U.S. patent application number 17/530664 was filed with the patent office on 2022-06-09 for cell penetrating peptides and methods of making and using thereof.
The applicant listed for this patent is Entrada Therapeutics, Inc.. Invention is credited to Dehua PEI, Ziqing QIAN.
Application Number | 20220177523 17/530664 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220177523 |
Kind Code |
A1 |
PEI; Dehua ; et al. |
June 9, 2022 |
CELL PENETRATING PEPTIDES AND METHODS OF MAKING AND USING
THEREOF
Abstract
Disclosed herein are compounds having activity as cell
penetrating peptides. In some examples, the compounds can comprise
a cell penetrating peptide moiety and a cargo moiety. The cargo
moiety can comprise one or more detectable moieties, one or more
therapeutic moieties, one or more targeting moieties, or any
combination thereof. In some examples, the cell penetrating peptide
moiety is cyclic. In some examples, the cell penetrating peptide
moiety and cargo moiety together are cyclic. In some examples, the
cell penetrating peptide moiety is cyclic and the cargo moiety is
appended to the cyclic cell penetrating peptide moiety structure.
In some examples, the cargo moiety is cyclic and the cell
penetrating peptide moiety is cyclic, and together they form a
fused bicyclic system.
Inventors: |
PEI; Dehua; (Columbus,
OH) ; QIAN; Ziqing; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Entrada Therapeutics, Inc. |
Boston |
MA |
US |
|
|
Appl. No.: |
17/530664 |
Filed: |
November 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16852615 |
Apr 20, 2020 |
11225506 |
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17530664 |
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15360719 |
Nov 23, 2016 |
10815276 |
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16852615 |
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15312878 |
Nov 21, 2016 |
10626147 |
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PCT/US2015/032043 |
May 21, 2015 |
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15360719 |
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62158351 |
May 7, 2015 |
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62001535 |
May 21, 2014 |
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International
Class: |
C07K 7/64 20060101
C07K007/64; A61K 49/00 20060101 A61K049/00; A61K 38/46 20060101
A61K038/46; A61K 38/05 20060101 A61K038/05; A61K 47/64 20060101
A61K047/64; A61K 38/12 20060101 A61K038/12; C07K 7/06 20060101
C07K007/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under grant
numbers GM062820, GM110208, and CA132855 awarded by the National
Institutes of Health. The government has certain rights in this
invention.
Claims
1-21. (canceled)
22. A compound comprising a cyclic peptide, the cyclic peptide
comprising 6-12 amino acids; wherein at least two amino acids are
phenylalanine and at least two amino acids are arginine; and
wherein two arginines are not consecutive and the two
non-consecutive arginines are separated by an intervening amino
acid, and wherein the two non-consecutive arginines have the same
chirality and the intervening amino acid does not have the
chirality of the two arginines.
23. The compound of claim 22, further comprising at least one amino
acid comprising a hydrophobic aromatic residue.
24. The compound of claim 23, wherein the amino acid comprising a
hydrophobic aromatic residue is naphthylalanine, tryptophan, or
tyrosine.
25. The compound of claim 22, wherein the cyclic peptide comprises
at least three arginines.
26. The compound of claim 22, wherein the cyclic peptide comprises
at least four arginines.
27. The compound of claim 22, further comprising a cargo conjugated
to the cyclic peptide.
28. The compound of claim 27, wherein the cargo is conjugated to an
amino group, side chain of an amino group, or carboxylate group of
an amino acid of the cyclic peptide.
29. The compound of claim 22, wherein the cyclic peptide comprises
Formula IIa, IIb, or IIc: ##STR00051## wherein: m, n and p are
independently selected from 0 and 1; AA.sup.1, AA.sup.2, AA.sup.3,
AA.sup.4, AA.sup.5, AA.sup.6, AA.sup.7, AA.sup.8, and AA.sup.9 are
each independently an amino acid; and the cargo comprises a
therapeutic moiety.
30. The compound of claim 27, wherein the cargo is a nucleic acid,
and enzyme or antibody.
31. The compound of claim 29, wherein the cargo is a nucleic acid,
and enzyme or antibody.
32. A method for delivering a cargo to cytoplasm of a cell,
comprising administering at least one compound of claim 29, thereby
delivering the cargo to the cytoplasm of the cell.
33. A method for delivering a cargo to cytoplasm of a cell,
comprising administering at least one compound, thereby delivering
the cargo to the cytoplasm of the cell, wherein the compound is of
formula IIA, IIb, or IIc ##STR00052## or a pharmaceutically
acceptable salt thereof, wherein: m, n, and p are independently
selected from 0 and 1, AA.sup.1 is phenylalanine, AA.sup.2 is
phenylalanine, AA.sup.3 is naphthylalanine, AA.sup.4 is arginine,
AA.sup.5 is arginine, AA.sup.6 is arginine, at least four
consecutive amino acids have alternating chirality, and the cargo
comprises a detectable moiety, a therapeutic moiety, a targeting
moiety or a combination thereof.
34. The method of claim 33, wherein: m is 0; n and p are each 1;
AA.sup.8 is arginine; and AA.sup.9 is glutamine.
35. The method of claim 34, wherein the compound is of formula
(IIa).
36. The method of claim 34, wherein the compound is of formula
(IIb).
37. The method of claim 34, wherein the compound is of formula
(IIc).
38. The method of claim 34, wherein the therapeutic moiety is a
therapeutic protein.
39. The method of claim 38, wherein the therapeutic protein
comprises an enzyme.
40. The method of claim 38, wherein the therapeutic protein
comprises an antibody or nucleic acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/852,615, filed Apr. 20, 2020, which is a
continuation of U.S. patent application Ser. No. 15/360,719, filed
Nov. 23, 2016, now U.S. Pat. No. 10,815,276, which is a
continuation in part of U.S. patent application Ser. No.
15/312,878, filed on Nov. 21, 2016, now U.S. Pat. No. 10,626,147,
which is a national phase application of International Patent
Application No. PCT/US2015/032043, filed May 21, 2015, which claims
priority to U.S. Provisional Application 62/158,351, filed May 7,
2015, and U.S. Provisional Application 62/001,535, filed May 21,
2014, the entire contents of each of which are herein incorporated
by reference in its entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
CYPT_001_05US_SubSeqList_ST25.txt, date recorded: Nov. 17, 2021,
file size 112 kilobytes).
BACKGROUND
[0004] The plasma membrane presents a major challenge in drug
discovery, especially for biologics such as peptides, proteins and
nucleic acids. One potential strategy to subvert the membrane
barrier and deliver the biologics into cells is to attach them to
"cell-penetrating peptides (CPPs)". Despite three decades of
investigation, the fundamental basis for CPP activity remains
elusive. CPPs that enter cells via endocytosis must exit from
endocytic vesicles in order to reach the cytosol. Unfortunately,
the endosomal membrane has proven to be a significant barrier
towards cytoplasmic delivery by these CPPs; often a negligible
fraction of the peptides escapes into the cell interior (El-Sayed,
A et al. AAPSJ., 2009, 11, 13-22; Varkouhi, A K et al. J.
Controlled Release, 2011, 151, 220-228; Appelbaum, J S et al. Chem.
Biol., 2012, 19, 819-830). What are thus needed are new cell
penetrating peptides and compositions comprising such peptides that
can be used to deliver agents to various cell types. The
compositions and methods disclosed herein address these and other
needs.
SUMMARY
[0005] Disclosed herein are compounds having activity as cell
penetrating peptides. In some examples, the compounds can comprise
a cell penetrating peptide moiety and a cargo moiety. The cargo
moiety can comprise one or more detectable moieties, one or more
therapeutic moieties, one or more targeting moieties, or any
combination thereof.
[0006] In some examples, the cell penetrating peptide moiety is
cyclic. In some examples, the cell penetrating peptide moiety and
cargo moiety together are cyclic; this is referred to herein as an
"endocyclic" configuration. In some examples, the cell penetrating
peptide moiety is cyclic and the cargo moiety is appended to the
cyclic cell penetrating peptide moiety structure; this is referred
to herein as an "exocyclic" configuration. In some examples, the
cargo moiety is cyclic and the cell penetrating peptide moiety is
cyclic, and together they form a fused bicyclic system; this is
referred to herein as a "bicyclic" configuration.
[0007] In some examples, the compounds can be of Formula I:
##STR00001##
wherein AA.sup.1, AA.sup.2, AA.sup.3, AA.sup.4, AA.sup.5, AA.sup.6,
AA.sup.7, AA.sup.8, and AA.sup.9 (i.e., AA.sup.1-AA.sup.9) are each
independently an amino acid; and m, n and p are independently
selected from 0 and 1. In other examples, of Formula I, there can
be more than 9 amino acids, such that when m and p are 1, n is 2 or
more. These larger peptides are disclosed with each of formula
herein, e.g., IA, II, IIa, IIb, and IIc. In some examples three or
more amino acids are arginine and one or more are phenylalanine. In
still other examples one or more amino acids is naphthylalanine or
tryptophan.
[0008] In some examples, the cell penetrating peptide moiety is
cyclic, and the compounds can be of Formula Ia:
##STR00002##
wherein AA.sup.1-AA.sup.9, m, n, and p are as defined in Formula I,
and wherein the curved line indicates a covalent bond.
[0009] In some examples, the compound further comprises a cargo
moiety, and the compounds can be of Formula II:
##STR00003##
wherein the cargo moiety can comprise a detectable moiety, a
therapeutic moiety, a targeting moiety, or a combination thereof
and AA.sup.1-AA.sup.9, m, n, and p are as defined in Formula I.
[0010] In some examples, the cell penetrating peptide moiety and
cargo moiety together are cyclic, and the compounds are of Formula
IIa:
##STR00004##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0011] In some examples, the cell penetrating peptide moiety is
cyclic and the cargo moiety is appended to the cyclic cell
penetrating peptide moiety structure, and the compounds are of
Formula IIb:
##STR00005##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0012] In some examples, the cargo moiety is cyclic and the cell
penetrating peptide moiety is cyclic, and together they form a
fused bicyclic system, and the compounds are of Formula IIc:
##STR00006##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0013] The amino acids can be coupled by a peptide bond. The amino
acids can be coupled to the cargo moiety at the amino group, the
carboxylate group, or the side chain.
[0014] In some examples, at least one amino acid comprises
napthylalanine or an analogue or derivative thereof. In some
examples, at least three of the amino acids independently comprise
arginine or an analogue or derivative thereof. In some examples, at
least one amino acid comprises phenylalanine or an analogue or
derivative thereof. In some examples of, at least one amino acid
comprises glutamine or an analogue or derivative thereof.
[0015] In some examples, the cell penetrating peptide moeity can by
any of SEQ ID NO:1 to SEQ ID NO:90. In some examples, the cell
penetrating peptide moiety can be a variant of any of SEQ ID NO:1
to SEQ ID NO:90.
[0016] The cargo moiety can comprise any cargo of interest, for
example a linker moiety, a detectable moiety, a therapeutic moiety,
a targeting moiety, and the like, or any combination thereof.
[0017] The cargo moiety can be attached to the cell penetrating
peptide moiety at the amino group, the carboxylate group, or the
side chain of any of the amino acids of the cell penetrating
peptide moiety (e.g., at the amino group, the carboxylate group, or
the side chain or any of AA.sup.1-AA.sup.9).
[0018] In some examples, the therapeutic moiety comprises a
targeting moiety. The targeting moiety can comprise, for example, a
sequence of amino acids that can target one or more enzyme domains.
In some examples, the targeting moiety can comprise an inhibitor
against a protein that can play a role in a disease, such as
cancer, cystic fibrosis, diabetes, obesity, or combinations
thereof. In some examples, the therapeutic moiety can comprise a
targeting moiety that can act as an inhibitor against Ras (e.g.,
K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, and the like, or
combinations thereof.
[0019] Also disclosed herein are compositions that comprise the
compounds described herein. Also disclosed herein are
pharmaceutically-acceptable salts and prodrugs of the disclosed
compounds.
[0020] Also provided herein are methods of use of the compounds or
compositions described herein. Also provided herein are methods for
treating a disease or pathology in a subject in need thereof
comprising administering to the subject an effective amount of any
of the compounds or compositions described herein.
[0021] Also provided herein are methods of treating, preventing, or
ameliorating cancer in a subject. The methods include administering
to a subject an effective amount of one or more of the compounds or
compositions described herein, or a pharmaceutically acceptable
salt thereof. The methods of treatment or prevention of cancer
described herein can further include treatment with one or more
additional agents (e.g., an anti-cancer agent or ionizing
radiation).
[0022] Also described herein are methods of killing a tumor cell in
a subject. The method includes contacting the tumor cell with an
effective amount of a compound or composition as described herein,
and optionally includes the step of irradiating the tumor cell with
an effective amount of ionizing radiation. Additionally, methods of
radiotherapy of tumors are provided herein. The methods include
contacting the tumor cell with an effective amount of a compound or
composition as described herein, and irradiating the tumor with an
effective amount of ionizing radiation.
[0023] In some examples of the methods of treating of treating,
preventing, or ameliorating cancer or a tumor in a subject, the
compound or composition administered to the subject can comprise a
therapeutic moiety that can comprise a targeting moiety that can
act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2
SH2, or combinations thereof.
[0024] The disclosed subject matter also concerns methods for
treating a subject having a metabolic disorder or condition. In one
embodiment, an effective amount of one or more compounds or
compositions disclosed herein is administered to a subject having a
metabolic disorder and who is in need of treatment thereof. In some
examples, the metabolic disorder can comprise type II diabetes. In
some examples of the methods of treating of treating, preventing,
or ameliorating the metabolic disorder in a subject, the compound
or composition administered to the subject can comprise a
therapeutic moiety that can comprise a targeting moiety that can
act as an inhibitor against PTP1B.
[0025] The disclosed subject matter also concerns methods for
treating a subject having an immune disorder or condition. In one
embodiment, an effective amount of one or more compounds or
compositions disclosed herein is administered to a subject having
an immune disorder and who is in need of treatment thereof. In some
examples of the methods of treating of treating, preventing, or
ameliorating the immune disorder in a subject, the compound or
composition administered to the subject can comprise a therapeutic
moiety that can comprise a targeting moiety that can act as an
inhibitor against Pin1.
[0026] The disclosed subject matter also concerns methods for
treating a subject having cystic fibrosis. In one embodiment, an
effective amount of one or more compounds or compositions disclosed
herein is administered to a subject having cystic fibrosis and who
is in need of treatment thereof. In some examples of the methods of
treating the cystic fibrosis in a subject, the compound or
composition administered to the subject can comprise a therapeutic
moiety that can comprise a targeting moiety that can act as an
inhibitor against CAL PDZ.
[0027] The details of one or more 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.
DESCRIPTION OF FIGURES
[0028] The accompanying Figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0029] FIGS. 1A, 1B, and 1C display structures showing cargo
attachment during endocyclic (A), exocyclic (B), and bicyclic (C)
delivery of cargos (shown in light grey) by cF.PHI.R.sub.4.
[0030] FIG. 2 displays the structures of some of the peptides used
in this study.
[0031] FIG. 3 displays a scheme showing the synthesis of
cF.PHI.R.sub.4-S-S-GFP.
[0032] FIG. 4 displays a scheme showing the synthesis of
cF.PHI.R.sub.4-PTP1B.
[0033] FIGS. 5A and 5B display the binding of FITC-labeled
cF.PHI.R.sub.4, R.sub.9 and Tat to (FIG. 5A) SUV and (FIG. 5B)
heparin sulfate.
[0034] FIGS. 6A and 6B display representative live-cell confocal
images of HEK293 cells treated for 2 h with rhodamine B-labeled
peptides and fluid-phase uptake marker, dextran.sup.FITC. (FIG. 6A)
Cells treated with 5 .mu.M cF.PHI.R.sub.4-A.sub.5 and
dextran.sup.FITC in the same Z-section. (FIG. 6B) Cells treated
with 5 .mu.M cF.PHI.R.sub.4-R.sub.5 and dextran.sup.FITC in the
same Z-section.
