclosed herein induces cell growth arrest or apoptosis.
[0375] 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.
[0376] In some embodiments, the peptidomimetic macrocycles can be
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 can be categorized
as pathologic, i.e., characterizing or constituting a disease
state, or can 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 peptidomimetic
macrocycles are novel therapeutic agents for controlling breast
cancer, ovarian cancer, colon cancer, lung cancer, metastasis of
such cancers and the like.
[0377] 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.
[0378] In some embodiments, the cancer is head and neck cancer,
melanoma, lung cancer, breast cancer, or glioma.
[0379] 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. The diseases can 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
(CML) (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.
[0380] 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.
[0381] Examples of cellular proliferative and/or differentiative
disorders of the skin include, but are not limited to proliferative
skin disease such as melanomas, including mucosal melanoma,
superficial spreading melanoma, nodular melanoma, lentigo (e.g.
lentigo maligna, lentigo maligna melanoma, or acral lentiginous
melanoma), amelanotic melanoma, desmoplastic melanoma, melanoma
with features of a Spitz nevus, melanoma with small nevus-like
cells, polypoid melanoma, and soft-tissue melanoma; basal cell
carcinomas including micronodular basal cell carcinoma, superficial
basal cell carcinoma, nodular basal cell carcinoma (rodent ulcer),
cystic basal cell carcinoma, cicatricial basal cell carcinoma,
pigmented basal cell carcinoma, aberrant basal cell carcinoma,
infiltrative basal cell carcinoma, nevoid basal cell carcinoma
syndrome, polypoid basal cell carcinoma, pore-like basal cell
carcinoma, and fibroepithelioma of Pinkus; squamus cell carcinomas
including acanthoma (large cell acanthoma), adenoid squamous cell
carcinoma, basaloid squamous cell carcinoma, clear cell squamous
cell carcinoma, signet-ring cell squamous cell carcinoma, spindle
cell squamous cell carcinoma, Marjolin's ulcer, erythroplasia of
Queyrat, and Bowen's disease; or other skin or subcutaneous
tumors.
[0382] 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.
[0383] 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.
[0384] 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.
[0385] 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.
[0386] 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 described herein can be employed in practicing the
invention. It is intended that the following claims define the
scope and that methods and structures within the scope of these
claims and their equivalents be covered thereby.
EXAMPLES
Example 1: Synthesis of 6-Chlorotryptophan Fmoc Amino Acids
##STR00034##
[0388] Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1. To
a stirred solution of dry DMF (12 mL) was added dropwise POCl.sub.3
(3.92 mL, 43 mmol, 1.3 equiv) at 0.degree. C. under Argon. The
solution was stirred at the same temperature for 20 min before a
solution of 6-chloroindole (5.0 g, 33 mmol, 1 eq.) in dry DMF (30
mL) was added dropwise. The resulting mixture was allowed to warm
to room temperature and stirred for an additional 2.5 h. Water (50
mL) was added and the solution was neutralized with 4M aqueous NaOH
(pH.about.8). The resulting solid was filtered off, washed with
water and dried under vacuum. This material was directly used in
the next step without additional purification. To a stirred
solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150
mL) was added successively Boc.sub.2O (7.91 g, 36.3 mmol, 1.1
equiv) and DMAP (0.4 g, 3.3 mmol, 0.1 equiv) at room temperature
under N.sub.2. The resulting mixture was stirred at room
temperature for 1.5 h and the solvent was evaporated under reduced
pressure. The residue was taken up in EtOAc and washed with IN HCl,
dried and concentrated to give the formyl indole 1 (9 g, 98% over 2
steps) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta.: 1.70 (s,
Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).
[0389] Tert-butyl
6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To a
solution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL)
was added NaBH.sub.4 (2.4 g, 63 mmol, 2 eq.). The reaction was
stirred for 3 h at room temperature. The reaction mixture was
concentrated and the residue was poured into diethyl ether and
water. The organic layer was separated, dried over magnesium
sulfate and concentrated to give a white solid (8.7 g, 98%). This
material was directly used in the next step without additional
purification. .sup.1H NMR (CDCl.sub.3) .delta.: 1.65 (s, Boc, 9H);
4.80 (s, 2H, CH.sub.2); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs,
1H).
[0390] Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate,
3. To a solution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in
dichloromethane (50 mL) under argon was added a solution of
triphenylphosphine (4.59 g, 17.5 mmol, 1.2 eq.) in dichloromethane
(50 mL) at -40.degree. C. The reaction solution was stirred an
additional 30 min at 40.degree. C. Then NBS (3.38 g, 19 mmol, 1.3
eq.) was added. The resulting mixture was allowed to warm to room
temperature and stirred overnight. Dichloromethane was evaporated,
Carbon Tetrachloride (100 mL) was added and the mixture was stirred
for 1 h and filtrated. The filtrate was concentrated, loaded in a
silica plug and quickly eluted with 25% EtOAc in Hexanes. The
solution was concentrated to give a white foam (3.84 g, 77%).
.sup.1H NMR (CDCl.sub.3) .delta.: 1.66 (s, Boc, 9H); 4.63 (s, 2H,
CH.sub.2); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs, 1H); 8.18 (bs,
1H).
[0391] .alpha.Me-6Cl-Trp(Boc)-Ni--S-BPB, 4. To S-Ala-Ni--S-BPB
(2.66 g, 5.2 mmol, 1 eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.)
was added 50 mL of DMF under argon. The bromide derivative compound
3 (2.68 g, 7.8 mmol, 1.5 eq.) in solution of DMF (5.0 mL) was added
via syringe. The reaction mixture was stirred at ambient
temperature for 1 h. The solution was then quenched with 5% aqueous
acetic acid and diluted with water. The desired product was
extracted in dichloromethane, dried and concentrated. The oily
product 4 was purified by flash chromatography (solid loading) on
normal phase using EtOAc and Hexanes as eluents to give a red solid
(1.78 g, 45% yield). .alpha.Me-6Cl-Trp(Boc)-Ni--S-BPB, 4: M+H calc.
775.21, M+H obs. 775.26; .sup.1H NMR (CDCl.sub.3) .delta.: 1.23 (s,
3H, .alpha.Me); 1.56 (m, 11H, Boc+CH.sub.2); 1.82-2.20 (m, 4H,
2CH.sub.2); 3.03 (m, 1H, CL); 3.24 (m, 2H, CH.sub.2); 3.57 and 4.29
(AB system, 2H, CH.sub.2 (benzyl), J=12.8 Hz); 6.62 (d, 2H); 6.98
(d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m,
2H); 7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).
[0392] Fmoc-.alpha.Me-6Cl-Trp(Boc)-OH, 6. To a solution of 3N
HCl/MeOH (1/3, 15 mL) at 50.degree. C. was added a solution of
compound 4 (1.75 g, 2.3 mmol, 1 eq.) in MeOH (5 ml) dropwise. The
starting material disappeared within 3-4 h. The acidic solution was
then cooled to 0.degree. C. with an ice bath and quenched with an
aqueous solution of Na.sub.2CO.sub.3 (1.21 g, 11.5 mmol, 5 eq.).
Methanol was removed and 8 more equivalents of Na.sub.2CO.sub.3
(1.95 g, 18.4 mmol) were added to the suspension. The Nickel
scavenging EDTA disodium salt dihydrate (1.68 g, 4.5 mmol, 2 eq.)
was then added and the suspension was stirred for 2 h. A solution
of Fmoc-OSu (0.84 g, 2.5 mmol, 1.1 eq.) in acetone (50 mL) was
added and the reaction was stirred overnight. Afterwards, the
reaction was diluted with diethyl ether and 1N HCl. The organic
layer was then dried over magnesium sulfate and concentrated in
vacuo. The desired product 6 was purified on normal phase using
acetone and dichloromethane as eluents to give a white foam (0.9 g,
70% yield). Fmoc-.alpha.Me-6Cl-Trp(Boc)-0H, 6: M+H calc. 575.19,
M+H obs. 575.37; .sup.1H NMR (CDCl.sub.3) .delta.: 1.59 (s, 9H,
Boc); 1.68 (s, 3H, Me); 3.48 (bs, 2H, CH.sub.2); 4.22 (m, 1H, CH);
4.39 (bs, 2H, CH.sub.2); 5.47 (s, 1H, NH); 7.10 (m, 1H); 7.18 (m,
2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H); 7.75 (d, 2H); 8.12
(bs, 1H).
[0393] 6Cl-Trp(Boc)-Ni--S-BPB, 5. To Gly-Ni--S-BPB (4.6 g, 9.2
mmol, 1 eq.) and KO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95
mL of DMF under argon. The bromide derivative compound 3 (3.5 g,
4.6 mmol, 1.1 eq.) in solution of DMF (10 mL) was added via
syringe. The reaction mixture was stirred at ambient temperature
for 1 h. The solution was then quenched with 5% aqueous acetic acid
and diluted with water. The desired product was extracted in
dichloromethane, dried and concentrated. The oily product 5 was
purified by flash chromatography (solid loading) on normal phase
using EtOAc and Hexanes as eluents to give a red solid (5 g, 71%
yield). 6Cl-Trp(Boc)-Ni--S-BPB, 5: M+H calc. 761.20, M+H obs.
761.34; .sup.1H NMR (CDCl.sub.3) .delta.: 1.58 (m, 11H,
Boc+CH.sub.2); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m, 2H, CH.sub.2);
3.00 (m, 1H, CH.sub..alpha.); 3.22 (m, 2H, CH.sub.2); 3.45 and 4.25
(AB system, 2H, CH.sub.2 (benzyl), J=12.8 Hz); 4.27 (m, 1H,
CH.sub..alpha.); 6.65 (d, 2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m,
2H); 7.28 (m, 3H); 7.35-7.39 (m, 2H); 7.52 (m, 2H); 7.96 (d, 2H);
8.28 (m, 2H).
[0394] Fmoc-6Cl-Trp(Boc)-OH, 7. To a solution of 3N HCl/MeOH (1/3,
44 mL) at 50.degree. C. was added a solution of compound 5 (5 g,
6.6 mmol, 1 eq.) in MeOH (10 ml) dropwise. The starting material
disappeared within 3-4 h. The acidic solution was then cooled to
0.degree. C. with an ice bath and quenched with an aqueous solution
of Na.sub.2CO.sub.3 (3.48 g, 33 mmol, 5 eq.). Methanol was removed
and 8 more equivalents of Na.sub.2CO.sub.3 (5.57 g, 52 mmol) were
added to the suspension. The Nickel scavenging EDTA disodium salt
dihydrate (4.89 g, 13.1 mmol, 2 eq.) and the suspension was stirred
for 2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in
acetone (100 mL) was added and the reaction was stirred overnight.
Afterwards, the reaction was diluted with diethyl ether and 1N HCl.
The organic layer was then dried over magnesium sulfate and
concentrated in vacuo. The desired product 7 was purified on normal
phase using acetone and dichloromethane as eluents to give a white
foam (2.6 g, 69% yield). Fmoc-6C1-Trp(Boc)-OH, 7: M+H calc. 561.17,
M+H obs. 561.37; .sup.1H NMR (CDCl.sub.3) .delta.: 1.63 (s, 9H,
Boc); 3.26 (m, 2H, CH.sub.2); 4.19 (m, 1H, CH); 4.39 (m, 2H,
CH.sub.2); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m,
2H); 7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).
Example 2: Peptidomimetic Macrocycles
[0395] Peptidomimetic macrocycles were synthesized, purified and
analyzed as previously described and as described below
(Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000);
Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005);
Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No.
7,192,713). Peptidomimetic macrocycles were designed by replacing
two or more naturally occurring amino acids with the corresponding
synthetic amino acids. Substitutions were made at i and i+4, and i
and i+7 positions. Peptide synthesis was 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 were employed. Non-natural
amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU
(Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic
peptides were acetylated, while the C-termini were amidated.
[0396] Purification of cross-linked compounds was achieved by 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 was confirmed by LC/MS
mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC
system) and amino acid analysis (Applied Biosystems, model
420A).
[0397] The following protocol was used in the synthesis of
dialkyne-crosslinked peptidomimetic macrocycles, including SP662,
SP663 and SP664. Fully protected resin-bound peptides were
synthesized on a PEG-PS resin (loading 0.45 mmol/g) on a 0.2 mmol
scale. Deprotection of the temporary Fmoc group was achieved by
3.times.10 min treatments of the resin bound peptide with 20% (v/v)
piperidine in DMF. After washing with NMP (3.times.),
dichloromethane (3.times.) and NMP (3.times.), coupling of each
successive amino acid was achieved with 1.times.60 min incubation
with the appropriate preactivated Fmoc-amino acid derivative. All
protected amino acids (0.4 mmol) were dissolved in NMP and
activated with HCTU (0.4 mmol) and DIEA (0.8 mmol) prior to
transfer of the coupling solution to the deprotected resin-bound
peptide. After coupling was completed, the resin was washed in
preparation for the next deprotection/coupling cycle. Acetylation
of the amino terminus was carried out in the presence of acetic
anhydride/DIEA in NMP. The LC-MS analysis of a cleaved and
deprotected sample obtained from an aliquot of the fully assembled
resin-bound peptide was accomplished in order to verifying the
completion of each coupling. In a typical example, tetrahydrofuran
(4 ml) and triethylamine (2 ml) were added to the peptide resin
(0.2 mmol) in a 40 ml glass vial and shaken for 10 minutes.
Pd(PPh.sub.3).sub.2Cl.sub.2 (0.014 g, 0.02 mmol) and copper iodide
(0.008 g, 0.04 mmol) were then added and the resulting reaction
mixture was mechanically shaken 16 hours while open to atmosphere.
The diyne-cyclized resin-bound peptides were deprotected and
cleaved from the solid support by treatment with TFA/H.sub.2O/TIS
(95/5/5 v/v) for 2.5 h at room temperature. After filtration of the
resin the TFA solution was precipitated in cold diethyl ether and
centrifuged to yield the desired product as a solid. The crude
product was purified by preparative HPLC.
[0398] The following protocol was used in the synthesis of single
alkyne-crosslinked peptidomimetic macrocycles, including SP665.
Fully protected resin-bound peptides were synthesized on a Rink
amide MBHA resin (loading 0.62 mmol/g) on a 0.1 mmol scale.
Deprotection of the temporary Fmoc group was achieved by 2.times.20
min treatments of the resin bound peptide with 25% (v/v) piperidine
in NMP. After extensive flow washing with NMP and dichloromethane,
coupling of each successive amino acid was achieved with 1.times.60
min incubation with the appropriate preactivated Fmoc-amino acid
derivative. All protected amino acids (1 mmol) were dissolved in
NMP and activated with HCTU (1 mmol) and DIEA (1 mmol) prior to
transfer of the coupling solution to the deprotected resin-bound
peptide. After coupling was completed, the resin was extensively
flow washed in preparation for the next deprotection/coupling
cycle. Acetylation of the amino terminus was carried out in the
presence of acetic anhydride/DIEA in NMP/NMM. The LC-MS analysis of
a cleaved and deprotected sample obtained from an aliquot of the
fully assembled resin-bound peptide was accomplished in order to
verifying the completion of each coupling. In a typical example,
the peptide resin (0.1 mmol) was washed with DCM. Resin was loaded
into a microwave vial. The vessel was evacuated and purged with
nitrogen. Molybdenumhexacarbonyl (0.01 eq, Sigma Aldrich 199959)
was added. Anhydrous chlorobenzene was added to the reaction
vessel. Then 2-fluorophenol (1 eq, Sigma Aldrich F12804) was added.
The reaction was then loaded into the microwave and held at
130.degree. C. for 10 minutes. Reaction may need to be pushed a
subsequent time for completion. The alkyne metathesized resin-bound
peptides were deprotected and cleaved from the solid support by
treatment with TFA/H.sub.2O/TIS (94/3/3 v/v) for 3 h at room
temperature. After filtration of the resin the TFA solution was
precipitated in cold diethyl ether and centrifuged to yield the
desired product as a solid. The crude product was purified by
preparative HPLC.
[0399] Table 1 shows a list of peptidomimetic macrocycles
prepared.
TABLE-US-00002 TABLE 1 SEQ ID Exact Found Calc Calc Calc SP
Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP1
Ac-F$r8AYWEAc3cL$AAA-NH2 10 1456.78 729.44 1457.79 729.4 486.6 SP2
Ac-F$r8AYWEAc3cL$AAibA-NH2 11 1470.79 736.4 1471.8 736.4 491.27 SP3
Ac-LTF$r8AYWAQL$SANle-NH2 12 1715.97 859.02 1716.98 858.99 573 SP4
Ac-LTF$r8AYWAQL$SAL-NH2 13 1715.97 859.02 1716.98 858.99 573 SP5
Ac-LTF$r8AYWAQL$SAM-NH2 14 1733.92 868.48 1734.93 867.97 578.98 SP6
Ac-LTF$r8AYWAQL$SAhL-NH2 15 1729.98 865.98 1730.99 866 577.67 SP7
Ac-LTF$r8AYWAQL$SAF-NH2 16 1749.95 876.36 1750.96 875.98 584.32 SP8
Ac-LTF$r8AYWAQL$SAI-NH2 17 1715.97 859.02 1716.98 858.99 573 SP9
Ac-LTF$r8AYWAQL$SAChg-NH2 18 1741.98 871.98 1742.99 872 581.67 SP10
Ac-LTF$r8AYWAQL$SAAib-NH2 19 1687.93 845.36 1688.94 844.97 563.65
SP11 Ac-LTF$r8AYWAQL$SAA-NH2 20 1673.92 838.01 1674.93 837.97
558.98 SP12 Ac-LTF$r8AYWA$L$S$Nle-NH2 21 1767.04 884.77 1768.05
884.53 590.02 SP13 Ac-LTF$r8AYWA$L$S$A-NH2 22 1724.99 864.23 1726
863.5 576 SP14 Ac-F$r8AYWEAc3cL$AANle-NH2 23 1498.82 750.46 1499.83
750.42 500.61 SP15 Ac-F$r8AYWEAc3cL$AAL-NH2 24 1498.82 750.46
1499.83 750.42 500.61 SP16 Ac-F$r8AYWEAc3cL$AAM-NH2 25 1516.78
759.41 1517.79 759.4 506.6 SP17 Ac-F$r8AYWEAc3cL$AAhL-NH2 26
1512.84 757.49 1513.85 757.43 505.29 SP18 Ac-F$r8AYWEAc3cL$AAF-NH2
27 1532.81 767.48 1533.82 767.41 511.94 SP19
Ac-F$r8AYWEAc3cL$AAI-NH2 28 1498.82 750.39 1499.83 750.42 500.61
SP20 Ac-F$r8AYWEAc3cL$AAChg-NH2 29 1524.84 763.48 1525.85 763.43
509.29 SP21 Ac-F$r8AYWEAc3cL$AACha-NH2 30 1538.85 770.44 1539.86
770.43 513.96 SP22 Ac-F$r8AYWEAc3cL$AAAib-NH2 31 1470.79 736.84
1471.8 736.4 491.27 SP23 Ac-LTF$r8AYWAQL$AAAibV-NH2 32 1771.01
885.81 1772.02 886.51 591.34 SP24 Ac-LTF$r8AYWAQL$AAAibV-NH2 33
iso2 1771.01 886.26 1772.02 886.51 591.34 SP25
Ac-LTF$r8AYWAQL$SAibAA-NH2 34 1758.97 879.89 1759.98 880.49 587.33
SP26 Ac-LTF$r8AYWAQL$SAibAA-NH2 35 iso2 1758.97 880.34 1759.98
880.49 587.33 SP27 Ac-HLTF$r8HHWHQL$AANleNle-NH2 36 2056.15 1028.86
2057.16 1029.08 686.39 SP28 Ac-DLTFF$r8HHWHQL$RRLV-NH2 37 2190.23
731.15 2191.24 1096.12 731.08 SP29 Ac-HHTF$r8HHWHQL$AAML-NH2 38
2098.08 700.43 2099.09 1050.05 700.37 SP30 Ac-F$r8HHWHQL$RRDCha-NH2
39 1917.06 959.96 1918.07 959.54 640.03 SP31 Ac-F$r8HHWHQL$HRFV-NH2
40 1876.02 938.65 1877.03 939.02 626.35 SP32
Ac-HLTF$r8HHWHQL$AAhLA-NH2 41 2028.12 677.2 2029.13 1015.07 677.05
SP33 Ac-DLTF$r8HHWHQL$RRChgl-NH2 42 2230.26 1115.89 2231.27 1116.14
744.43 SP34 Ac-DLTF$r8HHWHQL$RRChgl-NH2 43 iso2 2230.26 1115.96
2231.27 1116.14 744.43 SP35 Ac-HHTF$r8HHWHQL$AAChav-NH2 44 2106.14
1053.95 2107.15 1054.08 703.05 SP36 Ac-F$r8HHWHQL$RRDa-NH2 45
1834.99 918.3 1836 918.5 612.67 SP37 Ac-F$r8HHWHQL$HRAibG-NH2 46
1771.95 886.77 1772.96 886.98 591.66 SP38 Ac-F$r8AYWAQL$HHNleL-NH2
47 1730.97 866.57 1731.98 866.49 578 SP39 Ac-F$r8AYWSAL$HQANle-NH2
48 1638.89 820.54 1639.9 820.45 547.3 SP40 Ac-F$r8AYWVQL$QHChgl-NH2
49 1776.01 889.44 1777.02 889.01 593.01 SP41
Ac-F$r8AYWTAL$QQNlev-NH2 50 1671.94 836.97 1672.95 836.98 558.32
SP42 Ac-F$r8AYWYQL$HAibAa-NH2 51 1686.89 844.52 1687.9 844.45 563.3
SP43 Ac-LTF$r8AYWAQL$HHLa-NH2 52 1903.05 952.27 1904.06 952.53
635.36 SP44 Ac-LTF$r8AYWAQL$HHLa-NH2 53 iso2 1903.05 952.27 1904.06
952.53 635.36 SP45 Ac-LTF$r8AYWAQL$HQNlev-NH2 54 1922.08 962.48
1923.09 962.05 641.7 SP46 Ac-LTF$r8AYWAQL$HQNlev-NH2 55 iso2
1922.08 962.4 1923.09 962.05 641.7 SP47 Ac-LTF$r8AYWAQL$QQMl-NH2 56
1945.05 973.95 1946.06 973.53 649.36 SP48 Ac-LTF$r8AYWAQL$QQMl-NH2
57 iso2 1945.05 973.88 1946.06 973.53 649.36 SP49
Ac-LTF$r8AYWAQL$HAibhLV-NH2 58 1893.09 948.31 1894.1 947.55 632.04
SP50 Ac-LTF$r8AYWAQL$AHFA-NH2 59 1871.01 937.4 1872.02 936.51
624.68 SP51 Ac-HLTF$r8HHWHQL$AANlel-NH2 60 2056.15 1028.79 2057.16
1029.08 686.39 SP52 Ac-DLTF$r8HHWHQL$RRLa-NH2 61 2162.2 721.82
2163.21 1082.11 721.74 SP53 Ac-HHTF$r8HHWHQL$AAMv-NH2 62 2084.07
1042.92 2085.08 1043.04 695.7 SP54 Ac-F$r8HHWHQL$RRDA-NH2 63
1834.99 612.74 1836 918.5 612.67 SP55 Ac-F$r8HHWHQL$HRFCha-NH2 64
1930.06 966.47 1931.07 966.04 644.36 SP56 Ac-F$r8AYWEAL$AA-NHAm 65
1443.82 1445.71 1444.83 722.92 482.28 SP57 Ac-F$r8AYWEAL$AA-NHiAm
66 1443.82 723.13 1444.83 722.92 482.28 SP58
Ac-F$r8AYWEAL$AA-NHnPR3Ph 67 1491.82 747.3 1492.83 746.92 498.28
SP59 Ac-F$r8AYWEAL$AA-NHnBu33Me 68 1457.83 1458.94 1458.84 729.92
486.95 SP60 Ac-F$r8AYWEAL$AA-NHnPr 69 1415.79 709.28 1416.8 708.9
472.94 SP61 Ac-F$r8AYWEAL$AA-NHnEt2Ch 70 1483.85 1485.77 1484.86
742.93 495.62 SP62 Ac-F$r8AYWEAL$AA-NHnEt2Cp 71 1469.83 1470.78
1470.84 735.92 490.95 SP63 Ac-F$r8AYWEAL$AA-NHHex 72 1457.83 730.19
1458.84 729.92 486.95 SP64 Ac-LTF$r8AYWAQL$AAIA-NH2 73 1771.01
885.81 1772.02 886.51 591.34 SP65 Ac-LTF$r8AYWAQL$AAIA-NH2 74 iso2
1771.01 866.8 1772.02 886.51 591.34 SP66 Ac-LTF$r8AYWAAL$AAMA-NH2
75 1731.94 867.08 1732.95 866.98 578.32 SP67
Ac-LTF$r8AYWAAL$AAMA-NH2 76 iso2 1731.94 867.28 1732.95 866.98
578.32 SP68 Ac-LTF$r8AYWAQL$AANleA-NH2 77 1771.01 867.1 1772.02
886.51 591.34 SP69 Ac-LTF$r8AYWAQL$AANleA-NH2 78 iso2 1771.01
886.89 1772.02 886.51 591.34 SP70 Ac-LTF$r8AYWAQL$AAIa-NH2 79
1771.01 886.8 1772.02 886.51 591.34 SP71 Ac-LTF$r8AYWAQL$AAIa-NH2
80 iso2 1771.01 887.09 1772.02 886.51 591.34 SP72
Ac-LTF$r8AYWAAL$AAMa-NH2 81 1731.94 867.17 1732.95 866.98 578.32
SP73 Ac-LTF$r8AYWAAL$AAMa-NH2 82 iso2 1731.94 867.37 1732.95 866.98
578.32 SP74 Ac-LTF$r8AYWAQL$AANlea-NH2 83 1771.01 887.08 1772.02
886.51 591.34 SP75 Ac-LTF$r8AYWAQL$AANlea-NH2 84 iso2 1771.01
887.08 1772.02 886.51 591.34 SP76 Ac-LTF$r8AYWAAL$AAIv-NH2 85
1742.02 872.37 1743.03 872.02 581.68 SP77 Ac-LTF$r8AYWAAL$AAIv-NH2
86 iso2 1742.02 872.74 1743.03 872.02 581.68 SP78
Ac-LTF$r8AYWAQL$AAMv-NH2 87 1817 910.02 1818.01 909.51 606.67 SP79
Ac-LTF$r8AYWAAL$AANlev-NH2 88 1742.02 872.37 1743.03 872.02 581.68
SP80 Ac-LTF$r8AYWAAL$AANlev-NH2 89 iso2 1742.02 872.28 1743.03
872.02 581.68 SP81 Ac-LTF$r8AYWAQL$AAIl-NH2 90 1813.05 907.81
1814.06 907.53 605.36 SP82 Ac-LTF$r8AYWAQL$AAIl-NH2 91 iso2 1813.05
907.81 1814.06 907.53 605.36 SP83 Ac-LTF$r8AYWAAL$AAMl-NH2 92
1773.99 887.37 1775 888 592.34 SP84 Ac-LTF$r8AYWAQL$AANlel-NH2 93
1813.05 907.61 1814.06 907.53 605.36 SP85
Ac-LTF$r8AYWAQL$AANlel-NH2 94 iso2 1813.05 907.71 1814.06 907.53
605.36 SP86 Ac-F$r8AYWEAL$AAMA-NH2 95 1575.82 789.02 1576.83 788.92
526.28 SP87 Ac-F$r8AYWEAL$AANleA-NH2 96 1557.86 780.14 1558.87
779.94 520.29 SP88 Ac-F$r8AYWEAL$AAIa-NH2 97 1557.86 780.33 1558.87
779.94 520.29 SP89 Ac-F$r8AYWEAL$AAMa-NH2 98 1575.82 789.3 1576.83
788.92 526.28 SP90 Ac-F$r8AYWEAL$AANlea-NH2 99 1557.86 779.4
1558.87 779.94 520.29 SP91 Ac-F$r8AYWEAL$AAIv-NH2 100 1585.89
794.29 1586.9 793.95 529.64 SP92 Ac-F$r8AYWEAL$AAMv-NH2 101 1603.85
803.08 1604.86 802.93 535.62 SP93 Ac-F$r8AYWEAL$AANlev-NH2 102
1585.89 793.46 1586.9 793.95 529.64 SP94 Ac-F$r8AYWEAL$AAIl-NH2 103
1599.91 800.49 1600.92 800.96 534.31 SP95 Ac-F$r8AYWEAL$AAMl-NH2
104 1617.86 809.44 1618.87 809.94 540.29 SP96
Ac-F$r8AYWEAL$AANlel-NH2 105 1599.91 801.7 1600.92 800.96 534.31
SP97 Ac-F$r8AYWEAL$AANlel-NH2 106 iso2 1599.91 801.42 1600.92
800.96 534.31 SP98 Ac-LTF$r8AY6clWAQL$SAA-NH2 107 1707.88 855.72
1708.89 854.95 570.3 SP99 Ac-LTF$r8AY6clWAQL$SAA-NH2 108 iso2
1707.88 855.35 1708.89 854.95 570.3 SP100 Ac-WTF$r8FYWSQL$AVAa-NH2
109 1922.01 962.21 1923.02 962.01 641.68 SP101
Ac-WTF$r8FYWSQL$AVAa-NH2 110 iso2 1922.01 962.49 1923.02 962.01
641.68 SP102 Ac-WTF$r8VYWSQL$AVA-NH2 111 1802.98 902.72 1803.99
902.5 602 SP103 Ac-WTF$r8VYWSQL$AVA-NH2 112 iso2 1802.98 903
1803.99 902.5 602 SP104 Ac-WTF$r8FYWSQL$SAAa-NH2 113 1909.98 956.47
1910.99 956 637.67 SP105 Ac-WTF$r8FYWSQL$SAAa-NH2 114 iso2 1909.98
956.47 1910.99 956 637.67 SP106 Ac-WTF$r8VYWSQL$AVAaa-NH2 115
1945.05 974.15 1946.06 973.53 649.36 SP107
Ac-WTF$r8VYWSQL$AVAaa-NH2 116 iso2 1945.05 973.78 1946.06 973.53
649.36 SP108 Ac-LTF$r8AYWAQL$AVG-NH2 117 1671.94 837.52 1672.95
836.98 558.32 SP109 Ac-LTF$r8AYWAQL$AVG-NH2 118 iso2 1671.94 837.21
1672.95 836.98 558.32 SP110 Ac-LTF$r8AYWAQL$AVQ-NH2 119 1742.98
872.74 1743.99 872.5 582 SP111 Ac-LTF$r8AYWAQL$AVQ-NH2 120 iso2
1742.98 872.74 1743.99 872.5 582 SP112 Ac-LTF$r8AYWAQL$SAa-NH2 121
1673.92 838.23 1674.93 837.97 558.98 SP113 Ac-LTF$r8AYWAQL$SAa-NH2
122 iso2 1673.92 838.32 1674.93 837.97 558.98 SP114
Ac-LTF$r8AYWAQhL$SAA-NH2 123 1687.93 844.37 1688.94 844.97 563.65
SP115 Ac-LTF$r8AYWAQhL$SAA-NH2 124 iso2 1687.93 844.81 1688.94
844.97 563.65 SP116 Ac-LTF$r8AYWEQLStSA$-NH2 125 1826 905.27
1827.01 914.01 609.67 SP117 Ac-LTF$r8AYWAQL$SLA-NH2 126 1715.97
858.48 1716.98 858.99 573 SP118 Ac-LTF$r8AYWAQL$SLA-NH2 127 iso2
1715.97 858.87 1716.98 858.99 573 SP119 Ac-LTF$r8AYWAQL$SWA-NH2 128
1788.96 895.21 1789.97 895.49 597.33 SP120 Ac-LTF$r8AYWAQL$SWA-NH2
129 iso2 1788.96 895.28 1789.97 895.49 597.33 SP121
Ac-LTF$r8AYWAQL$SVS-NH2 130 1717.94 859.84 1718.95 859.98 573.65
SP122 Ac-LTF$r8AYWAQL$SAS-NH2 131 1689.91 845.85 1690.92 845.96
564.31 SP123 Ac-LTF$r8AYWAQL$SVG-NH2 132 1687.93 844.81 1688.94
844.97 563.65 SP124 Ac-ETF$r8VYWAQL$SAa-NH2 133 1717.91 859.76
1718.92 859.96 573.64 SP125 Ac-ETF$r8VYWAQL$SAA-NH2 134 1717.91
859.84 1718.92 859.96 573.64 SP126 Ac-ETF$r8VYWAQL$SVA-NH2 135
1745.94 873.82 1746.95 873.98 582.99 SP127 Ac-ETF$r8VYWAQL$SLA-NH2
136 1759.96 880.85 1760.97 880.99 587.66 SP128
Ac-ETF$r8VYWAQL$SWA-NH2 137 1832.95 917.34 1833.96 917.48 611.99
SP129 Ac-ETF$r8KYWAQL$SWA-NH2 138 1861.98 931.92 1862.99 932 621.67
SP130 Ac-ETF$r8VYWAQL$SVS-NH2 139 1761.93 881.89 1762.94 881.97
588.32 SP131 Ac-ETF$r8VYWAQL$SAS-NH2 140 1733.9 867.83 1734.91
867.96 578.97 SP132 Ac-ETF$r8VYWAQL$SVG-NH2 141 1731.92 866.87
1732.93 866.97 578.31 SP133 Ac-LTF$r8VYWAQL$SSa-NH2 142 1717.94
859.47 1718.95 859.98 573.65 SP134 Ac-ETF$r8VYWAQL$SSa-NH2 143
1733.9 867.83 1734.91 867.96 578.97 SP135 Ac-LTF$r8VYWAQL$SNa-NH2
144 1744.96 873.38 1745.97 873.49 582.66 SP136
Ac-ETF$r8VYWAQL$SNa-NH2 145 1760.91 881.3 1761.92 881.46 587.98
SP137 Ac-LTF$r8VYWAQL$SAa-NH2 146 1701.95 851.84 1702.96 851.98
568.32 SP138 Ac-LTF$r8VYWAQL$SVA-NH2 147 1729.98 865.53 1730.99 866
577.67 SP139 Ac-LTF$r8VYWAQL$SVA-NH2 148 iso2 1729.98 865.9 1730.99
866 577.67 SP140 Ac-LTF$r8VYWAQL$SWA-NH2 149 1816.99 909.42 1818
909.5 606.67 SP141 Ac-LTF$r8VYWAQL$SVS-NH2 150 1745.98 873.9
1746.99 874 583 SP142 Ac-LTF$r8VYWAQL$SVS-NH2 151 iso2 1745.98
873.9 1746.99 874 583 SP143 Ac-LTF$r8VYWAQL$SAS-NH2 152 1717.94
859.84 1718.95 859.98 573.65 SP144 Ac-LTF$r8VYWAQL$SAS-NH2 153 iso2
1717.94 859.91 1718.95 859.98 573.65 SP145 Ac-LTF$r8VYWAQL$SVG-NH2
154 1715.97 858.87 1716.98 858.99 573 SP146 Ac-LTF$r8VYWAQL$SVG-NH2
155 iso2 1715.97 858.87 1716.98 858.99 573 SP147
Ac-LTF$r8EYWAQCha$SAA-NH2 156 1771.96 886.85 1772.97 886.99 591.66
SP148 Ac-LTF$r8EYWAQCha$SAA-NH2 157 iso2 1771.96 886.85 1772.97
886.99 591.66 SP149 Ac-LTF$r8EYWAQCpg$SAA-NH2 158 1743.92 872.86
1744.93 872.97 582.31 SP150 Ac-LTF$r8EYWAQCpg$SAA-NH2 159 iso2
1743.92 872.86 1744.93 872.97 582.31 SP151 Ac-LTF$r8EYWAQF$SAA-NH2
160 1765.91 883.44 1766.92 883.96 589.64 SP152
Ac-LTF$r8EYWAQF$SAA-NH2 161 iso2 1765.91 883.89 1766.92 883.96
589.64 SP153 Ac-LTF$r8EYWAQCba$SAA-NH2 162 1743.92 872.42 1744.93
872.97 582.31 SP154 Ac-LTF$r8EYWAQCba$SAA-NH2 163 iso2 1743.92
873.39 1744.93 872.97 582.31 SP155 Ac-LTF3Cl$r8EYWAQL$SAA-NH2 164
1765.89 883.89 1766.9 883.95 589.64 SP156
Ac-LTF3Cl$r8EYWAQL$SAA-NH2 165 iso2 1765.89 883.96 1766.9 883.95
589.64 SP157 Ac-LTF34F2$r8EYWAQL$SAA-NH2 166 1767.91 884.48 1768.92
884.96 590.31 SP158 Ac-LTF34F2$r8EYWAQL$SAA-NH2 167 iso2 1767.91
884.48 1768.92 884.96 590.31 SP159 Ac-LTF34F2$r8EYWAQhL$SAA-NH2 168
1781.92 891.44 1782.93 891.97 594.98 SP160
Ac-LTF34F2$r8EYWAQhL$SAA-NH2 169 iso2 1781.92 891.88 1782.93 891.97
594.98 SP161 Ac-ETF$r8EYWAQL$SAA-NH2 170 1747.88 874.34 1748.89
874.95 583.63 SP162 Ac-LTF$r8AYWVQL$SAA-NH2 171 1701.95 851.4
1702.96 851.98 568.32 SP163 Ac-LTF$r8AHWAQL$SAA-NH2 172 1647.91
824.83 1648.92 824.96 550.31 SP164 Ac-LTF$r8AEWAQL$SAA-NH2 173
1639.9 820.39 1640.91 820.96 547.64 SP165 Ac-LTF$r8ASWAQL$SAA-NH2
174 1597.89 799.38 1598.9 799.95 533.64 SP166
Ac-LTF$r8AEWAQL$SAA-NH2 175 iso2 1639.9 820.39 1640.91 820.96
547.64 SP167 Ac-LTF$r8ASWAQL$SAA-NH2 176 iso2 1597.89 800.31 1598.9
799.95 533.64 SP168 Ac-LTF$r8AF4coohWAQL$SAA-NH2 177 1701.91 851.4
1702.92 851.96 568.31 SP169 Ac-LTF$r8AF4coohWAQL$SAA-NH2 178 iso2
1701.91 851.4 1702.92 851.96 568.31 SP170 Ac-LTF$r8AHWAQL$AAIa-NH2
179 1745 874.13 1746.01 873.51 582.67 SP171
Ac-ITF$r8FYWAQL$AAIa-NH2 180 1847.04 923.92 1848.05 924.53 616.69
SP172 Ac-ITF$r8EHWAQL$AAIa-NH2 181 1803.01 903.17 1804.02 902.51
602.01 SP173 Ac-ITF$r8EHWAQL$AAIa-NH2 182 iso2 1803.01 903.17
1804.02 902.51 602.01 SP174 Ac-ETF$r8EHWAQL$AAIa-NH2 183 1818.97
910.76 1819.98 910.49 607.33 SP175 Ac-ETF$r8EHWAQL$AAIa-NH2 184
iso2 1818.97 910.85 1819.98 910.49 607.33 SP176
Ac-LTF$r8AHWVQL$AAIa-NH2 185 1773.03 888.09 1774.04 887.52 592.02
SP177 Ac-ITF$r8FYWVQL$AAIa-NH2 186 1875.07 939.16 1876.08 938.54
626.03 SP178 Ac-ITF$r8EYWVQL$AAIa-NH2 187 1857.04 929.83 1858.05
929.53 620.02 SP179 Ac-ITF$r8EHWVQL$AAIa-NH2 188 1831.04 916.86
1832.05 916.53 611.35 SP180 Ac-LTF$r8AEWAQL$AAIa-NH2 189 1736.99
869.87 1738 869.5 580 SP181 Ac-LTF$r8AF4coohWAQL$AAIa-NH2 190 1799
900.17 1800.01 900.51 600.67 SP182 Ac-LTF$r8AF4coohWAQL$AAIa-NH2
191 iso2 1799 900.24 1800.01 900.51 600.67 SP183
Ac-LTF$r8AHWAQL$AHFA-NH2 192 1845.01 923.89 1846.02 923.51 616.01
SP184 Ac-ITF$r8FYWAQL$AHFA-NH2 193 1947.05 975.05 1948.06 974.53
650.02 SP185 Ac-ITF$r8FYWAQL$AHFA-NH2 194 iso2 1947.05 976.07
1948.06 974.53 650.02 SP186 Ac-ITF$r8FHWAQL$AEFA-NH2 195 1913.02
958.12 1914.03 957.52 638.68 SP187 Ac-ITF$r8FHWAQL$AEFA-NH2 196
iso2 1913.02 957.86 1914.03 957.52 638.68 SP188
Ac-ITF$r8EHWAQL$AHFA-NH2 197 1903.01 952.94 1904.02 952.51 635.34
SP189 Ac-ITF$r8EHWAQL$AHFA-NH2 198 iso2 1903.01 953.87 1904.02
952.51 635.34 SP190 Ac-LTF$r8AHWVQL$AHFA-NH2 199 1873.04 937.86
1874.05 937.53 625.35 SP191 Ac-ITF$r8FYWVQL$AHFA-NH2 200 1975.08
988.83 1976.09 988.55 659.37 SP192 Ac-ITF$r8EYWVQL$AHFA-NH2 201
1957.05 979.35 1958.06 979.53 653.36 SP193 Ac-ITF$r8EHWVQL$AHFA-NH2
202 1931.05 967 1932.06 966.53 644.69 SP194
Ac-ITF$r8EHWVQL$AHFA-NH2 203 iso2 1931.05 967.93 1932.06 966.53
644.69
SP195 Ac-ETF$r8EYWAAL$SAA-NH2 204 1690.86 845.85 1691.87 846.44
564.63 SP196 Ac-LTF$r8AYWVAL$SAA-NH2 205 1644.93 824.08 1645.94
823.47 549.32 SP197 Ac-LTF$r8AHWAAL$SAA-NH2 206 1509.89 796.88
1591.9 796.45 531.3 SP198 Ac-LTF$r8AEWAAL$SAA-NH2 207 1582.88 791.9
1583.89 792.45 528.63 SP199 Ac-LTF$r8AEWAAL$SAA-NH2 208 iso2
1582.88 791.9 1583.89 792.45 528.63 SP200 Ac-LTF$r8ASWAAL$SAA-NH2
209 1540.87 770.74 1541.88 771.44 514.63 SP201
Ac-LTF$r8ASWAAL$SAA-NH2 210 iso2 1540.87 770.88 1541.88 771.44
514.63 SP202 Ac-LTF$r8AYWAAL$AAIa-NH2 211 1713.99 857.39 1715 858
572.34 SP203 Ac-LTF$r8AYWAAL$AAIa-NH2 212 iso2 1713.99 857.84 1715
858 572.34 SP204 Ac-LTF$r8AYWAAL$AHFA-NH2 213 1813.99 907.86 1815
908 605.67 SP205 Ac-LTF$r8EHWAQL$AHIa-NH2 214 1869.03 936.1 1870.04
935.52 624.02 SP206 Ac-LTF$r8EHWAQL$AHIa-NH2 215 iso2 1869.03
937.03 1870.04 935.52 624.02 SP207 Ac-LTF$r8AHWAQL$AHIa-NH2 216
1811.03 906.87 1812.04 906.52 604.68 SP208 Ac-LTF$r8EYWAQL$AHIa-NH2
217 1895.04 949.15 1896.05 948.53 632.69 SP209
Ac-LTF$r8AYWAQL$AAFa-NH2 218 1804.99 903.2 1806 903.5 602.67 SP210
Ac-LTF$r8AYWAQL$AAFa-NH2 219 iso2 1804.99 903.28 1806 903.5 602.67
SP211 Ac-LTF$r8AYWAQL$AAWa-NH2 220 1844 922.81 1845.01 923.01
615.67 SP212 Ac-LTF$r8AYWAQL$AAVa-NH2 221 1756.99 878.86 1758 879.5
586.67 SP213 Ac-LTF$r8AYWAQL$AAVa-NH2 222 iso2 1756.99 879.3 1758
879.5 586.67 SP214 Ac-LTF$r8AYWAQL$AALa-NH2 223 1771.01 886.26
1772.02 886.51 591.34 SP215 Ac-LTF$r8AYWAQL$AALa-NH2 224 iso2
1771.01 886.33 1772.02 886.51 591.34 SP216 Ac-LTF$r8EYWAQL$AAIa-NH2
225 1829.01 914.89 1830.02 915.51 610.68 SP217
Ac-LTF$r8EYWAQL$AAIa-NH2 226 iso2 1829.01 915.34 1830.02 915.51
610.68 SP218 Ac-LTF$r8EYWAQL$AAFa-NH2 227 1863 932.87 1864.01
932.51 622.01 SP219 Ac-LTF$r8EYWAQL$AAFa-NH2 228 iso2 1863 932.87
1864.01 932.51 622.01 SP220 Ac-LTF$r8EYWAQL$AAVa-NH2 229 1815
908.23 1816.01 908.51 606.01 SP221 Ac-LTF$r8EYWAQL$AAVa-NH2 230
iso2 1815 908.31 1816.01 908.51 606.01 SP222
Ac-LTF$r8EHWAQL$AAIa-NH2 231 1803.01 903.17 1804.02 902.51 602.01
SP223 Ac-LTF$r8EHWAQL$AAIa-NH2 232 iso2 1803.01 902.8 1804.02
902.51 602.01 SP224 Ac-LTF$r8EHWAQL$AAWa-NH2 233 1876 939.34
1877.01 939.01 626.34 SP225 Ac-LTF$r8EHWAQL$AAWa-NH2 234 iso2 1876
939.62 1877.01 939.01 626.34 SP226 Ac-LTF$r8EHWAQL$AALa-NH2 235
1803.01 902.8 1804.02 902.51 602.01 SP227 Ac-LTF$r8EHWAQL$AALa-NH2
236 iso2 1803.01 902.9 1804.02 902.51 602.01 SP228
Ac-ETF$r8EHWVQL$AALa-NH2 237 1847 924.82 1848.01 924.51 616.67
SP229 Ac-LTF$r8AYWAQL$AAAa-NH2 238 1728.96 865.89 1729.97 865.49
577.33 SP230 Ac-LTF$r8AYWAQL$AAAa-NH2 239 iso2 1728.96 865.89
1729.97 865.49 577.33 SP231 Ac-LTF$r8AYWAQL$AAAibA-NH2 240 1742.98
872.83 1743.99 872.5 582 SP232 Ac-LTF$r8AYWAQL$AAAibA-NH2 241 iso2
1742.98 872.92 1743.99 872.5 582 SP233 Ac-LTF$r8AYWAQL$AAAAa-NH2
242 1800 901.42 1801.01 901.01 601.01 SP234
Ac-LTF$r5AYWAQL$s8AAIa-NH2 243 1771.01 887.17 1772.02 886.51 591.34
SP235 Ac-LTF$r5AYWAQL$s8SAA-NH2 244 1673.92 838.33 1674.93 837.97
558.98 SP236 Ac-LTF$r8AYWAQCba$AANleA-NH2 245 1783.01 892.64
1784.02 892.51 595.34 SP237 Ac-ETF$r8AYWAQCba$AANleA-NH2 246
1798.97 900.59 1799.98 900.49 600.66 SP238
Ac-LTF$r8EYWAQCba$AANleA-NH2 247 1841.01 922.05 1842.02 921.51
614.68 SP239 Ac-LTF$r8AYWAQCba$AWNleA-NH2 248 1898.05 950.46
1899.06 950.03 633.69 SP240 Ac-ETF$r8AYWAQCba$AWNleA-NH2 249
1914.01 958.11 1915.02 958.01 639.01 SP241
Ac-LTF$r8EYWAQCba$AWNleA-NH2 250 1956.06 950.62 1957.07 979.04
653.03 SP242 Ac-LTF$r8EYWAQCba$SAFA-NH2 251 1890.99 946.55 1892
946.5 631.34 SP243 Ac-LTF34F2$r8EYWAQCba$SANleaA-NH2 252 1892.99
947.57 1894 947.5 632 SP244 Ac-LTF$r8EF4coohWAQCba$SANleaA-NH2 253
1885 943.59 1886.01 943.51 629.34 SP245
Ac-LTF$r8EYWSQCba$SANleaA-NH2 254 1873 937.58 1874.01 937.51 625.34
SP246 Ac-LTF$r8EYWWQCba$SANleaA-NH2 255 1972.05 987.61 1973.06
987.03 658.36 SP247 Ac-LTF$r8EYWAQCba$AAIa-NH2 256 1841.01 922.05
1842.02 921.51 614.68 SP248 Ac-LTF34F2$r8EYWAQCba$AAIa-NH2 257
1876.99 939.99 1878 939.5 626.67 SP249
Ac-LTF$r8EF4coohWAQCba$AAIa-NH2 258 1869.01 935.64 1870.02 935.51
624.01 SP250 Pam-ETF$r8EYWAQCba$SAA-NH2 259 1956.1 979.57 1957.11
979.06 653.04 SP251 Ac-LThF$r8EFWAQCba$SAA-NH2 260 1741.94 872.11
1742.95 871.98 581.65 SP252 Ac-LTA$r8EYWAQCba$SAA-NH2 261 1667.89
835.4 1668.9 834.95 556.97 SP253 Ac-LTF$r8EYAAQCba$SAA-NH2 262
1628.88 815.61 1629.89 815.45 543.97 SP254
Ac-LTF$r8EY2NalAQCba$SAA-NH2 263 1754.93 879.04 1755.94 878.47
585.98 SP255 Ac-LTF$r8AYWAQCba$SAA-NH2 264 1685.92 844.71 1686.93
843.97 562.98 SP256 Ac-LTF$r8EYWAQCba$SAF-NH2 265 1819.96 911.41
1820.97 910.99 607.66 SP257 Ac-LTF$r8EYWAQCba$SAFa-NH2 266 1890.99
947.41 1892 946.5 631.34 SP258 Ac-LTF$r8AYWAQCba$SAF-NH2 267
1761.95 882.73 1762.96 881.98 588.32 SP259
Ac-LTF34F2$r8AYWAQCba$SAF-NH2 268 1797.93 900.87 1798.94 899.97
600.32 SP260 Ac-LTF$r8AF4coohWAQCba$SAF-NH2 269 1789.94 896.43
1790.95 895.98 597.65 SP261 Ac-LTF$r8EY6clWAQCba$SAF-NH2 270
1853.92 929.27 1854.93 927.97 618.98 SP262
Ac-LTF$r8AYWSQCba$SAF-NH2 271 1777.94 890.87 1778.95 889.98 593.65
SP263 Ac-LTF$r8AYWWQCba$SAF-NH2 272 1876.99 939.91 1878 939.5
626.67 SP264 Ac-LTF$r8AYWAQCba$AAIa-NH2 273 1783.01 893.19 1784.02
892.51 595.34 SP265 Ac-LTF34F2$r8AYWAQCba$AAIa-NH2 274 1818.99
911.23 1820 910.5 607.34 SP266 Ac-LTF$r8AY6clWAQCba$AAIa-NH2 275
1816.97 909.84 1817.98 909.49 606.66 SP267
Ac-LTF$r8AF4coohWAQCba$AAIa-NH2 276 1811 906.88 1812.01 906.51
604.67 SP268 Ac-LTF$r8EYWAQCba$AAFa-NH2 277 1875 938.6 1876.01
938.51 626.01 SP269 Ac-LTF$r8EYWAQCba$AAFa-NH2 278 iso2 1875 938.6
1876.01 938.51 626.01 SP270 Ac-ETF$r8AYWAQCba$AWNlea-NH2 279
1914.01 958.42 1915.02 958.01 639.01 SP271
Ac-LTF$r8EYWAQCba$AWNlea-NH2 280 1956.06 979.42 1957.07 979.04
653.03 SP272 Ac-ETF$r8EYWAQCba$AWNlea-NH2 281 1972.01 987.06
1973.02 987.01 658.34 SP273 Ac-ETF$r8EYWAQCba$AWNlea-NH2 282 iso2
1972.01 987.06 1973.02 987.01 658.34 SP274
Ac-LTF$r8AYWAQCba$SAFa-NH2 283 1832.99 917.89 1834 917.5 612 SP275
Ac-LTF$r8AYWAQCba$SAFa-NH2 284 iso2 1832.99 918.07 1834 917.5 612
SP276 Ac-ETF$r8AYWAQL$AWNlea-NH2 285 1902.01 952.22 1903.02 952.01
635.01 SP277 Ac-LTF$r8EYWAQL$AWNlea-NH2 286 1944.06 973.5 1945.07
973.04 649.03 SP278 Ac-ETF$r8EYWAQL$AWNlea-NH2 287 1960.01 981.46
1961.02 981.01 654.34 SP279 Dmaac-LTF$r8EYWAQhL$SAA-NH2 288 1788.98
896.06 1789.99 895.5 597.33 SP280 Hexac-LTF$r8EYWAQhL$SAA-NH2 289
1802 902.9 1803.01 902.01 601.67 SP281 Napac-LTF$r8EYWAQhL$SAA-NH2
290 1871.99 937.58 1873 937 625 SP282 Decac-LTF$r8EYWAQhL$SAA-NH2
291 1858.06 930.55 1859.07 930.04 620.36 SP283
Admac-LTF$r8EYWAQhL$SAA-NH2 292 1866.03 934.07 1867.04 934.02
623.02 SP284 Tmac-LTF$r8EYWAQhL$SAA-NH2 293 1787.99 895.41 1789 895
597 SP285 Pam-LTF$r8EYWAQhL$SAA-NH2 294 1942.16 972.08 1943.17
972.09 648.39 SP286 Ac-LTF$r8AYWAQCba$AANleA-NH2 295 iso2 1783.01
892.64 1784.02 892.51 595.34 SP287 Ac-LTF34F2$r8EYWAQCba$AAIa-NH2
296 iso2 1876.99 939.62 1878 939.5 626.67 SP288
Ac-LTF34F2$r8EYWAQCba$AA-NH2 297 1779.91 892.07 1780.92 890.96
594.31 SP289 Ac-LTF34F2$r8EYWAQCba$AA-NH2 298 iso2 1779.91 891.61
1780.92 890.96 594.31 SP290 Ac-LTF$r8EF4coohWAQCba$SAA-NH2 299
1771.92 887.54 1772.93 886.97 591.65 SP291
Ac-LTF$r8EF4coohWAQCba$SAA-NH2 300 iso2 1771.92 887.63 1772.93
886.97 591.65 SP292 Ac-LTF$r8EYWSQCba$SAA-NH2 301 1759.92 881.9
1760.93 880.97 587.65 SP293 Ac-LTF$r8EYWSQCba$SAA-NH2 302 iso2
1759.92 881.9 1760.93 880.97 587.65 SP294 Ac-LTF$r8EYWAQhL$SAA-NH2
303 1745.94 875.05 1746.95 873.98 582.99 SP295
Ac-LTF$r8AYWAQhL$SAF-NH2 304 1763.97 884.02 1764.98 882.99 589
SP296 Ac-LTF$r8AYWAQhL$SAF-NH2 305 iso2 1763.97 883.56 1764.98
882.99 589 SP297 Ac-LTF34F2$r8AYWAQhL$SAA-NH2 306 1723.92 863.67
1724.93 862.97 575.65 SP298 Ac-LTF34F2$r8AYWAQhL$SAA-NH2 307 iso2
1723.92 864.04 1724.93 862.97 575.65 SP299
Ac-LTF$r8AF4coohWAQhL$SAA-NH2 308 1715.93 859.44 1716.94 858.97
572.98 SP300 Ac-LTF$r8AF4coohWAQhL$SAA-NH2 309 iso2 1715.93 859.6
1716.94 858.97 572.98 SP301 Ac-LTF$r8AYWSQhL$SAA-NH2 310 1703.93
853.96 1704.94 852.97 568.98 SP302 Ac-LTF$r8AYWSQhL$SAA-NH2 311
iso2 1703.93 853.59 1704.94 852.97 568.98 SP303
Ac-LTF$r8EYWAQL$AANleA-NH2 312 1829.01 915.45 1830.02 915.51 610.68
SP304 Ac-LTF34F2$r8AYWAQL$AANleA-NH2 313 1806.99 904.58 1808 904.5
603.34 SP305 Ac-LTF$r8AF4coohWAQL$AANleA-NH2 314 1799 901.6 1800.01
900.51 600.67 SP306 Ac-LTF$r8AYWSQL$AANleA-NH2 315 1787 894.75
1788.01 894.51 596.67 SP307 Ac-LTF34F2$r8AYWAQhL$AANleA-NH2 316
1821 911.79 1822.01 911.51 608.01 SP308
Ac-LTF34F2$r8AYWAQhL$AANleA-NH2 317 iso2 1821 912.61 1822.01 911.51
608.01 SP309 Ac-LTF$r8AF4coohWAQhL$AANleA-NH2 318 1813.02 907.95
1814.03 907.52 605.35 SP310 Ac-LTF$r8AF4coohWAQhL$AANleA-NH2 319
iso2 1813.02 908.54 1814.03 907.52 605.35 SP311
Ac-LTF$r8AYWSQhL$AANleA-NH2 320 1801.02 901.84 1802.03 901.52
601.35 SP312 Ac-LTF$r8AYWSQhL$AANleA-NH2 321 iso2 1801.02 902.62
1802.03 901.52 601.35 SP313 Ac-LTF$r8AYWAQhL$AAAAa-NH2 322 1814.01
908.63 1815.02 908.01 605.68 SP314 Ac-LTF$r8AYWAQhL$AAAAa-NH2 323
iso2 1814.01 908.34 1815.02 908.01 605.68 SP315
Ac-LTF$r8AYWAQL$AAAAAa-NH2 324 1871.04 936.94 1872.05 936.53 624.69
SP316 Ac-LTF$r8AYWAQL$AAAAAAa-NH2 325 iso2 1942.07 972.5 1943.08
972.04 648.37 SP317 Ac-LTF$r8AYWAQL$AAAAAAa-NH2 326 iso1 1942.07
972.5 1943.08 972.04 648.37 SP318 Ac-LTF$r8EYWAQhL$AANleA-NH2 327
1843.03 922.54 1844.04 922.52 615.35 SP319
Ac-AATF$r8AYWAQL$AANleA-NH2 328 1800 901.39 1801.01 901.01 601.01
SP320 Ac-LTF$r8AYWAQL$AANleAA-NH2 329 1842.04 922.45 1843.05 922.03
615.02 SP321 Ac-ALTF$r8AYWAQL$AANleAA-NH2 330 1913.08 957.94
1914.09 957.55 638.7 SP322 Ac-LTF$r8AYWAQCba$AANleAA-NH2 331
1854.04 928.43 1855.05 928.03 619.02 SP323
Ac-LTF$r8AYWAQhL$AANleAA-NH2 332 1856.06 929.4 1857.07 929.04
619.69 SP324 Ac-LTF$r8EYWAQCba$SAAA-NH2 333 1814.96 909.37 1815.97
908.49 605.99 SP325 Ac-LTF$r8EYWAQCba$SAAA-NH2 334 iso2 1814.96
909.37 1815.97 908.49 605.99 SP326 Ac-LTF$r8EYWAQCba$SAAAA-NH2 335
1886 944.61 1887.01 944.01 629.67 SP327 Ac-LTF$r8EYWAQCba$SAAAA-NH2
336 iso2 1886 944.61 1887.01 944.01 629.67 SP328
Ac-ALTF$r8EYWAQCba$SAA-NH2 337 1814.96 909.09 1815.97 908.49 605.99
SP329 Ac-ALTF$r8EYWAQCba$SAAA-NH2 338 1886 944.61 1887.01 944.01
629.67 SP330 Ac-ALTF$r8EYWAQCba$SAA-NH2 339 iso2 1814.96 909.09
1815.97 908.49 605.99 SP331 Ac-LTF$r8EYWAQL$AAAAAa-NH2 340 iso2
1929.04 966.08 1930.05 965.53 644.02 SP332
Ac-LTF$r8EY6clWAQCba$SAA-NH2 341 1777.89 890.78 1778.9 889.95
593.64 SP333 Ac-LTF$r8EF4cooh6clWAQCba$SANleA-NH2 342 1918.96
961.27 1919.97 960.49 640.66 SP334
Ac-LTF$r8EF4cooh6clWAQCba$SANleA-NH2 343 iso2 1918.96 961.27
1919.97 960.49 640.66 SP335 Ac-LTF$r8EF4cooh6clWAQCba$AAIa-NH2 344
1902.97 953.03 1903.98 952.49 635.33 SP336
Ac-LTF$r8EF4cooh6clWAQCba$AAIa-NH2 345 iso2 1902.97 953.13 1903.98
952.49 635.33 SP337 Ac-LTF$r8AY6clWAQL$AAAAAa-NH2 346 1905 954.61
1906.01 953.51 636.01 SP338 Ac-LTF$r8AY6clWAQL$AAAAAa-NH2 347 iso2
1905 954.9 1906.01 953.51 636.01 SP339 Ac-F$r8AY6clWEAL$AAAAAAa-NH2
348 1762.89 883.01 1763.9 882.45 58.64 SP340
Ac-ETF$r8EYWAQL$AAAAAa-NH2 349 1945 974.31 1946.01 973.51 649.34
SP341 Ac-ETF$r8EYWAQL$AAAAAa-NH2 350 iso2 1945 974.49 1946.01
973.51 649.34 SP342 Ac-LTF$r8EYWAQL$AAAAAAa-NH2 351 2000.08 1001.6
2001.09 1001.05 667.7 SP343 Ac-LTF$r8EYWAQL$AAAAAAa-NH2 352 iso2
2000.08 1001.6 2001.09 1001.05 667.7 SP344
Ac-LTF$r8AYWAQL$AANleAAa-NH2 353 1913.08 958.58 1914.09 957.55
638.7 SP345 Ac-LTF$r8AYWAQL$AANleAAa-NH2 354 iso2 1913.08 958.58
1914.09 957.55 638.7 SP346 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 355 1941.04
972.55 1942.05 971.53 648.02 SP347 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 356
iso2 1941.04 972.55 1942.05 971.53 648.02 SP348
Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH2 357 1969.04 986.33 1970.05 985.53
657.35 SP349 Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH2 358 iso2 1969.04
986.06 1970.05 985.53 657.35
SP350 Ac-LTF$r8EYWSQCba$AAAAAa-NH2 359 1957.04 980.04 1958.05
979.53 653.35 SP351 Ac-LTF$r8EYWSQCba$AAAAAa-NH2 360 iso2 1957.04
980.04 1958.05 979.53 653.35 SP352 Ac-LTF$r8EYWAQCba$SAAa-NH2 361
1814.96 909 1815.97 908.49 605.99 SP353 Ac-LTF$r8EYWAQCba$SAAa-NH2
362 iso2 1814.96 909 1815.97 908.49 605.99 SP354
Ac-ALTF$r8EYWAQCba$SAAa-NH2 363 1886 944.52 1887.01 944.01 629.67