[0035] FIG. 7 displays the effect of cF.PHI.R.sub.4 on the
endocytosis of dextran.sup.Alexa488 by HeLa cells. HeLa cells were
treated with clear DMEM containing no supplement, 1 .mu.M
cF.PHI.R.sub.4 only, 100 .mu.M dextran.sup.Alexa488 only, or both 1
.mu.M cF.PHI.R.sub.4 and 100 .mu.M dextran.sup.Alexa488 MFI, mean
fluorescence intensity.
[0036] FIG. 8 displays the effect of pH on CAP fluorescence.
cF.PHI.R.sub.4-PCP was dephosphorylated by alkaline phosphatase and
purified by HPLC and its fluorescence at indicated pH's was
measured.
[0037] FIGS. 9A, 9B, and 9C display the internalization of
pCAP-containing peptides into cultured cells: I, untagged PCP; II,
cF.PHI.R.sub.4-PCP; III, cF.PHI.R.sub.4-PCP and Na.sub.3VO.sub.4;
IV, R.sub.9-PCP; V, Tat-PCP; and VI, Antp-PCP. (FIG. 9A)
Representative live-cell confocal images of HEK293 cells treated
with 5 .mu.M peptides. Top panel, nuclear stain with DRAQ5; bottom
panel, CAP fluorescence in the same Z-section. (FIG. 9B) Flow
cytometry of HeLa cells treated with 0 or 10 .mu.M peptides. (FIG.
9C) CAP fluorescence from (FIG. 9B) after subtraction of background
fluorescence (untreated cells). MFI, mean fluorescence
intensity.
[0038] FIGS. 10A, 10B, 10C, 10D, 10E, and 10F displays
representative live-cell confocal microscopic images of HEK293
cells treated for 2 h with rhodamine B-labeled peptides (5 .mu.M
each) and fluid-phase endocytosis marker, dextran.sup.FITC (0.5
mg/mL). The red fluorescence of rhodamine B and the green
fluorescence of dextran.sup.FITC from the same Z-section and their
merged image are shown in each panel. The enlarged images of a
typical cell(s) are shown in each case in order to show the
intracellular distribution of the internalized peptides. (FIG. 10A)
Cells treated with bicyclo(F.PHI.R.sub.4-A.sub.5).sup.Rho; (FIG.
10B) monocyclo(F.PHI.R.sub.4-A.sub.5).sup.Rho; (FIG. 10C)
bicyclo(F.PHI.R.sub.4-A.sub.7).sup.Rho; (FIG. 10D)
monocyclo(F.PHI.R.sub.4-A.sub.7).sup.Rho; (FIG. 10E)
bicyclo(F.PHI.R.sub.4-RARAR).sup.Rho; and (FIG. 10F)
bicyclo(F.PHI.R.sub.4-DADAD).sup.Rho.
[0039] FIGS. 11A and 11B display: (FIG. 11A) Structures of
CPP-S-S-GFP conjugates; and (FIG. 1B) Live-cell confocal images of
mammalian cells after 2-h treatment with 1 .mu.M GFP (I),
Tat-S-S-GFP (II), or cF.PHI.R.sub.4-S-S-GFP (III) and nuclear stain
DRAQ5. All images were recorded in the same Z-section.
[0040] FIGS. 12A and 12B display: (FIG. 12A) Western blot analysis
of the global pY protein levels of NIH 3T3 cells after treatment
with 0-500 nM PTP1B or cF.PHI.R.sub.4-PTP1B (IB: anti-pY antibody
4G10); and (FIG. 12B) Same samples as in (FIG. 12A) were analyzed
by SDS-PAGE and coomassie blue staining. M, molecular-weight
markers.
[0041] FIGS. 13A and 13B display: (FIG. 13A) Comparison of the
serum stability of cF.PHI.R.sub.4, Tat, R.sub.9, and Antp; and
(FIG. 13B) Cytotoxicity of cF.PHI.R.sub.4. The indicated cell lines
were treated with DMSO (control), 5 .mu.M, or 50 .mu.M
cF.PHI.R.sub.4 for 24 h and the percentage of live cells was
determined by MTT assay.
[0042] FIGS. 14A and 14B display MTT assay of various mammalian
cells after treatment with cF.PHI.R.sub.4 (5 or 50 .mu.M) for (FIG.
14A) 48 h or (FIG. 14B) 72 h.
[0043] FIG. 15 displays a diagram showing the points along the
endocytic pathway where cF.PHI.R.sub.4, R.sub.9, and Tat escape
into the cytoplasm and where specific inhibitors are proposed to
function.
[0044] FIG. 16 displays scheme showing the reversible cyclization
strategy for delivering linear peptidyl cargos into mammalian
cells. GSH, glutathione.
[0045] FIGS. 17A, 17B, 17C, 17D, 17E, and 17F display: (FIG. 17A)
Synthesis of disulfide-bond cyclized peptide. (FIG. 17B) Synthesis
of thioether-bond cyclized peptide. Reagents and conditions: (a)
Standard Fmoc/HATU chemistry; (b) piperidine/DMF; (c)
3,3'-dithiodipropionic acid/DIC; (d) .beta.-mercaptoethanol/DMF;
(e) modified reagent K; (f) trituration; (g) DMSO/DPBS (pH 7.4).
(h) 4-bromobutyric acid/DIC; (i) 1% TFA/DCM; (j) 1% DIPEA/DMF; PG,
protecting group. Trt, trityl; Mint, methoxytrityl. (FIG. 17C)
Structures of FITC labeled peptides 1 and 2. (FIG. 17D) Structures
of pCAP (phosphocoumaryl aminopropionic acid) containing peptides
1-PCP and 2-PCP. (FIG. 17E) Structures of Amc
(7-amino-4-methylcourmarin) containing caspase fluorogenic
substrates 3-7. (FIG. 17F) Structures of FITC labeled CAL-PDZ
domain ligands 9-11.
[0046] FIGS. 18A and 18B display (FIG. 18A) Live-cell confocal
microscopic images of HeLa cells treated with 5 .mu.M FITC-labeled
peptide 1 (I) or 2 (II), endocytosis marker Dextran.sup.Rho (0.5 mg
mL.sup.-1), and nuclear stain DRAQ5. Images in different
fluorescence channels were all recorded in the same Z-section.
(FIG. 18B) Flow cytometry of HeLa cells treated with 5 .mu.M
FITC-labeled peptides 1, 2, or FITC alone.
[0047] FIGS. 19A and 19B display: (FIG. 19A) FACS analysis of HeLa
cells treated with 0 or 5 .mu.M peptides 1-PCP, 2-PCP for 2 h; and
(FIG. 19B) CAP fluorescence from (FIG. 19A) after subtraction of
background fluorescence (untreated cell). MFI, mean fluorescence
intensity.
[0048] FIG. 20 displays a comparison of the proteolytic stability
of peptides 1 and 2.
[0049] FIG. 21 displays the time-dependent release of fluorogenic
coumarin product by Jurkat cells treated with peptides 3-7 (5
.mu.M) in the absence and presence of 100 .mu.M caspase inhibitor
Z-VAD(OMe)-FMK (FMK).
[0050] FIGS. 22A, 22B, 22C, 22D, and 22E display: (FIG. 22A)
Structure of CAL-PDZ inhibitor 8. (FIG. 22B) Binding of peptide 8
to CAL-PDZ domain in the presence or absence of reducing reagent.
(FIG. 22C) Live-cell microscopic images of HeLa cells treated with
peptide 8 (5 .mu.M) and DRAQ5 in the same Z-section. I, green
fluorescence of internalized peptide 8; II, overlay of green
peptide fluorescence and blue nuclear stain. (FIG. 22D)
Immunofluorescent staining showing the distribution of CFTR in the
presence or absence of Corr-4a (10 .mu.M) and unlabeled peptide 8
(50 .mu.M). (FIG. 22E) SPQ assays showing CFTR-specific
stimulation-induced fluorescence increase in slope in the absence
or presence of VX809 (20 .mu.M) and peptide 8 (50 .mu.M). P values
were calculated from two-tailed t-test.
[0051] FIG. 23 displays a schematic of the evolution of a
cell-permeable PTP1B inhibitor.
[0052] FIG. 24 displays a schematic of the design and synthesis of
cyclic peptide library. Reagents and conditions: (a) standard
Fmoc/HBTU chemistry; (b) soak in water; (c) 0.1 equiv
Fmoc-Glu(.delta.-NHS)-OAll, 0.4 equiv Boc-Met-OH in
Et.sub.2O/CH.sub.2Cl.sub.2; (d) piperidine; (e) split into two
parts; (f) split-and-pool synthesis by Fmoc/HATU chemistry; (g)
Pd(PPh.sub.3).sub.4; (h) PyBOP, HOBt; and (i) Reagent K. X.sup.2,
10% F.sub.2Pmp and 90% Tyr; X.sup.1 and X.sup.3--X.sup.5, random
positions; .PHI., L-2-naphthylalanine; CPP, cell-penetrating motif
F.PHI.R.sub.4 or R.sub.4.PHI.F.
[0053] FIGS. 25A and 25B display the competitive inhibition of
PTP1B by monocyclic peptide inhibitor 2. (FIG. 25A) Lineweaver-Burk
plots for PTP1B-catalyzed hydrolysis of pNPP (0-24 mM) in the
presence of varying concentrations of inhibitor 2 (0, 22.5, 45, and
90 nM). (FIG. 25B) Secondary plot of the Michaelis constant ratio
(K/K.sub.0) as a function of [I].
[0054] FIGS. 26A, 26B, and 26C display: (FIG. 26A) live-cell
confocal microscopic images (same Z-section) of A549 lung cancer
cells after treatment for 2 h with 5 .mu.M FITC-labeled inhibitor 2
(top panel) or 4 (bottom panel) and endocytosis marker
dextran.sup.Rho (1.0 mg/mL); (FIG. 26B) Lineweaver-Burk plot
showing competitive inhibition of PTP1B by 0, 28, 56, and 112 nM
inhibitor 4; and (FIG. 26C) Sensitivity of various PTPs to
inhibition by inhibitor 4 (all activities were relative to that in
the absence of inhibitor).
[0055] FIG. 27 displays the solid-phase synthesis of inhibitor 4.
Reagents and conditions: a) standard Fmoc chemistry; b) trimesic
acid, HBTU; c) Pd(PPh.sub.3).sub.4, N-methylaniline; d) PyBOP; e)
TFA.
[0056] FIG. 28 displays a comparison of the serum stability of
monocyclic PTP1B inhibitor 2 and bicyclic inhibitor 4.
[0057] FIGS. 29A, 29B, 29C, and 29D display: (FIG. 29A) Global pY
protein levels in A549 cells after treatment with 0-5 .mu.M
inhibitor 4 for 2 h; (FIG. 29B) SDS-PAGE analysis (Coomassie blue
staining) of the same samples from (FIG. 29A) shows uniform sample
loading in all lanes; (FIG. 29C) Effect of inhibitor 4 on insulin
receptor phosphorylation at Tyr.sup.1162 and Tyr.sup.1163 sites.
HepG2 cells were treated with indicated concentrations of inhibitor
4 for 2 h and then stimulated with insulin (100 nM) for 5 min,
followed by SDS-PAGE and immunoblotting with
anti-IRpY.sup.1162/pY.sup.1163 antibody; and (FIG. 29D)
Quantitation of IR pY levels from (FIG. 29C) (data shown are the
mean.+-.SD from five independent experiments).
[0058] FIG. 30 displays the conversion of impermeable Pin1
inhibitor into a cell-permeable bicyclic inhibitor.
[0059] FIGS. 31A, 31B, 31C, 31D, and 31E display the FA analysis of
the binding of Pin1 inhibitor 5-9 to Pin1, respectively.
[0060] FIGS. 32A and 32B display the competition for binding to
Pin1 by inhibitors 5 and 7. Each reaction contained 0.1 .mu.M
FITC-labeled inhibitor 5, 1 .mu.M Pin1, and 0-5 .mu.M unlabeled
inhibitor 5 (FIG. 3A) or inhibitor 7 (FIG. 32B) and the FA value
was measured and plotted against the competitor concentration.
[0061] FIGS. 33A, 33B, and 33C display the cellular uptake of Pin1
inhibitors. Live-cell confocal microscopic images of HEK293 cells
treated with 5 .mu.M FITC-labeled Pin1 inhibitor 5 (FIG. 33A) or 7
(FIG. 33B) and 1 mg/mL endocytosis marker Dextran.sup.Rho for 2 h.
All images were recorded at the same Z-section. (FIG. 33C) FACS
analysis of HeLa cells after 2-h treatment with DMSO or 5 .mu.M
FITC-labeled Pin1 inhibitor 5, 7, 8, or 9. MFI, mean fluorescence
intensity. Procedure: Hela cells were cultured in six-well plates
(2.times.10.sup.5 cells per well) for 24 h. On the day of
experiment, the cells were incubated with 5 .mu.M FITC labeled
bicyclic peptide or control monocyclic peptide in phenol red-free
DMEM supplemented with 1% FBS. After 2 h, the peptide solution was
removed, and the cells were washed with DPBS, treated with 0.25%
trypsin for 5 min, washed again with DPBS. Finally, the cells were
resuspended in the flow cytometry buffer and analyzed by flow
cytometry (BD FACS Aria), with excitation at 535 nm.
[0062] FIG. 34 displays the effect of Pin1 Inhibitors 5, 7, 8, and
9 on cancer cell proliferation. HeLa cells (100 .mu.L/each well,
5.times.10.sup.4 cells/mL) were seeded in a 96-well culture plate
and allowed to grow overnight in DMEM supplemented with 10% FBS.
Varying concentrations of Pin1 inhibitor (0-5 .mu.M) were added to
the wells and the cells were incubated at 37.degree. C. with 5% CO2
for 72 h. After that, 10 .mu.L of a MTT stock solution (5 mg/mL)
was added into each well. The plate was incubated at 37.degree. C.
for 4 h and 100 .mu.L of SDS-HCl solubilizing solution was added
into each well, followed by thorough mixing. The plate was
incubated at 37.degree. C. overnight and the absorbance of the
formazan product was measured at 570 nm on a Molecular Devices
Spectramax M5 plate reader. Each experiment was performed in
triplicates and the cells untreated with peptide were used as
control.
[0063] FIGS. 35A, 35B, 35C, and 35D display live cell confocal
images of mouse ventricular cardiac myocytes after treatment for 3
h with 5 .mu.M c(F.PHI.RRRRQ)-K(FITC) (FIG. 35A) and
c(f.PHI.RrRrQ)-K(FITC) (FIG. 35B). FIG. 35C displays labeling of
calmodulin (T5C) with cyclic cell penetrating peptide through a
disulfide bond. FIG. 35D displays live cell confocal images of
mouse ventricular cardiac myocytes after treatment for 3 h with 6
.mu.M cF.PHI.R.sub.4-conjugated Cy3-labeled calmodulin.
[0064] FIG. 36 displays the evolution of bicyclic peptide
inhibitors against Pin1. The structural moieties derived from
library screening are shown in grey, while the changes made during
optimization are shown in light grey.
[0065] FIGS. 37A, 37B, 37C, and 37D displays the characterization
of peptide 37. (FIG. 37A) Binding to FITC-labeled peptide 37 to
Pin1 as analyzed by fluorescent anisotropy (FA). (FIG. 37B)
Competition between peptide 37 and FITC-labeled peptide 1 (100 nM)
for binding to Pin1 (400 nM) as monitored by FA. (FIG. 37C) Effect
of peptide 37 on the cis-trans isomerase activity of Pin1, Pin4,
FKBP12, and cyclophilin A using Suc-Ala-Glu-Pro-Phe-pNA as
substrate. (FIG. 37D) Comparison of the serum stability of peptides
1 and 37.