SP355 Ac-ALTF$r8EYWAQCba$SAAa-NH2 364 iso2 1886 944.98 1887.01
944.01 629.67 SP356 Ac-ALTF$r8EYWAQCba$SAAAa-NH2 365 1957.04 980.04
1958.05 979.53 653.35 SP357 Ac-ALTF$r8EYWAQCba$SAAAa-NH2 366 iso2
1957.04 980.04 1958.05 979.53 653.35 SP358
Ac-AALTF$r8EYWAQCba$SAAAa-NH2 367 2028.07 1016.1 2029.08 1015.04
677.03 SP359 Ac-AALTF$r8EYWAQCba$SAAAa-NH2 368 iso2 2028.07 1015.57
2029.08 1015.04 677.03 SP360 Ac-RTF$r8EYWAQCba$SAA-NH2 369 1786.94
895.03 1787.95 894.48 596.65 SP361 Ac-LRF$r8EYWAQCba$SAA-NH2 370
1798.98 901.51 1799.99 900.5 600.67 SP362 Ac-LTF$r8EYWRQCba$SAA-NH2
371 1828.99 916.4 1830 915.5 610.67 SP363 Ac-LTF$r8EYWARCba$SAA-NH2
372 1771.97 887.63 1772.98 886.99 591.66 SP364
Ac-LTF$r8EYWAQCba$RAA-NH2 373 1812.99 908.08 1814 907.5 605.34
SP365 Ac-LTF$r8EYWAQCba$SRA-NH2 374 1828.99 916.12 1830 915.5
610.67 SP366 Ac-LTF$r8EYWAQCba$SAR-NH2 375 1828.99 916.12 1830
915.5 610.67 SP367 5-FAM-BaLTF$r8EYWAQCba$SAA-NH2 376 2131 1067.09
2132.01 1066.51 711.34 SP368 5-FAM-BaLTF$r8AYWAQL$AANleA-NH2 377
2158.08 1080.6 2159.09 1080.05 720.37 SP369
Ac-LAF$r8EYWAQL$AANleA-NH2 378 1799 901.05 1800.01 900.51 600.67
SP370 Ac-ATF$r8EYWAQL$AANleA-NH2 379 1786.97 895.03 1787.98 894.49
596.66 SP371 Ac-AAF$r8EYWAQL$AANleA-NH2 380 1756.96 880.05 1757.97
879.49 586.66 SP372 Ac-AAAF$r8EYWAQL$AANleA-NH2 381 1827.99 915.57
1829 915 610.34 SP373 Ac-AAAAF$r8EYWAQL$AANleA-NH2 382 1899.03
951.09 1900.04 950.52 634.02 SP374 Ac-AATF$r8EYWAQL$AANleA-NH2 383
1858 930.92 1859.01 930.01 620.34 SP375
Ac-AALTF$r8EYWAQL$AANleA-NH2 384 1971.09 987.17 1972.1 986.55
658.04 SP376 Ac-AAALTF$r8EYWAQL$AANleA-NH2 385 2042.12 1023.15
2043.13 1022.07 681.71 SP377 Ac-LTF$r8EYWAQL$AANleAA-NH2 386
1900.05 952.02 1901.06 951.03 634.36 SP378
Ac-ALTF$r8EYWAQL$AANleAA-NH2 387 1971.09 987.63 1972.1 986.55
658.04 SP379 Ac-AALTF$r8EYWAQL$AANleAA-NH2 388 2042.69 1022.69
2043.13 1022.07 681.71 SP380 Ac-LTF$r8EYWAQCba$AANleAA-NH2 389
1912.05 958.03 1913.06 957.03 638.36 SP381
Ac-LTF$r8EYWAQhL$AANleAA-NH2 390 1914.07 958.68 1915.08 958.04
639.03 SP382 Ac-ALTF$r8EYWAQhL$AANleAA-NH2 391 1985.1 994.1 1986.11
993.56 662.71 SP383 Ac-LTF$r8ANmYWAQL$AANleA-NH2 392 1785.02 894.11
1786.03 893.52 596.01 SP384 Ac-LTF$r8ANmYWAQL$AANleA-NH2 393 iso2
1785.02 894.11 1796.03 893.52 596.01 SP385
Ac-LTF$r8AYNmWAQL$AANleA-NH2 394 1785.02 941.11 1786.03 893.52
596.01 SP386 Ac-LTF$r8AYNmWAQL$AANleA-NH2 395 iso2 1785.02 894.11
1786.03 893.52 596.01 SP387 Ac-LTF$r8AYAmwAQL$AANleA-NH2 396
1785.02 894.01 1786.03 893.52 596.01 SP388
Ac-LTF$r8AYAmwAQL$AANleA-NH2 397 iso2 1785.02 894.01 1786.03 893.52
596.01 SP389 Ac-LTF$r8AYWAibQL$AANleA-NH2 398 1785.02 894.01
1786.03 893.52 596.01 SP390 Ac-LTF$r8AYWAibQL$AANleA-NH2 399 iso2
1785.02 894.01 1786.03 893.52 596.01 SP391
Ac-LTF$r8AYWAQL$AAibNleA-NH2 400 1785.02 894.38 1786.03 893.52
596.01 SP392 Ac-LTF$r8AYWAQL$AAibNleA-NH2 401 iso2 1785.02 894.38
1786.03 893.52 596.01 SP393 Ac-LTF$r8AYWAQL$AaNleA-NH2 402 1771.01
887.54 1772.02 886.51 591.34 SP394 Ac-LTF$r8AYWAQL$AaNleA-NH2 403
iso2 1771.01 887.54 1772.02 886.51 591.34 SP395
Ac-LTF$r8AYWAQL$ASarNleA-NH2 404 1771.01 887.35 1772.02 886.51
591.34 SP396 Ac-LTF$r8AYWAQL$ASarNleA-NH2 405 iso2 1771.01 887.35
1772.02 886.51 591.34 SP397 Ac-LTF$r8AYWAQL$AANleAib-NH2 406
1785.02 894.75 1786.03 893.52 596.01 SP398
Ac-LTF$r8AYWAQL$AANleAib-NH2 407 iso2 1785.02 894.75 1786.03 893.52
596.01 SP399 Ac-LTF$r8AYWAQL$AANleNmA-NH2 408 1785.02 894.6 1786.03
893.52 596.01 SP400 Ac-LTF$r8AYWAQL$AANleNmA-NH2 409 iso2 1785.02
894.6 1786.03 893.52 596.01 SP401 Ac-LTF$r8AYWAQL$AANleSar-NH2 410
1771.01 886.98 1772.02 886.51 591.34 SP402
Ac-LTF$r8AYWAQL$AANleSar-NH2 411 iso2 1771.01 886.98 1772.02 886.51
591.34 SP403 Ac-LTF$r8AYWAQL$AANleAAib-NH2 412 1856.06 1857.07
929.04 619.69 SP404 Ac-LTF$r8AYWAQL$AANleAAib-NH2 413 iso2 1856.06
1857.07 929.04 619.69 SP405 Ac-LTF$r8AYWAQL$AANleANmA-NH2 414
1856.06 930.37 1857.07 929.04 619.69 SP406
Ac-LTF$r8AYWAQL$AANleANmA-NH2 415 iso2 1856.06 930.37 1857.07
929.04 619.69 SP407 Ac-LTF$r8AYWAQL$AANleAa-NH2 416 1842.04 922.69
1843.05 922.03 615.02 SP408 Ac-LTF$r8AYWAQL$AANleAa-NH2 417 iso2
1842.04 922.69 1843.05 922.03 615.02 SP409
Ac-LTF$r8AYWAQL$AANleASar-NH2 418 1842.04 922.6 1843.05 922.03
615.02 SP410 Ac-LTF$r8AYWAQL$AANleASar-NH2 419 iso2 1842.04 922.6
1843.05 922.03 615.02 SP411 Ac-LTF$/r8AYWAQL$/AANleA-NH2 420
1799.04 901.14 1800.05 900.53 600.69 SP412
Ac-LTFAibAYWAQLAibAANleA-NH2 421 1648.9 826.02 1649.91 825.46
550.64 SP413 Ac-LTF$r8Cou4YWAQL$AANleA-NH2 422 1975.05 989.11
1976.06 988.53 659.36 SP414 Ac-LTF$r8Cou4YWAQL$AANleA-NH2 423 iso2
1975.05 989.11 1976.06 988.53 659.36 SP415
Ac-LTF$r8AYWCou4QL$AANleA-NH2 424 1975.05 989.11 1976.06 988.53
659.36 SP416 Ac-LTF$r8AYWAQL$Cou4ANleA-NH2 425 1975.05 989.57
1976.06 988.53 659.36 SP417 Ac-LTF$r8AYWAQL$Cou4ANleA-NH2 426 iso2
1975.05 989.57 1976.06 988.53 659.36 SP418
Ac-LTF$r8AYWAQL$ACou4NleA-NH2 427 1975.05 989.57 1976.06 988.53
659.36 SP419 Ac-LTF$r8AYWAQL$ACou4NleA-NH2 428 iso2 1975.05 989.57
1976.06 988.53 659.36 SP420 Ac-LTF$r8AYWAQL$AANleA-OH 429 1771.99
887.63 1773 887 591.67 SP421 Ac-LTF$r8AYWAQL$AANleA-OH 430 iso2
1771.99 887.63 1773 887 591.67 SP422 Ac-LTF$r8AYWAQL$AANleA-NHnPr
431 1813.05 908.08 1814.06 907.53 605.36 SP423
Ac-LTF$r8AYWAQL$AANleA-NHnPr 432 iso2 1813.05 908.08 1814.06 907.53
605.36 SP424 Ac-LTF$r8AYWAQL$AANleA-NHnBu33Me 433 1855.1 929.17
1856.11 928.56 619.37 SP425 Ac-LTF$r8AYWAQL$AANleA-NHnBu33Me 434
iso2 1855.1 929.17 1856.11 928.56 619.37 SP426
Ac-LTF$r8AYWAQL$AANleA-NHHex 435 1855.1 929.17 1856.11 928.56
619.37 SP427 Ac-LTF$r8AYWAQL$AANleA-NHHex 436 iso2 1855.1 929.17
1856.11 928.56 619.37 SP428 Ac-LTA$r8AYWAQL$AANleA-NH2 437 1694.98
849.33 1695.99 848.5 566 SP429 Ac-LThL$r8AYWAQL$AANleA-NH2 438
1751.04 877.09 1752.05 876.53 584.69 SP430
Ac-LTF$r8AYAAQL$AANleA-NH2 439 1655.97 829.54 1656.98 828.99 553
SP431 Ac-LTF$r8AY2NalAQL$AANleA-NH2 440 1782.01 892.63 1783.02
892.01 595.01 SP432 Ac-LTF$r8EYWCou4QCba$SAA-NH2 441 1947.97 975.8
1948.98 974.99 650.33 SP433 Ac-LTF$r8EYWCou7QCba$SAA-NH2 442 16.03
974.9 17.04 9.02 6.35 SP434 Ac-LTF%r8EYWAQCba%SAA-NH2 443 1745.94
874.8 1746.95 873.98 582.99 SP435 Dmaac-LTF$r8EYWAQCba$SAA-NH2 444
1786.97 894.8 1787.98 894.49 596.66 SP436
Dmaac-LTF$r8AYWAQL$AAAAAa-NH2 445 1914.08 958.2 1915.09 958.05
639.03 SP437 Dmaac-LTF$r8AYWAQL$AAAAAa-NH2 446 iso2 1914.08 958.2
1915.09 958.05 639.03 SP438 Dmaac-LTF$r8EYWAQL$AAAAAa-NH2 447
1972.08 987.3 1973.09 987.05 658.37 SP493
Dmaac-LTF$r8EYWAQL$AAAAAa-NH2 448 iso2 1972.08 987.3 1973.09 987.05
658.37 SP440 Dmaac-LTF$r8EF4coohWAQCba$AAIa-NH2 449 1912.05 957.4
1913.06 957.03 638.36 SP441 Dmaac-LTF$r8EF4coohWAQCba$AAIa-NH2 450
iso2 1912.05 957.4 1913.06 957.03 638.36 SP442
Dmaac-LTF$r8AYWAQL$AANleA-NH2 451 1814.05 908.3 1815.06 908.03
605.69 SP443 Dmaac-LTF$r8AYWAQL$AANleA-NH2 452 iso2 1814.05 908.3
1815.06 908.03 605.69 SP444 Ac-LTF%r8AYWAQL%AANleA-NH2 453 1773.02
888.37 1774.03 887.52 592.01 SP445 Ac-LTF%r8EYWAQL%AAAAAa-NH2 454
1931.06 966.4 1932.07 966.54 644.69 SP446
Cou6BaLTF$r8EYWAQhL$SAA-NH2 455 2018.05 1009.9 2019.06 1010.03
673.69 SP447 Cou8BaLTF$r8EYWAQhL$SAA-NH2 456 1962.96 982.34 1963.97
982.49 655.32 SP448 Ac-LTF4I$r8EYWAQL$AAAAAa-NH2 457 2054.93
1028.68 2055.94 1028.47 685.98 SP449 Ac-LTF$r8EYWAQL$AAAAAa-NH2 458
1929.04 966.17 1930.05 965.53 644.02 SP550
Ac-LTF$r8EYWAQL$AAAAAa-OH 459 1930.02 966.54 1931.03 966.02 644.35
SP551 Ac-LTF$r8EYWAQL$AAAAAa-OH 460 iso2 1930.02 965.89 1931.03
966.02 644.35 SP552 Ac-LTF$r8EYWAEL$AAAAAa-NH2 461 1930.02 966.82
1931.03 966.02 644.35 SP553 Ac-LTF$r8EYWAEL$AAAAAa-NH2 462 iso2
1930.02 966.91 1931.03 966.02 644.35 SP554
Ac-LTF$r8EYWAEL$AAAAAa-OH 463 1931.01 967.28 1932.02 966.51 644.68
SP555 Ac-LTF$r8EY6clWAQL$AAAAAa-NH2 464 1963 983.28 1964.01 982.51
655.34 SP556 Ac-LTF$r8EF4bOH2WAQL$AAAAAa-NH2 465 1957.05 980.04
1958.06 979.53 653.36 SP557 Ac-AAALTF$r8EYWAQL$AAAAAa-NH2 466
2142.15 1072.83 2143.16 1072.08 715.06 SP558
Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2 467 1965.02 984.3 1966.03 983.52
956.01 SP559 Ac-RTF$r8EYWAQL$AAAAAa-NH2 468 1972.06 987.81 1973.07
987.04 658.36 SP560 Ac-LTA$r8EYWAQL$AAAAAa-NH2 469 1853.01 928.33
1854.02 927.51 618.68 SP561 Ac-LTF$r8EYWAibQL$AAAAAa-NH2 470
1943.06 973.48 1944.07 972.54 648.69 SP562
Ac-LTF$r8EYWAQL$AAibAAAa-NH2 471 1943.06 973.11 1944.07 972.54
648.69 SP563 Ac-LTF$r8EYWAQL$AAAibAAa-NH2 472 1943.06 973.48
1944.07 972.54 648.69 SP564 Ac-LTF$r8EYWAQL$AAAAibAa-NH2 473
1943.06 973.48 1944.07 972.54 648.69 SP565
Ac-LTF$r8EYWAQL$AAAAAiba-NH2 474 1943.06 973.38 1944.07 972.54
648.69 SP566 Ac-LTF$r8EYWAQL$AAAAAiba-NH2 475 iso2 1943.06 973.38
1944.07 972.54 648.69 SP567 Ac-LTF$r8EYWAQL$AAAAAAib-NH2 476
1943.06 973.01 1944.07 972.54 648.69 SP568
Ac-LTF$r8EYWAQL$AaAAAa-NH2 477 1929.04 966.54 1930.05 965.53 644.02
SP569 Ac-LTF$r8EYWAQL$AAaAAa-NH2 478 1929.04 966.35 1930.05 965.53
644.02 SP570 Ac-LTF$r8EYWAQL$AAAaAa-NH2 479 1929.04 966.54 1930.05
965.53 644.02 SP571 Ac-LTF$r8EYWAQL$AAAaAa-NH2 480 iso2 1929.04
966.35 1930.05 965.53 644.02 SP572 Ac-LTF$r8EYWAQL$AAAAaa-NH2 481
1929.04 966.35 1930.05 965.53 644.02 SP573
Ac-LTF$r8EYWAQL$AAAAAA-NH2 482 1929.04 966.35 1930.05 965.53 644.02
SP574 Ac-LTF$r8EYWAQL$ASarAAAa-NH2 483 1929.04 966.54 1930.05
965.53 644.02 SP575 Ac-LTF$r8EYWAQL$AASarAAa-NH2 484 1929.04 966.35
1930.05 965.53 644.02 SP576 Ac-LTF$r8EYWAQL$AAASarAa-NH2 485
1929.04 966.35 1930.05 965.53 644.02 SP577
Ac-LTF$r8EYWAQL$AAAASara-NH2 486 1929.04 966.35 1930.05 965.53
644.02 SP578 Ac-LTF$r8EYWAQL$AAAAASar-NH2 487 1929.04 966.08
1930.05 965.53 644.02 SP579 Ac-7LTF$r8EYWAQL$AAAAAa-NH2 488 1918.07
951.99 1919.08 960.04 640.37 SP581 Ac-TF$r8EYWAQL$AAAAAa-NH2 489
1815.96 929.85 1816.97 908.99 606.33 SP582 Ac-F$r8EYWAQL$AAAAAa-NH2
490 1714.91 930.92 1715.92 858.46 572.64 SP583
Ac-LVF$r8EYWAQL$AAAAAa-NH2 491 1927.06 895.12 1928.07 964.54 643.36
SP584 Ac-AAF$r8EYWAQL$AAAAAa-NH2 492 1856.98 859.51 1857.99 929.5
620 SP585 Ac-LTF$r8EYWAQL$AAAAa-NH2 493 1858 824.08 1859.01 930.01
620.34 SP586 Ac-LTF$r8EYWAQL$AAAa-NH2 494 1786.97 788.56 1787.98
894.49 596.66 SP587 Ac-LTF$r8EYWAQL$AAa-NH2 495 1715.93 1138.57
1716.94 858.97 572.98 SP588 Ac-LTF$r8EYWAQL$Aa-NH2 496 1644.89
1144.98 1645.9 823.45 549.3 SP589 Ac-LTF$r8EYWAQL$a-NH2 497 1573.85
1113.71 1574.86 787.93 525.62 SP590 Ac-LTF$r8EYWAQL$AAA-OH 498
1716.91 859.55 1717.92 859.46 573.31 SP591 Ac-LTF$r8EYWAQL$A-NH2
499 1574.84 975.14 1575.85 788.43 525.95 SP592
Ac-LTF$r8EYWAQL$AAA-NH2 500 1715.93 904.75 1716.94 858.97
572.98
SP593 Ac-LTF$r8EYWAQCba$SAA-OH 501 1744.91 802.49 1745.92 973.46
582.64 SP594 Ac-LTF$r8EYWAQCba$S-OH 502 1602.83 913.53 1603.84
802.42 535.28 SP595 Ac-LTF$r8EYWAQCba$S-NH2 503 1601.85 979.58
1602.86 801.93 534.96 SP596 4-FBzl-LTF$r8EYWAQL$AAAAAa-NH2 504
2009.05 970.52 2010.06 1005.53 670.69 SP597
4-FBzl-LTF$r8EYWAQCba$SAA-NH2 505 1823.93 965.8 1824.94 912.97
608.98 SP598 Ac-LTF$r8RYWAQL$AAAAAa-NH2 506 1956.1 988.28 1957.11
979.06 653.04 SP599 Ac-LTF$r8HYWAQL$AAAAAa-NH2 507 1937.06 1003.54
1938.07 969.54 646.69 SP600 Ac-LTF$r8QYWAQL$AAAAAa-NH2 508 1928.06
993.92 1929.07 965.04 643.69 SP601 Ac-LTF$r8CitYWAQL$AAAAAa-NH2 509
1957.08 987 1958.09 979.55 653.37 SP602
Ac-LTF$r8GlaYWAQL$AAAAAa-NH2 510 1973.03 983 1974.04 987.52 658.68
SP603 Ac-LTF$r8F4gYWAQL$AAAAAa-NH2 511 2004.1 937.86 2005.11
1003.06 669.04 SP604 Ac-LTF$r82mRYWAQL$AAAAAa-NH2 512 1984.13
958.58 1985.14 993.07 662.38 SP605 Ac-LTF$r8ipKYWAQL$AAAAAa-NH2 513
1970.14 944.52 1971.15 986.08 657.72 SP606
Ac-LTF$r8F4NH2YWAQL$AAAAAa-NH2 514 1962.08 946 1963.09 982.05
655.03 SP607 Ac-LTF$r8EYWAAL$AAAAAa-NH2 515 1872.02 959.32 1873.03
937.02 625.01 SP608 Ac-LTF$r8EYWALL$AAAAAa-NH2 516 1914.07 980.88
1915.08 958.04 639.03 SP609 Ac-LTF$r8EYWAAibL$AAAAAa-NH2 517
1886.03 970.61 1887.04 944.02 629.68 SP610
Ac-LTF$r8EYWASL$AAAAAa-NH2 518 1888.01 980.51 1889.02 945.01 630.34
SP611 Ac-LTF$r8EYWANL$AAAAAa-NH2 519 1915.02 1006.41 1916.03 958.52
639.35 SP612 Ac-LTF$r8EYWACitL$AAAAAa-NH2 520 1958.07 1959.08
980.04 653.7 SP613 Ac-LTF$r8EYWAHL$AAAAAa-NH2 521 1938.04 966.24
1939.05 970.03 647.02 SP614 Ac-LTF$r8EYWARL$AAAAAa-NH2 522 1957.08
1958.09 979.55 653.37 SP615 Ac-LTF$r8EpYWAQL$AAAAAa-NH2 523 2009.01
2010.02 1005.51 670.68 SP616 Cbm-LTF$r8EYWAQCba$SAA-NH2 524 1590.85
1591.86 796.43 531.29 SP617 Cbm-LTF$r8EYWAQL$AAAAA-NH2 525 1930.04
1931.05 966.03 644.35 SP618 Ac-LTF$r8EYWAQL$SAAAAa-NH2 526 1945.04
1005.11 1946.05 973.53 649.35 SP619 Ac-LTF$r8EYWAQL$AAAASa-NH2 527
1945.04 986.52 1946.05 973.53 649.35 SP620
Ac-LTF$r8EYWAQL$SAAASa-NH2 528 1961.03 993.27 1962.04 981.52 654.68
SP621 Ac-LTF$r8EYWAQTba$AAAAAa-NH2 529 1943.06 983.1 1944.07 972.54
648.69 SP622 Ac-LTF$r8EYWAQAdm$AAAAAa-NH2 530 2007.09 990.31 2008.1
1004.55 670.04 SP623 Ac-LTF$r8EYWAQCha$AAAAAa-NH2 531 1969.07
987.17 1970.08 985.54 657.36 SP624 Ac-LTF$r8EYWAQhCha$AAAAAa-NH2
532 1983.09 1026.11 1984.1 992.55 662.04 SP625
Ac-LTF$r8EYWAQF$AAAAAa-NH2 533 1963.02 957.01 1964.03 982.52 655.35
SP626 Ac-LTF$r8EYWAQhF$AAAAAa-NH2 534 1977.04 1087.81 1978.05
989.53 660.02 SP627 Ac-LTF$r8EYWAQL$AANleAAa-NH2 535 1971.09 933.45
1972.1 986.55 658.04 SP628 Ac-LTF$r8EYWAQAdm$AANleAAa-NH2 536
2049.13 1017.97 2050.14 1025.57 684.05 SP629
4-FBz-BaLTF$r8EYWAQL$AAAAAa-NH2 537 2080.08 2081.09 1041.05 694.37
SP630 4-FBz-BaLTF$r8EYWAQCba$SAA-NH2 538 1894.97 1895.98 948.49
632.66 SP631 Ac-LTF$r5EYWAQL$s8AAAAAa-NH2 539 1929.04 1072.68
1930.05 965.53 644.02 SP632 Ac-LTF$r5EYWAQCba$s8SAA-NH2 540 1743.92
1107.79 1744.93 872.97 582.31 SP633 Ac-LTF$r8EYWAQL$AAhhLAAa-NH2
541 1999.12 2000.13 1000.57 667.38 SP634
Ac-LTF$r8EYWAQL$AAAAAAAa-NH2 542 2071.11 2072.12 1036.56 691.38
SP635 Ac-LTF$r8EYWAQL$AAAAAAAAa-NH2 543 2142.15 778.1 2143.16
1072.08 715.06 SP636 Ac-LTF$r8EYWAQL$AAAAAAAAAa-NH2 544 2213.19
870.53 2214.2 1107.6 738.74 SP637 Ac-LTA$r8EYAAQCba$SAA-NH2 545
1552.85 1553.86 777.43 518.62 SP638 Ac-LTA$r8EYAAQL$AAAAAa-NH2 546
1737.97 779.45 1738.98 869.99 580.33 SP639
Ac-LTF$r8EPmpWAQL$AAAAAa-NH2 547 2007.03 779.54 2008.04 1004.52
670.02 SP640 Ac-LTF$r8EPmpWAQCba$SAA-NH2 548 1821.91 838.04 1822.92
911.96 608.31 SP641 Ac-ATF$r8HYWAQL$S-NH2 549 1555.82 867.83
1556.83 778.92 519.61 SP642 Ac-LTF$r8HAWAQL$S-NH2 550 1505.84
877.91 1506.85 753.93 502.95 SP643 Ac-LTF$r8HYWAQA$S-NH2 551
1555.82 852.52 1556.83 778.92 519.61 SP644 Ac-LTF$r8EYWAQCba$SA-NH2
552 1672.89 887.18 1673.9 837.45 558.64 SP645
Ac-LTF$r8EYWAQL$SAA-NH2 553 1731.92 873.32 1732.93 866.97 578.31
SP646 Ac-LTF$r8HYWAQCba$SAA-NH2 554 1751.94 873.05 1752.95 876.98
584.99 SP647 Ac-LTF$r8SYWAQCba$SAA-NH2 555 1701.91 844.88 1702.92
851.96 568.31 SP648 Ac-LTF$r8RYWAQCba$SAA-NH2 556 1770.98 865.58
1771.99 886.5 591.33 SP649 Ac-LTF$r8KYWAQCba$SAA-NH2 557 1742.98
936.57 1743.99 872.5 582 SP650 Ac-LTF$r8QYWAQCba$SAA-NH2 558
1742.94 930.93 1743.95 872.48 581.99 SP651
Ac-LTF$r8EYWAACba$SAA-NH2 559 1686.9 1032.45 1687.91 844.46 563.31
SP652 Ac-LTF$r8EYWAQCba$AAA-NH2 560 1727.93 895.46 1728.94 864.97
576.98 SP653 Ac-LTF$r8EYWAQL$AAAAA-OH 561 1858.99 824.54 1860 930.5
620.67 SP654 Ac-LTF$r8EYWAQL$AAAA-OH 562 1787.95 894.48 1788.96
894.98 596.99 SP655 Ac-LTF$r8EYWAQL$AA-OH 563 1645.88 856 1646.89
823.95 549.63 SP656 Ac-LTF$r8AF4bOH2WAQL$AAAAAa-NH2 564 SP657
Ac-LTF$r8AF4bOH2WAAL$AAAAAa-NH2 565 SP658
Ac-LTF$r8EF4bOH2WAQCba$SAA-NH2 566 SP659
Ac-LTF$r8ApYWAQL$AAAAAa-NH2 567 SP660 Ac-LTF$r8ApYWAAL$AAAAAa-NH2
568 SP661 Ac-LTF$r8EpYWAQCba$SAA-NH2 569 SP662
Ac-LTF$rda6AYWAQL$da5AAAAAa-NH2 570 1974.06 934.44 SP663
Ac-LTF$rda6EYWAQCba$da5SAA-NH2 571 1846.95 870.52 869.94 SP664
Ac-LTF$rda6EYWAQL$da5AAAAAa-NH2 572 SP665
Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2 573 935.57 935.51 SP666
Ac-LTF$ra9EYWAQL$a6AAAAAa-NH2 574 SP667
Ac-LTF$ra9EYWAQCba$a6SAA-NH2 575 SP668 Ac-LTA$ra9EYWAQCba$a6SAA-NH2
576 SP669 5-FAM-BaLTF$ra9EYWAQCba$a6SAA-NH2 577 SP670
5-FAM-BaLTF$r8EYWAQL$AAAAAa-NH2 578 2316.11 SP671
5-FAM-BaLTF$/r8EYWAQL$/AAAAAa-NH2 579 2344.15 SP672
5-FAM-BaLTA$r8EYWAQL$AAAAAa-NH2 580 2240.08 SP673
5-FAM-BaLTF$r8AYWAQL$AAAAAa-NH2 581 2258.11 SP674
5-FAM-BaATF$r8EYWAQL$AAAAAa-NH2 582 2274.07 SP675
5-FAM-BaLAF$r8EYWAQL$AAAAAa-NH2 583 2286.1 SP676
5-FAM-BaLTF$r8EAWAQL$AAAAAa-NH2 584 2224.09 SP677
5-FAM-BaLTF$r8EYAAQL$AAAAAa-NH2 585 2201.07 SP678
5-FAM-BaLTA$r8EYAAQL$AAAAAa-NH2 586 2125.04 SP679
5-FAM-BaLTF$r8EYWAAL$AAAAAa-NH2 587 2259.09 SP680
5-FAM-BaLTF$r8EYWAQA$AAAAAa-NH2 588 2274.07 SP681
5-FAM-BaLTF$/r8EYWAQCba$/SAA-NH2 589 2159.03 SP682
5-FAM-BaLTA$r8EYWAQCba$SAA-NH2 590 2054.97 SP683
5-FAM-BaLTF$r8EYAAQCba$SAA-NH2 591 2015.96 SP684
5-FAM-BaLTA$r8EYAAQCba$SAA-NH2 592 1939.92 SP685
5-FAM-BaQSQQTF$r8NLWRLL$QN-NH2 593 2495.23 SP686
5-TAMRA-BaLTF$r8EYWAQCba$SAA-NH2 594 2186.1 SP687
5-TAMRA-BaLTA$r8EYWAQCba$SAA-NH2 595 2110.07 SP688
5-TAMRA-BaLTF$r8EYAAQCba$SAA-NH2 596 2071.06 SP689
5-TAMRA-BaLTA$r8EYAAQCba$SAA-NH2 597 1995.03 SP690
5-TAMRA-BaLTF$/r8EYWAQCba$/SAA-NH2 598 2214.13 SP691
5-TAMRA-BaLTF$r8EYWAQL$AAAAAa-NH2 599 2371.22 SP692
5-TAMRA-BaLTA$r8EYWAQL$AAAAAa-NH2 600 2295.19 SP693
5-TAMRA-BaLTF$/r8EYWAQL$/AAAAAa-NH2 601 2399.25 SP694
Ac-LTF$r8EYWCou7QCba$SAA-OH 602 1947.93 SP695
Ac-LTF$r8EYWCou7QCba$S-OH 603 1805.86 SP696
Ac-LTA$r8EYWCou7QCba$SAA-NH2 604 1870.91 SP697
Ac-LTF$r8EYACou7QCba$SAA-NH2 605 1831.9 SP698
Ac-LTA$r8EYACou7QCba$SAA-NH2 606 1755.87 SP699
Ac-LTF$/r8EYWCou7QCba$/SAA-NH2 607 1974.98 SP700
Ac-LTF$r8EYWCou7QL$AAAAAa-NH2 608 2132.06 SP701
Ac-LTF$/r8EYWCou7QL$/AAAAAa-NH2 609 2160.09 SP702
Ac-LTF$r8EYWCou7QL$AAAAA-OH 610 2062.01 SP703
Ac-LTF$r8EYWCou7QL$AAAA-OH 611 1990.97 SP704
Ac-LTF$r8EYWCou7QL$AAA-OH 612 1919.94 SP705
Ac-LTF$r8EYWCou7QL$AA-OH 613 1848.9 SP706 Ac-LTF$r8EYWCou7QL$A-OH
614 1777.86 SP707 Ac-LTF$r8EYWAQL$AAAASa-NH2 615 iso2 974.4 973.53
SP708 Ac-LTF$r8AYWAAL$AAAAAa-NH2 616 iso2 1814.01 908.82 1815.02
908.01 605.68 SP709 Biotin-BaLTF$r8EYWAQL$AAAAAa-NH2 617 2184.14
1093.64 2185.15 1093.08 729.05 SP710 Ac-LTF$r8HAWAQL$S-NH2 618 iso2
1505.84 754.43 1506.85 753.93 502.95 SP711 Ac-LTF$r8EYWAQCba$SA-NH2
619 iso2 1672.89 838.05 1673.9 837.45 558.64 SP712
Ac-LTF$r8HYWAQCba$SAA-NH2 620 iso2 1751.94 877.55 1752.95 876.98
584.99 SP713 Ac-LTF$r8SYWAQCba$SAA-NH2 621 iso2 1701.91 852.48
1702.92 851.96 568.31 SP714 Ac-LTF$r8RYWAQCba$SAA-NH2 622 iso2
1770.98 887.45 1771.99 886.5 591.33 SP715 Ac-LTF$r8KYWAQCba$SAA-NH2
623 iso2 1742.98 872.92 1743.99 872.5 582 SP716
Ac-LTF$r8EYWAQCba$AAA-NH2 624 iso2 1727.93 865.71 1728.94 864.97
576.98 SP717 Ac-LTF$r8EYWAQL$AAAAAaBaC-NH2 625 2103.09 1053.12
2104.1 1052.55 702.04 SP718 Ac-LTF$r8EYWAQL$AAAAAadPeg4C-NH2 626
2279.19 1141.46 2280.2 1140.6 760.74 SP719
Ac-LTA$r8AYWAAL$AAAAAa-NH2 627 1737.98 870.43 1738.99 870 580.33
SP720 Ac-LTF$r8AYAAAL$AAAAAa-NH2 628 1698.97 851 1699.98 850.49
567.33 SP721 5-FAM-BaLTF$r8AYWAAL$AAAAAa-NH2 629 2201.09 1101.87
2202.1 1101.55 734.7 SP722 Ac-LTA$r8AYWAQL$AAAAAa-NH2 630 1795
898.92 1796.01 898.51 599.34 SP723 Ac-LTF$r8AYAAQL$AAAAAa-NH2 631
1755.99 879.49 1757 879 586.34 SP724
Ac-LTF$rda6AYWAAL$da5AAAAAa-NH2 632 1807.97 1808.98 904.99 603.66
SP725 FITC-BaLTF$r8EYWAQL$AAAAAa-NH2 633 2347.1 1174.49 2348.11
1174.56 783.37 SP726 FITC-BaLTF$r8EYWAQCba$SAA-NH2 634 2161.99
1082.35 2163 1082 721.67 SP733 Ac-LTF$r8EYWAQL$EAAAAa-NH2 635
1987.05 995.03 1988.06 994.53 663.36 SP734
Ac-LTF$r8AYWAQL$EAAAAa-NH2 636 1929.04 966.35 1930.05 965.53 644.02
SP735 Ac-LTF$r8EYWAQL$AAAAAaBaKbio-NH2 637 2354.25 1178.47 2355.26
1178.13 785.76 SP736 Ac-LTF$r8AYWAAL$AAAAAa-NH2 638 1814.01 908.45
1815.02 908.01 605.68 SP737 Ac-LTF$r8AYAAAL$AAAAAa-NH2 639 iso2
1698.97 850.91 1699.98 850.49 567.33 SP738
Ac-LTF$r8AYAAQL$AAAAAa-NH2 640 iso2 1755.99 879.4 1757 879 586.34
SP739 Ac-LTF$r8EYWAQL$EAAAAa-NH2 641 iso2 1987.05 995.21 1988.06
994.53 663.36 SP740 Ac-LTF$r8AYWAQL$EAAAAa-NH2 642 iso2 1929.04
966.08 1930.05 965.53 644.02 SP741 Ac-LTF$r8EYWAQCba$SAAAAa-NH2 643
1957.04 980.04 1958.05 979.53 653.35 SP742
Ac-LTF$r8EYWAQLStAAA$r5AA-NH2 644 2023.12 1012.83 2024.13 1012.57
675.38 SP743 Ac-LTF$r8EYWAQL$A$AAA$A-NH2 645 2108.17 1055.44
2109.18 1055.09 703.73 SP744 Ac-LTF$r8EYWAQL$AA$AAA$A-NH2 646
2179.21 1090.77 2180.22 1090.61 727.41 SP745
Ac-LTF$r8EYWAQL$AAA$AAAA$A-NH2 647 2250.25 1126.69 2251.26 1126.13
751.09 SP746 Ac-AAALTF$r8EYWAQL$AAA-OH 648 1930.02 1931.03 966.02
644.35 SP747 Ac-AAALTF$r8EYWAQL$AAA-NH2 649 1929.04 965.85 1930.05
965.53 644.02 SP748 Ac-AAAALTF$r8EYWAQL$AAA-NH2 650 2000.08 1001.4
2001.09 1001.05 667.7 SP749 Ac-AAAAALTF$r8EYWAQL$AAA-NH2 651
2071.11 1037.13 2072.12 1036.56 691.38 SP750
Ac-AAAAAALTF$r8EYWAQL$AAA-NH2 652 2142.15 2143.16 1072.08 715.06
SP751 Ac-LTF$rda6EYWAQCba$da6SAA-NH2 653 iso2 1751.89 877.36 1752.9
876.95 584.97 SP752 Ac-t$r5wya$r5f4CF3ekllr-NH2 654 844.25 SP753
Ac-tawy$r5nf4CF3e$r5llr-NH2 655 837.03 SP754
Ac-tawya$r5f4CF3ek$r5lr-NH2 656 822.97 SP755
Ac-tawyanf4CF3e$r5llr$r5a-NH2 657 908.35 SP756
Ac-t$s8wyanf4CF3e$r5llr-NH2 658 858.03 SP757
Ac-tawy$s8nf4CF3ekll$r5a-NH2 659 879.86 SP758
Ac-tawya$s8f4CF3ekllr$r5a-NH2 660 936.38 SP759
Ac-tawy$s8naekll$r5a-NH2 661 844.25 SP760
5-FAM-Batawy$s8nf4CF3ekll$r5a-NH2 662 SP761
5-FAM-Batawy$s8naekll$r5a-NH2 663 SP762
Ac-tawy$s8nf4CF3eall$r5a-NH2 664 SP763
Ac-tawy$s8nf4CF3ekll$r5aaaaa-NH2 665 SP764
Ac-tawy$s8nf4CF3eall$r5aaaaa-NH2 666
[0400] Table 1a shows a selection of peptidomimetic
macrocycles.
TABLE-US-00003 TABLE 1a SEQ ID Exact Found Calc Calc Calc SP
Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP244
Ac-LTF$r8EF4coohWAQCba$SANleA-NH2 667 1885 943.59 1886.01 943.51
629.34 SP331 Ac-LTF$r8EYWAQL$AAAAAa-NH2 668 iso2 1929.04 966.08
1930.05 965.53 644.02 SP555 Ac-LTF$r8EY6clWAQL$AAAAAa-NH2 669 1963
983.28 1964.01 982.51 655.34 SP557 Ac-AAALTF$r8EYWAQL$AAAAAa-NH2
670 2142.15 1072.83 2143.16 1072.08 715.06 SP558
Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2 671 1965.02 984.3 1966.03 983.52
656.01 SP562 Ac-LTF$r8EYWAQL$AAibAAAa-NH2 672 1943.06 973.11
1944.07 972.54 648.69 SP564 Ac-LTF$r8EYWAQL$AAAAibAa-NH2 673
1943.06 973.48 1944.07 972.54 648.69 SP566
Ac-LTF$r8EYWAQL$AAAAAiba-NH2 674 iso2 1943.06 973.38 1944.07 972.54
648.69 SP567 Ac-LTF$r8EYWAQL$AAAAAAib-NH2 675 1943.06 973.01
1944.07 972.54 648.69 SP572 Ac-LTF$r8EYWAQL$AAAAaa-NH2 676 1929.04
966.35 1930.05 965.53 644.02 SP573 Ac-LTF$r8EYWAQL$AAAAAA-NH2 677
1929.04 966.35 1930.05 965.53 644.02 SP578
Ac-LTF$r8EYWAQL$AAAAASar-NH2 678 1929.04 966.08 1930.05 965.53
644.02 SP551 Ac-LTF$r8EYWAQL$AAAAAa-OH 679 iso2 1930.02 965.89
1931.03 966.02 644.35 SP662 Ac-LTF$rda6AYWAQL$da5AAAAAa-NH2 680
1974.06 934.44 933.49 SP367 5-FAM-BaLTF$r8EYWAQCba$SAA-NH2 681 2131
1067.09 2132.01 1066.51 711.34 SP349
Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH2 682 iso2 1969.04 986.06 1970.05
985.53 657.35 SP347 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 683 iso2 1941.04
972.55 1942.05 971.53 648.02
[0401] Table 1b shows a further selection of peptidomimetic
macrocycles.
TABLE-US-00004 TABLE 1b SEQ ID Exact Found Calc Calc Calc SP
Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP581
Ac-TF$r8EYWAQL$AAAAAa-NH2 684 1815.96 929.85 1816.97 908.99 606.33
SP582 Ac-F$r8EYWAQL$AAAAAa-NH2 685 1714.91 930.92 1715.92 858.46
572.64 SP583 Ac-LVF$r8EYWAQL$AAAAAa-NH2 686 1927.06 895.12 1928.07
964.54 643.36 SP584 Ac-AAF$r8EYWAQL$AAAAAa-NH2 687 1856.98 859.51
1857.99 929.5 620 SP585 Ac-LTF$r8EYWAQL$AAAAa-NH2 688 1858 824.08
1859.01 930.01 620.34 SP586 Ac-LTF$r8EYWAQL$AAAa-NH2 689 1786.97
788.56 1787.98 894.49 596.66 SP587 Ac-LTF$r8EYWAQL$AAa-NH2 690
1715.93 1138.57 1716.94 858.97 572.98 SP588 Ac-LTF$r8EYWAQL$Aa-NH2
691 1644.89 1144.98 1645.9 823.45 549.3 SP589 Ac-LTF$r8EYWAQL$a-NH2
692 1573.85 1113.71 1574.86 787.93 525.62
[0402] In the sequences shown above and elsewhere, the following
abbreviations are used: "Nle" represents norleucine, "Aib"
represents 2-aminoisobutyric acid, "Ac" represents acetyl, and "Pr"
represents propionyl. Amino acids represented as "$" are alpha-Me
S5-pentenyl-alanine olefin amino acids connected by an all-carbon
crosslinker comprising one double bond. Amino acids represented as
"$r5" are alpha-Me R.sub.5-pentenyl-alanine olefin amino acids
connected by an all-carbon comprising one double bond. Amino acids
represented as "$s8" are alpha-Me S8-octenyl-alanine olefin amino
acids connected by an all-carbon crosslinker comprising one double
bond. Amino acids represented as "$r8" are alpha-Me
R.sub.8-octenyl-alanine olefin amino acids connected by an
all-carbon crosslinker comprising one double bond. "Ahx" represents
an aminocyclohexyl linker. The crosslinkers are linear all-carbon
crosslinker comprising eight or eleven carbon atoms between the
alpha carbons of each amino acid. Amino acids represented as "V"
are alpha-Me S5-pentenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "$/r5" are
alpha-Me R.sub.5-pentenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "$/s8" are
alpha-Me S8-octenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "$/r8" are
alpha-Me R.sub.8-octenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "Amw" are
alpha-Me tryptophan amino acids. Amino acids represented as "Aml"
are alpha-Me leucine amino acids. Amino acids represented as "Amf"
are alpha-Me phenylalanine amino acids. Amino acids represented as
"2ff" are 2-fluoro-phenylalanine amino acids. Amino acids
represented as "3ff" are 3-fluoro-phenylalanine amino acids. Amino
acids represented as "St" are amino acids comprising two
pentenyl-alanine olefin side chains, each of which is crosslinked
to another amino acid as indicated. Amino acids represented as
"St//" are amino acids comprising two pentenyl-alanine olefin side
chains that are not crosslinked. Amino acids represented as "% St"
are amino acids comprising two pentenyl-alanine olefin side chains,
each of which is crosslinked to another amino acid as indicated via
fully saturated hydrocarbon crosslinks. Amino acids represented as
"Ba" are beta-alanine. The lower-case character "e" or "z" within
the designation of a crosslinked amino acid (e.g. "$er8" or "$zr8")
represents the configuration of the double bond (E or Z,
respectively). In other contexts, lower-case letters such as "a" or
"f" represent D amino acids (e.g. D-alanine, or D-phenylalanine,
respectively). Amino acids designated as "NmW" represent
N-methyltryptophan. Amino acids designated as "NmY" represent
N-methyltyrosine. Amino acids designated as "NmA" represent
N-methylalanine. "Kbio" represents a biotin group attached to the
side chain amino group of a lysine residue. Amino acids designated
as "Sar" represent sarcosine. Amino acids designated as "Cha"
represent cyclohexyl alanine. Amino acids designated as "Cpg"
represent cyclopentyl glycine. Amino acids designated as "Chg"
represent cyclohexyl glycine. Amino acids designated as "Cba"
represent cyclobutyl alanine. Amino acids designated as "F41"
represent 4-iodo phenylalanine. "7L" represents N15 isotopic
leucine. Amino acids designated as "F3C1" represent 3-chloro
phenylalanine. Amino acids designated as "F4cooh" represent
4-carboxy phenylalanine Amino acids designated as "F34F2" represent
3,4-difluoro phenylalanine. Amino acids designated as "6clW"
represent 6-chloro tryptophan. Amino acids designated as "$rda6"
represent alpha-Me R.sub.6-hexynyl-alanine alkynyl amino acids,
crosslinked via a dialkyne bond to a second alkynyl amino acid.
Amino acids designated as "$da5" represent alpha-Me
S5-pentynyl-alanine alkynyl amino acids, wherein the alkyne forms
one half of a dialkyne bond with a second alkynyl amino acid. Amino
acids designated as "$ra9" represent alpha-Me
R.sub.9-nonynyl-alanine alkynyl amino acids, crosslinked via an
alkyne metathesis reaction with a second alkynyl amino acid. Amino
acids designated as "$a6" represent alpha-Me S6-hexynyl-alanine
alkynyl amino acids, crosslinked via an alkyne metathesis reaction
with a second alkynyl amino acid. The designation "iso1" or "iso2"
indicates that the peptidomimetic macrocycle is a single
isomer.
[0403] Amino acids designated as "Cit" represent citrulline. Amino
acids designated as "Cou4", "Cou6", "Cou7" and "Cou8",
respectively, represent the following structures:
##STR00035## ##STR00036##
[0404] In some embodiments, a peptidomimetic macrocycle is obtained
in more than one isomer, for example due to the configuration of a
double bond within the structure of the crosslinker (E vs Z). Such
isomers can or can not be separable by conventional chromatographic
methods. In some embodiments, one isomer has improved biological
properties relative to the other isomer. In one embodiment, an E
crosslinker olefin isomer of a peptidomimetic macrocycle has better
solubility, better target affinity, better in vivo or in vitro
efficacy, higher helicity, or improved cell permeability relative
to its Z counterpart. In another embodiment, a Z crosslinker olefin
isomer of a peptidomimetic macrocycle has better solubility, better
target affinity, better in vivo or in vitro efficacy, higher
helicity, or improved cell permeability relative to its E
counterpart.
[0405] Table 1c shows exemplary peptidomimetic macrocycle:
TABLE-US-00005 TABLE 1c Structure SP154 (SEQ ID NO: 163)
##STR00037## SP155 (SEQ ID NO: 124) ##STR00038## SP114 (SEQ ID NO:
123) ##STR00039## SP99 (SEQ ID NO: 108) ##STR00040## SP388 (SEQ ID
NO: 397) ##STR00041## SP331 (SEQ ID NO: 340) ##STR00042## SP445
(SEQ ID NO: 454) ##STR00043## SP351 (SEQ ID NO: 360) ##STR00044##
SP71 (SEQ ID NO: 80) ##STR00045## SP69 (SEQ ID NO: 78) ##STR00046##
SP7 (SEQ ID NO: 16) ##STR00047## SP160 (SEQ ID NO: 169)
##STR00048## SP315 (SEQ ID NO: 324) ##STR00049## SP249 (SEQ ID NO:
258) ##STR00050## SP437 (SEQ ID NO: 446) ##STR00051## SP349 (SEQ ID
NO: 358) ##STR00052## SP555 (SEQ ID NO: 464) ##STR00053## SP557
(SEQ ID NO: 466) ##STR00054## SP558 (SEQ ID NO: 467) ##STR00055##
SP367 (SEQ ID NO: 376) ##STR00056## SP562 (SEQ ID NO: 471)
##STR00057## SP564 (SEQ ID NO: 473) ##STR00058## SP566 (SEQ ID NO:
475) ##STR00059## SP567 (SEQ ID NO: 476) ##STR00060## SP572 (SEQ ID
NO: 480) ##STR00061## SP573 (SEQ ID NO: 482) ##STR00062## SP578
(SEQ ID NO: 487) ##STR00063## SP664 (SEQ ID NO: 572) ##STR00064##
SP662 (SEQ ID NO: 570) ##STR00065## (SEQ ID NO: 1500)
##STR00066##
[0406] In some embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 2a:
TABLE-US-00006 TABLE 2A Number Sequence SEQ ID NO: 1
L$r5QETFSD$s8WKLLPEN 693 2 LSQ$r5TFSDLW$s8LLPEN 694 3
LSQE$r5FSDLWK$s8LPEN 695 4 LSQET$r5SDLWKL$s8PEN 696 5
LSQETF$r5DLWKLL$s8EN 697 6 LXQETFS$r5LWKLLP$s8N 698 7
LSQETFSD$r5WKLLPE$s8 699 8 LSQQTF$r5DLWKLL$s8EN 700 9
LSQETF$r5DLWKLL$s8QN 701 10 LSQQTF$r5DLWKLL$s8QN 702 11
LSQETF$r5NLWKLL$s8QN 703 12 LSQQTF$r5NLWKLL$s8QN 704 13
LSQQTF$r5NLWRLL$s8QN 705 14 QSQQTF$r5NLWKLL$s8QN 706 15
QSQQTF$r5NLWRLL$s8QN 707 16 QSQQTA$r5NLWRLL$s8QN 708 17
L$r8QETFSD$WKLLPEN 709 18 LSQ$r8TFSDLW$LLPEN 710 19
LSQE$r8FSDLWK$LPEN 711 20 LSQET$r8SDLWKL$PEN 712 21
LSQETF$r8DLWKLL$EN 713 22 LXQETFS$r8LWKLLP$N 714 23
LSQETFSD$r8WKLLPE$ 715 24 LSQQTF$r8DLWKLL$EN 716 25
LSQETF$r8DLWKLL$QN 717 26 LSQQTF$r8DLWKLL$QN 718 27
LSQETF$r8NLWKLL$QN 719 28 LSQQTF$r8NLWKLL$QN 720 29
LSQQTF$r8NLWRLL$QN 721 30 QSQQTF$r8NLWKLL$QN 722 31
QSQQTF$r8NLWRLL$QN 723 32 QSQQTA$r8NLWRLL$QN 724 33
QSQQTF$r8NLWRKK$QN 725 34 QQTF$r8DLWRLL$EN 726 35 QQTF$r8DLWRLL$
727 36 LSQQTF$DLW$LL 728 37 QQTF$DLW$LL 729 38 QQTA$r8DLWRLL$EN 730
39 QSQQTF$r5NLWRLL$s8QN 731 (dihydroxylated olefin) 40
QSQQTA$r5NLWRLL$s8QN 732 (dihydroxylated olefin) 41
QSQQTF$r8DLWRLL$QN 733 42 QTF$r8NLWRLL$ 734 43 QSQQTF$NLW$LLPQN 735
44 QS$QTF$NLWRLLPQN 736 45 $TFS$LWKLL 737 46 ETF$DLW$LL 738 47
QTF$NLW$LL 739 48 $SQE$FSNLWKLL 740
[0407] In Table 2a, X represents S or any amino acid. Peptides
shown can comprise an N-terminal capping group such as acetyl or an
additional linker such as beta-alanine between the capping group
and the start of the peptide sequence.
[0408] In some embodiments, peptidomimetic macrocycles do not
comprise a peptidomimetic macrocycle structure as shown in Table
2a.
[0409] In other embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 2b:
TABLE-US-00007 TABLE 2b SEQ Observed ID Exact mass Number Sequenece
NO: Mass M + 2 (m/e) 1 Ac-LSQETF$r8DLWKLL$EN-NH2 741 2068.13
1035.07 1035.36 2 Ac-LSQETF$r8NLWKLL$QN-NH2 742 2066.16 1034.08
1034.31 3 Ac-LSQQTF$r8NLWRLL$QN-NH2 743 2093.18 1047.59 1047.73 4
Ac-QSQQTF$r8NLWKLL$QN-NH2 744 2080.15 1041.08 1041.31 5
Ac-QSQQTF$r8NLWRLL$QN-NH2 745 2108.15 1055.08 1055.32 6
Ac-QSQQTA$r8NLWRLL$QN-NH2 746 2032.12 1017.06 1017.24 7
Ac-QAibQQTF$r8NLWRLL$QN-NH2 747 2106.17 1054.09 1054.34 8
Ac-QSQQTFSNLWRLLPQN-NH2 748 2000.02 1001.01 1001.26 9
Ac-QSQQTF$/r8NLWRLLS/QN-NH2 749 2136.18 1069.09 1069.37 10
Ac-QSQAibTF$r8NLWRLL$QN-NH2 750 2065.15 1033.58 1033.71 11
Ac-QSQQTF$r8NLWRLL$AN-NH2 751 2051.13 1026.57 1026.70 12
Ac-ASQQTF$r8NLWRLL$QN-NH2 752 2051.13 1026.57 1026.90 13
Ac-QSQQTF$r8ALWRLL$QN-NH2 753 2065.15 1033.58 1033.41 14
Ac-QSQETF$r8NLWRLL$QN-NH2 754 2109.14 1055.57 1055.70 15
Ac-RSQQTF$r8NLWRLL$QN-NH2 755 2136.20 1069.10 1069.17 16
Ac-RSQQTF$r8NLWRLL$EN-NH2 756 2137.18 1069.59 1069.75 17
Ac-LSQETFSDLWKLLPEN-NH2 757 1959.99 981.00 981.24 18
Ac-QSQ$TFS$LWRLLPQN-NH2 758 2008.09 1005.05 1004.97 19
Ac-QSQQ$FSN$WRLLPQN-NH2 759 2036.06 1019.03 1018.86 20
Ac-QSQQT$SNL$RLLPQN-NH2 760 1917.04 959.52 959.32 21
Ac-QSQQTF$NLW$LLPQN-NH2 761 2007.06 1004.53 1004.97 22
Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2 762 2310.26 1156.13 1156.52 23
Ac-QSQQTF$r8NLWRLL$RN-NH2 763 2136.20 1069.10 1068.94 24
Ac-QSQRTF$r8NLWRLL$QN-NH2 764 2136.20 1069.10 1068.94 25
Ac-QSQQTF$r8NNleWRLL$QN-NH2 765 2108.15 1055.08 1055.44 26
Ac-QSQQTF$r8NLWRNleL$QN-NH2 766 2108.15 1055.08 1055.84 27
Ac-QSQQTF$r8NLWRLNle$QN-NH2 767 2108.15 1055.08 1055.12 28
Ac-QSQQTY$r8NLWRLL$QN-NH2 768 2124.15 1063.08 1062.92 29
Ac-RAibQQTF$r8NLWRLL$QN-NH2 769 2134.22 1068.11 1068.65 30
Ac-MPRFMDYWEGLN-NH2 770 1598.70 800.35 800.45 31
Ac-RSQQRF$r8NLWRLL$QN-NH2 771 2191.25 1096.63 1096.83 32
Ac-QSQQRF$r8NLWRLL$QN-NH2 772 2163.21 1082.61 1082.87 33
Ac-RAibQQRF$r8NLWRLL$QN-NH2 773 2189.27 1095.64 1096.37 34
Ac-RSQQRF$r8NFWRLL$QN-NH2 774 2225.23 1113.62 1114.37 35
Ac-RSQQRF$r8NYWRLL$QN-NH2 775 2241.23 1121.62 1122.37 36
Ac-RSQQTF$r8NLWQLL$QN-NH2 776 2108.15 1055.08 1055.29 37
Ac-QSQQTF$r8NLWQAmlL$QN-NH2 777 2094.13 1048.07 1048.32 38
Ac-QSQQTF$r8NAmlWRLL$QN-NH2 778 2122.17 1062.09 1062.35 39
Ac-NlePRF$r8DYWEGL$QN-NH2 779 1869.98 935.99 936.20 40
Ac-NlePRF$r8NYWRLL$QN-NH2 780 1952.12 977.06 977.35 41
Ac-RF$r8NLWRLL$Q-NH2 781 1577.96 789.98 790.18 42
Ac-QSQQTF$r8N2ffWRLL$QN-NH2 782 2160.13 1081.07 1081.40 43
Ac-QSQQTF$r8N3ffWRLL$QN-NH2 783 2160.13 1081.07 1081.34 44
Ac-QSQQTF#r8NLWRLL#QN-NH2 784 2080.12 1041.06 1041.34 45
Ac-RSQQTA$r8NLWRLL$QN-NH2 785 2060.16 1031.08 1031.38 46
Ac-QSQQTF%r8NLWRLL%QN-NH2 786 2110.17 1056.09 1056.55 47
HepQSQ$TFSNLWRLLPQN-NH2 787 2051.10 1026.55 1026.82 48
HepQSQ$TF$r8NLWRLL$QN-NH2 788 2159.23 1080.62 1080.89 49
Ac-QSQQTF$r8NL6clWRLL$QN-NH2 789 2142.11 1072.06 1072.35 50
Ac-QSQQTF$r8NLMe6clwRLL$QN-NH2 790 2156.13 1079.07 1079.27 51
Ac-LTFEHYWAQLTS-NH2 791 1535.74 768.87 768.91 52
Ac-LTF$HYWSQLTS-NH2 792 1585.83 793.92 794.17 53
Ac-LTFE$YWA$LTS-NH2 793 1520.79 761.40 761.67 54
Ac-LTF$zr8HYWAQL$zS-NH2 794 1597.87 799.94 800.06 55
Ac-LTF$r8HYWRQL$S-NH2 795 1682.93 842.47 842.72 56
Ac-QS$QTFStNLWRLL$s8QN-NH2 796 2145.21 1073.61 1073.90 57
Ac-QSQQTASNLWRLLPQN-NH2 797 1923.99 963.00 963.26 58
Ac-QSQQTA$/r8NLWRLL$/QN-NH2 798 2060.15 1031.08 1031.24 59
Ac-ASQQTF$/r8NLWRLL$/QN-NH2 799 2079.16 1040.58 1040.89 60
Ac-$SQQ$FSNLWRLLAibQN-NH2 800 2009.09 1005.55 1005.86 61
Ac-QS$QTF$NLWRLLAibQN-NH2 801 2023.10 1012.55 1012.79 62
Ac-QSQQ$FSN$WRLLAibQN-NH2 802 2024.06 1013.03 1013.31 63
Ac-QSQQTF$NLW$LLAibQN-NH2 803 1995.06 998.53 998.87 64
Ac-QSQQTFS$LWR$LAibQN-NH2 804 2011.06 1006.53 1006.83 65
Ac-QSQQTFSNLW$LLA$N-NH2 805 1940.02 971.01 971.29 66
Ac-$/SQQ$/FSNLWRLLAibQN-NH2 806 2037.12 1019.56 1019.78 67
Ac-QS$/QTFS/NLWRLLAibQN-NH2 807 2051.13 1026.57 1026.90 68
Ac-QSQQS/FSNS/WRLLAibQN-NH2 808 2052.09 1027.05 1027.36 69
Ac-QSQQTFS/NLW$/LLAibQN-NH2 809 2023.09 1012.55 1013.82 70
Ac-QSQ$TFS$LWRLLAibQN-NH2 810 1996.09 999.05 999.39 71
Ac-QSQ$/TFS$/LWRLLAibQN-NH2 811 2024.12 1013.06 1013.37 72
Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2 812 2201.27 1101.64 1102.00 73
Ac-$r8SQQTFS$LWRLLAibQN-NH2 813 2038.14 1020.07 1020.23 74
Ac-QSQ$r8TFSNLW$LLAibQN-NH2 814 1996.08 999.04 999.32 75
Ac-QSQQTFS$r8LWRLLA$N-NH2 815 2024.12 1013.06 1013.37 76
Ac-QS$r5QTFStNLW$LLAibQN-NH2 816 2032.12 1017.06 1017.39 77
Ac-$/r8SQQTFSS/LWRLLAibQN-NH2 817 2066.17 1034.09 1034.80 78
Ac-QSQ$/r8TFSNLM/LLAibQN-NH2 818 2024.11 1013.06 1014.34 79
Ac-QSQQTFS$/r8LWRLLA$/N-NH2 819 2052.15 1027.08 1027.16 80
Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2 820 2088.18 1045.09 1047.10 81
Ac-QSQQTFSNLWRLLAibQN-NH2 821 1988.02 995.01 995.31 82
Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2 822 2215.29 1108.65 1108.93 83
Ac-ASQQTF$r8NLRWLL$QN-NH2 823 2051.13 1026.57 1026.90 84
Ac-QSQQTF$/r8NLWRLL$/Q-NH2 824 2022.14 1012.07 1012.66 85
Ac-QSQQTF$r8NLWRLL$Q-NH2 825 1994.11 998.06 998.42 86
Ac-AAARAA$r8AAARAA$AA-NH2 826 1515.90 758.95 759.21 87
Ac-LTFEHYWAQLTSA-NH2 827 1606.78 804.39 804.59 88
Ac-LTF$r8HYWAQL$SA-NH2 828 1668.90 835.45 835.67 89
Ac-ASQQTFSNLWRLLPQN-NH2 829 1943.00 972.50 973.27 90
Ac-QS$QTFStNLW$r5LLAibQN-NH2 830 2032.12 1017.06 1017.30 91
Ac-QSQQTFAibNLWRLLAibQN-NH2 831 1986.04 994.02 994.19 92
Ac-QSQQTFNleNLWRLLNleQN-NH2 832 2042.11 1022.06 1022.23 93
Ac-QSQQTF$/r8NLWRLLAibQN-NH2 833 2082.14 1042.07 1042.23 94
Ac-QSQQTF$/r8NLWRLLNleQN-NH2 834 2110.17 1056.09 1056.29 95
Ac-QSQQTFAibNLWRLL$/QN-NH2 835 2040.09 1021.05 1021.25 96
Ac-QSQQTFNleNLWRLL$/QN-NH2 836 2068.12 1035.06 1035.31 97
Ac-QSQQTF%r8NL6clWRNleL%QN-NH2 837 2144.13 1073.07 1073.32 98
Ac-QSQQTF%r8NLMe6clWRLL%QN-NH2 838 2158.15 1080.08 1080.31 101
Ac-FNle$YWE$L-NH2 839 1160.63 -- 1161.70 102 Ac-F$r8AYWELL$A-NH2
840 1344.75 -- 1345.90 103 Ac-F$r8AYWQLL$A-NH2 841 1343.76 --
1344.83 104 Ac-NlePRF$r8NYWELL$QN-NH2 842 1925.06 963.53 963.69 105
Ac-NlePRF$r8DYWRLL$QN-NH2 843 1953.10 977.55 977.68 106
Ac-NlePRF$r8NYWRLL$Q-NH2 844 1838.07 920.04 920.18 107
Ac-NlePRF$r8NYWRLL$-NH2 845 1710.01 856.01 856.13 108
Ac-QSQQTF$r8DLWRLL$QN-NH2 846 2109.14 1055.57 1055.64 109
Ac-QSQQTF$r8NLWRLL$EN-NH2 847 2109.14 1055.57 1055.70 110
Ac-QSQQTF$r8NLWRLL$QD-NH2 848 2109.14 1055.57 1055.64 111
Ac-QSQQTF$r8NLWRLL$S-NH2 849 1953.08 977.54 977.60 112
Ac-ESQQTF$r8NLWRLL$QN-NH2 850 2109.14 1055.57 1055.70 113
Ac-LTF$r8NLWRNleL$Q-NH2 851 1635.99 819.00 819.10 114
Ac-LRF$r8NLWRNleL$Q-NH2 852 1691.04 846.52 846.68 115
Ac-QSQQTF$r8NWWRNleL$QN-NH2 853 2181.15 1091.58 1091.64 116
Ac-QSQQTF$r8NLWRNleL$Q-NH2 854 1994.11 998.06 998.07 117
Ac-QTF$r8NLWRNleL$QN-NH2 855 1765.00 883.50 883.59 118
Ac-NlePRF$r8NWWRLL$QN-NH2 856 1975.13 988.57 988.75 119
Ac-NlePRF$r8NWWRLL$A-NH2 857 1804.07 903.04 903.08 120
Ac-TSFAEYWNLLNH2 858 1467.70 734.85 734.90 121