[0066] FIGS. 38A, 38B, 38C, and 38D display cellular activity of
peptide 37. (FIG. 38A) Cellular uptake of peptides 1, 37, and 46 (5
.mu.M) by HeLa cells as analyzed by flow cytometry. MFI, mean
fluorescence intensity; none, untreated cells (no peptide). (FIG.
38B) Anti-proliferative effect of peptides 37, 46, and 47 on HeLa
cells as measured by MTT assay. (FIG. 38C) Western blots showing
the effect of peptides 1, 37 and 47 on the protein level of PML in
HeLa cells. .beta.-Actin was used as loading control. (FIG. 38D)
Quantification of western blot results from (FIG. 38C). Data
reported were after background subtraction and represent the
mean.+-.SD from 3 independent experiments.
DETAILED DESCRIPTION
[0067] The compounds, compositions, and methods described herein
may be understood more readily by reference to the following
detailed description of specific aspects of the disclosed subject
matter and the Examples and Figures included therein.
[0068] Before the present compounds, compositions, and methods are
disclosed and described, it is to be understood that the aspects
described below are not limited to specific synthetic methods or
specific reagents, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular aspects only and is not intended to be
limiting.
[0069] Also, throughout this specification, various publications
are referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which the disclosed matter pertains. The references disclosed are
also individually and specifically incorporated by reference herein
for the material contained in them that is discussed in the
sentence in which the reference is relied upon.
General Definitions
[0070] In this specification and in the claims that follow,
reference will be made to a number of terms, which shall be defined
to have the following meanings.
[0071] Throughout the description and claims of this specification
the word "comprise" and other forms of the word, such as
"comprising" and "comprises," means including but not limited to,
and is not intended to exclude, for example, other additives,
components, integers, or steps.
[0072] As used in the description and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a composition" includes mixtures of two or more such
compositions, reference to "an agent" includes mixtures of two or
more such agents, reference to "the component" includes mixtures of
two or more such components, and the like.
[0073] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0074] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. By
"about" is meant within 5% of the value, e.g., within 4, 3, 2, or
10% of the value. When such a range is expressed, another aspect
includes from the one particular value and/or to the other
particular value. Similarly, when values are expressed as
approximations, by use of the antecedent "about," it will be
understood that the particular value forms another aspect. It will
be further understood that the endpoints of each of the ranges are
significant both in relation to the other endpoint, and
independently of the other endpoint.
[0075] As used herein, by a "subject" is meant an individual. Thus,
the "subject" can include domesticated animals (e.g., cats, dogs,
etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),
laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.),
and birds. "Subject" can also include a mammal, such as a primate
or a human. Thus, the subject can be a human or veterinary patient.
The term "patient" refers to a subject under the treatment of a
clinician, e.g., physician.
[0076] The term "inhibit" refers to a decrease in an activity,
response, condition, disease, or other biological parameter. This
can include but is not limited to the complete ablation of the
activity, response, condition, or disease. This can also include,
for example, a 10% reduction in the activity, response, condition,
or disease as compared to the native or control level. Thus, the
reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any
amount of reduction in between as compared to native or control
levels.
[0077] By "reduce" or other forms of the word, such as "reducing"
or "reduction," is meant lowering of an event or characteristic
(e.g., tumor growth). It is understood that this is typically in
relation to some standard or expected value, in other words it is
relative, but that it is not always necessary for the standard or
relative value to be referred to. For example, "reduces tumor
growth" means reducing the rate of growth of a tumor relative to a
standard or a control.
[0078] By "prevent" or other forms of the word, such as
"preventing" or "prevention," is meant to stop a particular event
or characteristic, to stabilize or delay the development or
progression of a particular event or characteristic, or to minimize
the chances that a particular event or characteristic will occur.
Prevent does not require comparison to a control as it is typically
more absolute than, for example, reduce. As used herein, something
could be reduced but not prevented, but something that is reduced
could also be prevented. Likewise, something could be prevented but
not reduced, but something that is prevented could also be reduced.
It is understood that where reduce or prevent are used, unless
specifically indicated otherwise, the use of the other word is also
expressly disclosed. For example, the terms "prevent" or "suppress"
can refer to a treatment that forestalls or slows the onset of a
disease or condition or reduced the severity of the disease or
condition. Thus, if a treatment can treat a disease in a subject
having symptoms of the disease, it can also prevent or suppress
that disease in a subject who has yet to suffer some or all of the
symptoms.
[0079] The term "treatment" refers to the medical management of a
patient with the intent to cure, ameliorate, stabilize, or prevent
a disease, pathological condition, or disorder. This term includes
active treatment, that is, treatment directed specifically toward
the improvement of a disease, pathological condition, or disorder,
and also includes causal treatment, that is, treatment directed
toward removal of the cause of the associated disease, pathological
condition, or disorder. In addition, this term includes palliative
treatment, that is, treatment designed for the relief of symptoms
rather than the curing of the disease, pathological condition, or
disorder; preventative treatment, that is, treatment directed to
minimizing or partially or completely inhibiting the development of
the associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0080] The term "anticancer" refers to the ability to treat or
control cellular proliferation and/or tumor growth at any
concentration.
[0081] The term "therapeutically effective" refers to the amount of
the composition used is of sufficient quantity to ameliorate one or
more causes or symptoms of a disease or disorder. Such amelioration
only requires a reduction or alteration, not necessarily
elimination.
[0082] The term "pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other
problems or complications commensurate with a reasonable
benefit/risk ratio.
[0083] The term "carrier" means a compound, composition, substance,
or structure that, when in combination with a compound or
composition, aids or facilitates preparation, storage,
administration, delivery, effectiveness, selectivity, or any other
feature of the compound or composition for its intended use or
purpose. For example, a carrier can be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject.
[0084] The terms "peptide," "protein," and "polypeptide" are used
interchangeably to refer to a natural or synthetic molecule
comprising two or more amino acids linked by the carboxyl group of
one amino acid to the alpha amino group of another.
[0085] Unless stated to the contrary, a formula with chemical bonds
shown only as solid lines and not as wedges or dashed lines
contemplates each possible isomer, e.g., each enantiomer,
diastereomer, and meso compound, and a mixture of isomers, such as
a racemic or scalemic mixture.
[0086] Reference will now be made in detail to specific aspects of
the disclosed materials, compounds, compositions, articles, and
methods, examples of which are illustrated in the accompanying
Examples and Figures.
[0087] Compounds
[0088] Disclosed herein are compounds having activity as cell
penetrating peptides. In some examples, the compounds can comprise
a cell penetrating peptide moiety and a cargo moiety. The cargo
moiety can comprise one or more detectable moieties, one or more
therapeutic moieties, one or more targeting moieties, or any
combination thereof.
[0089] In some examples, the cell penetrating peptide moiety is
cyclic. In some examples, the cell penetrating peptide moiety and
cargo moiety together are cyclic. In some examples, the cell
penetrating peptide moiety is cyclic and the cargo moiety is
appended to the cyclic cell penetrating peptide moiety structure.
In some examples, the cargo moiety is cyclic and the cell
penetrating peptide moiety is cyclic, and together they form a
fused bicyclic system.
[0090] The cell penetrating peptide moiety can comprise five or
more, more specifically six or more, for example, six to twelve, or
six to nine amino acids. When there are six to nine amino acids the
compounds can be of Formula I.
##STR00007##
wherein AA.sup.1, AA.sup.2, AA.sup.3, AA.sup.4, AA.sup.5, AA.sup.6,
AA.sup.7, AA.sup.8, and AA.sup.9 (i.e., AA.sup.1-AA.sup.9) are each
independently an amino acid; and m, n and p are independently
selected from 0 and 1. Wherein there are more than 9 amino acids,
Formula I can have m and p each be 1 and n can be 2 or more, e.g.,
2 to 10 or 2 to 5. In some examples three or more amino acids are
arginine and one or more are phenylalanine. In still other examples
one or more amino acids is naphthylalanine or tryptophan.
[0091] In some examples, the compounds can be of Formula I:
##STR00008##
wherein AA.sup.1, AA.sup.2, AA.sup.3, AA.sup.4, AA.sup.5, AA.sup.6,
AA.sup.7, AA.sup.8, and AA.sup.9 (i.e., AA.sup.1-AA.sup.9) are each
independently an amino acid; and m, n and p are independently
selected from 0 and 1.
[0092] In some examples, the cell penetrating peptide moiety is
cyclic, and the compounds can be of Formula Ia:
##STR00009##
wherein AA.sup.1-AA.sup.9, m, n, and p are as defined in Formula I,
and wherein the curved line indicates a covalent bond. The curved
line can be a covalent bond in the backbone of the peptide (i.e.,
the carboxylic acid of one AA forming an amide bond with the
.alpha.-amine of another AA), a bond between the side chains of two
AAs, a bond from one side chain of an AA to either the backbone
carboxylic acid or .alpha.-amine of another AA, or a disulfide bond
between two AAs.
[0093] In some examples, the compound further comprises a cargo
moiety, and the compounds can be of Formula II:
##STR00010##
wherein the cargo moiety can comprise a detectable moiety, a
therapeutic moiety, a targeting moiety, or a combination thereof
and AA.sup.1-AA.sup.9, m, n, and p are as defined in Formula I.
[0094] In some examples, the cell penetrating peptide moiety and
cargo moiety together are cyclic, and the compounds are of Formula
IIa:
##STR00011##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0095] In some examples, the cell penetrating peptide moiety is
cyclic and the cargo moiety is appended to the cyclic cell
penetrating peptide moiety structure, and the compounds are of
Formula IIb:
##STR00012##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0096] In some examples, the cargo moiety is cyclic and the cell
penetrating peptide moiety is cyclic, and together they form a
fused bicyclic system, and the compounds are of Formula IIc:
##STR00013##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0097] Cell Penetrating Peptide
[0098] The cell penetrating peptide moeity comprises at least 5,
more specifically, at least 6 amino acids, even more specifically
from 6 to 12, from 6 to 9, from 6 to 7, from 7 to 8, from 8 to 9,
and more specially 6, 7, 8, or 9 amino acids. For the endocyclic
motif, at least 5 amino acids can be used. It is also disclosed
herein that for the endocyclic structure, some amino acids in the
penetrating peptide moiety can also be part of the cargo moiety.
For example, a peptide penetrating moiety FNalRR can be formed when
from FNal and an cargo moiety with two Args. In this case, the two
Arg residues perform dual functions. Thus, in some cases the
sequence of the cargo moiety is taken into account when referring
to the peptide penetrating moiety.
[0099] For the exocyclic motif, at least 6 amino acids can be used
with, for example, glutamine being used to attach the cargo.
[0100] Each amino acid can be a natural or non-natural amino acid.
The term "non-natural amino acid" refers to an organic compound
that is a congener of a natural amino acid in that it has a
structure similar to a natural amino acid so that it mimics the
structure and reactivity of a natural amino acid. The non-natural
amino acid can be a modified amino acid, and/or amino acid analog,
that is not one of the 20 common naturally occurring amino acids or
the rare natural amino acids selenocysteine or pyrrolysine.
Non-natural amino acids can also be the D-isomer of the natural
amino acids. Examples of suitable amino acids include, but are not
limited to, alanine, allosoleucine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, napthylalanine,
phenylalanine, proline, pyroglutamic acid, serine, threonine,
tryptophan, tyrosine, valine, a derivative, or combinations
thereof. These, and others, are listed in the Table 1 along with
their abbreviations used herein.
TABLE-US-00001 TABLE 1 Amino Acid Abbreviations Amino Acid
Abbreviations* alanine Ala (A) allosoleucine AIle arginine Arg (R)
asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C)
cyclohexylalanine Cha 2,3-diaminopropionic acid Dap
4-fluorophenylalanine Fpa (.SIGMA.) glutamic acid Glu (E) glutamine
Gln (Q) glycine Gly (G) histidine His (H) homoproline Pip (.THETA.)
isoleucine Ile (I) leucine Leu (L) lysine Lys (K) methionine Met
(M) napthylalanine Nal (.PHI.) norleucine Nle (.OMEGA.)
phenylalanine Phe (F) phenylglycine Phg (.PSI.)
4-(phosphonodifluoromethyl)phenylalanine F.sub.2Pmp (.LAMBDA.)
pipecolic acid PP ( ) proline Pro (P) sarcosine Sar (.XI.)
selenocysteine Sec (U) serine Ser (S) threonine Thr (T) tyrosine
Tyr (Y) tryptophan Trp (W) valine Val (V) *single letter
abbreviations: when shown incapital letters herein it indicates the
L-amino acid form, when shown in lower case herein it indicates the
D-amino acid form
[0101] The amino acids can be coupled by a peptide bond. The amino
acids can be coupled to the cargo moiety at the amino group, the
carboxylate group, or the side chain.
[0102] In some examples of Formula I, at least one amino acid
comprises napthylalanine or tryptophan, or analogues or derivatives
thereof. In some examples of Formula I, at least three of the amino
acids independently comprise arginine or an analogue or derivative
thereof. In some examples of Formula I, at least one amino acid
comprises phenylalanine, phenylglycine, or histidine, or analogues
or derivatives thereof. In some examples of Formula I, at least one
amino acid comprises glutamine or an analogue or derivative
thereof.
[0103] In some examples, the cell penetrating peptide (CPP) moiety
can be any of the sequences listed in Table 2. In some examples,
the cell penetrating peptide can be the reverse of any of the
sequences listed in Table 2. In some examples, the cell penetrating
peptide sequence can be a cyclic form of any of the sequences
listed in Table 2.
TABLE-US-00002 TABLE 2 CPP sequences - linear or cyclic SEQ ID NO
CPP sequence # AA's #R residues 1 F.PHI.RRRQ 6 3 2 F.PHI.RRRC 6 3 3
F.PHI.RRRU 6 3 4 RRR.PHI.FQ 6 3 5 RRRR.PHI.F 6 4 6 F.PHI.RRRR 6 4 7
F.PHI.RrRq 7 3 8 F.PHI.RrRQ 7 3 9 F.PHI.RRRRQ 7 4 10 f.PHI.RrRrQ 7
4 11 RRFR.PHI.RQ 7 4 12 FRRRR.PHI.Q 7 4 13 rRFR.PHI.RQ 7 4 14
RR.PHI.FRRQ 7 4 15 CRRRRFWQ 7 4 16 Ff.PHI.RrRrQ 8 4 17 FF.PHI.RRRRQ
8 4 18 RFRFR.PHI.RQ 8 4 19 URRRRFWQ 8 4 20 CRRRRFWQ 8 4 21
F.PHI.RRRRQK 8 4 22 F.PHI.RRRRQC 8 4 23 f.PHI.RrRrRQ 8 5 24
F.PHI.RRRRRQ 8 5 25 RRRR.PHI.FD.OMEGA.C 9 4 26 F.PHI.RRR 5 3 27
FWRRR 5 3 28 RRR.PHI.F 5 3 29 RRRWF 5 3 .PHI. = L-naphthylalanine;
.PHI. = D-naphthylalanine; .OMEGA. = L-norleucine
[0104] Certain embodiments of the invention include amino acid
sequences wherein at least four consecutive amino acids have
alternating chirality. As used herein, chirality refers to the "D"
and "L" isomers of amino acids. In particular embodiments of the
invention, at least four consecutive amino acids have alternating
chirality and the remaining amino acids are L-amino acids. In other
embodiments, the peptides of the invention comprise a four amino
acid sequence having D-L-D-L chirality. In still other embodiments,
the peptides of the invention comprise a four amino acid sequence
having L-D-L-D chirality.
[0105] In embodiments, peptides of the invention comprise two
consecutive L-amino acids.
[0106] In further embodiments, peptides of the invention comprise
two consecutive L-amino acids separating two D-amino acids. In yet
further embodiments, peptides of the invention comprise two
consecutive L-amino acids separating two D-amino acids and at least
four consecutive amino acids having alternating chirality, such as,
but not limited to peptide sequences with D-L-L-D-L-D or
L-D-L-L-D-L-D chirality. In even further embodiments, peptides of
the invention comprise two consecutive L-amino acids separating two
D-amino acids and at least five consecutive amino acid having
alternating chirality, such as, but not limited to peptide
sequences with D-L-L-D-L-D-L or L-D-L-L-D-L-D-L chirality.