Ac-QTF$r8HWWSQL$S-NH2 859 1651.85 826.93 827.12 122 Ac-FM$YWE$L-NH2
860 1178.58 -- 1179.64 123 Ac-QTFEHWWSQLLS-NH2 861 1601.76 801.88
801.94 124 Ac-QSQQTF$r8NLAmwRLNle$QN-NH2 862 2122.17 1062.09
1062.24 125 Ac-FMAibY6clWEAc3cL-NH2 863 1130.47 -- 1131.53 126
Ac-FNle$Y6clWE$L-NH2 864 1194.59 -- 1195.64 127
Ac-F$zr8AY6clWEAc3cL$z-NH2 865 1277.63 639.82 1278.71 128
Ac-F$r8AY6clWEAc3cL$A-NH2 866 1348.66 -- 1350.72 129
Ac-NlePRF$r8NY6clWRLL$QN-NH2 867 1986.08 994.04 994.64 130
Ac-AF$r8AAWALA$A-NH2 868 1223.71 -- 1224.71 131
Ac-TF$r8AAWRLA$Q-NH2 869 1395.80 698.90 399.04 132
Pr-TF$r8AAWRLA$Q-NH2 870 1409.82 705.91 706.04 133
Ac-QSQQTF%r8NLWRNleL%QN-NH2 871 2110.17 1056.09 1056.22 134
Ac-LTF%r8HYWAQL%SA-NH2 872 1670.92 836.46 836.58 135
Ac-NlePRF%r8NYWRLL%QN-NH2 873 1954.13 978.07 978.19 136
Ac-NlePRF%r8NY6clWRLL%QN-NH2 874 1988.09 995.05 995.68 137
Ac-LTF%r8HY6clWAQL%S-NH2 875 1633.84 817.92 817.93 138
Ac-QS%QTF%StNLWRLL%s8QN-NH2 876 2149.24 1075.62 1075.65 139
Ac-LTF%r8HY6clWRQL%S-NH2 877 1718.91 860.46 860.54 140
Ac-QSQQTF%r8NL6clWRLL%QN-NH2 878 2144.13 1073.07 1073.64 141
Ac-%r8SQQTFS%LWRLLAibQN-NH2 879 2040.15 1021.08 1021.13 142
Ac-LTF%r8HYWAQL%S-NH2 880 1599.88 800.94 801.09 143
Ac-TSF%r8QYWNLL%P-NH2 881 1602.88 802.44 802.58 147
Ac-LTFEHYWAQLTS-NH2 882 1535.74 768.87 769.5 152
Ac-F$er8AY6clWEAc3cL$e-NH2 883 1277.63 639.82 1278.71 153
Ac-AF$r8AAWALA$A-NH2 884 1277.63 639.82 1277.84 154
Ac-TF$r8AAWRLA$Q-NH2 885 1395.80 698.90 699.04 155
Pr-TF$r8AAWRLA$Q-NH2 886 1409.82 705.91 706.04 156
Ac-LTF$er8HYWAQL$eS-NH2 887 1597.87 799.94 800.44 159
Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2 888 2745.30 1373.65 1372.99 160
Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-NH2 889 2669.27 1335.64 1336.09 161
Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2 890 2589.26 1295.63 1296.2 162
Ac-LTF$/r8HYWAQL$/S-NH2 891 1625.90 813.95 814.18 163
Ac-F%r8HY6clWRAc3cL%-NH2 892 1372.72 687.36 687.59 164
Ac-QTF%r8HWWSQL%S-NH2 893 1653.87 827.94 827.94 165
Ac-LTA$r8HYWRQL$S-NH2 894 1606.90 804.45 804.66 166
Ac-Q$r8QQTFSN$WRLLAibQN-NH2 895 2080.12 1041.06 1041.61 167
Ac-QSQQ$r8FSNLWR$LAibQN-NH2 896 2066.11 1034.06 1034.58 168
Ac-F$r8AYWEAc3cL$A-NH2 897 1314.70 658.35 1315.88 169
Ac-F$r8AYWEAc3cL$S-NH2 898 1330.70 666.35 1331.87 170
Ac-F$r8AYWEAc3cL$Q-NH2 899 1371.72 686.86 1372.72 171
Ac-F$r8AYWEAibL$S-NH2 900 1332.71 667.36 1334.83 172
Ac-F$r8AYWEAL$S-NH2 901 1318.70 660.35 1319.73 173
Ac-F$r8AYWEQL$S-NH2 902 1375.72 688.86 1377.53 174
Ac-F$r8HYWEQL$S-NH2 903 1441.74 721.87 1443.48 175
Ac-F$r8HYWAQL$S-NH2 904 1383.73 692.87 1385.38 176
Ac-F$r8HYWAAc3cL$S-NH2 905 1338.71 670.36 1340.82 177
Ac-F$r8HYWRAc3cL$S-NH2 906 1423.78 712.89 713.04 178
Ac-F$r8AYWEAc3cL#A-NH2 907 1300.69 651.35 1302.78 179
Ac-NlePTF%r8NYWRLL%QN-NH2 908 1899.08 950.54 950.56 180
Ac-TF$r8AAWRAL$Q-NH2 909 1395.80 698.90 699.13 181
Ac-TSF%r8HYWAQL%S-NH2 910 1573.83 787.92 787.98 184
Ac-F%r8AY6clWEAc3cL%A-NH2 911 1350.68 676.34 676.91 185
Ac-LTF$r8HYWAQI$S-NH2 912 1597.87 799.94 800.07 186
Ac-LTF$r8HYWAQNle$S-NH2 913 1597.87 799.94 800.07 187
Ac-LTF$r8HYWAQL$A-NH2 914 1581.87 791.94 792.45 188
Ac-LTF$r8HYWAQL$Abu-NH2 915 1595.89 798.95 799.03 189
Ac-LTF$r8HYWAbuQL$S-NH2 916 1611.88 806.94 807.47 190
Ac-LTF$er8AYWAQL$eS-NH2 917 1531.84 766.92 766.96 191
Ac-LAF$r8HYWAQL$S-NH2 918 1567.86 784.93 785.49 192
Ac-LAF$r8AYWAQL$S-NH2 919 1501.83 751.92 752.01 193
Ac-LTF$er8AYWAQL$eA-NH2 920 1515.85 758.93 758.97 194
Ac-LAF$r8AYWAQL$A-NH2 921 1485.84 743.92 744.05 195
Ac-LTF$r8NLWANleL$Q-NH2 922 1550.92 776.46 776.61 196
Ac-LTF$r8NLWANleL$A-NH2 923 1493.90 747.95 1495.6 197
Ac-LTF$r8ALWANleL$Q-NH2 924 1507.92 754.96 755 198
Ac-LAF$r8NLWANleL$Q-NH2 925 1520.91 761.46 761.96 199
Ac-LAF$r8ALWANleL$A-NH2 926 1420.89 711.45 1421.74 200
Ac-A$r8AYWEAc3cL$A-NH2 927 1238.67 620.34 1239.65 201
Ac-F$r8AYWEAc3cL$AA-NH2 928 1385.74 693.87 1386.64 202
Ac-F$r8AYWEAc3cL$Abu-NH2 929 1328.72 665.36 1330.17 203
Ac-F$r8AYWEAc3cL$Nle-NH2 930 1356.75 679.38 1358.22 204
Ac-F$r5AYWEAc3cL$s8A-NH2 931 1314.70 658.35 1315.51 205
Ac-F$AYWEAc3cL$r8A-NH2 932 1314.70 658.35 1315.66 206
Ac-F$r8AYWEAc3cI$A-NH2 933 1314.70 658.35 1316.18 207
Ac-F$r8AYWEAc3cNle$A-NH2 934 1314.70 658.35 1315.66 208
Ac-F$r8AYWEAmlL$A-NH2 935 1358.76 680.38 1360.21 209
Ac-F$r8AYWENleL$A-NH2 936 1344.75 673.38 1345.71 210
Ac-F$r8AYWQAc3cL$A-NH2 937 1313.72 657.86 1314.7 211
Ac-F$r8AYWAAc3cL$A-NH2 938 1256.70 629.35 1257.56 212
Ac-F$r8AYWAbuAc3cL$A-NH2 939 1270.71 636.36 1272.14 213
Ac-F$r8AYWNleAc3cL$A-NH2 940 1298.74 650.37 1299.67 214
Ac-F$r8AbuYWEAc3cL$A-NH2 941 1328.72 665.36 1329.65 215
Ac-F$r8NleYWEAc3cL$A-NH2 942 1356.75 679.38 1358.66 216
5-FAM-BaLTFEHYWAQLTS-NH2 943 1922.82 962.41 962.87 217
5-FAM-BaLTF%r8HYWAQL%S-NH2 944 1986.96 994.48 994.97 218
Ac-LTF$r8HYWAQhL$S-NH2 945 1611.88 806.94 807 219
Ac-LTF$r8HYWAQTle$S-NH2 946 1597.87 799.94 799.97 220
Ac-LTF$r8HYWAQAdm$S-NH2 947 1675.91 838.96 839.09 221
Ac-LTF$r8HYWAQhCha$S-NH2 948 1651.91 826.96 826.98 222
Ac-LTF$r8HYWAQCha$S-NH2 949 1637.90 819.95 820.02 223
Ac-LTF$r8HYWAc6cQL$S-NH2 950 1651.91 826.96 826.98 224
Ac-LTF$r8HYWAc5cQL$S-NH2 951 1637.90 819.95 820.02 225
Ac-LThF$r8HYWAQL$S-NH2 952 1611.88 806.94 807 226
Ac-LTIgl$r8HYWAQL$S-NH2 953 1625.90 813.95 812.99 227
Ac-LTF$r8HYWAQChg$S-NH2 954 1623.88 812.94 812.99 228
Ac-LTF$r8HYWAQF$S-NH2 955 1631.85 816.93 816.99 229
Ac-LTF$r8HYWAQIgl$S-NH2 956 1659.88 830.94 829.94 230
Ac-LTF$r8HYWAQCba$S-NH2 957 1609.87 805.94 805.96 231
Ac-LTF$r8HYWAQCpg$S-NH2 958 1609.87 805.94 805.96 232
Ac-LTF$r8HhYWAQL$S-NH2 959 1611.88 806.94 807 233
Ac-F$r8AYWEAc3chL$A-NH2 960 1328.72 665.36 665.43 234
Ac-F$r8AYWEAc3cTle$A-NH2 961 1314.70 658.35 1315.62 235
Ac-F$r8AYWEAc3cAdm$A-NH2 962 1392.75 697.38 697.47 236
Ac-F$r8AYWEAc3chCha$A-NH2 963 1368.75 685.38 685.34 237
Ac-F$r8AYWEAc3cCha$A-NH2 964 1354.73 678.37 678.38 238
Ac-F$r8AYWEAc6cL$A-NH2 965 1356.75 679.38 679.42 239
Ac-F$r8AYWEAc5cL$A-NH2 966 1342.73 672.37 672.46 240
Ac-hF$r8AYWEAc3cL$A-NH2 967 1328.72 665.36 665.43 241
Ac-Igl$r8AYWEAc3cL$A-NH2 968 1342.73 672.37 671.5 243
Ac-F$r8AYWEAc3cF$A-NH2 969 1348.69 675.35 675.35 244
Ac-F$r8AYWEAc3cIgl$A-NH2 970 1376.72 689.36 688.37 245
Ac-F$r8AYWEAc3cCba$A-NH2 971 1326.70 664.35 664.47 246
Ac-F$r8AYWEAc3cCpg$A-NH2 972 1326.70 664.35 664.39 247
Ac-F$r8AhYWEAc3cL$A-NH2 973 1328.72 665.36 665.43 248
Ac-F$r8AYWEAc3cL$Q-NH2 974 1371.72 686.86 1372.87 249
Ac-F$r8AYWEAibL$A-NH2 975 1316.72 659.36 1318.18 250
Ac-F$r8AYWEAL$A-NH2 976 1302.70 652.35 1303.75 251
Ac-LAF$r8AYWAAL$A-NH2 977 1428.82 715.41 715.49 252
Ac-LTF$r8HYWAAc3cL$S-NH2 978 1552.84 777.42 777.5 253
Ac-NleTF$r8HYWAQL$S-NH2 979 1597.87 799.94 800.04 254
Ac-VTF$r8HYWAQL$S-NH2 980 1583.85 792.93 793.04 255
Ac-FTF$r8HYWAQL$S-NH2 981 1631.85 816.93 817.02 256
Ac-WTF$r8HYWAQL$S-NH2 982 1670.86 836.43 836.85 257
Ac-RTF$r8HYWAQL$S-NH2 983 1640.88 821.44 821.9
258 Ac-KTF$r8HYWAQL$S-NH2 984 1612.88 807.44 807.91 259
Ac-LNleF$r8HYWAQL$S-NH2 985 1609.90 805.95 806.43 260
Ac-LVF$r8HYWAQL$S-NH2 986 1595.89 798.95 798.93 261
Ac-LFF$r8HYWAQL$S-NH2 987 1643.89 822.95 823.38 262
Ac-LWF$r8HYWAQL$S-NH2 988 1682.90 842.45 842.55 263
Ac-LRF$r8HYWAQL$S-NH2 989 1652.92 827.46 827.52 264
Ac-LKF$r8HYWAQL$S-NH2 990 1624.91 813.46 813.51 265
Ac-LTF$r8NleYWAQL$S-NH2 991 1573.89 787.95 788.05 266
Ac-LTF$r8VYWAQL$S-NH2 992 1559.88 780.94 780.98 267
Ac-LTF$r8FYWAQL$S-NH2 993 1607.88 804.94 805.32 268
Ac-LTF$r8WYWAQL$S-NH2 994 1646.89 824.45 824.86 269
Ac-LTF$r8RYWAQL$S-NH2 995 1616.91 809.46 809.51 270
Ac-LTF$r8KYWAQL$S-NH2 996 1588.90 795.45 795.48 271
Ac-LTF$r8HNleWAQL$S-NH2 997 1547.89 774.95 774.98 272
Ac-LTF$r8HVWAQL$S-NH2 998 1533.87 767.94 767.95 273
Ac-LTF$r8HFWAQL$S-NH2 999 1581.87 791.94 792.3 274
Ac-LTF$r8HWWAQL$S-NH2 1000 1620.88 811.44 811.54 275
Ac-LTF$r8HRWAQL$S-NH2 1001 1590.90 796.45 796.52 276
Ac-LTF$r8HKWAQL$S-NH2 1002 1562.90 782.45 782.53 277
Ac-LTF$r8HYWNleQL$S-NH2 1003 1639.91 820.96 820.98 278
Ac-LTF$r8HYWVQL$S-NH2 1004 1625.90 813.95 814.03 279
Ac-LTF$r8HYWFQL$S-NH2 1005 1673.90 837.95 838.03 280
Ac-LTF$r8HYWWQL$S-NH2 1006 1712.91 857.46 857.5 281
Ac-LTF$r8HYWKQL$S-NH2 1007 1654.92 828.46 828.49 282
Ac-LTF$r8HYWANleL$S-NH2 1008 1582.89 792.45 792.52 283
Ac-LTF$r8HYWAVL$S-NH2 1009 1568.88 785.44 785.49 284
Ac-LTF$r8HYWAFL$S-NH2 1010 1616.88 809.44 809.47 285
Ac-LTF$r8HYWAWL$S-NH2 1011 1655.89 828.95 829 286
Ac-LTF$r8HYWARL$S-NH2 1012 1625.91 813.96 813.98 287
Ac-LTF$r8HYWAQL$Nle-NH2 1013 1623.92 812.96 813.39 288
Ac-LTF$r8HYWAQL$V-NH2 1014 1609.90 805.95 805.99 289
Ac-LTF$r8HYWAQL$F-NH2 1015 1657.90 829.95 830.26 290
Ac-LTF$r8HYWAQL$W-NH2 1016 1696.91 849.46 849.5 291
Ac-LTF$r8HYWAQL$R-NH2 1017 1666.94 834.47 834.56 292
Ac-LTF$r8HYWAQL$K-NH2 1018 1638.93 820.47 820.49 293
Ac-Q$r8QQTFSN$WRLLAibQN-NH2 1019 2080.12 1041.06 1041.54 294
Ac-QSQQ$r8FSNLWR$LAibQN-NH2 1020 2066.11 1034.06 1034.58 295
Ac-LT2Pal$r8HYWAQL$S-NH2 1021 1598.86 800.43 800.49 296
Ac-LT3Pal$r8HYWAQL$S-NH2 1022 1598.86 800.43 800.49 297
Ac-LT4Pal$r8HYWAQL$S-NH2 1023 1598.86 800.43 800.49 298
Ac-LTF2CF3$r8HYWAQL$S-NH2 1024 1665.85 833.93 834.01 299
Ac-LTF2CN$r8HYWAQL$S-NH2 1025 1622.86 812.43 812.47 300
Ac-LTF2Me$r8HYWAQL$S-NH2 1026 1611.88 806.94 807 301
Ac-LTF3Cl$r8HYWAQL$S-NH2 1027 1631.83 816.92 816.99 302
Ac-LTF4CF3$r8HYWAQL$S-NH2 1028 1665.85 833.93 833.94 303
Ac-LTF4tBu$r8HYWAQL$S-NH2 1029 1653.93 827.97 828.02 304
Ac-LTF5F$r8HYWAQL$S-NH2 1030 1687.82 844.91 844.96 305
Ac-LTF$r8HY3BthAAQL$S-NH2 1031 1614.83 808.42 808.48 306
Ac-LTF2Br$r8HYWAQL$S-NH2 1032 1675.78 838.89 838.97 307
Ac-LTF4Br$r8HYWAQL$S-NH2 1033 1675.78 838.89 839.86 308
Ac-LTF2Cl$r8HYWAQL$S-NH2 1034 1631.83 816.92 816.99 309
Ac-LTF4Cl$r8HYWAQL$S-NH2 1035 1631.83 816.92 817.36 310
Ac-LTF3CN$r8HYWAQL$S-NH2 1036 1622.86 812.43 812.47 311
Ac-LTF4CN$r8HYWAQL$S-NH2 1037 1622.86 812.43 812.47 312
Ac-LTF34Cl2$r8HYWAQL$S-NH2 1038 1665.79 833.90 833.94 313
Ac-LTF34F2$r8HYWAQL$S-NH2 1039 1633.85 817.93 817.95 314
Ac-LTF35F2$r8HYWAQL$S-NH2 1040 1633.85 817.93 817.95 315
Ac-LTDip$r8HYWAQL$S-NH2 1041 1673.90 837.95 838.01 316
Ac-LTF2F$r8HYWAQL$S-NH2 1042 1615.86 808.93 809 317
Ac-LTF3F$r8HYWAQL$S-NH2 1043 1615.86 808.93 809 318
Ac-LTF4F$r8HYWAQL$S-NH2 1044 1615.86 808.93 809 319
Ac-LTF4I$r8HYWAQL$S-NH2 1045 1723.76 862.88 862.94 320
Ac-LTF3Me$r8HYWAQL$S-NH2 1046 1611.88 806.94 807.07 321
Ac-LTF4Me$r8HYWAQL$S-NH2 1047 1611.88 806.94 807 322
Ac-LT1Nal$r8HYWAQL$S-NH2 1048 1647.88 824.94 824.98 323
Ac-LT2Nal$r8HYWAQL$S-NH2 1049 1647.88 824.94 825.06 324
Ac-LTF3CF3$r8HYWAQL$S-NH2 1050 1665.85 833.93 834.01 325
Ac-LTF4NO2$r8HYWAQL$S-NH2 1051 1642.85 822.43 822.46 326
Ac-LTF3NO2$r8HYWAQL$S-NH2 1052 1642.85 822.43 822.46 327
Ac-LTF$r82ThiYWAQL$S-NH2 1053 1613.83 807.92 807.96 328
Ac-LTF$r8HBipWAQL$S-NH2 1054 1657.90 829.95 830.01 329
Ac-LTF$r8HF4tBuWAQL$S-NH2 1055 1637.93 819.97 820.02 330
Ac-LTF$r8HF4CF3WAQL$S-NH2 1056 1649.86 825.93 826.02 331
Ac-LTF$r8HF4ClWAQL$S-NH2 1057 1615.83 808.92 809.37 332
Ac-LTF$r8HF4MeWAQL$S-NH2 1058 1595.89 798.95 799.01 333
Ac-LTF$r8HF4BrWAQL$S-NH2 1059 1659.78 830.89 830.98 334
Ac-LTF$r8HF4CNWAQL$S-NH2 1060 1606.87 804.44 804.56 335
Ac-LTF$r8HF4NO2WAQL$S-NH2 1061 1626.86 814.43 814.55 336
Ac-LTF$r8H1NalWAQL$S-NH2 1062 1631.89 816.95 817.06 337
Ac-LTF$r8H2NalWAQL$S-NH2 1063 1631.89 816.95 816.99 338
Ac-LTF$r8HWAQL$S-NH2 1064 1434.80 718.40 718.49 339
Ac-LTF$r8HY1NalAQL$S-NH2 1065 1608.87 805.44 805.52 340
Ac-LTF$r8HY2NalAQL$S-NH2 1066 1608.87 805.44 805.52 341
Ac-LTF$r8HYWAQI$S-NH2 1067 1597.87 799.94 800.07 342
Ac-LTF$r8HYWAQNle$S-NH2 1068 1597.87 799.94 800.44 343
Ac-LTF$er8HYWAQL$eA-NH2 1069 1581.87 791.94 791.98 344
Ac-LTF$r8HYWAQL$Abu-NH2 1070 1595.89 798.95 799.03 345
Ac-LTF$r8HYWAbuQL$S-NH2 1071 1611.88 806.94 804.47 346
Ac-LAF$r8HYWAQL$S-NH2 1072 1567.86 784.93 785.49 347
Ac-LTF$r8NLWANleL$Q-NH2 1073 1550.92 776.46 777.5 348
Ac-LTF$r8ALWANleL$Q-NH2 1074 1507.92 754.96 755.52 349
Ac-LAF$r8NLWANleL$Q-NH2 1075 1520.91 761.46 762.48 350
Ac-F$r8AYWAAc3cL$A-NH2 1076 1256.70 629.35 1257.56 351
Ac-LTF$r8AYWAAL$S-NH2 1077 1474.82 738.41 738.55 352
Ac-LVF$r8AYWAQL$S-NH2 1078 1529.87 765.94 766 353
Ac-LTF$r8AYWAbuQL$S-NH2 1079 1545.86 773.93 773.92 354
Ac-LTF$r8AYWNleQL$S-NH2 1080 1573.89 787.95 788.17 355
Ac-LTF$r8AbuYWAQL$S-NH2 1081 1545.86 773.93 773.99 356
Ac-LTF$r8AYWHQL$S-NH2 1082 1597.87 799.94 799.97 357
Ac-LTF$r8AYWKQL$S-NH2 1083 1588.90 795.45 795.53 358
Ac-LTF$r8AYWOQL$S-NH2 1084 1574.89 788.45 788.5 359
Ac-LTF$r8AYWRQL$S-NH2 1085 1616.91 809.46 809.51 360
Ac-LTF$r8AYWSQL$S-NH2 1086 1547.84 774.92 774.96 361
Ac-LTF$r8AYWRAL$S-NH2 1087 1559.89 780.95 780.95 362
Ac-LTF$r8AYWRQL$A-NH2 1088 1600.91 801.46 801.52 363
Ac-LTF$r8AYWRAL$A-NH2 1089 1543.89 772.95 773.03 364
Ac-LTF$r5HYWAQL$s8S-NH2 1090 1597.87 799.94 799.97 365
Ac-LTF$HYWAQL$r8S-NH2 1091 1597.87 799.94 799.97 366
Ac-LTF$r8HYWAAL$S-NH2 1092 1540.84 771.42 771.48 367
Ac-LTF$r8HYWAAbuL$S-NH2 1093 1554.86 778.43 778.51 368
Ac-LTF$r8HYWALL$S-NH2 1094 1582.89 792.45 792.49 369
Ac-F$r8AYWHAL$A-NH2 1095 1310.72 656.36 656.4 370
Ac-F$r8AYWAAL$A-NH2 1096 1244.70 623.35 1245.61 371
Ac-F$r8AYWSAL$A-NH2 1097 1260.69 631.35 1261.6 372
Ac-F$r8AYWRAL$A-NH2 1098 1329.76 665.88 1330.72 373
Ac-F$r8AYWKAL$A-NH2 1099 1301.75 651.88 1302.67 374
Ac-F$r8AYWOAL$A-NH2 1100 1287.74 644.87 1289.13 375
Ac-F$r8VYWEAc3cL$A-NH2 1101 1342.73 672.37 1343.67 376
Ac-F$r8FYWEAc3cL$A-NH2 1102 1390.73 696.37 1392.14 377
Ac-F$r8WYWEAc3cL$A-NH2 1103 1429.74 715.87 1431.44 378
Ac-F$r8RYWEAc3cL$A-NH2 1104 1399.77 700.89 700.95 379
Ac-F$r8KYWEAc3cL$A-NH2 1105 1371.76 686.88 686.97 380
Ac-F$r8ANleWEAc3cL$A-NH2 1106 1264.72 633.36 1265.59 381
Ac-F$r8AVWEAc3cL$A-NH2 1107 1250.71 626.36 1252.2 382
Ac-F$r8AFWEAc3cL$A-NH2 1108 1298.71 650.36 1299.64 383
Ac-F$r8AWWEAc3cL$A-NH2 1109 1337.72 669.86 1338.64 384
Ac-F$r8ARWEAc3cL$A-NH2 1110 1307.74 654.87 655 385
Ac-F$r8AKWEAc3cL$A-NH2 1111 1279.73 640.87 641.01 386
Ac-F$r8AYWVAc3cL$A-NH2 1112 1284.73 643.37 643.38 387
Ac-F$r8AYWFAc3cL$A-NH2 1113 1332.73 667.37 667.43 388
Ac-F$r8AYWWAc3cL$A-NH2 1114 1371.74 686.87 686.97 389
Ac-F$r8AYWRAc3cL$A-NH2 1115 1341.76 671.88 671.94 390
Ac-F$r8AYWKAc3cL$A-NH2 1116 1313.75 657.88 657.88 391
Ac-F$r8AYWEVL$A-NH2 1117 1330.73 666.37 666.47 392
Ac-F$r8AYWEFL$A-NH2 1118 1378.73 690.37 690.44 393
Ac-F$r8AYWEWL$A-NH2 1119 1417.74 709.87 709.91 394
Ac-F$r8AYWERL$A-NH2 1120 1387.77 694.89 1388.66 395
Ac-F$r8AYWEKL$A-NH2 1121 1359.76 680.88 1361.21 396
Ac-F$r8AYWEAc3cL$V-NH2 1122 1342.73 672.37 1343.59 397
Ac-F$r8AYWEAc3cL$F-NH2 1123 1390.73 696.37 1392.58 398
Ac-F$r8AYWEAc3cL$W-NH2 1124 1429.74 715.87 1431.29 399
Ac-F$r8AYWEAc3cL$R-NH2 1125 1399.77 700.89 700.95 400
Ac-F$r8AYWEAc3cL$K-NH2 1126 1371.76 686.88 686.97 401
Ac-F$r8AYWEAc3cL$AV-NH2 1127 1413.77 707.89 707.91 402
Ac-F$r8AYWEAc3cL$AF-NH2 1128 1461.77 731.89 731.96 403
Ac-F$r8AYWEAc3cL$AW-NH2 1129 1500.78 751.39 751.5 404
Ac-F$r8AYWEAc3cL$AR-NH2 1130 1470.80 736.40 736.47 405
Ac-F$r8AYWEAc3cL$AK-NH2 1131 1442.80 722.40 722.41 406
Ac-F$r8AYWEAc3cL$AH-NH2 1132 1451.76 726.88 726.93 407
Ac-LTF2NO2$r8HYWAQL$S-NH2 1133 1642.85 822.43 822.54 408
Ac-LTA$r8HYAAQL$S-NH2 1134 1406.79 704.40 704.5 409
Ac-LTF$r8HYAAQL$S-NH2 1135 1482.82 742.41 742.47 410
Ac-QSQQTF$r8NLWALL$AN-NH2 1136 1966.07 984.04 984.38 411
Ac-QAibQQTF$r8NLWALL$AN-NH2 1137 1964.09 983.05 983.42 412
Ac-QAibQQTF$r8ALWALL$AN-NH2 1138 1921.08 961.54 961.59 413
Ac-AAAATF$r8AAWAAL$AA-NH2 1139 1608.90 805.45 805.52 414
Ac-F$r8AAWRAL$Q-NH2 1140 1294.76 648.38 648.48 415
Ac-TF$r8AAWAAL$Q-NH2 1141 1310.74 656.37 1311.62 416
Ac-TF$r8AAWRAL$A-NH2 1142 1338.78 670.39 670.46 417
Ac-VF$r8AAWRAL$Q-NH2 1143 1393.82 697.91 697.99 418
Ac-AF$r8AAWAAL$A-NH2 1144 1223.71 612.86 1224.67 420
Ac-TF$r8AAWKAL$Q-NH2 1145 1367.80 684.90 684.97 421
Ac-TF$r8AAWOAL$Q-NH2 1146 1353.78 677.89 678.01 422
Ac-TF$r8AAWSAL$Q-NH2 1147 1326.73 664.37 664.47 423
Ac-LTF$r8AAWRAL$Q-NH2 1148 1508.89 755.45 755.49 424
Ac-F$r8AYWAQL$A-NH2 1149 1301.72 651.86 651.96 425
Ac-F$r8AWWAAL$A-NH2 1150 1267.71 634.86 634.87 426
Ac-F$r8AWWAQL$A-NH2 1151 1324.73 663.37 663.43 427
Ac-F$r8AYWEAL$-NH2 1152 1231.66 616.83 1232.93 428
Ac-F$r8AYWAAL$-NH2 1153 1173.66 587.83 1175.09 429
Ac-F$r8AYWKAL$-NH2 1154 1230.72 616.36 616.44 430
Ac-F$r8AYWOAL$-NH2 1155 1216.70 609.35 609.48 431
Ac-F$r8AYWQAL$-NH2 1156 1230.68 616.34 616.44 432
Ac-F$r8AYWAQL$-NH2 1157 1230.68 616.34 616.37 433
Ac-F$r8HYWDQL$S-NH2 1158 1427.72 714.86 714.86 434
Ac-F$r8HFWEQL$S-NH2 1159 1425.74 713.87 713.98 435
Ac-F$r8AYWHQL$S-NH2 1160 1383.73 692.87 692.96 436
Ac-F$r8AYWKQL$S-NH2 1161 1374.77 688.39 688.45 437
Ac-F$r8AYWOQL$S-NH2 1162 1360.75 681.38 681.49 438
Ac-F$r8HYWSQL$S-NH2 1163 1399.73 700.87 700.95 439
Ac-F$r8HWWEQL$S-NH2 1164 1464.76 733.38 733.44 440
Ac-F$r8HWWAQL$S-NH2 1165 1406.75 704.38 704.43 441
Ac-F$r8AWWHQL$S-NH2 1166 1406.75 704.38 704.43 442
Ac-F$r8AWWKQL$S-NH2 1167 1397.79 699.90 699.92 443
Ac-F$r8AWWOQL$S-NH2 1168 1383.77 692.89 692.96 444
Ac-F$r8HWWSQL$S-NH2 1169 1422.75 712.38 712.42 445
Ac-LTF$r8NYWANleL$Q-NH2 1170 1600.90 801.45 801.52 446
Ac-LTF$r8NLWAQL$Q-NH2 1171 1565.90 783.95 784.06 447
Ac-LTF$r8NYWANleL$A-NH2 1172 1543.88 772.94 773.03 448
Ac-LTF$r8NLWAQL$A-NH2 1173 1508.88 755.44 755.49 449
Ac-LTF$r8AYWANleL$Q-NH2 1174 1557.90 779.95 780.06 450
Ac-LTF$r8ALWAQL$Q-NH2 1175 1522.89 762.45 762.45 451
Ac-LAF$r8NYWANleL$Q-NH2 1176 1570.89 786.45 786.5 452
Ac-LAF$r8NLWAQL$Q-NH2 1177 1535.89 768.95 769.03 453
Ac-LAF$r8AYWANleL$A-NH2 1178 1470.86 736.43 736.47 454
Ac-LAF$r8ALWAQL$A-NH2 1179 1435.86 718.93 719.01 455
Ac-LAF$r8AYWAAL$A-NH2 1180 1428.82 715.41 715.41 456
Ac-F$r8AYWEAc3cL$AAib-NH2 1181 1399.75 700.88 700.95 457
Ac-F$r8AYWAQL$AA-NH2 1182 1372.75 687.38 687.78 458
Ac-F$r8AYWAAc3cL$AA-NH2 1183 1327.73 664.87 664.84 459
Ac-F$r8AYWSAc3cL$AA-NH2 1184 1343.73 672.87 672.9 460
Ac-F$r8AYWEAc3cL$AS-NH2 1185 1401.73 701.87 701.84 461
Ac-F$r8AYWEAc3cL$AT-NH2 1186 1415.75 708.88 708.87 462
Ac-F$r8AYWEAc3cL$AL-NH2 1187 1427.79 714.90 714.94 463
Ac-F$r8AYWEAc3cL$AQ-NH2 1188 1442.76 722.38 722.41 464
Ac-F$r8AFWEAc3cL$AA-NH2 1189 1369.74 685.87 685.93 465
Ac-F$r8AWWEAc3cL$AA-NH2 1190 1408.75 705.38 705.39 466
Ac-F$r8AYWEAc3cL$SA-NH2 1191 1401.73 701.87 701.99 467
Ac-F$r8AYWEAL$AA-NH2 1192 1373.74 687.87 687.93 468
Ac-F$r8AYWENleL$AA-NH2 1193 1415.79 708.90 708.94 469
Ac-F$r8AYWEAc3cL$AbuA-NH2 1194 1399.75 700.88 700.95 470
Ac-F$r8AYWEAc3cL$NleA-NH2 1195 1427.79 714.90 714.86 471
Ac-F$r8AYWEAibL$NleA-NH2 1196 1429.80 715.90 715.97 472
Ac-F$r8AYWEAL$NleA-NH2 1197 1415.79 708.90 708.94 473
Ac-F$r8AYWENleL$NleA-NH2 1198 1457.83 729.92 729.96 474
Ac-F$r8AYWEAibL$Abu-NH2 1199 1330.73 666.37 666.39 475
Ac-F$r8AYWENleL$Abu-NH2 1200 1358.76 680.38 680.39 476
Ac-F$r8AYWEAL$Abu-NH2 1201 1316.72 659.36 659.36 477
Ac-LTF$r8AFWAQL$S-NH2 1202 1515.85 758.93 759.12 478
Ac-LTF$r8AWWAQL$S-NH2 1203 1554.86 778.43 778.51 479
Ac-LTF$r8AYWAQI$S-NH2 1204 1531.84 766.92 766.96 480
Ac-LTF$r8AYWAQNle$S-NH2 1205 1531.84 766.92 766.96 481
Ac-LTF$r8AYWAQL$SA-NH2 1206 1602.88 802.44 802.48 482
Ac-LTF$r8AWWAQL$A-NH2 1207 1538.87 770.44 770.89 483
Ac-LTF$r8AYWAQI$A-NH2 1208 1515.85 758.93 759.42 484
Ac-LTF$r8AYWAQNle$A-NH2 1209 1515.85 758.93 759.42 485
Ac-LTF$r8AYWAQL$AA-NH2 1210 1586.89 794.45 794.94 486
Ac-LTF$r8HWWAQL$S-NH2 1211 1620.88 811.44 811.47 487
Ac-LTF$r8HRWAQL$S-NH2 1212 1590.90 796.45 796.52 488
Ac-LTF$r8HKWAQL$S-NH2 1213 1562.90 782.45 782.53 489
Ac-LTF$r8HYWAQL$W-NH2 1214 1696.91 849.46 849.5 491
Ac-F$r8AYWAbuAL$A-NH2 1215 1258.71 630.36 630.5 492
Ac-F$r8AbuYWEAL$A-NH2 1216 1316.72 659.36 659.51 493
Ac-NlePRF%r8NYWRLL%QN-NH2 1217 1954.13 978.07 978.54 494
Ac-TSF%r8HYWAQL%S-NH2 1218 1573.83 787.92 787.98 495
Ac-LTF%r8AYWAQL%S-NH2 1219 1533.86 767.93 768 496
Ac-HTF$r8HYWAQL$S-NH2 1220 1621.84 811.92 811.96 497
Ac-LHF$r8HYWAQL$S-NH2 1221 1633.88 817.94 818.02 498
Ac-LTF$r8HHWAQL$S-NH2 1222 1571.86 786.93 786.94 499
Ac-LTF$r8HYWHQL$S-NH2 1223 1663.89 832.95 832.38 500
Ac-LTF$r8HYWAHL$S-NH2 1224 1606.87 804.44 804.48 501
Ac-LTF$r8HYWAQL$H-NH2 1225 1647.89 824.95 824.98 502
Ac-LTF$r8HYWAQL$S-NHPr 1226 1639.91 820.96 820.98 503
Ac-LTF$r8HYWAQL$S-NHsBu 1227 1653.93 827.97 828.02 504
Ac-LTF$r8HYWAQL$S-NHiBu 1228 1653.93 827.97 828.02 505
Ac-LTF$r8HYWAQL$S-NHBn 1229 1687.91 844.96 844.44 506
Ac-LTF$r8HYWAQL$S-NHPe 1230 1700.92 851.46 851.99 507
Ac-LTF$r8HYWAQL$S-NHChx 1231 1679.94 840.97 841.04 508
Ac-ETF$r8AYWAQL$S-NH2 1232 1547.80 774.90 774.96 509
Ac-STF$r8AYWAQL$S-NH2 1233 1505.79 753.90 753.94 510
Ac-LEF$r8AYWAQL$S-NH2 1234 1559.84 780.92 781.25
511 Ac-LSF$r8AYWAQL$S-NH2 1235 1517.83 759.92 759.93 512
Ac-LTF$r8EYWAQL$S-NH2 1236 1589.85 795.93 795.97 513
Ac-LTF$r8SYWAQL$S-NH2 1237 1547.84 774.92 774.96 514
Ac-LTF$r8AYWEQL$S-NH2 1238 1589.85 795.93 795.9 515
Ac-LTF$r8AYWAEL$S-NH2 1239 1532.83 767.42 766.96 516
Ac-LTF$r8AYWASL$S-NH2 1240 1490.82 746.41 746.46 517
Ac-LTF$r8AYWAQL$E-NH2 1241 1573.85 787.93 787.98 518
Ac-LTF2CN$r8HYWAQL$S-NH2 1242 1622.86 812.43 812.47 519
Ac-LTF3Cl$r8HYWAQL$S-NH2 1243 1631.83 816.92 816.99 520
Ac-LTDip$r8HYWAQL$S-NH2 1244 1673.90 837.95 838.01 521
Ac-LTF$r8HYWAQTle$S-NH2 1245 1597.87 799.94 800.04 522
Ac-F$r8AY6clWEAL$A-NH2 1246 1336.66 669.33 1338.56 523
Ac-F$r8AYdl6brWEAL$A-NH2 1247 1380.61 691.31 692.2 524
Ac-F$r8AYdl6fWEAL$A-NH2 1248 1320.69 661.35 1321.61 525
Ac-F$r8AYdl4mWEAL$A-NH2 1249 1316.72 659.36 659.36 526
Ac-F$r8AYdl5clWEAL$A-NH2 1250 1336.66 669.33 669.35 527
Ac-F$r8AYdl7mWEAL$A-NH2 1251 1316.72 659.36 659.36 528
Ac-LTF%r8HYWAQL%A-NH2 1252 1583.89 792.95 793.01 529
Ac-LTF$r8HCouWAQL$S-NH2 1253 1679.87 840.94 841.38 530
Ac-LTFEHCouWAQLTS-NH2 1254 1617.75 809.88 809.96 531
Ac-LTA$r8HCouWAQL$S-NH2 1255 1603.84 802.92 803.36 532
Ac-F$r8AYWEAL$AbuA-NH2 1256 1387.75 694.88 694.88 533
Ac-F$r8AYWEAI$AA-NH2 1257 1373.74 687.87 687.93 534
Ac-F$r8AYWEANle$AA-NH2 1258 1373.74 687.87 687.93 535
Ac-F$r8AYWEAmlL$AA-NH2 1259 1429.80 715.90 715.97 536
Ac-F$r8AYWQAL$AA-NH2 1260 1372.75 687.38 687.48 537
Ac-F$r8AYWAAL$AA-NH2 1261 1315.73 658.87 658.92 538
Ac-F$r8AYWAbuAL$AA-NH2 1262 1329.75 665.88 665.95 539
Ac-F$r8AYWNleAL$AA-NH2 1263 1357.78 679.89 679.94 540
Ac-F$r8AbuYWEAL$AA-NH2 1264 1387.75 694.88 694.96 541
Ac-F$r8NleYWEAL$AA-NH2 1265 1415.79 708.90 708.94 542
Ac-F$r8FYWEAL$AA-NH2 1266 1449.77 725.89 725.97 543
Ac-LTF$r8HYWAQhL$S-NH2 1267 1611.88 806.94 807 544
Ac-LTF$r8HYWAQAdm$S-NH2 1268 1675.91 838.96 839.04 545
Ac-LTF$r8HYWAQIgl$S-NH2 1269 1659.88 830.94 829.94 546
Ac-F$r8AYWAQL$AA-NH2 1270 1372.75 687.38 687.48 547
Ac-LTF$r8ALWAQL$Q-NH2 1271 1522.89 762.45 762.52 548
Ac-F$r8AYWEAL$AA-NH2 1272 1373.74 687.87 687.93 549
Ac-F$r8AYWENleL$AA-NH2 1273 1415.79 708.90 708.94 550
Ac-F$r8AYWEAibL$Abu-NH2 1274 1330.73 666.37 666.39 551
Ac-F$r8AYWENleL$Abu-NH2 1275 1358.76 680.38 680.38 552
Ac-F$r8AYWEAL$Abu-NH2 1276 1316.72 659.36 659.36 553
Ac-F$r8AYWEAc3cL$AbuA-NH2 1277 1399.75 700.88 700.95 554
Ac-F$r8AYWEAc3cL$NleA-NH2 1278 1427.79 714.90 715.01 555
H-LTF$r8AYWAQL$S-NH2 1279 1489.83 745.92 745.95 556
mdPEG3-LTF$r8AYWAQL$S-NH2 1280 1679.92 840.96 840.97 557
mdPEG7-LTF$r8AYWAQL$S-NH2 1281 1856.02 929.01 929.03 558
Ac-F$r8ApmpEt6clWEAL$A-NH2 1282 1470.71 736.36 788.17 559
Ac-LTF3Cl$r8AYWAQL$S-NH2 1283 1565.81 783.91 809.18 560
Ac-LTF3Cl$r8HYWAQL$A-NH2 1284 1615.83 808.92 875.24 561
Ac-LTF3Cl$r8HYWWQL$S-NH2 1285 1746.87 874.44 841.65 562
Ac-LTF3Cl$r8AYWWQL$S-NH2 1286 1680.85 841.43 824.63 563
Ac-LTF$r8AYWWQL$S-NH2 1287 1646.89 824.45 849.98 564
Ac-LTF$r8HYWWQL$A-NH2 1288 1696.91 849.46 816.67 565
Ac-LTF$r8AYWWQL$A-NH2 1289 1630.89 816.45 776.15 566
Ac-LTF4F$r8AYWAQL$S-NH2 1290 1549.83 775.92 776.15 567
Ac-LTF2F$r8AYWAQL$S-NH2 1291 1549.83 775.92 776.15 568
Ac-LTF3F$r8AYWAQL$S-NH2 1292 1549.83 775.92 785.12 569
Ac-LTF34F2$r8AYWAQL$S-NH2 1293 1567.83 784.92 785.12 570
Ac-LTF35F2$r8AYWAQL$S-NH2 1294 1567.83 784.92 1338.74 571
Ac-F3Cl$r8AYWEAL$A-NH2 1295 1336.66 669.33 705.28 572
Ac-F3Cl$r8AYWEAL$AA-NH2 1296 1407.70 704.85 680.11 573
Ac-F$r8AY6clWEAL$AA-NH2 1297 1407.70 704.85 736.83 574
Ac-F$r8AY6clWEAL$-NH2 1298 1265.63 633.82 784.1 575
Ac-LTF$r8HYWAQLSt/S-NH2 1299 16.03 9.02 826.98 576
Ac-LTF$r8HYWAQL$S-NHsBu 1300 1653.93 827.97 828.02 577
Ac-STF$r8AYWAQL$S-NH2 1301 1505.79 753.90 753.94 578
Ac-LTF$r8AYWAEL$S-NH2 1302 1532.83 767.42 767.41 579
Ac-LTF$r8AYWAQL$E-NH2 1303 1573.85 787.93 787.98 580
mdPEG3-LTF$r8AYWAQL$S-NH2 1304 1679.92 840.96 840.97 581
Ac-LTF$r8AYWAQhL$S-NH2 1305 1545.86 773.93 774.31 583
Ac-LTF$r8AYWAQCha$S-NH2 1306 1571.88 786.94 787.3 584
Ac-LTF$r8AYWAQChg$S-NH2 1307 1557.86 779.93 780.4 585
Ac-LTF$r8AYWAQCba$S-NH2 1308 1543.84 772.92 780.13 586
Ac-LTF$r8AYWAQF$S-NH2 1309 1565.83 783.92 784.2 587
Ac-LTF4F$r8HYWAQhL$S-NH2 1310 1629.87 815.94 815.36 588
Ac-LTF4F$r8HYWAQCha$S-NH2 1311 1655.89 828.95 828.39 589
Ac-LTF4F$r8HYWAQChg$S-NH2 1312 1641.87 821.94 821.35 590
Ac-LTF4F$r8HYWAQCba$S-NH2 1313 1627.86 814.93 814.32 591
Ac-LTF4F$r8AYWAQhL$S-NH2 1314 1563.85 782.93 782.36 592
Ac-LTF4F$r8AYWAQCha$S-NH2 1315 1589.87 795.94 795.38 593
Ac-LTF4F$r8AYWAQChg$S-NH2 1316 1575.85 788.93 788.35 594
Ac-LTF4F$r8AYWAQCba$S-NH2 1317 1561.83 781.92 781.39 595
Ac-LTF3Cl$r8AYWAQhL$S-NH2 1318 1579.82 790.91 790.35 596
Ac-LTF3Cl$r8AYWAQCha$S-NH2 1319 1605.84 803.92 803.67 597
Ac-LTF3Cl$r8AYWAQChg$S-NH2 1320 1591.82 796.91 796.34 598
Ac-LTF3Cl$r8AYWAQCba$S-NH2 1321 1577.81 789.91 789.39 599
Ac-LTF$r8AYWAQhF$S-NH2 1322 1579.84 790.92 791.14 600
Ac-LTF$r8AYWAQF3CF3$S-NH2 1323 1633.82 817.91 818.15 601
Ac-LTF$r8AYWAQF3Me$S-NH2 1324 1581.86 791.93 791.32 602
Ac-LTF$r8AYWAQ1Nal$S-NH2 1325 1615.84 808.92 809.18 603
Ac-LTF$r8AYWAQBip$S-NH2 1326 1641.86 821.93 822.13 604
Ac-LTF$r8FYWAQL$A-NH2 1327 1591.88 796.94 797.33 605
Ac-LTF$r8HYWAQL$S-NHAm 1328 1667.94 834.97 835.92 606
Ac-LTF$r8HYWAQL$S-NHiAm 1329 1667.94 834.97 835.55 607
Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1330 1715.94 858.97 859.79 608
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1331 1681.96 841.98 842.49 610
Ac-LTF$r8HYWAQL$S-NHnPr 1332 1639.91 820.96 821.58 611
Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1333 1707.98 854.99 855.35 612
Ac-LTF$r8HYWAQL$S-NHHex 1334 1681.96 841.98 842.4 613
Ac-LTF$r8AYWAQL$S-NHmdPeg2 1335 1633.91 817.96 818.35 614
Ac-LTF$r8AYWAQL$A-NHmdPeg2 1336 1617.92 809.96 810.3 615
Ac-LTF$r8AYWAQL$A-NHmdPeg4 1337 1705.97 853.99 854.33 616
Ac-F$r8AYdl4mWEAL$A-NH2 1338 1316.72 659.36 659.44 617
Ac-F$r8AYdl5clWEAL$A-NH2 1339 1336.66 669.33 669.43 618
Ac-LThF$r8AYWAQL$S-NH2 1340 1545.86 773.93 774.11 619
Ac-LT2Nal$r8AYWAQL$S-NH2 1341 1581.86 791.93 792.43 620
Ac-LTA$r8AYWAQL$S-NH2 1342 1455.81 728.91 729.15 621
Ac-LTF$r8AYWVQL$S-NH2 1343 1559.88 780.94 781.24 622
Ac-LTF$r8HYWAAL$A-NH2 1344 1524.85 763.43 763.86 623
Ac-LTF$r8VYWAQL$A-NH2 1345 1543.88 772.94 773.37 624
Ac-LTF$r8IYWAQL$S-NH2 1346 1573.89 787.95 788.17 625
Ac-FTF$r8VYWSQL$S-NH2 1347 1609.85 805.93 806.22 626
Ac-ITF$r8FYWAQL$S-NH2 1348 1607.88 804.94 805.2 627
Ac-2NalTF$r8VYWSQL$S-NH2 1349 1659.87 830.94 831.2 628
Ac-ITF$r8LYWSQL$S-NH2 1350 1589.89 795.95 796.13 629
Ac-FTF$r8FYWAQL$S-NH2 1351 1641.86 821.93 822.13 630
Ac-WTF$r8VYWAQL$S-NH2 1352 1632.87 817.44 817.69 631
Ac-WTF$r8WYWAQL$S-NH2 1353 1719.88 860.94 861.36 632
Ac-VTF$r8AYWSQL$S-NH2 1354 1533.82 767.91 768.19 633
Ac-WTF$r8FYWSQL$S-NH2 1355 1696.87 849.44 849.7 634
Ac-FTF$r8IYWAQL$S-NH2 1356 1607.88 804.94 805.2 635
Ac-WTF$r8VYWSQL$S-NH2 1357 1648.87 825.44 824.8 636
Ac-FTF$r8LYWSQL$S-NH2 1358 1623.87 812.94 812.8 637
Ac-YTF$r8FYWSQL$S-NH2 1359 1673.85 837.93 837.8 638
Ac-LTF$r8AY6clWEAL$A-NH2 1360 1550.79 776.40 776.14 639
Ac-LTF$r8AY6clWSQL$S-NH2 1361 1581.80 791.90 791.68 640
Ac-F$r8AY6clWSAL$A-NH2 1362 1294.65 648.33 647.67 641
Ac-F$r8AY6clWQAL$AA-NH2 1363 1406.72 704.36 703.84 642
Ac-LHF$r8AYWAQL$S-NH2 1364 1567.86 784.93 785.21 643
Ac-LTF$r8AYWAQL$S-NH2 1365 1531.84 766.92 767.17 644
Ac-LTF$r8AHWAQL$S-NH2 1366 1505.84 753.92 754.13 645
Ac-LTF$r8AYWAHL$S-NH2 1367 1540.84 771.42 771.61 646
Ac-LTF$r8AYWAQL$H-NH2 1368 1581.87 791.94 792.15 647
H-LTF$r8AYWAQL$A-NH2 1369 1473.84 737.92 737.29 648
Ac-HHF$r8AYWAQL$S-NH2 1370 1591.83 796.92 797.35 649
Ac-aAibWTF$r8VYWSQL$S-NH2 1371 1804.96 903.48 903.64 650
Ac-AibWTF$r8HYWAQL$S-NH2 1372 1755.91 878.96 879.4 651
Ac-AibAWTF$r8HYWAQL$S-NH2 1373 1826.95 914.48 914.7 652
Ac-fWTF$r8HYWAQL$S-NH2 1374 1817.93 909.97 910.1 653
Ac-AibWWTF$r8HYWAQL$S-NH2 1375 1941.99 972.00 972.2 654
Ac-WTF$r8LYWSQL$S-NH2 1376 1662.88 832.44 832.8 655
Ac-WTF$r8NleYWSQL$S-NH2 1377 1662.88 832.44 832.6 656
Ac-LTF$r8AYWSQL$a-NH2 1378 1531.84 766.92 767.2 657
Ac-LTF$r8EYWARL$A-NH2 1379 1601.90 801.95 802.1 658
Ac-LTF$r8EYWAHL$A-NH2 1380 1582.86 792.43 792.6 659
Ac-aTF$r8AYWAQL$S-NH2 1381 1489.80 745.90 746.08 660
Ac-AibTF$r8AYWAQL$S-NH2 1382 1503.81 752.91 753.11 661
Ac-AmfTF$r8AYWAQL$S-NH2 1383 1579.84 790.92 791.14 662
Ac-AmwTF$r8AYWAQL$S-NH2 1384 1618.86 810.43 810.66 663
Ac-NmLTF$r8AYWAQL$S-NH2 1385 1545.86 773.93 774.11 664
Ac-LNmTF$r8AYWAQL$S-NH2 1386 1545.86 773.93 774.11 665
Ac-LSarF$r8AYWAQL$S-NH2 1387 1501.83 751.92 752.18 667
Ac-LGF$r8AYWAQL$S-NH2 1388 1487.82 744.91 745.15 668
Ac-LTNmF$r8AYWAQL$S-NH2 1389 1545.86 773.93 774.2 669
Ac-TF$r8AYWAQL$S-NH2 1390 1418.76 710.38 710.64 670
Ac-ETF$r8AYWAQL$A-NH2 1391 1531.81 766.91 767.2 671
Ac-LTF$r8EYWAQL$A-NH2 1392 1573.85 787.93 788.1 672
Ac-LT2Nal$r8AYWSQL$S-NH2 1393 1597.85 799.93 800.4 673
Ac-LTF$r8AYWAAL$S-NH2 1394 1474.82 738.41 738.68 674
Ac-LTF$r8AYWAQhCha$S-NH2 1395 1585.89 793.95 794.19 675
Ac-LTF$r8AYWAQChg$S-NH2 1396 1557.86 779.93 780.97 676
Ac-LTF$r8AYWAQCba$S-NH2 1397 1543.84 772.92 773.19 677
Ac-LTF$r8AYWAQF3CF3$S-NH2 1398 1633.82 817.91 818.15 678
Ac-LTF$r8AYWAQ1Nal$S-NH2 1399 1615.84 808.92 809.18 679
Ac-LTF$r8AYWAQBip$S-NH2 1400 1641.86 821.93 822.32 680
Ac-LT2Nal$r8AYWAQL$S-NH2 1401 1581.86 791.93 792.15 681
Ac-LTF$r8AYWVQL$S-NH2 1402 1559.88 780.94 781.62 682
Ac-LTF$r8AWWAQL$S-NH2 1403 1554.86 778.43 778.65 683
Ac-FTF$r8VYWSQL$S-NH2 1404 1609.85 805.93 806.12 684
Ac-ITF$r8FYWAQL$S-NH2 1405 1607.88 804.94 805.2 685
Ac-ITF$r8LYWSQL$S-NH2 1406 1589.89 795.95 796.22 686
Ac-FTF$r8FYWAQL$S-NH2 1407 1641.86 821.93 822.41 687
Ac-VTF$r8AYWSQL$S-NH2 1408 1533.82 767.91 768.19 688
Ac-LTF$r8AHWAQL$S-NH2 1409 1505.84 753.92 754.31 689
Ac-LTF$r8AYWAQL$H-NH2 1410 1581.87 791.94 791.94 690
Ac-LTF$r8AYWAHL$S-NH2 1411 1540.84 771.42 771.61 691
Ac-aAibWTF$r8VYWSQL$S-NH2 1412 1804.96 903.48 903.9 692
Ac-AibWTF$r8HYWAQL$S-NH2 1413 1755.91 878.96 879.5 693
Ac-AibAWTF$r8HYWAQL$S-NH2 1414 1826.95 914.48 914.7 694
Ac-fWTF$r8HYWAQL$S-NH2 1415 1817.93 909.97 910.2 695
Ac-AibWWTF$r8HYWAQL$S-NH2 1416 1941.99 972.00 972.7 696
Ac-WTF$r8LYWSQL$S-NH2 1417 1662.88 832.44 832.7 697
Ac-WTF$r8NleYWSQL$S-NH2 1418 1662.88 832.44 832.7 698
Ac-LTF$r8AYWSQL$a-NH2 1419 1531.84 766.92 767.2 699
Ac-LTF$r8EYWARL$A-NH2 1420 1601.90 801.95 802.2 700
Ac-LTF$r8EYWAHL$A-NH2 1421 1582.86 792.43 792.6 701
Ac-aTF$r8AYWAQL$S-NH2 1422 1489.80 745.90 746.1 702
Ac-AibTF$r8AYWAQL$S-NH2 1423 1503.81 752.91 753.2 703
Ac-AmfTF$r8AYWAQL$S-NH2 1424 1579.84 790.92 791.2 704
Ac-AmwTF$r8AYWAQL$S-NH2 1425 1618.86 810.43 810.7 705
Ac-NmLTF$r8AYWAQL$S-NH2 1426 1545.86 773.93 774.1 706
Ac-LNmTF$r8AYWAQL$S-NH2 1427 1545.86 773.93 774.4 707
Ac-LSarF$r8AYWAQL$S-NH2 1428 1501.83 751.92 752.1 708
Ac-TF$r8AYWAQL$S-NH2 1429 1418.76 710.38 710.8 709
Ac-ETF$r8AYWAQL$A-NH2 1430 1531.81 766.91 767.4 710
Ac-LTF$r8EYWAQL$A-NH2 1431 1573.85 787.93 788.2 711
Ac-WTF$r8VYWSQL$S-NH2 1432 1648.87 825.44 825.2 713
Ac-YTF$r8FYWSQL$S-NH2 1433 1673.85 837.93 837.3 714
Ac-F$r8AY6clWSAL$A-NH2 1434 1294.65 648.33 647.74 715
Ac-ETF$r8EYWVQL$S-NH2 1435 1633.84 817.92 817.36 716
Ac-ETF$r8EHWAQL$A-NH2 1436 1563.81 782.91 782.36 717
Ac-ITF$r8EYWAQL$S-NH2 1437 1589.85 795.93 795.38 718
Ac-ITF$r8EHWVQL$A-NH2 1438 1575.88 788.94 788.42 719
Ac-ITF$r8EHWAQL$S-NH2 1439 1563.85 782.93 782.43 720
Ac-LTF4F$r8AYWAQCba$S-NH2 1440 1561.83 781.92 781.32 721
Ac-LTF3Cl$r8AYWAQhL$S-NH2 1441 1579.82 790.91 790.64 722
Ac-LTF3Cl$r8AYWAQCha$S-NH2 1442 1605.84 803.92 803.37 723
Ac-LTF3C$r8AYWAQChg$S-NH2 1443 1591.82 796.91 796.27 724
Ac-LTF3Cl$r8AYWAQCba$S-NH2 1444 1577.81 789.91 789.83 725
Ac-LTF$r8AY6clWSQL$S-NH2 1445 1581.80 791.90 791.75 726
Ac-LTF4F$r8HYWAQhL$S-NH2 1446 1629.87 815.94 815.36 727
Ac-LTF4F$r8HYWAQCba$S-NH2 1447 1627.86 814.93 814.32 728
Ac-LTF4F$r8AYWAQhL$S-NH2 1448 1563.85 782.93 782.36 729
Ac-LTF4F$r8AYWAQChg$S-NH2 1449 1575.85 788.93 788.35 730
Ac-ETF$r8EYWVAL$S-NH2 1450 1576.82 789.41 788.79 731
Ac-ETF$r8EHWAAL$A-NH2 1451 1506.79 754.40 754.8 732
Ac-ITF$r8EYWAAL$S-NH2 1452 1532.83 767.42 767.75 733
Ac-ITF$r8EHWVAL$A-NH2 1453 1518.86 760.43 760.81 734
Ac-ITF$r8EHWAAL$S-NH2 1454 1506.82 754.41 754.8 735
Pam-LTF$r8EYWAQL$S-NH2 1455 1786.07 894.04 894.48 736
Pam-ETF$r8EYWAQL$S-NH2 1456 1802.03 902.02 902.34 737
Ac-LTF$r8AYWLQL$S-NH2 1457 1573.89 787.95 787.39 738
Ac-LTF$r8EYWLQL$S-NH2 1458 1631.90 816.95 817.33 739
Ac-LTF$r8EHWLQL$S-NH2 1459 1605.89 803.95 804.29 740
Ac-LTF$r8VYWAQL$S-NH2 1460 1559.88 780.94 781.34 741
Ac-LTF$r8AYWSQL$S-NH2 1461 1547.84 774.92 775.33 742
Ac-ETF$r8AYWAQL$S-NH2 1462 1547.80 774.90 775.7 743
Ac-LTF$r8EYWAQL$S-NH2 1463 1589.85 795.93 796.33 744
Ac-LTF$r8HYWAQL$S-NHAm 1464 1667.94 834.97 835.37 745
Ac-LTF$r8HYWAQL$S-NHiAm 1465 1667.94 834.97 835.27 746
Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1466 1715.94 858.97 859.42 747
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1467 1681.96 841.98 842.67 748
Ac-LTF$r8HYWAQL$S-NHnBu 1468 1653.93 827.97 828.24 749
Ac-LTF$r8HYWAQL$S-NHnPr 1469 1639.91 820.96 821.31 750
Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1470 1707.98 854.99 855.35 751
Ac-LTF$r8HYWAQL$S-NHHex 1471 1681.96 841.98 842.4 752
Ac-LTF$r8AYWAQL$S-NHmdPeg2 1472 1633.91 817.96 855.35 753
Ac-LTF$r8AYWAQL$A-NHmdPeg2 1473 1617.92 809.96 810.58 754
Ac-LTF$r5AYWAAL$s8S-NH2 1474 1474.82 738.41 738.79 755
Ac-LTF$r8AYWCouQL$S-NH2 1475 1705.88 853.94 854.61 756
Ac-LTF$r8CouYWAQL$S-NH2 1476 1705.88 853.94 854.7 757
Ac-CouTF$r8AYWAQL$S-NH2 1477 1663.83 832.92 833.33 758
H-LTF$r8AYWAQL$A-NH2 1478 1473.84 737.92 737.29 759
Ac-FIHF$r8AYWAQL$S-NH2 1479 1591.83 796.92 797.72 760
Ac-LT2Nal$r8AYWSQL$S-NH2 1480 1597.85 799.93 800.68 761
Ac-LTF$r8HCouWAQL$S-NH2 1481 1679.87 840.94 841.38 762
Ac-LTF$r8AYWCou2QL$S-NH2 1482 1789.94 895.97 896.51 763
Ac-LTF$r8Cou2YWAQL$S-NH2 1483 1789.94 895.97 896.5 764
Ac-Cou2TF$r8AYWAQL$S-NH2 1484 1747.90 874.95 875.42 765
Ac-LTF$r8ACou2WAQL$S-NH2 1485 1697.92 849.96 850.82
766 Dmaac-LTF$r8AYWAQL$S-NH2 1486 1574.89 788.45 788.82 767
Hexac-LTF$r8AYWAQL$S-NH2 1487 1587.91 794.96 795.11 768
Napac-LTF$r8AYWAQL$S-NH2 1488 1657.89 829.95 830.36 769
Pam-LTF$r8AYWAQL$S-NH2 1489 1728.06 865.03 865.45 770
Ac-LT2Nal$r8HYAAQL$S-NH2 1490 1532.84 767.42 767.61 771
Ac-LT2Nal$/r8HYWAQLS/S-NH2 1491 1675.91 838.96 839.1 772
Ac-LT2Nal$r8HYFAQL$S-NH2 1492 1608.87 805.44 805.9 773
Ac-LT2Nal$r8HWAAQL$S-NH2 1493 1555.86 778.93 779.08 774
Ac-LT2Nal$r8HYAWQL$S-NH2 1494 1647.88 824.94 825.04 775
Ac-LT2Nal$r8HYAAQW$S-NH2 1495 1605.83 803.92 804.05 776
Ac-LTW$r8HYWAQL$S-NH2 1496 1636.88 819.44 819.95 777
Ac-LT1Nal$r8HYWAQL$S-NH2 1497 1647.88 824.94 825.41 778
Ac-F$r8ApmpEt6clWEAL$A-NH2 1502 1470.71 736.36 788.17
[0410] In some embodiments, a peptidomimetic macrocycles disclosed
herein do not comprise a peptidomimetic macrocycle structure as
shown in Table 2b.
[0411] Table 2c shows examples of non-crosslinked polypeptides
comprising D-amino acids.