[0107] In embodiments, peptides of the invention comprise two
consecutive D-amino acids. In further embodiments, peptides of the
invention comprise two consecutive D-amino acids separating two
L-amino acids. In still further embodiments of the invention,
peptides of the invention comprise two consecutive D-amino acids
separating two L-amino acids and at least four consecutive amino
acids having alternating chirality, such as, but not limited to
peptide sequences with L-D-D-L-D-L. In even further embodiments of
the invention, peptides of the invention comprise two consecutive
D-amino acids separating two L-amino acids and at least five
consecutive amino acids having alternating chirality, such as, but
not limited to peptide sequences with L-D-D-L-D-L-D.
[0108] In some embodiments, the amino acid sequence with
alternating chirality comprises about at least about 4 amino acids,
at least about 5 amino acids, at least about 6 amino acids, at
least about 7 amino acids, at least about 8 amino acids or at least
about 9 amino acids. In embodiments, the amino acid sequence with
alternating chirality comprises of from about 4 amino acids to
about 9 amino acids, or about 5 amino acids to about 6 amino acids,
or about 7 amino acids to about 9 amino acids, or about 8 amino
acids to about 9 amino acids, or about 4 amino acids to about 8
amino acids, or about 4 amino acids to about 7 amino acids, or
about 4 amino acids to about 6 amino acids, or about 4 amino acids
to about 5 amino acids.
[0109] In particular embodiments, the cyclic cell-penetrating
peptides of the invention demonstrate improved cellular uptake
efficiency as compared to c(F.PHI.RRRRQ) (290-1F).
[0110] As used herein cellular uptake efficiency refers to the
ability of a cyclic peptide sequence to traverse a cell membrane.
In embodiments, cellular uptake of the cyclic, cell penetrating
peptide is not dependent on a receptor or a cell type.
[0111] In particular embodiments, uptake efficiency is determined
by comparing (i) the amount of a cyclic cell-penetrating peptide of
the invention internalized by a cell type (e.g., HeLa cells) to
(ii) the amount of c(F.PHI.RRRRQ) (290-1F) internalized by the same
cell type. To measure cellular uptake efficiency, the cell type may
be incubated in the presence of a cell-penetrating peptide of the
invention for a specified period of time (e.g., 30 minutes, 1 hour,
2 hours, etc.) after which the amount of the cell-penetrating
peptide internalized by the cell is quantified. Separately, the
same concentration of c(F.PHI.RRRRQ) (290-1F) is incubated in the
presence of the cell type over the same period of time, and the
amount of the second peptide internalized by the cell is
quantified. Quantification can be achieved by fluorescently
labeling the cell-penetrating peptide (e.g., with a FTIC dye) and
measuring the fluorescence intensity using techniques well-known in
the art.
[0112] In certain embodiments, peptides of the invention comprising
at least four consecutive amino acid having alternating chirality
have an uptake efficiency that is superior to that of a second
cyclic peptide wherein the second cyclic peptide has an otherwise
identical amino acid sequence consisting of L-amino acids. In some
embodiments, uptake efficiency can be improved by at least about
1.5 fold, at least about 2 fold, at least about 2.5 fold, at least
about 3 fold, at least about 3.5 fold, at least about 4 fold, at
least about 4.5 fold, at least about 5 fold, at least about 5.5
fold, at least about 6 fold, at least about 6.5 fold, at least
about 7 fold, at least about 7.5 fold, at least about 8 fold, at
least about 8.5 fold, at least about 9 fold, at 9.5 fold, or at
least about 10 fold. In other embodiments, the uptake efficiency
can be improved within the range of from about 1.5 fold to about 10
fold, or about 2 fold to about 10 fold, or about 2 fold to about
9.5 fold, or about 2 fold to about 9 fold, or about 2 fold to about
8.5 fold, or about 2 fold to about 8 fold, or about 2 fold to about
7.5 fold, or about 2 fold to about 7 fold, or about 2 fold to about
6.5 fold, or about 2 fold to about 6 fold, or about 2.5 fold to
about 7 fold, or about 3 fold to about 7 fold, or about 3.5 fold to
about 7 fold, or about 4 to about 7, or about 4.5 fold to about 7
fold, or about 5 fold to about 7 fold, or about 5.5 fold to about 7
fold, or about 6 fold to about 7 fold.
[0113] In certain embodiments, peptides of the invention comprising
at least four consecutive amino acid having alternating chirality
have a superior uptake efficiency as compared to c(F.PHI.RRRRQ)
(290-1F). In some embodiments, uptake efficiency can be improved by
at least about 1.5 fold, at least about 2 fold, at least about 2.5
fold, at least about 3 fold, at least about 3.5 fold, at least
about 4 fold, at least about 4.5 fold, at least about 5 fold, at
least about 5.5 fold, at least about 6 fold, at least about 6.5
fold, at least about 7 fold, at least about 7.5 fold, at least
about 8 fold, at least about 8.5 fold, at least about 9 fold, at
9.5 fold, or at least about 10 fold. In other embodiments, the
uptake efficiency can be improved within the range of from about
1.5 fold to about 10 fold, or about 2 fold to about 10 fold, or
about 2 fold to about 9.5 fold, or about 2 fold to about 9 fold, or
about 2 fold to about 8.5 fold, or about 2 fold to about 8 fold, or
about 2 fold to about 7.5 fold, or about 2 fold to about 7 fold, or
about 2 fold to about 6.5 fold, or about 2 fold to about 6 fold, or
about 2.5 fold to about 7 fold, or about 3 fold to about 7 fold, or
about 3.5 fold to about 7 fold, or about 4 to about 7, or about 4.5
fold to about 7 fold, or about 5 fold to about 7 fold, or about 5.5
fold to about 7 fold, or about 6 fold to about 7 fold.
[0114] In certain embodiments, the peptides of the invention
comprise at least one hydrophobic residue. In further embodiments,
the peptides of the invention comprise two hydrophobic residues. In
still further embodiments, the peptides of the invention comprise
at least two hydrophobic residues. In certain embodiments, at least
one hydrophobic residue is an aromatic hydrophobic residue. In
particular embodiments, at least one hydrophobic residue is
selected from the group consisting of naphthylalanine,
phenylalanine, tryptophan, and tyrosine. In further embodiments, at
least one hydrophobic residue is selected from the group consisting
of naphthylalanine and phenylalanine. In certain embodiments,
peptides of the invention comprise at least one naphthylalanine. In
yet other embodiments, peptides of the invention comprise at least
one phenylalanine. In still other embodiments, peptides of the
invention comprise at least one phenylalanine and at least one
naphthylalanine. In certain embodiments of the invention, the
peptide comprises at least one hydrophobic residue in the AA.sup.1,
AA.sup.2, or AA.sup.3 position. In certain embodiments of the
invention, the peptide comprises at least one aromatic hydrophobic
residue in the AA.sup.1, AA.sup.2, or AA.sup.3 position. In further
embodiments of the invention, the peptide comprises at least one
hydrophobic residue selected from the group consisting of
naphthylalanine and phenylalanine in the AA.sup.1, AA.sup.2, or
AA.sup.3 position.
[0115] In some examples, the cell penetrating peptide moeity can by
any of SEQ ID NO:1 to SEQ ID NO:29. In some examples, the cell
penetrating peptide moiety can be a variant of any of SEQ ID NO:1
to SEQ ID NO:29. Peptide variants are well understood to those of
skill in the art and can involve amino acid sequence modifications.
For example, amino acid sequence modifications typically fall into
one or more of three classes: substitutional, insertional, or
deletional variants. Insertions include amino and/or carboxyl
terminal fusions as well as intrasequence insertions of single or
multiple amino acid residues. Insertions ordinarily will be smaller
insertions than those of amino or carboxyl terminal fusions, for
example, on the order of 1 to 3 residues. Deletions are
characterized by the removal of one or more amino acid residues
from the peptide sequence. Typically, no more than from 1 to 3
residues are deleted at any one site within the peptide. Amino acid
substitutions are typically of single residues, but can occur at a
number of different locations at once; insertions usually will be
on the order of about from 1 to 3 amino acid residues; and
deletions will range about from 1 to 3 residues. Deletions or
insertions preferably are made in adjacent pairs, i.e. a deletion
of 2 residues or insertion of 2 residues. Substitutions, deletions,
insertions or any combination thereof can be combined to arrive at
a final construct. Substitutional variants are those in which at
least one residue has been removed and a different residue inserted
in its place. Such substitutions generally are made in accordance
with the following Table 3 and are referred to as conservative
substitutions.
TABLE-US-00003 TABLE 3 Amino Acid Substitutions Exemplary
Conservative Substitutions Ala replaced by ser Leu replaced by ile
or val Arg replaced by lys or gln Lys replaced by arg or gln Asn
replaced by gln or his Met replaced by leu or ile Asp replaced by
glu Phe replaced by met, leu, tyr, or fpa Cys replaced by ser Ser
replaced by thr Gln replaced by asn or lys Thr replaced by ser Glu
replaced by asp Trp replaced by tyr Gly replaced by pro Tyr
replaced by trp or phe His replaced by asn or gln Val replaced by
ile or leu Ile replaced by leu or val Nal replaced by Trp or
Phe
[0116] Substantial changes in function are made by selecting
substitutions that are less conservative than those in Table 3,
i.e., selecting residues that differ more significantly in their
effect on maintaining (a) the structure of the peptide backbone in
the area of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the protein properties will be those in which (a) a
hydrophilic residue, e.g., seryl or threonyl, is substituted for
(or by) a hydrophobic residue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g., lysyl, argininyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g., glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted for (or by) one not having a side
chain, e.g., glycine, in this case, (e) by increasing the number of
sites for sulfation and/or glycosylation.
[0117] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the peptides
provided herein.
[0118] It is understood that one way to define the variants of the
disclosed cell penetrating peptide moieties is through defining the
variants in terms of homology/identity to specific known sequences.
For example, SEQ ID NO:1 to SEQ ID NO:29 each sets forth a
particular sequence. Specifically disclosed are variants of these
peptide that have at least, 85%, 90%, 95%, or 97% homology to SEQ
ID NO:1 to SEQ ID NO:29. Those of skill in the art readily
understand how to determine the homology of two proteins. For
example, the homology can be calculated after aligning the two
sequences so that the homology is at its highest level.
[0119] In addition to variants of SEQ ID NO:1 to SEQ ID NO:29 are
derivatives of these peptides which also function in the disclosed
methods and compositions. Derivatives are formed by replacing one
or more residues with a modified residue, where the side chain of
the residue has been modified. Additional examples are shown in
Tables 6 and 18 and include variants thereof.
[0120] Cargo Moiety
[0121] The cargo moiety can comprise any cargo of interest, for
example a linker moiety, a detectable moiety, a therapeutic moiety,
a targeting moiety, and the like, or any combination thereof. In
some examples, the cargo moiety can comprise one or more additional
amino acids (e.g., K, UK, TRV); a linker (e.g., bifunctional linker
LC-SMCC); coenzyme A; phosphocoumaryl amino propionic acid (pCAP);
8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic
acid (Dap or J); L-.beta.-naphthylalanine; L-pipecolic acid (Pip);
sarcosine; trimesic acid; 7-amino-4-methylcourmarin (Amc);
fluorescein isothiocyanate (FITC); L-2-naphthylalanine; norleucine;
2-aminobutyric acid; Rhodamine B (Rho); Dexamethasone (DEX); or
combinations thereof.
[0122] In some examples the cargo moiety can comprise any of those
listed in Table 4, or derivatives or combinations thereof.
TABLE-US-00004 TABLE 4 Example cargo moieties SEQ ID NO
Abbreviation Sequence* 30 R.sub.5 RRRRR 31 A.sub.5 AAAAA 32 F.sub.4
FFFF 33 PCP DE(pCAP)LI 34 A.sub.7 AAAAAAA 35 RARAR 36 DADAD 37 DQUD
38 UTRV *pCAP, phosphocoumaryl amino propionic acid; .OMEGA.,
norleucine; U, 2-aminobutyric acid.
[0123] Detectable Moiety
[0124] The detectable moiety can comprise any detectable label.
Examples of suitable detectable labels include, but are not limited
to, a UV-Vis label, a near-infrared label, a luminescent group, a
phosphorescent group, a magnetic spin resonance label, a
photosensitizer, a photocleavable moiety, a chelating center, a
heavy atom, a radioactive isotope, a isotope detectable spin
resonance label, a paramagnetic moiety, a chromophore, or any
combination thereof. In some embodiments, the label is detectable
without the addition of further reagents.
[0125] In some embodiments, the detectable moiety is a
biocompatible detectable moiety, such that the compounds can be
suitable for use in a variety of biological applications.
"Biocompatible" and "biologically compatible", as used herein,
generally refer to compounds that are, along with any metabolites
or degradation products thereof, generally non-toxic to cells and
tissues, and which do not cause any significant adverse effects to
cells and tissues when cells and tissues are incubated (e.g.,
cultured) in their presence.
[0126] The detectable moiety can contain a luminophore such as a
fluorescent label or near-infrared label. Examples of suitable
luminophores include, but are not limited to, metal porphyrins;
benzoporphyrins; azabenzoporphyrine; napthoporphyrin;
phthalocyanine; polycyclic aromatic hydrocarbons such as perylene,
perylene diimine, pyrenes; azo dyes; xanthene dyes; boron
dipyoromethene, aza-boron dipyoromethene, cyanine dyes,
metal-ligand complex such as bipyridine, bipyridyls,
phenanthroline, coumarin, and acetylacetonates of ruthenium and
iridium; acridine, oxazine derivatives such as benzophenoxazine;
aza-annulene, squaraine; 8-hydroxyquinoline, polymethines,
luminescent producing nanoparticle, such as quantum dots,
nanocrystals; carbostyril; terbium complex; inorganic phosphor;
ionophore such as crown ethers affiliated or derivatized dyes; or
combinations thereof. Specific examples of suitable luminophores
include, but are not limited to, Pd (II) octaethylporphyrin; Pt
(II)-octaethylporphyrin; Pd (II) tetraphenylporphyrin; Pt (II)
tetraphenylporphyrin; Pd (II) meso-tetraphenylporphyrin
tetrabenzoporphine; Pt (II) meso-tetrapheny metrylbenzoporphyrin;
Pd (II) octaethylporphyrin ketone; Pt (II) octaethylporphyrin
ketone; Pd (II) meso-tetra(pentafluorophenyl)porphyrin; Pt (II)
meso-tetra (pentafluorophenyl) porphyrin; Ru (II)
tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp).sub.3); Ru (II)
tris(1,10-phenanthroline) (Ru(phen).sub.3),
tris(2,2'-bipyridine)rutheniurn (II) chloride hexahydrate
(Ru(bpy).sub.3); erythrosine B; fluorescein; fluorescein
isothiocyanate (FITC); eosin; iridium (III)
((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium
(III)
((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate);
Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent
yellow; Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine;
m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue;
bromocresol green; bromcresol red; bromothymol blue; Cy2; a Cy3; a
Cy5; a Cy5.5; Cy7; 4-nitirophenol; alizarin; phenolphthalein;
o-cresolphthalein; chlorophenol red; calmagite; bromo-xylenol;
phenol red; neutral red; nitrazine;
3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein; eosin;
2',7'-dichlorofluorescein; 5(6)-carboxy-fluorecsein;
carboxynaphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid;
semi-naphthorhodafluor; semi-naphthofluorescein; tris
(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride;
(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron;
platinum (II) octaethylporphyin; dialkylcarbocyanine;
dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl
chloride; 7-amino-4-methylcourmarin (Amc); green fluorescent
protein (GFP); and derivatives or combinations thereof.
[0127] In some examples, the detectable moiety can comprise
Rhodamine B (Rho), fluorescein isothiocyanate (FITC),
7-amino-4-methylcourmarin (Amc), green fluorescent protein (GFP),
or derivatives or combinations thereof.
[0128] The detectible moiety can be attached to the cell
penetrating peptide moiety at the amino group, the carboxylate
group, or the side chain of any of the amino acids of the cell
penetrating peptide moiety (e.g., at the amino group, the
carboxylate group, or the side chain or any of
AA.sup.1-AA.sup.x).
[0129] Therapeutic Moiety
[0130] The disclosed compounds can also comprise a therapeutic
moiety. In some examples, the cargo moiety comprises a therapeutic
moiety. The detectable moiety can be linked to a therapeutic moiety
or the detectable moiety can also serve as the therapeutic moiety.
Therapeutic moiety refers to a group that when administered to a
subject will reduce one or more symptoms of a disease or
disorder.
[0131] The therapeutic moiety can comprise a wide variety of drugs,
including antagonists, for example enzyme inhibitors, and agonists,
for example a transcription factor which results in an increase in
the expression of a desirable gene product (although as will be
appreciated by those in the art, antagonistic transcription factors
can also be used), are all included. In addition, therapeutic
moiety includes those agents capable of direct toxicity and/or
capable of inducing toxicity towards healthy and/or unhealthy cells
in the body. Also, the therapeutic moiety can be capable of
inducing and/or priming the immune system against potential
pathogens.
[0132] The therapeutic moiety can, for example, comprise an
anticancer agent, antiviral agent, antimicrobial agent,
anti-inflammatory agent, immunosuppressive agent, anesthetics, or
any combination thereof.
[0133] The therapeutic moiety can comprise an anticancer agent.
Example anticancer agents include 13-cis-Retinoic Acid,
2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine,
5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane,
Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin,
Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic
acid, Alpha interferon, Altretamine, Amethopterin, Amifostine,
Aminoglutethimide, Anagrelide, Anandron, Anastrozole,
Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic
trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab,
Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib,
Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar,
Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine,
Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine,
cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine,
Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren,
Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine,
Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin,
Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome,
Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt,
Dexamethasone, Dexamethasone acetate, Dexamethasone sodium
phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,
Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome,
Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt,
Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase,
Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate,
Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara,
Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex,
Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide,
Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine,
Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot,
Matulane, Maxidex, Mechlorethamine, -Mechlorethamine Hydrochlorine,
Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan,
Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium,
Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega,
Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex,
Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin,
Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin,
Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG
Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON,
PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ,
Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT,
Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol,
Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon
alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin,
Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571,
Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar,
Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys,
Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex,
Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,
Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,
Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,
Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,
Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid,
Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte
colony stimulating factor, Halotestin, Herceptin, Hexadrol,
Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort
Acetate, Hydrocortisone, Hydrocortisone sodium phosphate,
Hydrocortisone sodium succinate, Hydrocortone phosphate,
Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,
Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib
mesylate, Imidazole Carboxamide, Interferon alfa, Interferon
Alfa-2b (PEG conjugate), Interleukin 2, Interleukin-11, Intron A
(interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide,
Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine,
L-PAM, L-Sarcolysin, Meticorten, Mitomycin, Mitomycin-C,
Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin,
Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort,
L-asparaginase, and LCR. The therapeutic moiety can also comprise a
biopharmaceutical such as, for example, an antibody.
[0134] In some examples, the therapeutic moiety can comprise an
antiviral agent, such as ganciclovir, azidothymidine (AZT),
lamivudine (3TC), etc.
[0135] In some examples, the therapeutic moiety can comprise an
antibacterial agent, such as acedapsone; acetosulfone sodium;
alamecin; alexidine; amdinocillin; amdinocillin pivoxil;
amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin
sulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin;
amphomycin; ampicillin; ampicillin sodium; apalcillin sodium;
apramycin; aspartocin; astromicin sulfate; avilamycin; avoparcin;
azithromycin; azlocillin; azlocillin sodium; bacampicillin
hydrochloride; bacitracin; bacitracin methylene disalicylate;
bacitracin zinc; bambermycins; benzoylpas calcium; berythromycin;
betamicin sulfate; biapenem; biniramycin; biphenamine
hydrochloride; bispyrithione magsulfex; butikacin; butirosin
sulfate; capreomycin sulfate; carbadox; carbenicillin disodium;
carbenicillin indanyl sodium; carbenicillin phenyl sodium;
carbenicillin potassium; carumonam sodium; cefaclor; cefadroxil;
cefamandole; cefamandole nafate; cefamandole sodium; cefaparole;
cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium;
cefbuperazone; cefdinir; cefepime; cefepime hydrochloride;
cefetecol; cefixime; cefmenoxime hydrochloride; cefmetazole;
cefmetazole sodium; cefonicid monosodium; cefonicid sodium;
cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan;
cefotetan disodium; cefotiam hydrochloride; cefoxitin; cefoxitin
sodium; cefpimizole; cefpimizole sodium; cefpiramide; cefpiramide
sodium; cefpirome sulfate; cefpodoxime proxetil; cefprozil;
cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten;
ceftizoxime sodium; ceftriaxone sodium; cefuroxime; cefuroxime
axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrile
sodium; cephalexin; cephalexin hydrochloride; cephaloglycin;
cephaloridine; cephalothin sodium; cephapirin sodium; cephradine;
cetocycline hydrochloride; cetophenicol; chloramphenicol;
chloramphenicol palmitate; chloramphenicol pantothenate complex;
chloramphenicol sodium succinate; chlorhexidine phosphanilate;
chloroxylenol; chlortetracycline bisulfate; chlortetracycline
hydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin
hydrochloride; cirolemycin; clarithromycin; clinafloxacin
hydrochloride; clindamycin; clindamycin hydrochloride; clindamycin
palmitate hydrochloride; clindamycin phosphate; clofazimine;
cloxacillin benzathine; cloxacillin sodium; cloxyquin;
colistimethate sodium; colistin sulfate; coumermycin; coumermycin
sodium; cyclacillin; cycloserine; dalfopristin; dapsone;
daptomycin; demeclocycline; demeclocycline hydrochloride;
demecycline; denofungin; diaveridine; dicloxacillin; dicloxacillin
sodium; dihydrostreptomycin sulfate; dipyrithione; dirithromycin;
doxycycline; doxycycline calcium; doxycycline fosfatex; doxycycline
hyclate; droxacin sodium; enoxacin; epicillin; epitetracycline
hydrochloride; erythromycin; erythromycin acistrate; erythromycin
estolate; erythromycin ethylsuccinate; erythromycin gluceptate;
erythromycin lactobionate; erythromycin propionate; erythromycin
stearate; ethambutol hydrochloride; ethionamide; fleroxacin;
floxacillin; fludalanine; flumequine; fosfomycin; fosfomycin
tromethamine; fumoxicillin; furazolium chloride; furazolium
tartrate; fusidate sodium; fusidic acid; gentamicin sulfate;
gloximonam; gramicidin; haloprogin; hetacillin; hetacillin
potassium; hexedine; ibafloxacin; imipenem; isoconazole;
isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin;
levofuraltadone; levopropylcillin potassium; lexithromycin;
lincomycin; lincomycin hydrochloride; lomefloxacin; Lomefloxacin
hydrochloride; lomefloxacin mesylate; loracarbef; mafenide;
meclocycline; meclocycline sulfosalicylate; megalomicin potassium
phosphate; mequidox; meropenem; methacycline; methacycline
hydrochloride; methenamine; methenamine hippurate; methenamine
mandelate; methicillin sodium; metioprim; metronidazole
hydrochloride; metronidazole phosphate; mezlocillin; mezlocillin
sodium; minocycline; minocycline hydrochloride; mirincamycin
hydrochloride; monensin; monensin sodiumr; nafcillin sodium;
nalidixate sodium; nalidixic acid; natainycin; nebramycin; neomycin
palmitate; neomycin sulfate; neomycin undecylenate; netilmicin
sulfate; neutramycin; nifuiradene; nifuraldezone; nifuratel;
nifuratrone; nifurdazil; nifurimide; nifiupirinol; nifurquinazol;
nifurthiazole; nitrocycline; nitrofurantoin; nitromide;
norfloxacin; novobiocin sodium; ofloxacin; onnetoprim; oxacillin;
oxacillin sodium; oximonam; oximonam sodium; oxolinic acid;
oxytetracycline; oxytetracycline calcium; oxytetracycline
hydrochloride; paldimycin; parachlorophenol; paulomycin;
pefloxacin; pefloxacin mesylate; penamecillin; penicillin G
benzathine; penicillin G potassium; penicillin G procaine;
penicillin G sodium; penicillin V; penicillin V benzathine;
penicillin V hydrabamine; penicillin V potassium; pentizidone
sodium; phenyl aminosalicylate; piperacillin sodium; pirbenicillin
sodium; piridicillin sodium; pirlimycin hydrochloride;
pivampicillin hydrochloride; pivampicillin pamoate; pivampicillin
probenate; polymyxin B sulfate; porfiromycin; propikacin;
pyrazinamide; pyrithione zinc; quindecamine acetate; quinupristin;
racephenicol; ramoplanin; ranimycin; relomycin; repromicin;
rifabutin; rifametane; rifamexil; rifamide; rifampin; rifapentine;
rifaximin; rolitetracycline; rolitetracycline nitrate; rosaramicin;
rosaramicin butyrate; rosaramicin propionate; rosaramicin sodium
phosphate; rosaramicin stearate; rosoxacin; roxarsone;
roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin;
sarpicillin; scopafungin; sisomicin; sisomicin sulfate;
sparfloxacin; spectinomycin hydrochloride; spiramycin; stallimycin
hydrochloride; steffimycin; streptomycin sulfate; streptonicozid;
sulfabenz; sulfabenzamide; sulfacetamide; sulfacetamide sodium;
sulfacytine; sulfadiazine; sulfadiazine sodium; sulfadoxine;
sulfalene; sulfamerazine; sulfameter; sulfamethazine;
sulfamethizole; sulfamethoxazole; sulfamonomethoxine; sulfamoxole;
sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole;
sulfathiazole; sulfazamet; sulfisoxazole; sulfisoxazole acetyl;
sulfisboxazole diolamine; sulfomyxin; sulopenem; sultamricillin;
suncillin sodium; talampicillin hydrochloride; teicoplanin;
temafloxacin hydrochloride; temocillin; tetracycline; tetracycline
hydrochloride; tetracycline phosphate complex; tetroxoprim;
thiamphenicol; thiphencillin potassium; ticarcillin cresyl sodium;
ticarcillin disodium; ticarcillin monosodium; ticlatone; tiodonium
chloride; tobramycin; tobramycin sulfate; tosufloxacin;
trimethoprim; trimethoprim sulfate; trisulfapyrimidines;
troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin;
vancomycin hydrochloride; virginiamycin; or zorbamycin.
[0136] In some examples, the therapeutic moiety can comprise an
anti-inflammatory agent.
[0137] In some examples, the therapeutic moiety can comprise
dexamethasone (Dex).
[0138] In other examples, the therapeutic moiety comprises a
therapeutic protein. For example, some people have defects in
certain enzymes (e.g., lysosomal storage disease). It is disclosed
herein to deliver such enzymes/proteins to human cells by linking
to the enzyme/protein to one of the disclosed cell penetrating
peptides. The disclosed cell penetrating peptides have been tested
with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C) and
shown to work.
[0139] In some examples, the therapeutic moiety comprises a
targeting moiety. The targeting moiety can comprise, for example, a
sequence of amino acids that can target one or more enzyme domains.
In some examples, the targeting moiety can comprise an inhibitor
against an enzyme that can play a role in a disease, such as
cancer, cystic fibrosis, diabetes, obesity, or combinations
thereof. For example, the targeting moiety can comprise any of the
sequences listed in Table 5.
TABLE-US-00005 TABLE 5 Example targeting moieties SEQ ID NO
Abbreviation * Sequence 39 P.THETA.G.LAMBDA.YR
Pro-Pip-Gly-F.sub.2Pmp-Tyr- 40 S.THETA.I.LAMBDA..LAMBDA.R
Ser-Pip-Ile-F.sub.2Pmp-F.sub.2Pmp- 41 IHI.LAMBDA.IR
Ile-His-Ile-F.sub.2Pmp-Ile- 42 AaI.LAMBDA..THETA.R
Ala-(D-Ala)-Ile-F.sub.2Pmp-Pip- 43 .SIGMA.S.THETA..LAMBDA.vR
Fpa-Ser-Pip-F.sub.2Pmp-(D-Val)- 44 .THETA.nP.LAMBDA.AR
Pip-(D-Asn)-Pro-F.sub.2Pmp-Ala- 45 T.PSI.A.LAMBDA.GR
Tyr-Phg-Ala-F.sub.2Pmp-Gly- 46 AHI.LAMBDA.aR
Ala-His-Ile-F.sub.2Pmp-(D-Ala)- 47 GnG.LAMBDA.pR
Gly-(D-Asn)-Gly-F.sub.2Pmp- (D-Pro)- 48 fQ.THETA..LAMBDA.IR
(D-Phe)-Gln-Pip-F.sub.2Pmp-Ile- 49 SPG.LAMBDA.HR
Ser-Pro-Gly-F.sub.2Pmp-His- 50 .THETA.YI.LAMBDA.HR
Pip-Tyr-Ile-F.sub.2Pmp-His- 51 SvP.LAMBDA.HR
Ser-(D-Val)-Pro-F.sub.2Pmp-His- 52 AIP.LAMBDA.nR
Ala-Ile-Pro-F.sub.2Pmp-(D-Asn)- 53 .SIGMA.SI.LAMBDA.QF
Fpa-Ser-Ile-F.sub.2Pmp-Gln- 54 Aa.PSI..LAMBDA.fR
Ala-(D-Ala)-Phg-F.sub.2Pmp- (D-Phe)- 55 nt.PSI..LAMBDA..PSI.R
(D-Asn)-(D-Thr)-Phg-F.sub.2Pmp- Phg- 56 IP.PSI..LAMBDA..OMEGA.R
Ile-Pro-Phg-F.sub.2Pmp-Nle- 57 Q.THETA..SIGMA..LAMBDA..THETA.R
Gln-Pip-Fpa-F.sub.2Pmp-Pip- 58 nA.SIGMA..LAMBDA.GR
(D-Asn)-Ala-Fpa-F.sub.2Pmp-Gly- 59 ntY.LAMBDA.AR
(D-Asn)-(D-Thr)-Tyr-F.sub.2Pmp- Ala- 60 eA.PSI..LAMBDA.vR
(D-Glu)-Ala-Phg-F.sub.2Pmp- (D-Val)- 61 Iv.PSI..LAMBDA.AR
Ile-(D-Val)-Phg-F.sub.2Pmp-Ala- 62 Yt.PSI..LAMBDA.AR
Tyr-(D-Thr)-Phg-F.sub.2Pmp-Ala- 63 n.THETA..PSI..LAMBDA.IR
(D-Asn)-Pip-Phg-F.sub.2Pmp-Ile- 64 .THETA.nW.LAMBDA.HR
Pip-(D-Asn)-Trp-F.sub.2Pmp-His- 65 Y.THETA.v.LAMBDA.IR
Tyr-Pip-(D-Val)-F.sub.2Pmp-Ile- 66 nSA.LAMBDA.GR
(D-Asn)-Ser-(D-Ala)-F.sub.2Pmp- Gly- 67 tnv.LAMBDA.aR
(D-Thr)-(D-Asn)-(D-Val)- F.sub.2Pmp-(D-Ala)- 68 ntv.LAMBDA.tR
(D-Asn)-(D-Thr)-(D-Val)- F.sub.2Pmp-(D-Thr)- 69 SIt.LAMBDA.YR
Ser-Ile-(D-Thr)-F.sub.2Pmp-Tyr- 70 n.SIGMA.n.LAMBDA.lR
(D-Asn)-Fpa-(D-Asn)-F.sub.2Pmp- (D-Leu)- 71 Ynn.LAMBDA..OMEGA.R
Tyr-(D-Asn)-(D-Asn)-F.sub.2Pmp- Nle- 72 nYn.LAMBDA.GR
(D-Asn)-Tyr-(D-Asn)-F.sub.2Pmp- Gly- 73 AWn.LAMBDA.AR
Ala-Trp-(D-Asn)-F.sub.2Pmp-Ala- 74 vtH.LAMBDA.YR
(D-Val)-(D-Thr)-His-F.sub.2Pmp- Tyr- 75 P.PSI.H.LAMBDA..THETA.R
Pro-Phg-His-F.sub.2Pmp-Pip- 76 n.PSI.H.LAMBDA.GR
(D-Asn)-Phg-His-F.sub.2Pmp-Gly- 77 PAH.LAMBDA.GR
Pro-Ala-His-F.sub.2Pmp-Gly- 78 AYH.LAMBDA.IR
Ala-Tyr-His-F.sub.2Pmp-Ile- 79 n.THETA.e.LAMBDA.YR
(D-Asn)-Pip-(D-Glu)-F.sub.2Pmp- Tyr- 80 vSS.LAMBDA.tR
(D-Val)-Ser-Ser-F.sub.2Pmp- (D-Thr)- 81 a.XI.t' .PHI.'YNK
((D-Ala)-Sar-(D-pThr)-Pp- Nal-Tyr-Gln)-Lys 82 Tm(a.XI.t'
.PHI.'RA)Dap Tm((D-Ala)-Sar-(D-pThr)- Pp-Nal-Arg-Ala)-Dap 83
Tm(a.XI.t' .PHI.'RAa)Dap Tm((D-Ala)-Sar-(D-pThr)-
Pp-Nal-Arg-Ala-(D-Ala))- Dap 84 Tm(a.XI.t .PHI.'RAa)Dap
Tm((D-Ala)-Sar-(D-Thr)- Pp-Nal-Arg-Ala-(D-Ala))- Dap 85
Tm(a.XI.ta.PHI.'RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-
(D-Ala)-Nal-Arg-Ala- (D-Ala))-Dap *Fpa, .SIGMA.:
L-4-fluorophenylalanine; Pip, .THETA.: L-homoproline; Nle, .OMEGA.:
L-norleucine; Phg, .PSI. L-phenylglycine; F.sub.2Pmp, .LAMBDA.:
L-4-(phosphonodifluoromethyl)phenylalanine; Dap,
L-2,3-diaminopropionic acid; Nal, .PHI.': L-.beta.-naphthylalanine;
Pp, : L-pipecolic acid; Sar, .XI.: sarcosine; Tm, trimesic
acid.