TABLE-US-00008 SEQ ID Exact Found Calc Calc Calc SP Sequence NO:
Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP765
Ac-tawyanfekllr-NH2 1498 777.46 SP766 Ac-tawyanf4CF3ekllr-NH2 1499
811.41
Example 3: X-Ray Co-Crystallography of Peptidomimetic Macrocycles
in Complex with MDMX
[0412] For co-crystallization with peptide 46 (Table 2b), a
stoichiometric amount of compound from a 100 mM stock solution in
DMSO was added to the zebrafish MDMX protein solution and allowed
to sit overnight at 4.degree. C. before setting up crystallization
experiments. Procedures were similar to those described by Popowicz
et al. with some variations, as noted below. Protein (residues
15-129, L46VN95L) was obtained from an E. coli BL21(DE3) expression
system using the pET15b vector. Cells were grown at 37.degree. C.
and induced with 1 mM IPTG at an OD.sub.600 of 0.7. Cells were
allowed to grow an additional 18 hr at 23.degree. C. Protein was
purified using Ni-NT Agarose followed by Superdex 75 buffered with
50 mM NaPO.sub.4, pH 8.0, 150 mM NaCl, 2 mM TCEP and then
concentrated to 24 mg/ml. The buffer was exchanged to 20 mM Tris,
pH 8.0, 50 mM NaCl, 2 mM DTT for crystallization experiments.
Initial crystals were obtained with the Nextal (Qiagen) AMS screen
#94 and the final optimized reservoir was 2.6 M AMS, 75 mM Hepes,
pH 7.5. Crystals grew routinely as thin plates at 4.degree. C. and
were cryo-protected by pulling them through a solution containing
concentrated (3.4 M) malonate followed by flash cooling, storage,
and shipment in liquid nitrogen.
[0413] Data collection was performed at the APS at beamline 31-ID
(SGX-CAT) at 100.degree. K and wavelength 0.97929A. The beamline
was equipped with a Rayonix 225-HE detector. For data collection,
crystals were rotated through 180.degree. in 1.degree. increments
using 0.8 second exposure times. Data were processed and reduced
using Mosflm/scala (CCP4; see The CCP4 Suite: Programs for Protein
Crystallography. Acta Crystallogr. D50, 760-763 (1994); P. R.
Evans. Joint CCP4 and ESF-EACBM Newsletter 33, 22-24 (1997)) in
space group C2 (unit cell: a=109.2786, b=81.0836, c=30.9058 .ANG.,
.alpha.=90, .beta.=89.8577, .gamma.=90.degree.). Molecular
replacement with program Molrep (CCP4; see A. Vagin & A.
Teplyakov. J. Appl. Cryst. 30, 1022-1025 (1997)) was performed with
the MDMX component of the structure determined by Popowicz et al.
(2Z5S; see G. M. Popowicz, A. Czarna, U. Rothweiler, A.
Szwagierczak, M. Krajewski, L. Weber & T. A. Holak. Cell Cycle
6, 2386-2392 (2007)) and identified two molecules in the asymmetric
unit. Initial refinement of just the two molecules of the zebrafish
MDMX with program Refmac (CCP4; see G. N. Murshudov, A. A. Vagin
& E. J. Dodson. Acta Crystallogr. D53, 240-255 (1997)) resulted
in an R-factor of 0.3424 (R.sub.free=0.3712) and rmsd values for
bonds (0.018 .ANG.) and angles (1.698.degree.). The electron
density for the stapled peptide components, starting with
Gln.sup.19 and including all of the aliphatic staple, was very
clear. Further refinement with CNX (Accelrys) using data to 2.3
.ANG. resolution resulted in a model (comprised of 1448 atoms from
MDMX, 272 atoms from the stapled peptides and 46 water molecules)
that is well refined (R.sub.f=0.2601, R.sub.free=0.3162, rmsd
bonds=0.007 .ANG. and rmsd angles=0.916.degree.).
[0414] Results from this Example are shown in FIGS. 1 and 2.
Example 4: Circular Dichroism (CD) Analysis of Alpha-Helicity
[0415] Peptide solutions were analyzed by CD spectroscopy using a
Jasco J-815 spectropolarimeter (Jasco Inc., Easton, Md.) with the
Jasco Spectra Manager Ver.2 system software. A Peltier temperature
controller was used to maintain temperature control of the optical
cell. Results are expressed as mean molar ellipticity [.theta.]
(deg cm2 dmol-1) as calculated from the equation
[.theta.]=.theta.obsMRW/10*l*c where .theta.obs is the observed
ellipticity in millidegrees, MRW is the mean residue weight of the
peptide (peptide molecular weight/number of residues), l is the
optical path length of the cell in centimeters, and c is the
peptide concentration in mg/ml. Peptide concentrations were
determined by amino acid analysis. Stock solutions of peptides were
prepared in benign CD buffer (20 mM phosphoric acid, pH 2). The
stocks were used to prepare peptide solutions of 0.05 mg/ml in
either benign CD buffer or CD buffer with 50% trifluoroethanol
(TFE) for analyses in a 10 mm path length cell. Variable wavelength
measurements of peptide solutions were scanned at 4.degree. C. from
195 to 250 nm, in 0.2 nm increments, and a scan rate 50 nm per
minute. The average of six scans was reported.
[0416] Table 3 shows circular dichroism data for selected
peptidomimetic macrocycles:
TABLE-US-00009 TABLE 3 Molar Molar Molar Ellipticity % Helix %
Helix Ellipticity Ellipticity TFE- 50% TFE benign Benign 50% TFE
Molar compared to compared to (222 in (222 in Ellipticity 50% TFE
50% TFE SP# 0% TFE) 50% TFE) Benign parent (CD) parent (CD) 7 124
-19921.4 -20045.4 137.3 -0.9 11 -398.2 -16623.4 16225.2 106.1 2.5
41 -909 -21319.4 20410.4 136 5.8 43 -15334.5 -18247.4 2912.9 116.4
97.8 69 -102.6 -21509.7 -21407.1 148.2 0.7 71 -121.2 -17957
-17835.9 123.7 0.8 154 -916.2 -30965.1 -30048.9 213.4 6.3 230
-213.2 -17974 -17760.8 123.9 1.5 233 -477.9 -19032.6 -18554.7 131.2
3.3
Example 5: Direct Binding Assay MDM2 with Fluorescence Polarization
(FP)
[0417] The assay was performed according to the following general
protocol: [0418] 1. Dilute MDM2 (In-house, 41 kD) into FP buffer
(High salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5) to make 10 .mu.M
working stock solution. [0419] 2. Add 30 .mu.l of 10 .mu.M of
protein stock solution into A1 and B1 well of 96-well black HE
microplate (Molecular Devices). [0420] 3. Fill in 30 .mu.l of FP
buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12.
[0421] 4. 2 or 3 fold series dilution of protein stock from A1, B1
into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM
concentration at the last dilution point. [0422] 5. Dilute 1 mM (in
100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M
(dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water
(dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40
nM (dilution 1:250). This is the working solution which will be a
10 nM concentration in well (dilution 1:4). Keep the diluted FAM
labeled peptide in the dark until use. [0423] 6. Add 10 .mu.l of 10
nM of FAM labeled peptide into each well and incubate, and read at
different time points. Kd with 5-FAM-BaLTFEHYWAQLTS-NH.sub.2 (SEQ
ID NO: 943) is .about.13.38 nM.
Example 6: Competitive Fluorescence Polarization Assay for MDM2
[0424] The assay was performed according to the following general
protocol: [0425] 1. Dilute MDM2 (In-house, 41 kD) into FP buffer
(High salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5) to make 84 nM
(2.times.) working stock solution. [0426] 2. Add 20 .mu.l of 84 nM
(2.times.) of protein stock solution into each well of 96-well
black HE microplate (Molecular Devices) [0427] 3. Dilute 1 mM (in
100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M
(dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water
(dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40
nM (dilution 1:250). This is the working solution which will be a
10 nM concentration in well (dilution 1:4). Keep the diluted FAM
labeled peptide in the dark until use. [0428] 4. Make unlabeled
peptide dose plate with FP buffer starting with 1 .mu.M (final) of
peptide and making 5 fold serial dilutions for 6 points using
following dilution scheme. [0429] Dilute 10 mM (in 100% DMSO) with
DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500 .mu.M
with H.sub.2O (dilution 1:10) and then dilute with FP buffer from
500 .mu.M to 20 .mu.M (dilution 1:25). Making 5 fold serial
dilutions from 4 .mu.M (4.times.) for 6 points. [0430] 5. Transfer
10 .mu.l of serial diluted unlabeled peptides to each well which is
filled with 20 .mu.l of 84 nM of protein. [0431] 6. Add 10 .mu.l of
10 nM (4.times.) of FAM labeled peptide into each well and incubate
for 3 hr to read.
Example 7: Direct Binding Assay MDMX with Fluorescence Polarization
(FP)
[0432] The assay was performed according to the following general
protocol: [0433] 1. Dilute MDMX (In-house, 40 kD) into FP buffer
(High salt buffer-200 mM NaCl, 5 mM CHAPS, pH 7.5) to make 10 .mu.M
working stock solution. [0434] 2. Add 30 .mu.l of 10 .mu.M of
protein stock solution into A1 and B1 well of 96-well black HE
microplate (Molecular Devices). [0435] 3. Fill in 30 .mu.l of FP
buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12.
[0436] 4. 2 or 3 fold series dilution of protein stock from A1, B1
into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM
concentration at the last dilution point. [0437] 5. Dilute 1 mM (in
100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M
(dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water
(dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40
nM (dilution 1:250). This is the working solution which will be a
10 nM concentration in well (dilution 1:4). Keep the diluted FAM
labeled peptide in the dark until use. [0438] 6. Add 10 .mu.l of 10
nM of FAM labeled peptide into each well and incubate, and read at
different time points. [0439] Kd with 5-FAM-BaLTFEHYWAQLTS-NH.sub.2
(SEQ ID NO: 943) is -51 nM.
Example 8: Competitive Fluorescence Polarization Assay for MDMX
[0440] The assay was performed according to the following general
protocol: [0441] 1. Dilute MDMX (In-house, 40 kD) into FP buffer
(High salt buffer-200 mM NaCl. 5 mM CHAPS, pH 7.5.) to make 300 nM
(2.times.) working stock solution. [0442] 2. Add 20 .mu.l of 300 nM
(2.times.) of protein stock solution into each well of 96-well
black HE microplate (Molecular Devices) [0443] 3. Dilute 1 mM (in
100% DMSO) of FAM labeled linear peptide with DMSO to 100 .mu.M
(dilution 1:10). Then, dilute from 100 .mu.M to 10 .mu.M with water
(dilution 1:10) and then dilute with FP buffer from 10 .mu.M to 40
nM (dilution 1:250). This is the working solution which will be a
10 nM concentration in well (dilution 1:4). Keep the diluted FAM
labeled peptide in the dark until use. [0444] 4. Make unlabeled
peptide dose plate with FP buffer starting with 5 .mu.M (final) of
peptide and making 5 fold serial dilutions for 6 points using
following dilution scheme. [0445] 5. Dilute 10 mM (in 100% DMSO)
with DMSO to 5 mM (dilution 1:2). Then, dilute from 5 mM to 500
.mu.M with H.sub.2O (dilution 1:10) and then dilute with FP buffer
from 500 .mu.M to 20 .mu.M (dilution 1:25). Making 5 fold serial
dilutions from 20 .mu.M (4.times.) for 6 points. [0446] 6. Transfer
10 .mu.l of serial diluted unlabeled peptides to each well which is
filled with 20 .mu.l of 300 nM of protein. [0447] 7. Add 10 .mu.l
of 10 nM (4.times.) of FAM labeled peptide into each well and
incubate for 3 hr to read. Results from Examples 5-8 are shown in
Table 4. The following scale is used: "+" represents a value
greater than 1000 nM, "++" represents a value greater than 100 and
less than or equal to 1000 nM, "+++" represents a value greater
than 10 nM and less than or equal to 100 nM, and "++++" represents
a value of less than or equal to 10 nM.
TABLE-US-00010 [0447] TABLE 4 SP# IC50 (MDM2) IC50 (MDMX) Ki (MDM2)
Ki (MDMS) 3 ++ ++ +++ +++ 4 +++ ++ ++++ +++ 5 +++ ++ ++++ +++ 6 ++
++ +++ +++ 7 +++ +++ ++++ +++ 8 ++ ++ +++ +++ 9 ++ ++ +++ +++ 10 ++
++ +++ +++ 11 +++ ++ ++++ +++ 12 + + +++ ++ 13 ++ ++ +++ ++ 14 +++
+++ ++++ ++++ 15 +++ ++ +++ +++ 16 +++ +++ ++++ +++ 17 +++ +++ ++++
+++ 18 +++ +++ ++++ ++++ 19 ++ +++ +++ +++ 20 ++ ++ +++ +++ 21 ++
+++ +++ +++ 22 +++ +++ ++++ +++ 23 ++ ++ +++ +++ 24 +++ ++ ++++ +++
26 +++ ++ ++++ +++ 28 +++ +++ ++++ +++ 30 ++ ++ +++ +++ 32 +++ ++
++++ +++ 38 + ++ ++ +++ 39 + ++ ++ ++ 40 ++ ++ ++ +++ 41 ++ +++ +++
+++ 42 ++ ++ +++ ++ 43 +++ +++ ++++ +++ 45 +++ +++ ++++ ++++ 46 +++
+++ ++++ +++ 47 ++ ++ +++ +++ 48 ++ ++ +++ +++ 49 ++ ++ +++ +++ 50
+++ ++ ++++ +++ 52 +++ +++ ++++ ++++ 54 ++ ++ +++ +++ 55 + + ++ ++
65 +++ ++ ++++ +++ 68 ++ ++ +++ +++ 69 +++ ++ ++++ +++ 70 ++ ++
++++ +++ 71 +++ ++ ++++ +++ 75 +++ ++ ++++ +++ 77 +++ ++ ++++ +++
80 +++ ++ ++++ +++ 81 ++ ++ +++ +++ 82 ++ ++ +++ +++ 85 +++ ++ ++++
+++ 99 ++++ ++ ++++ +++ 100 ++ ++ ++++ +++ 101 +++ ++ ++++ +++ 102
++ ++ ++++ +++ 103 ++ ++ ++++ +++ 104 +++ ++ ++++ +++ 105 +++ ++
++++ +++ 106 ++ ++ +++ +++ 107 ++ ++ +++ +++ 108 +++ ++ ++++ +++
109 +++ ++ ++++ +++ 110 ++ ++ ++++ +++ 111 ++ ++ ++++ +++ 112 ++ ++
+++ +++ 113 ++ ++ +++ +++ 114 +++ ++ ++++ +++ 115 ++++ ++ ++++ +++
116 + + ++ ++ 118 ++++ ++ ++++ +++ 120 +++ ++ ++++ +++ 121 ++++ ++
++++ +++ 122 ++++ ++ ++++ +++ 123 ++++ ++ ++++ +++ 124 ++++ ++ ++++
+++ 125 ++++ ++ ++++ +++ 126 ++++ ++ ++++ +++ 127 ++++ ++ ++++ +++
128 ++++ ++ ++++ +++ 129 ++++ ++ ++++ +++ 130 ++++ ++ ++++ +++ 133
++++ ++ ++++ +++ 134 ++++ ++ ++++ +++ 135 ++++ ++ ++++ +++ 136 ++++
++ ++++ +++ 137 ++++ ++ ++++ +++ 139 ++++ ++ ++++ +++ 142 ++++ +++
++++ +++ 144 ++++ ++ ++++ +++ 146 ++++ ++ ++++ +++ 148 ++++ ++ ++++
+++ 150 ++++ ++ ++++ +++ 153 ++++ +++ ++++ +++ 154 ++++ +++ ++++
++++ 156 ++++ ++ ++++ +++ 158 ++++ ++ ++++ +++ 160 ++++ ++ ++++ +++
161 ++++ ++ ++++ +++ 166 ++++ ++ ++++ +++ 167 +++ ++ ++++ ++ 169
++++ +++ ++++ +++ 170 ++++ ++ ++++ +++ 173 ++++ ++ ++++ +++ 175
++++ ++ ++++ +++ 177 +++ ++ ++++ +++ 180 +++ ++ ++++ +++ 182 ++++
++ ++++ +++ 185 +++ + ++++ ++ 186 +++ ++ ++++ +++ 189 +++ ++ ++++
+++ 192 +++ ++ ++++ +++ 194 +++ ++ ++++ ++ 196 +++ ++ ++++ +++ 197
++++ ++ ++++ +++ 199 +++ ++ ++++ ++ 201 +++ ++ ++++ ++ 203 +++ ++
++++ +++ 204 +++ ++ ++++ +++ 206 +++ ++ ++++ +++ 207 ++++ ++ ++++
+++ 210 ++++ ++ ++++ +++ 211 ++++ ++ ++++ +++ 213 ++++ ++ ++++ +++
215 +++ ++ ++++ +++ 217 ++++ ++ ++++ +++ 218 ++++ ++ ++++ +++ 221
++++ +++ ++++ +++ 227 ++++ ++ ++++ +++ 230 ++++ +++ ++++ ++++ 232
++++ ++ ++++ +++ 233 ++++ +++ ++++ +++ 236 +++ ++ ++++ +++ 237 +++
++ ++++ +++ 238 +++ +++ ++++ +++ 239 +++ ++ +++ +++ 240 +++ ++ ++++
+++ 241 +++ ++ ++++ +++ 242 +++ ++ ++++ +++ 243 +++ +++ ++++ +++
244 +++ +++ ++++ ++++ 245 +++ +++ ++++ +++ 246 +++ ++ ++++ +++ 247
+++ +++ ++++ +++ 248 +++ +++ ++++ +++ 249 +++ +++ ++++ ++++ 250 ++
+ ++ + 252 ++ + ++ + 254 +++ ++ ++++ +++ 255 +++ +++ ++++ +++ 256
+++ +++ ++++ +++ 257 +++ +++ ++++ +++ 258 +++ ++ ++++ +++ 259 +++
+++ ++++ +++ 260 +++ +++ ++++ +++ 261 +++ ++ ++++ +++ 262 +++ ++
++++ +++ 263 +++ ++ ++++ +++ 264 +++ +++ ++++ +++ 266 +++ ++ ++++
+++ 267 +++ +++ ++++ ++++ 270 ++++ +++ ++++ +++ 271 ++++ +++ ++++
++++ 272 ++++ +++ ++++ ++++ 276 +++ +++ ++++ ++++ 277 +++ +++ ++++
++++ 278 +++ +++ ++++ ++++ 279 ++++ +++ ++++ +++ 280 +++ ++ ++++
+++ 281 +++ + +++ ++ 282 ++ + +++ + 283 +++ ++ +++ ++ 284 +++ ++
++++ +++ 289 +++ +++ ++++ +++ 291 +++ +++ ++++ ++++ 293 ++++ +++
++++ +++ 306 ++++ ++ ++++ +++ 308 ++ ++ +++ +++ 310 +++ +++ ++++
+++ 312 +++ ++ +++ +++ 313 ++++ ++ ++++ +++ 314 ++++ +++ ++++ ++++
315 +++ +++ ++++ +++ 316 ++++ ++ ++++ +++ 317 +++ ++ +++ +++ 318
+++ ++ +++ +++ 319 +++ ++ +++ ++ 320 +++ ++ +++ ++ 321 +++ ++ ++++
+++ 322 +++ ++ +++ ++ 323 +++ + +++ ++ 328 +++ +++ ++++ +++ 329 +++
+++ ++++ +++ 331 ++++ +++ ++++ ++++ 332 ++++ +++ ++++ ++++ 334 ++++
+++ ++++ ++++ 336 ++++ +++ ++++ ++++ 339 ++++ ++ ++++ +++ 341 +++
+++ ++++ ++++ 343 +++ +++ ++++ ++++ 347 +++ +++ ++++ +++ 349 ++++
+++ ++++ ++++ 351 ++++ +++ ++++ ++++ 353 ++++ +++ ++++ ++++ 355
++++ +++ ++++ ++++ 357 ++++ +++ ++++ ++++ 359 ++++ +++ ++++ +++ 360
++++ ++++ ++++ ++++ 363 +++ +++ ++++ ++++ 364 +++ +++ ++++ ++++ 365
+++ +++ ++++ ++++ 366 +++ +++ ++++ +++ 369 ++ ++ +++ +++ 370 +++
+++ ++++ +++ 371 ++ ++ +++ +++ 372 ++ ++ +++ +++ 373 +++ +++ +++
+++ 374 +++ +++ ++++ ++++ 375 +++ +++ ++++ ++++ 376 +++ +++ ++++
++++ 377 +++ +++ ++++ +++ 378 +++ +++ ++++ +++ 379 +++ +++ ++++ +++
380 +++ +++ ++++ +++ 381 +++ +++ ++++ +++ 382 +++ +++ ++++ ++++ 384
++ + ++ + 386 ++ + ++ + 388 ++ +++ +++ ++++ 390 +++ +++ ++++ +++
392 +++ +++ ++++ ++++ 394 ++++ +++ ++++ ++++ 396 ++++ ++++ ++++
++++ 398 +++ +++ ++++ +++ 402 ++++ ++++ ++++ ++++ 404 +++ +++ ++++
++++ 408 +++ +++ ++++ +++ 410 ++++ ++++ ++++ ++++ 411 ++ + ++ + 412
++++ +++ ++++ ++++ 415 ++++ ++++ ++++ ++++ 416 +++ +++ ++++ +++ 417
+++ +++ ++++ +++ 418 ++++ +++ ++++ ++++ 419 +++ +++ +++ ++++
421 ++++ ++++ ++++ ++++ 423 +++ +++ ++++ +++ 425 +++ +++ +++ +++
427 ++ ++ +++ +++ 432 ++++ +++ ++++ ++++ 434 +++ +++ ++++ +++ 435
++++ +++ ++++ ++++ 437 +++ +++ ++++ +++ 439 ++++ +++ ++++ ++++ 441
++++ ++++ ++++ ++++ 443 +++ +++ ++++ +++ 445 +++ ++ ++++ +++ 446
+++ + ++++ + 447 ++ + ++ + 551 N/A N/A ++++ +++ 555 N/A N/A ++++
+++ 556 N/A N/A ++++ +++ 557 N/A N/A +++ +++ 558 N/A N/A +++ +++
559 N/A N/A +++ +++ 560 N/A N/A + + 561 N/A N/A ++++ +++ 562 N/A
N/A +++ +++ 563 N/A N/A +++ +++ 564 N/A N/A ++++ +++ 565 N/A N/A
+++ +++ 566 N/A N/A ++++ +++ 567 N/A N/A ++++ +++ 568 N/A N/A ++++
++++ 569 N/A N/A ++++ +++ 570 N/A N/A ++++ +++ 571 N/A N/A ++++ +++
572 N/A N/A +++ +++ 573 N/A N/A +++ +++ 574 N/A N/A ++++ +++ 575
N/A N/A ++++ +++ 576 N/A N/A ++++ +++ 577 N/A N/A ++++ +++ 578 N/A
N/A ++++ +++ 585 N/A N/A +++ +++ 586 N/A N/A ++++ +++ 587 N/A N/A
++++ ++++ 589 N/A N/A ++++ 594 N/A N/A ++++ ++++ 596 N/A N/A ++++
+++ 597 N/A N/A ++++ +++ 598 N/A N/A ++++ +++ 600 N/A N/A ++++ ++++
602 N/A N/A ++++ ++++ 603 N/A N/A ++++ ++++ 604 N/A N/A +++ +++ 608
N/A N/A ++++ +++ 609 N/A N/A ++++ +++ 610 N/A N/A ++++ +++ 611 N/A
N/A ++++ +++ 612 N/A N/A ++++ +++ 613 N/A N/A ++++ +++ 615 N/A N/A
++++ ++++ 433 N/A N/A ++++ +++ 686 N/A N/A ++++ +++ 687 N/A N/A ++
++ 595 N/A N/A + N/A 665 N/A N/A +++ N/A 708 N/A N/A +++ +++ 710
N/A N/A +++ +++ 711 N/A N/A +++ ++ 712 N/A N/A ++++ ++++ 713 N/A
N/A ++++ ++++ 716 N/A N/A ++++ ++++ 765 + + 766 +++ + 752 ++ + 753
+++ + 754 ++ + 755 ++++ + 756 +++ + 757 ++++ + 758 +++ +
Example 9: Competition Binding ELISA (MDM2 & MDMX)
[0448] p53-His6 protein ("His6" disclosed as SEQ ID NO: 1501) (30
nM/well) is coated overnight at room temperature in the wells of a
96-well Immulon plates. On the day of the experiment, plates are
washed with 1.times.PBS-Tween 20 (0.05%) using an automated ELISA
plate washer, blocked with ELISA Micro well Blocking for 30 minutes
at room temperature; excess blocking agent is washed off by washing
plates with 1.times.PBS-Tween 20 (0.05%). Peptides are diluted from
10 mM DMSO stocks to 500 .mu.M working stocks in sterile water,
further dilutions made in 0.5% DMSO to keep the concentration of
DMSO constant across the samples. The peptides are added to wells
at 2.times. desired concentrations in 50 .mu.M volumes, followed by
addition of diluted GST-MDM2 or GST-HMDX protein (final
concentration: 10 nM). Samples are incubated at room temperature
for 2 h, plates are washed with PBS-Tween 20 (0.05%) prior to
adding 100 .mu.M of HRP-conjugated anti-GST antibody [Hypromatrix,
INC] diluted to 0.5 .mu.g/ml in HRP-stabilizing buffer. Post 30 min
incubation with detection antibody, plates are washed and incubated
with 100 .mu.M per well of TMB-E Substrate solution up to 30
minutes; reactions are stopped using 1M HCL and absorbance measured
at 450 nm on micro plate reader. Data is analyzed using Graph Pad
PRISM software.
Example 10: Cell Viability Assay
[0449] The assay was performed according to the following general
protocol:
[0450] Cell Plating: Trypsinize, count and seed cells at the
pre-determined densities in 96-well plates a day prior to assay.
Following cell densities are used for each cell line in use: [0451]
SJSA-1: 7500 cells/well [0452] RKO: 5000 cells/well [0453] RKO-E6:
5000 cells/well [0454] HCT-116: 5000 cells/well [0455] SW-480: 2000
cells/well [0456] MCF-7: 5000 cells/well
[0457] On the day of study, replace media with fresh media with 11%
FBS (assay media) at room temperature. Add 180 .mu.L of the assay
media per well. Control wells with no cells, receive 200 .mu.L
media.
[0458] Peptide dilution: all dilutions are made at room temperature
and added to cells at room temperature. [0459] Prepare 10 mM stocks
of the peptides in DMSO. Serially dilute the stock using 1:3
dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM
solutions using DMSO as diluents. Dilute the serially DMSO-diluted
peptides 33.3 times using sterile water. This gives range of
10.times. working stocks. Also prepare DMSO/sterile water (3% DMSO)
mix for control wells. [0460] Thus the working stocks concentration
range .mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix
well at each dilution step using multichannel [0461] Row H has
controls. H1-H3 will receive 20 .mu.L of assay media. H4-H9 will
receive 20 .mu.L of 3% DMSO-water vehicle. H10-H12 will have media
alone control with no cells. [0462] Positive control: MDM2 small
molecule inhibitor, Nutlin-3a (10 mM) is used as positive control.
Nutlin was diluted using the same dilution scheme as peptides.
[0463] Addition of Working Stocks to Cells: [0464] Add 20 .mu.L of
10.times. desired concentration to appropriate well to achieve the
final concentrations in total 200 .mu.L volume in well. (20 .mu.L
of 300 .mu.M peptide+180 .mu.L of cells in media=30 .mu.M final
concentration in 200 .mu.L volume in wells). Mix gently a few times
using pipette. Thus final concentration range used will be 30, 10,
3, 1, 0.3, 0.1, 0.03 & 0 .mu.M (for potent peptides further
dilutions are included). [0465] Controls include wells that get no
peptides but contain the same concentration of DMSO as the wells
containing the peptides, and wells containing NO CELLS. [0466]
Incubate for 72 hours at 37.degree. C. in humidified 5% CO.sub.2
atmosphere. [0467] The viability of cells is determined using MTT
reagent from Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116
cells is determined on day 3, MCF-7 cells on day 5 and SW-480 cells
on day 6. At the end of designated incubation time, allow the
plates to come to room temperature. Remove 80 .mu.M of assay media
from each well. Add 15 .mu.M of thawed MTT reagent to each well.
[0468] Allow plate to incubate for 2 h at 37.degree. C. in
humidified 5% CO.sub.2 atmosphere and add 100 .mu.l solubilization
reagent as per manufacturer's protocol. Incubate with agitation for
1 h at room temperature and read on Synergy Biotek multiplate
reader for absorbance at 570 nM. [0469] Analyze the cell viability
against the DMSO controls using GraphPad PRISM analysis tools.
[0470] Reagents: [0471] Invitrogen cell culture Media [0472]
i.Falcon 96-well clear cell culture treated plates (Nunc 353072)
[0473] DMSO (Sigma D 2650) [0474] RPMI 1640 (Invitrogen 72400)
[0475] MTT (Promega G4000)
[0476] Instruments:
[0477] Multiplate Reader for Absorbance readout (Synergy 2).
[0478] Results from cell viability assays are shown in Tables 5 and
6. The following scale is used: "+" represents a value greater than
30 .mu.M, "++" represents a value greater than 15 .mu.M and less
than or equal to 30 .mu.M, "+++" represents a value greater than 5
.mu.M and less than or equal to 15 .mu.M, and "++++" represents a
value of less than or equal to 5 .mu.M. "IC50 ratio" represents the
ratio of average IC50 in p53+/+ cells relative to average IC50 in
p53-/- cells.