[0140] The targeting moeity and cell penetrating peptide moiety can
overlap, that is residues that form the cell penetrating peptide
moiety can also be part of the sequence that forms the targeting
moiety, and vice a versa.
[0141] The therapeutic moiety can be attached to the cell
penetrating peptide moiety at the amino group, the carboxylate
group, or the side chain of any of the amino acids of the cell
penetrating peptide moiety (e.g., at the amino group, the
carboxylate group, or the side chain or any of AA.sup.1-AA.sup.x).
In some examples, the therapeutic moiety can be attached to the
detectable moiety.
[0142] In some examples, the therapeutic moiety can comprise a
targeting moiety that can act as an inhibitor against Ras (e.g.,
K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, and the like, or
combinations thereof.
[0143] Ras is a protein that in humans is encoded by the RAS gene.
The normal Ras protein performs an essential function in normal
tissue signaling, and the mutation of a Ras gene is implicated in
the development of many cancers. Ras can act as a molecular on/off
switch, once it is turned on Ras recruits and activates proteins
necessary for the propagation of growth factor and other receptors'
signal. Mutated forms of Ras have been implicated in various
cancers, including lung cancer, colon cancer, pancreatic cancer,
and various leukemias.
[0144] Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical
member of the PTP superfamily and plays numerous roles during
eukaryotic cell signaling. PTP1B is a negative regulator of the
insulin signaling pathway, and is considered a promising potential
therapeutic target, in particular for the treatment of type II
diabetes. PIP1B has also been implicated in the development of
breast cancer.
[0145] Pin1 is an enzyme that binds to a subset of proteins and
plays a role as a post phosphorylation control in regulating
protein function. Pin1 activity can regulate the outcome of
proline-directed kinase signaling and consequently can regulate
cell proliferation and cell survival. Deregulation of Pin1 can play
a role in various diseases. The up-regulation of Pin1 may be
implicated in certain cancers, and the down-regulation of Pin1 may
be implicated in Alzheimer's disease. Inhibitors of Pin1 can have
therapeutic implications for cancer and immune disorders.
[0146] Grb2 is an adaptor protein involved in signal transduction
and cell communication. The Grb2 protein contains one SH2 domain,
which can bind tyrosine phosphorylated sequences. Grb2 is widely
expressed and is essential for multiple cellular functions.
Inhibition of Grb2 function can impair developmental processes and
can block transformation and proliferation of various cell
types.
[0147] It was recently reported that the activity of cystic
fibrosis membrane conductance regulator (CFTR), a chloride ion
channel protein mutated in cystic fibrosis (CF) patients, is
negatively regulated by CFTR-associated ligand (CAL) through its
PDZ domain (CAL-PDZ) (Wolde, M et al. J. Biol. Chem. 2007, 282,
8099). Inhibition of the CFTR/CAL-PDZ interaction was shown to
improve the activity of APhe508-CFTR, the most common form of CFTR
mutation (Cheng, S H et al. Cell 1990, 63, 827; Kerem, B S et al.
Science 1989, 245, 1073), by reducing its proteasome-mediated
degradation (Cushing, P R et al. Angew. Chem. Int. Ed. 2010, 49,
9907). Thus, disclosed herein is a method for treating a subject
having cystic fibrosis by administering an effective amount of a
compound or composition disclosed herein. The compound or
composition administered to the subject can comprise a therapeutic
moiety that can comprise a targeting moiety that can act as an
inhibitor against CAL PDZ. Also, the decompositions or compositions
disclosed herein can be administered with a molecule that corrects
the CFTR function.
SPECIFIC EXAMPLES
[0148] In some examples, the compounds can be of Formula I:
##STR00014##
wherein AA.sup.1, AA.sup.2, AA.sup.3, AA.sup.4, AA.sup.5, AA.sup.6,
AA.sup.7, AA.sup.8, and AA.sup.9 (i.e., AA.sup.1-AA.sup.9) are each
independently an amino acid; and m, n and p are independently
selected from 0 and 1.
[0149] In some examples of Formula I, m, n, and p are 0 and the
compounds are of Formula I-1:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6 I-1
wherein AA.sup.1-AA.sup.6 are as defined in Formula I.
[0150] In some examples of Formula I, m is 1, and n and p are 0,
and the compounds are of Formula I-2:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-AA.sup.7
I-2
wherein AA.sup.1-AA.sup.7 are as defined in Formula I.
[0151] In some examples of Formula I, m and n are 1, p is 0, and
the compounds are of Formula I-3:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-AA.sup.7-AA.sup.8
I-3
wherein AA.sup.1-AA.sup.8 are as defined in Formula I.
[0152] In some examples of Formula I, m, n, and p are 1, and the
compounds are of Formula I-4:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-AA.sup.7-AA.sup.8--
AA.sup.9 I-4
wherein AA.sup.1-AA.sup.9 are as defined in Formula I.
[0153] In some examples, the cell penetrating peptide moiety is
cyclic, and the compounds can be of Formula Ia:
##STR00015##
wherein AA.sup.1-AA.sup.9, m, n, and p are as defined in Formula I,
and wherein the curved line indicates a covalent bond.
[0154] In some examples of Formula Ia, m, n, and p are 0 and the
compounds are of Formula Ia-1:
##STR00016##
wherein AA.sup.1-AA.sup.6 are as defined in Formula I.
[0155] In some examples of Formula Ia, m is 1, and n and p are 0,
and the compounds are of Formula Ia-2:
##STR00017##
wherein AA.sup.1-AA.sup.7 are as defined in Formula I.
[0156] In some examples of Formula Ia, m and n are 1, p is 0, and
the compounds are of Formula Ia-3:
##STR00018##
wherein AA.sup.1-AA.sup.8 are as defined in Formula I.
[0157] In some examples of Formula Ia, m, n, and p are 1, and the
compounds are of Formula Ia-4:
##STR00019##
wherein AA.sup.1-AA.sup.9 are as defined in Formula I.
[0158] In some examples, the compound further comprises a cargo
moiety, and the compounds can be of Formula II:
##STR00020##
wherein the cargo moiety can comprise a detectable moiety, a
therapeutic moiety, a targeting moiety, or a combination thereof
and AA.sup.1-AA.sup.9, m, n, and p are as defined in Formula I.
[0159] In some examples of Formula II, m, n, and p are 0 and the
compounds are of Formula II-1:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-cargo
II-1
wherein AA.sup.1-AA.sup.6 are as defined in Formula I and cargo is
as defined in Formula II.
[0160] In some examples of Formula II, m is 1, and n and p are 0,
and the compounds are of Formula II-2:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-AA.sup.7-cargo
II-2
wherein AA.sup.1-AA.sup.7 are as defined in Formula I and cargo is
as defined in Formula II.
[0161] In some examples of Formula II, m and n are 1, p is 0, and
the compounds are of Formula II-3:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-AA.sup.7-AA.sup.8--
cargo II-3
wherein AA.sup.1-AA.sup.8 are as defined in Formula I and cargo is
as defined in Formula II.
[0162] In some examples of Formula II, m, n, and p are 1, and the
compounds are of Formula II-4:
AA.sup.1-AA.sup.2-AA.sup.3-AA.sup.4-AA.sup.5-AA.sup.6-AA.sup.7-AA.sup.8--
AA.sup.9-cargo II-4
wherein AA.sup.1-AA.sup.9 are as defined in Formula I and cargo is
as defined in Formula II.
[0163] In some examples, the cell penetrating peptide moiety and
cargo moiety together are cyclic, and the compounds are of Formula
IIa:
##STR00021##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0164] In some examples of Formula IIa, m, n, and p are 0 and the
compounds are of Formula IIa-1:
##STR00022##
wherein AA.sup.1-AA.sup.6 are as defined in Formula I and cargo is
as defined in Formula II. Also disclosed herein is Formula IIa-1
wherein one of AA.sup.1-AA.sup.6 is absent (i.e., 5 amino acids in
the endocyclic structure.
[0165] In some examples of Formula IIa, m is 1, and n and p are 0,
and the compounds are of Formula IIa-2:
##STR00023##
wherein AA.sup.1-AA.sup.7 are as defined in Formula I and cargo is
as defined in Formula II.
[0166] In some examples of Formula IIa, m and n are 1, p is 0, and
the compounds are of Formula IIa-3:
##STR00024##
wherein AA.sup.1-AA.sup.8 are as defined in Formula I and cargo is
as defined in Formula II.
[0167] In some examples of Formula IIa, m, n, and p are 1, and the
compounds are of Formula IIa-4:
##STR00025##
wherein AA.sup.1-AA.sup.9 are as defined in Formula I and cargo is
as defined in Formula II.
[0168] In some examples, the cell penetrating peptide moiety is
cyclic and the cargo moiety is appended to the cyclic cell
penetrating peptide moiety structure, and the compounds are of
Formula IIb:
##STR00026##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0169] In some examples of Formula IIb, m, n, and p are 0 and the
compounds are of Formula IIb-1:
##STR00027##
wherein AA.sup.1-AA.sup.6 are as defined in Formula I and cargo is
as defined in Formula II.
[0170] In some examples of Formula IIb, m is 1, and n and p are 0,
and the compounds are of Formula IIb-2:
##STR00028##
wherein AA.sup.1-AA.sup.7 are as defined in Formula I and cargo is
as defined in Formula II.
[0171] In some examples of Formula IIb, m and n are 1, p is 0, and
the compounds are of Formula IIb-3:
##STR00029##
wherein AA.sup.1-AA.sup.8 are as defined in Formula I and cargo is
as defined in Formula II.
[0172] In some examples of Formula IIb, m, n, and p are 1, and the
compounds are of Formula IIb-4:
##STR00030##
wherein AA.sup.1-AA.sup.9 are as defined in Formula I and cargo is
as defined in Formula II.
[0173] In some examples, the cargo moiety is cyclic and the cell
penetrating peptide moiety is cyclic, and together they form a
fused bicyclic system, and the compounds are of Formula
##STR00031##
wherein the cargo moiety is as defined in Formula II and
AA.sup.1-AA.sup.9, m, n and p are as defined in Formula I.
[0174] In some examples of Formula IIc, m, n, and p are 0 and the
compounds are of Formula IIc-1:
##STR00032##
wherein AA.sup.1-AA.sup.6 are as defined in Formula I and cargo is
as defined in Formula II.
[0175] In some examples of Formula IIc, m is 1, and n and p are 0,
and the compounds are of Formula IIc-2:
##STR00033##
wherein AA.sup.1-AA.sup.7 are as defined in Formula I and cargo is
as defined in Formula II.
[0176] In some examples of Formula IIc, m and n are 1, p is 0, and
the compounds are of Formula IIc-3:
##STR00034##
wherein AA.sup.1-AA.sup.8 are as defined in Formula I and cargo is
as defined in Formula II.
[0177] In some examples of Formula IIc, m, n, and p are 1, and the
compounds are of Formula IIc-4:
##STR00035##
wherein AA.sup.1-AA.sup.9 are as defined in Formula I and cargo is
as defined in Formula II.
[0178] In some examples, the compounds can comprise any of the
compounds in Table 6. Further examples are shown in Table 18
below.
TABLE-US-00006 TABLE 6 Example compounds SEQ ID NO Abbreviation
Sequence 86 cF.PHI.R.sub.4.sup.Rho cyclo(F.PHI.RRRRQ)-K(Rho) 87
cF.PHI.R.sub.4.sup.Dex cyclo(F.PHI.RRRRQ)-K(Dex) 88 Tat.sup.Dex
K(Dex)-GRKKRRQRRRPPQY 89 cF.PHI.R.sub.4.sup.FITC
cyclo(F.PHI.RRRRQ)-K(FITC) 90 cF.PHI.R.sub.4-R.sub.5
cyclo(F.PHI.RRRRQ)-RRRRR-K(Rho) 91 cF.PHI.R.sub.4-A.sub.5
cyclo(F.PHI.RRRRQ)-AAAAA-K(Rho) 92 cF.PHI.R.sub.4-F.sub.4
cyclo(F.PHI.RRRRQ)-FFFF-K(Rho) 93 cF.PHI.R.sub.4-PCP
cyclo(F.PHI.RRRRQ)-miniPEG-DE(pCAP)LI 94 R.sub.9-PCP
RRRRRRRRR-miniPEG-DE(pCAP)LI 95 Tat-PCP
RKKRRQRRR-miniPEG-DE(pCAP)LI 96 Antp-PCP RQIKIWFQNRRMKWKK-miniPEG-
DE(pCAP)LI 97 bicyclo(F.PHI.R.sub.4-A.sub.5).sup.Rho
[Tm(AAAAA)K(RRRR.PHI.F)J]-K(Rho) 98
bicyclo(F.PHI.R.sub.4-A.sub.7).sup.Rho
[Tm(AAAAAAA)K(RRRR.PHI.F)J]-K(Rho) 99
bicyclo(F.PHI.R.sub.4-RARAR).sup.Rho
[Tm(RARAR)K(RRRR.PHI.F)J]-K(Rho) 100
bicyclo(F.PHI.R.sub.4-DADAD).sup.Rho
[Tm(DADAD)K(RRRR.PHI.F)J]-K(Rho) 101
monocyclo(F.PHI.R.sub.4-A.sub.5).sup.Rho
cyclo(AAAAARRRR.PHI.F)-K(Rho) 102
monocyc1o(F.PHI.R.sub.4-A.sub.7).sup.Rho
cyclo(AAAAAAARRRR.PHI.F)-K(Rho) 103 ##STR00036## 104
CH.sub.3CH.sub.2CH.sub.2CO-F.PHI.RRRRUK(FITC) 105 D.OMEGA.UD-Amc
106 ##STR00037## 107 ##STR00038## 108
CH.sub.3CH.sub.2CH.sub.2CO-RRRR.PHI.FD.OMEGA.UD-Amc 109
RRRRRRRRRD.OMEGA.UC-Amc 110 ##STR00039## 111 FITC-URRRRFWQUTRV 112
##STR00040## 113 cF.PHI.R.sub.4-PTP1B 114 cF.PHI.R.sub.4-PCP 115
cyclo((D-Thr)-(D-Asn)-(D-Val)- cyclo(tnv.LAMBDA.aRRRR.PHI.'FQ)
F.sub.2Pmp-(D-Ala)-Arg-Arg-Arg- Arg-Nal-Phe-Gln) 116
cyclo(Ser-(D-Val)-Pro-F.sub.2Pmp- cyclo(SvP.LAMBDA.HRRRR .PHI.'FQ)
His-Arg-Arg-Arg-Arg-Nal-Phe- Gln) 117
cyclo(Ile-Pro-Phg-F.sub.2Pmp-Nle-
cyclo(IP.PSI..LAMBDA..OMEGA.RRRR.PHI.'FQ)
Arg-Arg-Arg-Arg-Nal-Phe-Gln) 118 cyclo((D-Ala)-Sar-(D-pThr)-Pp-
cyclo(a.XI.t' .PHI.'YQ)-K Nal-Tyr-Gln)-Lys 119
bicyclo[Tm((D-Ala)-Sar-(D- bicyclo[Tm(a.XI.t'
.PHI.'RA)J(F.PHI.'RRRRJ)]-K pThr)-Pp-Nal-Arg-Ala)-Dap-
(Phe-Nal-Arg-Arg-Arg-Arg- Dap)]-Lys 120 bicyclo[Tm((D-Ala)-Sar-(D-
bicyclo[Tm(a.XI.t' .PHI.'RAa)J(FN.PHI.'RRRRJ)]-K
pThr)-Pp-Nal-Arg-Ala-(D-Ala))- Dap-(Phe-Nal-Arg-Arg-Arg-Arg-
Dap)]-Lys 121 bicyclo[Tm((D-Ala)-Sar-(D- bicyclo[Tm(a.XI.t
.PHI.'RAa)J(F.PHI.'RRRRJ)]-K Thr)-Pp-Nal-Arg-Ala-(D-Ala))-
Dap-(Phe-Nal-Arg-Arg-Arg-Arg- Dap)]-Lys 122
bicyclo[Tm((D-Ala)-Sar-(D-
bicyclo[Tm(a.XI.ta.PHI.'RAa)J(F.PHI.'RRRRJ)]-K
Thr)-(D-Ala)-Nal-Arg-Ala-(D- Ala))-Dap-(Phe-Nal-Arg-Arg-
Arg-Arg-Dap)]-Lys 123 Peptide 1 ##STR00041## 124 Peptide 2
CH.sub.3CH.sub.2CH.sub.2CO-F.PHI.RRRRUK(FITC)-NH.sub.2 125 Peptide
3 Ac-DMUD-Amc 126 Peptide 4 ##STR00042## 127 Peptide 5 ##STR00043##
128 Peptide 6 CH.sub.3CH.sub.2CH.sub.2CO-RRRR.PHI.FD.OMEGA.UD-Amc
129 Peptide 7 Ac-RRRRRRRRRD.OMEGA.UD-Amc 130 Peptide 8 ##STR00044##
131 Peptide 9 FITC-URRRRFWQUTRV-OH 132 Peptide 11 ##STR00045## 178
Monocyclic Inhibitor 1 cyclo(D-Thr-D-Asn-D-Val-F.sub.2Pmp-D-Ala-
Arg-Arg-Arg-Arg-Nal-Phe-Gln) 179 Monocyclic Inhibitor 2
cyclo(Ser-D-Val-Pro-F.sub.2Pmp-His-Arg-Arg- Arg-Arg-Nal-Phe-Gln)
180 Monocyclic Inhibitor 3
cyclo(Ile-Pro-Phg-F.sub.2Pmp-Nle-Arg-Arg-Arg- Arg-Nal-Phe-Gln) 181
Pin1 inhibitor 5 cyclo(D-Ala-Sar-D-pThr-Pip-Nal-Tyr-
Gln)-Lys-NH.sub.2 182 Pin1 inhibitor 6
bicyclo[Tm(D-Ala-Sar-D-pThr-Pip-Nal-
Arg-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg- Arg-Dap)]-Lys-NH.sub.2 183 Pin1
inhibitor 7 bicyclo[Tm(D-Ala-Sar-D-pThr-Pip-Nal-
Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys-NH.sub.2 184
Pin1 inhibitor 8 bicyclo[Tm(D-Ala-Sar-D-Thr-Pip-Nal-
Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys-NH.sub.2 185
Pin1 inhibitor 9 bicyclo[Tm(D-Ala-Sar-D-Thr-D-Ala-
Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg- Arg-Arg-Arg-Dap)]-Lys-NH.sub.2
*Fpa, .SIGMA.: L-4-fluorophenylalanine; Pip, .THETA.:
L-homoproline; Nle, .OMEGA.: L-norleucine; Phg, .PSI.
L-phenylglycine; F.sub.2Pmp, .LAMBDA.:
L-4-(phosphonodifluoromethyl)phenylalanine; Dap, J:
L-2,3-diaminopropionic acid; Nal, .PHI.': L-.beta.-naphthylalanine;
Pp, : L-pipecolic acid; Sar, .XI.: sarcosine; Tm, trimesic acid;
.PHI., L-2-naphthylalanine; Rho, rhodamine B; Dex, dexamethasone;
FITC, fluorescein isothiocyanate; miniPEG,
8-amino-3,6-dioxaoctanoic acid; pCAP, phosphocoumaryl amino
propionic acid; Amc, 7-amino-4-methylcourmarin; FITC, fluorescein
isothiocyanate; U, 2-aminobutyric acid.
TABLE-US-00007 TABLE 7 Previously reported cell penetrating
peptides SEQ ID NO Abbreviation Sequence 133 R.sub.9 RRRRRRRRR 134
Tat YGRKKRRQRRR 135 Antp RQIKIWFQNRRMKWKK
[0179] Also disclosed herein are compositions comprising the
compounds described herein.
[0180] Also disclosed herein are pharmaceutically-acceptable salts
and prodrugs of the disclosed compounds.
Pharmaceutically-acceptable salts include salts of the disclosed
compounds that are prepared with acids or bases, depending on the
particular substituents found on the compounds. Under conditions
where the compounds disclosed herein are sufficiently basic or
acidic to form stable nontoxic acid or base salts, administration
of the compounds as salts can be appropriate. Examples of
pharmaceutically-acceptable base addition salts include sodium,
potassium, calcium, ammonium, or magnesium salt. Examples of
physiologically-acceptable acid addition salts include
hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric,
and organic acids like acetic, propionic, benzoic, succinic,
fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic,
alpha-ketoglutaric, alpha-glycophosphoric, maleic, tosyl acid,
methanesulfonic, and the like. Thus, disclosed herein are the
hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate,
acetate, propionate, benzoate, succinate, fumarate, mandelate,
oxalate, citrate, tartarate, malonate, ascorbate,
alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate, and
mesylate salts. Pharmaceutically acceptable salts of a compound can
be obtained using standard procedures well known in the art, for
example, by reacting a sufficiently basic compound such as an amine
with a suitable acid affording a physiologically acceptable anion.
Alkali metal (for example, sodium, potassium or lithium) or
alkaline earth metal (for example calcium) salts of carboxylic
acids can also be made.
[0181] Methods of Making
[0182] The compounds described herein can be prepared in a variety
of ways known to one skilled in the art of organic synthesis or
variations thereon as appreciated by those skilled in the art. The
compounds described herein can be prepared from readily available
starting materials. Optimum reaction conditions can vary with the
particular reactants or solvents used, but such conditions can be
determined by one skilled in the art.
[0183] Variations on the compounds described herein include the
addition, subtraction, or movement of the various constituents as
described for each compound. Similarly, when one or more chiral
centers are present in a molecule, the chirality of the molecule
can be changed. Additionally, compound synthesis can involve the
protection and deprotection of various chemical groups. The use of
protection and deprotection, and the selection of appropriate
protecting groups can be determined by one skilled in the art. The
chemistry of protecting groups can be found, for example, in Wuts
and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley
& Sons, 2006, which is incorporated herein by reference in its
entirety.
[0184] The starting materials and reagents used in preparing the
disclosed compounds and compositions are either available from
commercial suppliers such as Aldrich Chemical Co., (Milwaukee,
Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific
(Pittsburgh, Pa.), Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.),
GlaxoSmithKline (Raleigh, N.C.), Merck (Whitehouse Station, N.J.),
Johnson & Johnson (New Brunswick, N.J.), Aventis (Bridgewater,
N.J.), AstraZeneca (Wilmington, Del.), Novartis (Basel,
Switzerland), Wyeth (Madison, N.J.), Bristol-Myers-Squibb (New
York, N.Y.), Roche (Basel, Switzerland), Lilly (Indianapolis,
Ind.), Abbott (Abbott Park, Ill.), Schering Plough (Kenilworth,
N.J.), or Boehringer Ingelheim (Ingelheim, Germany), or are
prepared by methods known to those skilled in the art following
procedures set forth in references such as Fieser and Fieser's
Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,
1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989); Organic
Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's
Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and
Larock's Comprehensive Organic Transformations (VCH Publishers
Inc., 1989). Other materials, such as the pharmaceutical carriers
disclosed herein can be obtained from commercial sources.
[0185] Reactions to produce the compounds described herein can be
carried out in solvents, which can be selected by one of skill in
the art of organic synthesis. Solvents can be substantially
nonreactive with the starting materials (reactants), the
intermediates, or products under the conditions at which the
reactions are carried out, i.e., temperature and pressure.
Reactions can be carried out in one solvent or a mixture of more
than one solvent. Product or intermediate formation can be
monitored according to any suitable method known in the art. For
example, product formation can be monitored by spectroscopic means,
such as nuclear magnetic resonance spectroscopy (e.g., .sup.1H or
.sup.13C) infrared spectroscopy, spectrophotometry (e.g.,
UV-visible), or mass spectrometry, or by chromatography such as
high performance liquid chromatography (HPLC) or thin layer
chromatography.
[0186] The disclosed compounds can be prepared by solid phase
peptide synthesis wherein the amino acid .alpha.-N-terminal is
protected by an acid or base protecting group. Such protecting
groups should have the properties of being stable to the conditions
of peptide linkage formation while being readily removable without
destruction of the growing peptide chain or racemization of any of
the chiral centers contained therein. Suitable protecting groups
are 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),
benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl,
t-amyloxycarbonyl, isobornyloxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like.
The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is
particularly preferred for the synthesis of the disclosed
compounds. Other preferred side chain protecting groups are, for
side chain amino groups like lysine and arginine,
2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro,
p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and
adamantyloxycarbonyl; for tyrosine, benzyl,
o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl
(t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine,
t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl,
benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for
tryptophan, formyl; for asparticacid and glutamic acid, benzyl and
t-butyl and for cysteine, triphenylmethyl (trityl). In the solid
phase peptide synthesis method, the .alpha.-C-terminal amino acid
is attached to a suitable solid support or resin. Suitable solid
supports useful for the above synthesis are those materials which
are inert to the reagents and reaction conditions of the stepwise
condensation-deprotection reactions, as well as being insoluble in
the media used. Solid supports for synthesis of .alpha.-C-terminal
carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1%
divinylbenzene) or
4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl
resin available from Applied Biosystems (Foster City, Calif.). The
.alpha.-C-terminal amino acid is coupled to the resin by means of
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DIC) or
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate
(HBTU), with or without 4-dimethylaminopyridine (DMAP),
1-hydroxybenzotriazole (HOBT),
benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate
(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl),
mediated coupling for from about 1 to about 24 hours at a
temperature of between 10.degree. C. and 50.degree. C. in a solvent
such as dichloromethane or DMF. When the solid support is
4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl
resin, the Fmoc group is cleaved with a secondary amine, preferably
piperidine, prior to coupling with the .alpha.-C-terminal amino
acid as described above. One method for coupling to the deprotected
4 (2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl
resin is
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate
(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in
DMF. The coupling of successive protected amino acids can be
carried out in an automatic polypeptide synthesizer. In one
example, the .alpha.-N-terminal in the amino acids of the growing
peptide chain are protected with Fmoc. The removal of the Fmoc
protecting group from the .alpha.-N-terminal side of the growing
peptide is accomplished by treatment with a secondary amine,
preferably piperidine. Each protected amino acid is then introduced
in about 3-fold molar excess, and the coupling is preferably
carried out in DMF. The coupling agent can be
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate
(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At
the end of the solid phase synthesis, the polypeptide is removed
from the resin and deprotected, either in successively or in a
single operation. Removal of the polypeptide and deprotection can
be accomplished in a single operation by treating the resin-bound
polypeptide with a cleavage reagent comprising thianisole, water,
ethanedithiol and trifluoroacetic acid. In cases wherein the
.alpha.-C-terminal of the polypeptide is an alkylamide, the resin
is cleaved by aminolysis with an alkylamine. Alternatively, the
peptide can be removed by transesterification, e.g. with methanol,
followed by aminolysis or by direct transamidation. The protected
peptide can be purified at this point or taken to the next step
directly. The removal of the side chain protecting groups can be
accomplished using the cleavage cocktail described above. The fully
deprotected peptide can be purified by a sequence of
chromatographic steps employing any or all of the following types:
ion exchange on a weakly basic resin (acetate form); hydrophobic
adsorption chromatography on underivitized
polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel
adsorption chromatography; ion exchange chromatography on
carboxymethylcellulose; partition chromatography, e.g. on Sephadex
G-25, LH-20 or countercurrent distribution; high performance liquid
chromatography (HPLC), especially reverse-phase HPLC on octyl- or
octadecylsilyl-silica bonded phase column packing.
[0187] Methods of Use
[0188] Also provided herein are methods of use of the compounds or
compositions described herein. Also provided herein are methods for
treating a disease or pathology in a subject in need thereof
comprising administering to the subject an effective amount of any
of the compounds or compositions described herein.
[0189] Also provided herein are methods of treating, preventing, or
ameliorating cancer in a subject. The methods include administering
to a subject an effective amount of one or more of the compounds or
compositions described herein, or a pharmaceutically acceptable
salt thereof. The compounds and compositions described herein or
pharmaceutically acceptable salts thereof are useful for treating
cancer in humans, e.g., pediatric and geriatric populations, and in
animals, e.g., veterinary applications. The disclosed methods can
optionally include identifying a patient who is or can be in need
of treatment of a cancer. Examples of cancer types treatable by the
compounds and compositions described herein include bladder cancer,
brain cancer, breast cancer, colorectal cancer, cervical cancer,
gastrointestinal cancer, genitourinary cancer, head and neck
cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate
cancer, renal cancer, skin cancer, and testicular cancer. Further
examples include cancer and/or tumors of the anus, bile duct, bone,
bone marrow, bowel (including colon and rectum), eye, gall bladder,
kidney, mouth, larynx, esophagus, stomach, testis, cervix,
mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid,
vagina, vulva, uterus, liver, muscle, blood cells (including
lymphocytes and other immune system cells). Further examples of
cancers treatable by the compounds and compositions described
herein include carcinomas, Karposi's sarcoma, melanoma,
mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer,
leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic,
chronic myeloid, and other), and lymphoma (Hodgkin's and
non-Hodgkin's), and multiple myeloma.