TABLE-US-00011 TABLE 5 SP # SJSA-1 EC50 (72 h) 3 +++ 4 +++ 5 ++++ 6
++ 7 ++++ 8 +++ 9 +++ 10 +++ 11 ++++ 12 ++ 13 +++ 14 + 15 ++ 16 +
17 + 18 + 19 ++ 20 + 21 + 22 + 24 +++ 26 ++++ 28 + 29 + 30 + 32 ++
38 + 39 + 40 + 41 + 42 + 43 ++ 45 + 46 + 47 + 48 + 49 +++ 50 ++++
52 + 54 + 55 + 65 ++++ 68 ++++ 69 ++++ 70 ++++ 71 ++++ 72 ++++ 74
++++ 75 ++++ 77 ++++ 78 ++ 80 ++++ 81 +++ 82 +++ 83 +++ 84 + 85 +++
99 ++++ 102 +++ 103 +++ 104 +++ 105 +++ 108 +++ 109 +++ 110 +++ 111
++ 114 ++++ 115 ++++ 118 ++++ 120 ++++ 121 ++++ 122 ++++ 123 ++++
124 +++ 125 ++++ 126 ++++ 127 ++++ 128 +++ 129 ++ 130 ++++ 131 +++
132 ++++ 133 +++ 134 +++ 135 +++ 136 ++ 137 +++ 139 ++++ 142 +++
144 ++++ 147 ++++ 148 ++++ 149 ++++ 150 ++++ 152 +++ 153 ++++ 154
++++ 155 ++ 156 +++ 157 +++ 158 +++ 160 ++++ 161 ++++ 162 +++ 163
+++ 166 ++ 167 +++ 168 ++ 169 ++++ 170 ++++ 171 ++ 173 +++ 174 ++++
175 +++ 176 +++ 177 ++++ 179 +++ 180 +++ 181 +++ 182 ++++ 183 ++++
184 +++ 185 +++ 186 ++ 188 ++ 190 ++++ 192 +++ 193 ++ 194 + 195
++++ 196 ++++ 197 ++++ 198 ++ 199 +++ 200 +++ 201 ++++ 202 +++ 203
++++ 204 ++++ 205 ++ 206 ++ 207 +++ 208 +++ 209 ++++ 210 +++ 211
++++ 213 ++++ 214 ++++ 215 ++++ 216 ++++ 217 ++++ 218 ++++ 219 ++++
220 +++ 221 ++++ 222 +++ 223 ++++ 224 ++ 225 +++ 226 ++ 227 +++ 228
++++ 229 ++++ 230 ++++ 231 ++++ 232 ++++ 233 ++++ 234 ++++ 235 ++++
236 ++++ 237 ++++ 238 ++++ 239 +++ 240 ++ 241 +++ 242 ++++ 243 ++++
244 ++++ 245 ++++ 246 +++ 247 ++++ 248 ++++ 249 ++++ 250 ++ 251 +
252 + 253 + 254 +++ 255 +++ 256 ++ 257 +++ 258 +++ 259 ++ 260 ++
261 ++ 262 +++ 263 ++ 264 ++++ 266 +++ 267 ++++ 270 ++ 271 ++ 272
++ 276 ++ 277 ++ 278 ++ 279 ++++ 280 +++ 281 ++ 282 ++ 283 ++ 284
++++ 289 ++++ 290 +++ 291 ++++ 292 ++++ 293 ++++ 294 ++++ 295 +++
296 ++++ 297 +++ 298 ++++ 300 ++++ 301 ++++ 302 ++++ 303 ++++ 304
++++ 305 ++++ 306 ++++ 307 +++ 308 ++++ 309 +++ 310 ++++ 312 ++++
313 ++++ 314 ++++ 315 ++++ 316 ++++ 317 ++++ 318 ++++ 319 ++++ 320
++++ 321 ++++ 322 ++++ 323 ++++
324 ++++ 326 ++++ 327 ++++ 328 ++++ 329 ++++ 330 ++++ 331 ++++ 332
++++ 333 ++ 334 +++ 335 ++++ 336 ++++ 337 ++++ 338 ++++ 339 ++++
340 ++++ 341 ++++ 342 ++++ 343 ++++ 344 ++++ 345 ++++ 346 ++++ 347
++++ 348 ++++ 349 ++++ 350 ++++ 351 ++++ 352 ++++ 353 ++++ 355 ++++
357 ++++ 358 ++++ 359 ++++ 360 ++++ 361 +++ 362 ++++ 363 ++++ 364
++++ 365 +++ 366 ++++ 367 ++++ 368 + 369 ++++ 370 ++++ 371 ++++ 372
+++ 373 +++ 374 ++++ 375 ++++ 376 ++++ 377 ++++ 378 ++++ 379 ++++
380 ++++ 381 ++++ 382 ++++ 386 +++ 388 ++ 390 ++++ 392 +++ 394 +++
396 +++ 398 +++ 402 +++ 404 +++ 408 ++++ 410 +++ 411 +++ 412 + 421
+++ 423 ++++ 425 ++++ 427 ++++ 434 +++ 435 ++++ 436 ++++ 437 ++++
438 ++++ 439 ++++ 440 ++++ 441 ++++ 442 ++++ 443 ++++ 444 +++ 445
++++ 449 ++++ 551 ++++ 552 ++++ 554 + 555 ++++ 557 ++++ 558 ++++
560 + 561 ++++ 562 ++++ 563 ++++ 564 ++++ 566 ++++ 567 ++++ 568 +++
569 ++++ 571 ++++ 572 ++++ 573 ++++ 574 ++++ 575 ++++ 576 ++++ 577
++++ 578 ++++ 585 ++++ 586 ++++ 587 ++++ 588 ++++ 589 +++ 432 ++++
672 + 673 ++ 682 + 686 + 687 + 662 ++++ 663 ++++ 553 +++ 559 ++++
579 ++++ 581 ++++ 582 ++ 582 ++++ 584 +++ 675 ++++ 676 ++++ 677 +
679 ++++ 700 +++ 704 +++ 591 + 706 ++ 695 ++ 595 ++++ 596 ++++ 597
+++ 598 +++ 599 ++++ 600 ++++ 601 +++ 602 +++ 603 +++ 604 +++ 606
++++ 607 ++++ 608 ++++ 610 ++++ 611 ++++ 612 ++++ 613 +++ 614 +++
615 ++++ 618 ++++ 619 ++++ 707 ++++ 620 ++++ 621 ++++ 622 ++++ 623
++++ 624 ++++ 625 ++++ 626 +++ 631 ++++ 633 ++++ 634 ++++ 635 +++
636 +++ 638 + 641 +++ 665 ++++ 708 ++++ 709 +++ 710 + 711 ++++ 712
++++ 713 ++++ 714 +++ 715 +++ 716 ++++ 765 + 753 + 754 + 755 + 756
+ 757 ++++ 758 +++
TABLE-US-00012 TABLE 6 SW480 HCT-116 EC50 RKO EC50 RKO-E6 EC50 EC50
IC50 SP # (72 h) (72 h) (72 h) (6 days) Ratio 4 ++++ ++++ +++ ++++
5 ++++ ++++ +++ ++++ 7 ++++ ++++ +++ ++++ 10 ++++ +++ +++ +++ 11
++++ ++++ ++ +++ 50 ++++ ++++ ++ +++ 65 +++ +++ +++ +++ 69 ++++
++++ + ++++ 70 ++++ ++++ ++ +++ 71 ++++ ++++ +++ +++ 81 +++ +++ +++
+++ 99 ++++ ++++ +++ ++++ 109 ++++ ++++ ++ +++ 114 +++ + +++ 115
+++ + +++ 1-29 118 +++ ++++ + ++++ 120 ++++ ++++ + ++++ 121 ++++
++++ + ++++ 122 +++ + +++ 1-29 125 +++ +++ + + 126 + + + + 148 ++ +
+ 150 ++ + + 153 +++ + 154 +++ +++ + + 30-49 158 + + + + 160 +++ +
+ + 1-29 161 +++ + + + 175 + + + + 196 ++++ ++++ +++ ++++ 219 ++++
+++ + + 1-29 233 ++++ 237 ++++ + + 238 ++++ + + 243 ++++ + + 244
++++ + + .gtoreq.50 245 ++++ + + 247 ++++ + + 249 ++++ ++++ + +
.gtoreq.50 255 ++++ + 291 + 293 +++ + 303 +++ + 1-29 305 + 306 ++++
+ 310 ++++ + 312 ++++ 313 ++++ ++ 314 + 315 ++++ ++++ ++ ++++
.gtoreq.50 316 ++++ ++++ + +++ .gtoreq.50 317 +++ + ++ 321 ++++ +
324 +++ + 325 +++ 326 +++ + 327 +++ + 328 +++ ++ 329 ++++ + 330 +
331 ++++ ++++ + + .gtoreq.50 338 ++++ ++++ ++ +++ 341 +++ ++ + +
343 +++ + + 346 ++++ + + 347 +++ + + 349 ++++ +++ + + 30-49 350
++++ + + 351 ++++ +++ + + 30-49 353 ++ ++ + + 355 ++++ ++ + + 1-29
357 ++++ ++++ + + 358 ++++ ++ + + 359 ++++ ++ + + 367 ++++ + +
30-49 386 ++++ ++++ ++++ ++++ 388 ++ ++ + +++ 1-29 390 ++++ ++++
+++ ++++ 435 +++ ++ + 436 ++++ ++++ ++ 437 ++++ ++++ ++ ++++ 30-49
440 ++ ++ + 442 ++++ ++++ ++ 444 ++++ ++++ +++ 445 ++++ +++ + +
.gtoreq.50 555 .gtoreq.50 557 .gtoreq.50 558 30-49 562 30-49 564
30-49 566 30-49 567 .gtoreq.50 572 .gtoreq.50 573 30-49 578 30-49
662 .gtoreq.50 379 1-29 375 1-29 559 .gtoreq.50 561 1-29 563 1-29
568 1-29 569 1-29 571 1-29 574 1-29 575 1-29 576 1-29 577 30-49 433
1-29 551 30-49 553 1-29 710 + 711 + 712 ++ 713 ++ 714 +++ 715 +++
716 +
Example 11: P21 ELISA Assay
[0479] The assay was performed according to the following general
protocol:
[0480] Cell Plating: [0481] Trypsinize, count and seed SJSA1 cells
at the density of 7500 cells/100 .mu.l/well in 96-well plates a day
prior to assay. [0482] On the day of study, replace media with
fresh RPMI-11% FBS (assay media). Add 904 of the assay media per
well. Control wells with no cells, receive 100 .mu.l media.
[0483] Peptide Dilution: [0484] Prepare 10 mM stocks of the
peptides in DMSO. Serially dilute the stock using 1:3 dilution
scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions
using DMSO as diluents. Dilute the serially DMSO-diluted peptides
33.3 times using sterile water This gives range of 10.times.
working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for
control wells. [0485] Thus the working stocks concentration range
.mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at
each dilution step using multichannel [0486] Row H has controls.
H1-H3 will receive 10 .mu.l of assay media. H4-H9 will receive 10
.mu.l of 3% DMSO-water vehicle. H10-H12 will have media alone
control with no cells. [0487] Positive control: MDM2 small molecule
inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin
was diluted using the same dilution scheme as peptides.
[0488] Addition of Working Stocks to Cells: [0489] Add 10 .mu.L of
10.times. desired concentration to appropriate well to achieve the
final concentrations in total 100 .mu.l volume in well. (10 .mu.l
of 300 .mu.M peptide+90 .mu.l of cells in media=30 .mu.M final
concentration in 100 .mu.l volume in wells). Thus final
concentration range used will be 30, 10, 3, 1, 0.3& 0 .mu.M.
[0490] Controls will include wells that get no peptides but contain
the same concentration of DMSO as the wells containing the
peptides, and wells containing NO CELLS. [0491] 20 h-post
incubation, aspirate the media; wash cells with 1.times.PBS
(without C.sup.++/Mg.sup.++) and lyse in 60 .mu.l of 1.times. Cell
lysis buffer (Cell Signaling technologies 10.times. buffer diluted
to 1.times. and supplemented with protease inhibitors and
Phosphatase inhibitors) on ice for 30 min. [0492] Centrifuge plates
in at 5000 rpm speed in at 4.degree. C. for 8 min; collect clear
supernatants and freeze at -80.degree. C. till further use.
[0493] Protein Estimation: [0494] Total protein content of the
lysates is measured using BCA protein detection kit and BSA
standards from Thermofisher. Typically about 6-7 .mu.g protein is
expected per well. [0495] Use 50 .mu.L of the lysate per well to
set up p21 ELISA.
[0496] Human Total p21 ELISA:
[0497] The ELISA assay protocol is followed as per the
manufacturer's instructions. 50 .mu.L lysate is used for each well,
and each well is set up in triplicate.
[0498] Reagents: [0499] Cell-Based Assay (-)-Nutlin-3 (10 mM):
Cayman Chemicals, catalog #600034 [0500] OptiMEM, Invitrogen
catalog #51985 [0501] Cell Signaling Lysis Buffer (10.times.), Cell
signaling technology, Catalog #9803 [0502] Protease inhibitor
Cocktail tablets(mini), Roche Chemicals, catalog #04693124001
[0503] Phosphatase inhibitor Cocktail tablet, Roche Chemicals,
catalog #04906837001 [0504] Human total p21 ELISA kit, R&D
Systems, DYC1047-5 [0505] STOP Solution (1M HCL), Cell Signaling
Technologies, Catalog #7002
[0506] Instruments: Micro centrifuge-Eppendorf 5415D and Multiplate
Reader for Absorbance readout (Synergy 2).
Example 12: Caspase 3 Detection Assay
[0507] The assay was performed according to the following general
protocol:
[0508] Cell Plating:
[0509] Trypsinize, count and seed SJSA1 cells at the density of
7500 cells/100 .mu.l/well in 96-well plates a day prior to assay.
On the day of study, replace media with fresh RPMI-11% FBS (assay
media). Add 1804 of the assay media per well. Control wells with no
cells, receive 200 media.
[0510] Peptide Dilution: [0511] Prepare 10 mM stocks of the
peptides in DMSO. Serially dilute the stock using 1:3 dilution
scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions
using DMSO as diluents. Dilute the serially DMSO-diluted peptides
33.3 times using sterile water This gives range of 10.times.
working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for
control wells. [0512] Thus the working stocks concentration range
.mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at
each dilution step using multichannel Add 20 .mu.l of 10.times.
working stocks to appropriate wells. [0513] Row H has controls.
H1-H3 will receive 20 .mu.l of assay media. H4-H9 will receive 20
.mu.l of 3% DMSO-water vehicle. H10-H12 will have media alone
control with no cells. [0514] Positive control: MDM2 small molecule
inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin
was diluted using the same dilution scheme as peptides.
[0515] Addition of Working Stocks to Cells: [0516] Add 10 .mu.l of
10.times. desired concentration to appropriate well to achieve the
final concentrations in total 100 .mu.l volume in well. (10 .mu.L
of 300 .mu.M peptide+90 .mu.L of cells in media=30 .mu.M final
concentration in 100 .mu.L volume in wells). Thus final
concentration range used will be 30, 10, 3, 1, 0.3& 0 .mu.M.
[0517] Controls will include wells that get no peptides but contain
the same concentration of DMSO as the wells containing the
peptides, and wells containing NO CELLS. [0518] 48 h-post
incubation, aspirate 80 .mu.L media from each well; add 100 .mu.L
Caspase3/7Glo assay reagent (Promega Caspase 3/7 glo assay system,
G8092) per well, incubate with gentle shaking for 1 h at room
temperature. [0519] read on Synergy Biotek multiplate reader for
luminescence. [0520] Data is analyzed as Caspase 3 activation over
DMSO-treated cells.
[0521] Results from Examples 11 and 12 are shown in Table 7:
TABLE-US-00013 TABLE 7 caspase caspase caspase caspase caspase p21
p21 p21 p21 p21 SP# 0.3 .mu.M 1 .mu.M 3 .mu.M 10 .mu.M 30 .mu.M 0.3
.mu.M 1 .mu.M 3 .mu.M 10 .mu.M 30 .mu.M 4 9 37 35 317 3049 3257 7
0.93 1.4 5.08 21.7 23.96 18 368 1687 2306 8 1 19 25 34 972 2857 10
1 1 17 32 10 89 970 2250 11 1 5 23 33.5 140 350 2075.5 3154 26 1 1
3 14 50 8 29 29 44 646 1923 1818 65 1 6 28 34 -69 -24 122 843 1472
69 4.34 9.51 16.39 26.59 26.11 272 458.72 1281.39 2138.88 1447.22
70 1 9 26 -19 68 828 1871 71 0.95 1.02 3.68 14.72 23.52 95 101 1204
2075 72 1 1 4 10 -19 57 282 772 1045 77 1 2 19 23 80 1 2 13 20 81 1
1 6 21 0 0 417 1649 99 1 7 31 33 -19 117 370 996 1398 109 4 16 25
161 445 1221 1680 114 1 6 28 34 -21 11 116 742 910 115 1 10 26 32
-10 36 315 832 1020 118 1 2 18 27 -76 -62 -11 581 1270 120 2 11 20
30 -4 30 164 756 1349 121 1 5 19 30 9 33 81 626 1251 122 1 2 15 30
-39 -18 59 554 1289 123 1 1 6 14 125 1 3 9 29 50 104 196 353 1222
126 1 1 6 30 -47 -10 90 397 1443 127 1 1 4 13 130 1 2 6 17 139 1 2
9 18 142 1 2 15 20 144 1 4 10 16 148 1 11 23 31 -23 55 295 666 820
149 1 2 4 10 35 331 601 1164 1540 150 2 11 19 35 -37 24 294 895 906
153 2 10 15 20 154 2.68 4 13.93 19.86 30.14 414.04 837.45 1622.42
2149.51 2156.98 158 1 1.67 5 16.33 -1.5 95 209.5 654 1665.5 160 2
10 16 31 -43 46 373 814 1334 161 2 8 14 22 13 128 331 619 1078 170
1 1 16 20 175 1 5 12 21 -65 1 149 543 1107 177 1 1 8 20 183 1 1 4 8
-132 -119 -14 1002 818 196 1 4 33 26 -49 -1 214 1715 687 197 1 1 10
20 203 1 3 12 10 77 329 534 1805 380 204 1 4 10 10 3 337 928 1435
269 218 1 2 8 18 219 1 5 17 34 28 53 289 884 1435 221 1 3 6 12 127
339 923 1694 1701 223 1 1 5 18 230 1 2 3 11 245.5 392 882 1549 2086
233 6 8 17 22 23 2000 2489 3528 3689 2481 237 1 5 9 15 0 0 2 284
421 238 1 2 4 21 0 149 128 825 2066 242 1 4 5 18 0 0 35 577 595 243
1 2 5 23 0 0 0 456 615 244 1 2 7 17 0 178 190 708 1112 245 1 3 9 16
0 0 0 368 536 247 1 3 11 24 0 0 49 492 699 248 0 50 22 174 1919 249
2 5 11 23 0 0 100 907 1076 251 0 0 0 0 0 252 0 0 0 0 0 253 0 0 0 0
0 254 1 3 7 14 22 118 896 1774 3042 3035 286 1 4 11 20 22 481 1351
2882 3383 2479 287 1 1 3 11 23 97 398 986 2828 3410 315 11 14.5
25.5 32 34 2110 2209 2626 2965 2635 316 6.5 10.5 21 32 32.5 1319
1718 2848 2918 2540 317 3 4 9 26 35 551 624 776 1367 1076 331 4.5 8
11 14.5 30.5 1510 1649 2027 2319 2509 338 1 5 23 20 29 660.37
1625.38 3365.87 2897.62 2727 341 3 8 11 14 21 1325.62 1873 2039.75
2360.75 2574 343 1 1 2 5 29 262 281 450 570 1199 346 235.86 339.82
620.36 829.32 1695.78 347 2 3 5 8 29 374 622 659 905 1567 349 1 8
11 16 24 1039.5 1598.88 1983.75 2191.25 2576.38 351 3 9 13 15 24
1350.67 1710.67 2030.92 2190.67 2668.54 353 1 2 5 7 30 390 490 709
931 1483 355 1 4 11 13 30 191 688 1122 1223 1519 357 2 7 11 15 23
539 777 1080 1362 1177 358 1 2 3 6 24 252 321 434 609 1192 359 3 9
11 13 23 1163.29 1508.79 1780.29 2067.67 2479.29 416 33.74 39.82
56.57 86.78 1275.28 417 0 0 101.13 639.04 2016.58 419 58.28 97.36
221.65 1520.69 2187.94 432 54.86 68.86 105.11 440.28 1594.4
Example 13. Cell Lysis by Peptidomimetic Macrocycles
[0522] SJSA-1 cells were plated out one day in advance in clear
flat-bottom plates (Costar, catalog number 353072) at 7500
cells/well with 100 ul/well of growth media, leaving row H columns
10-12 empty for media alone. On the day of the assay, media was
exchanged with RPMI 1% FBS media, 90 uL of media per well.
[0523] 10 mM stock solutions of the peptidomimetic macrocycles were
prepared in 100% DMSO. Peptidomimetic macrocycles were then diluted
serially in 100% DMSO, and then further diluted 20-fold in sterile
water to prepare working stock solutions in 5% DMSO/water of each
peptidomimetic macrocycle at concentrations ranging from 500 .mu.M
to 62.5 .mu.M.
[0524] 10 .mu.L of each compound was added to the 90 .mu.L of
SJSA-1 cells to yield final concentrations of 50 .mu.M to 6.25
.mu.M in 0.5% DMSO-containing media. The negative control
(non-lytic) sample was 0.5% DMSO alone and positive control (lytic)
samples include 10 .mu.M Melittin and 1% Triton X-100.
[0525] Cell plates were incubated for 1 hour at 37 C. After the 1
hour incubation, the morphology of the cells is examined by
microscope and then the plates were centrifuged at 1200 rpm for 5
minutes at room temperature. 40 uL of supernatant for each
peptidomimetic macrocycle and control sample is transferred to
clear assay plates. LDH release is measured using the LDH
cytotoxicity assay kit from Caymen, catalog #1000882.
[0526] Results are shown in Table 8:
TABLE-US-00014 TABLE 8 6.25 .mu.M % 12.5 .mu.M % 25 .mu.M % 50
.mu.M % Lysed cells Lysed cells Lysed cells Lysed cells SP # (1 h
LDH) (1 h LDH) (1 h LDH) (1 h LDH) 3 1 0 1 3 4 -2 1 1 2 6 1 1 1 1 7
0 0 0 0 8 -1 0 1 1 9 -3 0 0 2 11 -2 1 2 3 15 1 2 2 5 18 0 1 2 4 19
2 2 3 21 22 0 -1 0 0 26 2 5 -1 0 32 0 0 2 0 39 0 -1 0 3 43 0 0 -1
-1 55 1 5 9 13 65 0 0 0 2 69 1 0.5 -0.5 5 71 0 0 0 0 72 2 1 0 3 75
-1 3 1 1 77 -2 -2 1 -1 80 0 1 1 5 81 1 1 0 0 82 0 0 0 1 99 1.5 3 2
3.5 108 0 0 0 1 114 3 -1 4 9 115 0 1 -1 6 118 4 2 2 4 120 0 -1 0 6
121 1 0 1 7 122 1 3 0 6 123 -2 2 5 3 125 0 1 0 2 126 1 2 1 1 130 1
3 0 -1 139 -2 -3 -1 -1 142 1 0 1 3 144 1 2 -1 2 147 8 9 16 55 148 0
1 -1 0 149 6 7 7 21 150 -1 -2 0 2 153 4 3 2 3 154 -1 -1.5 -1 -1 158
0 -6 -2 160 -1 0 -1 1 161 1 1 -1 0 169 2 3 3 7 170 2 2 1 -1 174 5 3
2 5 175 3 2 1 0 177 -1 -1 0 1 182 0 2 3 6 183 2 1 0 3 190 -1 -1 0 1
196 0 -2 0 3 197 1 -4 -1 -2 203 0 -1 2 2 204 4 3 2 0 211 5 4 3 1
217 2 1 1 2 218 0 -3 -4 1 219 0 0 -1 2 221 3 3 3 11 223 -2 -2 -4 -1
230 0.5 -0.5 0 3 232 6 6 5 5 233 2.5 4.5 3.5 6 237 0 3 7 55 243 4
23 39 64 244 0 1 0 4 245 1 14 11 56 247 0 0 0 4 249 0 0 0 0 254 11
34 60 75 279 6 4 5 6 280 5 4 6 18 284 5 4 5 6 286 0 0 0 0 287 0 6
11 56 316 0 1 0 1 317 0 1 0 0 331 0 0 0 0 335 0 0 0 1 336 0 0 0 0
338 0 0 0 1 340 0 2 0 0 341 0 0 0 0 343 0 1 0 0 347 0 0 0 0 349 0 0
0 0 351 0 0 0 0 353 0 0 0 0 355 0 0 0 0 357 0 0 0 0 359 0 0 0 0 413
5 3 3 3 414 3 3 2 2 415 4 4 2 2
Example 14: p53 GRIP Assay
[0527] Thermo Scientific* BioImage p53-MDM2 Redistribution Assay
monitors the protein interaction with MDM2 and cellular
translocation of GFP-tagged p53 in response to drug compounds or
other stimuli. Recombinant CHO-hIR cells stably express human
p53(1-312) fused to the C-terminus of enhanced green fluorescent
protein (EGFP) and PDE4A4-MDM2(1-124), a fusion protein between
PDE4A4 and MDM2(1-124). They provide a ready-to-use assay system
for measuring the effects of experimental conditions on the
interaction of p53 and MDM2. Imaging and analysis is performed with
a HCS platform.
[0528] CHO-hIR cells are regularly maintained in Ham's F12 media
supplemented with 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin,
1 mg/ml Zeocin and 10% FBS. Cells seeded into 96-well plates at the
density of 7000 cells/100 .mu.M per well 18-24 hours prior to
running the assay using culture media. The next day, media is
refreshed and PD177 is added to cells to the final concentration of
3 .mu.M to activate foci formation. Control wells are kept without
PD-177 solution. 24 h post stimulation with PD177, cells are washed
once with Opti-MEM Media and 50 .mu.L of the Opti-MEM Media
supplemented with PD-177(6 .mu.M) is added to cells. Peptides are
diluted from 10 mM DMSO stocks to 500 .mu.M working stocks in
sterile water, further dilutions made in 0.5% DMSO to keep the
concentration of DMSO constant across the samples. Final highest
DMSO concentration is 0.5% and is used as the negative control.
Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as
positive control. Nutlin was diluted using the same dilution scheme
as peptides. 50 .mu.l of 2.times. desired concentrations is added
to the appropriate well to achieve the final desired
concentrations. Cells are then incubated with peptides for 6 h at
37.degree. C. in humidified 5% CO.sub.2 atmosphere. Post-incubation
period, cells are fixed by gently aspirating out the media and
adding 150 .mu.l of fixing solution per well for 20 minutes at room
temperature. Fixed cells are washed 4 times with 200 .mu.l PBS per
well each time. At the end of last wash, 100 .mu.M of 1 .mu.M
Hoechst staining solution is added. Sealed plates incubated for at
least 30 min in dark, washed with PBS to remove excess stain and
PBS is added to each well. Plates can be stored at 4.degree. C. in
dark up to 3 days. The translocation of p53/MDM2 is imaged using
Molecular translocation module on Cellomics Arrayscan instrument
using 10.times. objective, XF-100 filter sets for Hoechst and GFP.
The output parameters was Mean-CircRINGAveIntenRatio (the ratio of
average fluorescence intensities of nucleus and cytoplasm, (well
average)). The minimally acceptable number of cells per well used
for image analysis was set to 500 cells.
Example 15: MCF-7 Breast Cancer Study Using SP315, SP249 and
SP154
[0529] A xenograft study was performed to test the efficacy of
SP315, SP249 and SP154 in inhibiting tumor growth in athymic mice
in the MCF-7 breast cancer xenograft model. A negative control
stapled peptide. SP252, a point mutation of SP154 (F to A at
position 19) was also tested in one group; this peptide had shown
no activity in the SJSA-1 in vitro viability assay. Slow release 90
day 0.72 mg 17.beta.-estradiol pellets (Innovative Research,
Sarasota, Fla.) were implanted subcutaneously (sc) on the nape of
the neck one day prior to tumor cell implantation (Day -1). On Day
0, MCF-7 tumor cells were implanted sc in the flank of female nude
(Crl:NU-Foxnlnu) mice. On Day 18, the resultant sc tumors were
measured using calipers to determine their length and width and the
mice were weighed. The tumor sizes were calculated using the
formula (length.times.width.sup.2)/2 and expressed as cubic
millimeters (mm.sup.3). Mice with tumors smaller than 85.3 mm.sup.3
or larger than 417.4 mm.sup.3 were excluded from the subsequent
group formation. Thirteen groups of mice, 10 mice per group, were
formed by randomization such that the group mean tumor sizes were
essentially equivalent (mean of groups.+-.standard deviation of
groups=180.7.+-.17.5 mm.sup.3).
[0530] SP315, SP249, SP154 and SP252 dosing solutions were prepared
from peptides formulated in a vehicle containing MPEG(2K)-DSPE at
50 mg/mL concentration in a 10 mM Histidine buffered saline at pH
7. This formulation was prepared once for the duration of the
study. This vehicle was used as the vehicle control in the
subsequent study.
[0531] Each group was assigned to a different treatment regimen.
Group 1, as the vehicle negative control group, received the
vehicle administered at 8 mL/kg body weight intravenously(iv) three
times per week from Days 18-39. Groups 2 and 3 received SP154 as an
iv injection at 30 mg/kg three times per week or 40 mg/kg twice a
week, respectively. Group 4 received 6.7 mg/kg SP249 as an iv
injection three times per week. Groups 5, 6, 7 and 8 received SP315
as an iv injection of 26.7 mg/kg three times per week, 20 mg/kg
twice per week, 30 mg/kg twice per week, or 40 mg/kg twice per
week, respectively. Group 9 received 30 mg/kg SP252 as an iv
injection three times per week.
[0532] During the dosing period the mice were weighed and tumors
measured 1-2 times per week. Results in terms of tumor volume are
shown in FIGS. 3-6 and tumor growth inhibition compared with the
vehicle group, body weight change and number of mice with
.gtoreq.20% body weight loss or death are shown in Table 9. Tumor
growth inhibition (TGI) was calculated as %
TGI=100-[(TuVol.sup.Treated-day x-TuVol.sup.Treated-day
18)/(TuVol.sup.Vehicle negative control-day x-TuVol.sup.Vehicle
negative control day 18)*100, where x=day that effect of treatment
is being assessed. Group 1, the vehicle negative control group,
showed good tumor growth rate for this tumor model.
[0533] For SP154, in the group dosed with 40 mg/kg twice a week 2
mice died during treatment, indicating that this dosing regimen was
not tolerable. The dosing regimen of 30 mg/kg of SP154 three times
per week was well-tolerated and yielded a TGI of 84%.
[0534] For SP249, the group dosed with 6.7 mg/kg three times per
week 4 mice died during treatment, indicating that this dosing
regimen was not tolerable.
[0535] All dosing regimens used for SP315 showed good tolerability,
with no body weight loss or deaths noted. Dosing with 40 mg/kg of
SP315 twice per week produced the highest TGI (92%). The dosing
regimens of SP315 of 26.7 mg/kg three times per week, 20 mg/kg
twice per week, 30 mg/kg twice per week produced TGI of 86, 82, and
85%, respectively.
[0536] For SP252, the point mutation of SP154 which shows no
appreciable activity in in vitro assays, dosing with 30 mg/kg three
times per week was well-tolerated with no body weight loss or
deaths noted. While TGI of 88% was noted by Day 32, that TGI was
reduced to 41% by Day 39.
[0537] Results from this Example are shown in FIGS. 3-6 and are
summarized in Table 9.
TABLE-US-00015 TABLE 9 No. with .gtoreq.20% Group % BW No. with BW
Loss or Number Treatment Group Change .gtoreq.10% BW Loss death %
TGI 1 Vehicle +8.6 0/10 0/10 -- 2 SP154 30 mg/kg 3x/wk iv +5.7 0/10
0/10 *84 3 SP154 40 mg/kg 2x/wk iv N/A 0/10 2/10 (2 deaths) Regimen
not tolerated 4 SP249 6.7 mg/kg 3x/wk iv N/A 6/10 4/10 Regimen not
tolerated 5 SP315 26.7 mg/kg 3x/wk iv +3.7 0/10 0/10 *86 6 SP315 20
mg/kg 2x/wk iv +3.9 0/10 0/10 *82 7 SP315 30 mg/kg 2x/wk iv +8.0
0/10 0/10 *85 8 SP315 40 mg/kg 2x/wk iv +2.1 0/10 0/10 *92 9 SP252
30 mg/kg 3x/wk iv +3.3 0/10 0/10 *41 *p .ltoreq.0.05 Vs Vehicle
Control
Example 21: Solubility Determination for Peptidomimetic
Macrocycles
[0538] Peptidomimetic macrocycles are first dissolved in neat N,
N-dimethylacetamide (DMA, Sigma-Aldrich, 38840-1L-F) to make
20.times. stock solutions over a concentration range of 20-140
mg/mL. The DMA stock solutions are diluted 20-fold in an aqueous
vehicle containing 2% Solutol-HS-15, 25 mM Histidine, 45 mg/mL
Mannitol to obtain final concentrations of 1-7 mg/ml of the
peptidomimetic macrocycles in 5% DMA, 2% Solutol-HS-15, 25 mM
Histidine, 45 mg/mL Mannitol. The final solutions are mixed gently
by repeat pipetting or light vortexing, and then the final
solutions are sonicated for 10 min at room temperature in an
ultrasonic water bath. Careful visual observation is then performed
under hood light using a 7x visual amplifier to determine if
precipitate exists on the bottom or as a suspension. Additional
concentration ranges are tested as needed to determine the maximum
solubility limit for each peptidomimetic macrocycle.
[0539] Results from this Example are shown in FIG. 7.
Example 22: Preparation of Peptidomimetic Macrocycles Using a
Boc-Protected Amino Acid
[0540] Peptidomimetic macrocycle precursors were prepared as
described in Example 2 comprising an R8 amino acid at position "i"
and an S5 amino acid at position "i+7". The amino acid at position
"i+3" was a Boc-protected tryptophan which was incorporated during
solid-phase synthesis. Specifically, the Boc-protected tryptophan
amino acid shown below (and commercially available, for example,
from Novabiochem) was using during solid phase synthesis:
##STR00067##
[0541] Metathesis was performed using a ruthenium catalyst prior to
the cleavage and deprotection steps. The composition obtained
following cyclization was determined by HPLC analysis to contain
primarily peptidomimetic macrocycles having a crosslinker
comprising a trans olefin ("iso2", comprising the double bond in an
E configuration). Unexpectedly, a ratio of 90:10 was observed for
the trans and cis products, respectively.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220000968A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220000968A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References