[0190] The methods of treatment or prevention of cancer described
herein can further include treatment with one or more additional
agents (e.g., an anti-cancer agent or ionizing radiation). The one
or more additional agents and the compounds and compositions or
pharmaceutically acceptable salts thereof as described herein can
be administered in any order, including simultaneous
administration, as well as temporally spaced order of up to several
days apart. The methods can also include more than a single
administration of the one or more additional agents and/or the
compounds and compositions or pharmaceutically acceptable salts
thereof as described herein. The administration of the one or more
additional agents and the compounds and compositions or
pharmaceutically acceptable salts thereof as described herein can
be by the same or different routes. When treating with one or more
additional agents, the compounds and compositions or
pharmaceutically acceptable salts thereof as described herein can
be combined into a pharmaceutical composition that includes the one
or more additional agents.
[0191] For example, the compounds or compositions or
pharmaceutically acceptable salts thereof as described herein can
be combined into a pharmaceutical composition with an additional
anti-cancer agent, such as 13-cis-Retinoic Acid,
2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine,
5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane,
Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin,
Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic
acid, Alpha interferon, Altretamine, Amethopterin, Amifostine,
Aminoglutethimide, Anagrelide, Anandron, Anastrozole,
Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic
trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab,
Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib,
Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar,
Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine,
Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine,
cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine,
Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren,
Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine,
Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin,
Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome,
Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt,
Dexamethasone, Dexamethasone acetate, Dexamethasone sodium
phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,
Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome,
Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt,
Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase,
Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate,
Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara,
Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex,
Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide,
Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine,
Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot,
Matulane, Maxidex, Mechlorethamine, -Mechlorethamine Hydrochlorine,
Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan,
Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium,
Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega,
Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex,
Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin,
Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin,
Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG
Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON,
PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ,
Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT,
Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol,
Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon
alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin,
Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571,
Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar,
Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys,
Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex,
Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,
Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,
Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,
Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,
Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid,
Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte
colony stimulating factor, Halotestin, Herceptin, Hexadrol,
Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort
Acetate, Hydrocortisone, Hydrocortisone sodium phosphate,
Hydrocortisone sodium succinate, Hydrocortone phosphate,
Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,
Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib
mesylate, Imidazole Carboxamide, Interferon alfa, Interferon
Alfa-2b (PEG conjugate), Interleukin 2, Interleukin-11, Intron A
(interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide,
Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine,
L-PAM, L-Sarcolysin, Meticorten, Mitomycin, Mitomycin-C,
Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin,
Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort,
L-asparaginase, and LCR. The additional anti-cancer agent can also
include biopharmaceuticals such as, for example, antibodies.
[0192] Many tumors and cancers have viral genome present in the
tumor or cancer cells. For example, Epstein-Barr Virus (EBV) is
associated with a number of mammalian malignancies. The compounds
disclosed herein can also be used alone or in combination with
anticancer or antiviral agents, such as ganciclovir, azidothymidine
(AZT), lamivudine (3TC), etc., to treat patients infected with a
virus that can cause cellular transformation and/or to treat
patients having a tumor or cancer that is associated with the
presence of viral genome in the cells. The compounds disclosed
herein can also be used in combination with viral based treatments
of oncologic disease.
[0193] Also described herein are methods of killing a tumor cell in
a subject. The method includes contacting the tumor cell with an
effective amount of a compound or composition as described herein,
and optionally includes the step of irradiating the tumor cell with
an effective amount of ionizing radiation. Additionally, methods of
radiotherapy of tumors are provided herein. The methods include
contacting the tumor cell with an effective amount of a compound or
composition as described herein, and irradiating the tumor with an
effective amount of ionizing radiation. As used herein, the term
ionizing radiation refers to radiation comprising particles or
photons that have sufficient energy or can produce sufficient
energy via nuclear interactions to produce ionization. An example
of ionizing radiation is x-radiation. An effective amount of
ionizing radiation refers to a dose of ionizing radiation that
produces an increase in cell damage or death when administered in
combination with the compounds described herein. The ionizing
radiation can be delivered according to methods as known in the
art, including administering radiolabeled antibodies and
radioisotopes.
[0194] The methods and compounds as described herein are useful for
both prophylactic and therapeutic treatment. As used herein the
term treating or treatment includes prevention; delay in onset;
diminution, eradication, or delay in exacerbation of signs or
symptoms after onset; and prevention of relapse. For prophylactic
use, a therapeutically effective amount of the compounds and
compositions or pharmaceutically acceptable salts thereof as
described herein are administered to a subject prior to onset
(e.g., before obvious signs of cancer), during early onset (e.g.,
upon initial signs and symptoms of cancer), or after an established
development of cancer. Prophylactic administration can occur for
several days to years prior to the manifestation of symptoms of an
infection. Prophylactic administration can be used, for example, in
the chemopreventative treatment of subjects presenting precancerous
lesions, those diagnosed with early stage malignancies, and for
subgroups with susceptibilities (e.g., family, racial, and/or
occupational) to particular cancers. Therapeutic treatment involves
administering to a subject a therapeutically effective amount of
the compounds and compositions or pharmaceutically acceptable salts
thereof as described herein after cancer is diagnosed.
[0195] In some examples of the methods of treating of treating,
preventing, or ameliorating cancer or a tumor in a subject, the
compound or composition administered to the subject can comprise a
therapeutic moiety that can comprise a targeting moiety that can
act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2
SH2, or combinations thereof.
[0196] The disclosed subject matter also concerns methods for
treating a subject having a metabolic disorder or condition. In one
embodiment, an effective amount of one or more compounds or
compositions disclosed herein is administered to a subject having a
metabolic disorder and who is in need of treatment thereof. In some
examples, the metabolic disorder can comprise type II diabetes. In
some examples of the methods of treating of treating, preventing,
or ameliorating the metabolic disorder in a subject, the compound
or composition administered to the subject can comprise a
therapeutic moiety that can comprise a targeting moiety that can
act as an inhibitor against PTP1B. In one particular example of
this method the subject is obese and the method comprises treating
the subject for obesity by administering a composition as disclosed
herein.
[0197] The disclosed subject matter also concerns methods for
treating a subject having an immune disorder or condition. In one
embodiment, an effective amount of one or more compounds or
compositions disclosed herein is administered to a subject having
an immune disorder and who is in need of treatment thereof. In some
examples of the methods of treating of treating, preventing, or
ameliorating the immune disorder in a subject, the compound or
composition administered to the subject can comprise a therapeutic
moiety that can comprise a targeting moiety that can act as an
inhibitor against Pin1.
[0198] The disclosed subject matter also concerns methods for
treating a subject having cystic fibrosis. In one embodiment, an
effective amount of one or more compounds or compositions disclosed
herein is administered to a subject having cystic fibrosis and who
is in need of treatment thereof. In some examples of the methods of
treating the cystic fibrosis in a subject, the compound or
composition administered to the subject can comprise a therapeutic
moiety that can comprise a targeting moiety that can act as an
inhibitor against CAL PDZ.
[0199] Compositions, Formulations and Methods of Administration
[0200] In vivo application of the disclosed compounds, and
compositions containing them, can be accomplished by any suitable
method and technique presently or prospectively known to those
skilled in the art. For example, the disclosed compounds can be
formulated in a physiologically- or pharmaceutically-acceptable
form and administered by any suitable route known in the art
including, for example, oral, nasal, rectal, topical, and
parenteral routes of administration. As used herein, the term
parenteral includes subcutaneous, intradermal, intravenous,
intramuscular, intraperitoneal, and intrasternal administration,
such as by injection. Administration of the disclosed compounds or
compositions can be a single administration, or at continuous or
distinct intervals as can be readily determined by a person skilled
in the art.
[0201] The compounds disclosed herein, and compositions comprising
them, can also be administered utilizing liposome technology, slow
release capsules, implantable pumps, and biodegradable containers.
These delivery methods can, advantageously, provide a uniform
dosage over an extended period of time. The compounds can also be
administered in their salt derivative forms or crystalline
forms.
[0202] The compounds disclosed herein can be formulated according
to known methods for preparing pharmaceutically acceptable
compositions. Formulations are described in detail in a number of
sources which are well known and readily available to those skilled
in the art. For example, Remington's Pharmaceutical Science by E.
W. Martin (1995) describes formulations that can be used in
connection with the disclosed methods. In general, the compounds
disclosed herein can be formulated such that an effective amount of
the compound is combined with a suitable carrier in order to
facilitate effective administration of the compound. The
compositions used can also be in a variety of forms. These include,
for example, solid, semi-solid, and liquid dosage forms, such as
tablets, pills, powders, liquid solutions or suspension,
suppositories, injectable and infusible solutions, and sprays. The
preferred form depends on the intended mode of administration and
therapeutic application. The compositions also preferably include
conventional pharmaceutically-acceptable carriers and diluents
which are known to those skilled in the art. Examples of carriers
or diluents for use with the compounds include ethanol, dimethyl
sulfoxide, glycerol, alumina, starch, saline, and equivalent
carriers and diluents. To provide for the administration of such
dosages for the desired therapeutic treatment, compositions
disclosed herein can advantageously comprise between about 0.10%
and 100% by weight of the total of one or more of the subject
compounds based on the weight of the total composition including
carrier or diluent.
[0203] Formulations suitable for administration include, for
example, aqueous sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient; and
aqueous and nonaqueous sterile suspensions, which can include
suspending agents and thickening agents. The formulations can be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and can be stored in a freeze dried
(lyophilized) condition requiring only the condition of the sterile
liquid carrier, for example, water for injections, prior to use.
Extemporaneous injection solutions and suspensions can be prepared
from sterile powder, granules, tablets, etc. It should be
understood that in addition to the ingredients particularly
mentioned above, the compositions disclosed herein can include
other agents conventional in the art having regard to the type of
formulation in question.
[0204] Compounds disclosed herein, and compositions comprising
them, can be delivered to a cell either through direct contact with
the cell or via a carrier means. Carrier means for delivering
compounds and compositions to cells are known in the art and
include, for example, encapsulating the composition in a liposome
moiety. Another means for delivery of compounds and compositions
disclosed herein to a cell comprises attaching the compounds to a
protein or nucleic acid that is targeted for delivery to the target
cell. U.S. Pat. No. 6,960,648 and U.S. Application Publication Nos.
20030032594 and 20020120100 disclose amino acid sequences that can
be coupled to another composition and that allows the composition
to be translocated across biological membranes. U.S. Application
Publication No. 20020035243 also describes compositions for
transporting biological moieties across cell membranes for
intracellular delivery. Compounds can also be incorporated into
polymers, examples of which include poly (D-L lactide-co-glycolide)
polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)
propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);
chondroitin; chitin; and chitosan.
[0205] For the treatment of oncological disorders, the compounds
disclosed herein can be administered to a patient in need of
treatment in combination with other antitumor or anticancer
substances and/or with radiation and/or photodynamic therapy and/or
with surgical treatment to remove a tumor. These other substances
or treatments can be given at the same as or at different times
from the compounds disclosed herein. For example, the compounds
disclosed herein can be used in combination with mitotic inhibitors
such as taxol or vinblastine, alkylating agents such as
cyclophosamide or ifosfamide, antimetabolites such as
5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin
or bleomycin, topoisomerase inhibitors such as etoposide or
camptothecin, antiangiogenic agents such as angiostatin,
antiestrogens such as tamoxifen, and/or other anti-cancer drugs or
antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals
Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an
immunotherapeutic such as ipilimumab and bortezomib.
[0206] In certain examples, compounds and compositions disclosed
herein can be locally administered at one or more anatomical sites,
such as sites of unwanted cell growth (such as a tumor site or
benign skin growth, e.g., injected or topically applied to the
tumor or skin growth), optionally in combination with a
pharmaceutically acceptable carrier such as an inert diluent.
Compounds and compositions disclosed herein can be systemically
administered, such as intravenously or orally, optionally in
combination with a pharmaceutically acceptable carrier such as an
inert diluent, or an assimilable edible carrier for oral delivery.
They can be enclosed in hard or soft shell gelatin capsules, can be
compressed into tablets, or can be incorporated directly with the
food of the patient's diet. For oral therapeutic administration,
the active compound can be combined with one or more excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and
the like.
[0207] The disclosed compositions are bioavailable and can be
delivered orally. Oral compositions can be tablets, troches, pills,
capsules, and the like, and can also contain the following: binders
such as gum tragacanth, acacia, corn starch or gelatin; excipients
such as dicalcium phosphate; a disintegrating agent such as corn
starch, potato starch, alginic acid and the like; a lubricant such
as magnesium stearate; and a sweetening agent such as sucrose,
fructose, lactose or aspartame or a flavoring agent such as
peppermint, oil of wintergreen, or cherry flavoring can be added.
When the unit dosage form is a capsule, it can contain, in addition
to materials of the above type, a liquid carrier, such as a
vegetable oil or a polyethylene glycol. Various other materials can
be present as coatings or to otherwise modify the physical form of
the solid unit dosage form. For instance, tablets, pills, or
capsules can be coated with gelatin, wax, shellac, or sugar and the
like. A syrup or elixir can contain the active compound, sucrose or
fructose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or orange flavor.
Of course, any material used in preparing any unit dosage form
should be pharmaceutically acceptable and substantially non-toxic
in the amounts employed. In addition, the active compound can be
incorporated into sustained-release preparations and devices.
[0208] Compounds and compositions disclosed herein, including
pharmaceutically acceptable salts or prodrugs thereof, can be
administered intravenously, intramuscularly, or intraperitoneally
by infusion or injection. Solutions of the active agent or its
salts can be prepared in water, optionally mixed with a nontoxic
surfactant. Dispersions can also be prepared in glycerol, liquid
polyethylene glycols, triacetin, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
can contain a preservative to prevent the growth of
microorganisms.
[0209] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient, which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. The ultimate dosage form should be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium
comprising, for example, water, ethanol, a polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycols, and the
like), vegetable oils, nontoxic glyceryl esters, and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or by the use of
surfactants. Optionally, the prevention of the action of
microorganisms can be brought about by various other antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
buffers or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the inclusion of agents that
delay absorption, for example, aluminum monostearate and
gelatin.
[0210] Sterile injectable solutions are prepared by incorporating a
compound and/or agent disclosed herein in the required amount in
the appropriate solvent with various other ingredients enumerated
above, as required, followed by filter sterilization. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and the freeze drying techniques, which yield a powder of the
active ingredient plus any additional desired ingredient present in
the previously sterile-filtered solutions.
[0211] For topical administration, compounds and agents disclosed
herein can be applied in as a liquid or solid. However, it will
generally be desirable to administer them topically to the skin as
compositions, in combination with a dermatologically acceptable
carrier, which can be a solid or a liquid. Compounds and agents and
compositions disclosed herein can be applied topically to a
subject's skin to reduce the size (and can include complete
removal) of malignant or benign growths, or to treat an infection
site. Compounds and agents disclosed herein can be applied directly
to the growth or infection site. Preferably, the compounds and
agents are applied to the growth or infection site in a formulation
such as an ointment, cream, lotion, solution, tincture, or the
like.
[0212] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers, for example.
[0213] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0214] Useful dosages of the compounds and agents and
pharmaceutical compositions disclosed herein can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art.
[0215] The dosage ranges for the administration of the compositions
are those large enough to produce the desired effect in which the
symptoms or disorder are affected. The dosage should not be so
large as to cause adverse side effects, such as unwanted
cross-reactions, anaphylactic reactions, and the like. Generally,
the dosage will vary with the age, condition, sex and extent of the
disease in the patient and can be determined by one of skill in the
art. The dosage can be adjusted by the individual physician in the
event of any counterindications. Dosage can vary, and can be
administered in one or more dose administrations daily, for one or
several days.
[0216] Also disclosed are pharmaceutical compositions that comprise
a compound disclosed herein in combination with a pharmaceutically
acceptable carrier. Pharmaceutical compositions adapted for oral,
topical or parenteral administration, comprising an amount of a
compound constitute a preferred aspect. The dose administered to a
patient, particularly a human, should be sufficient to achieve a
therapeutic response in the patient over a reasonable time frame,
without lethal toxicity, and preferably causing no more than an
acceptable level of side effects or morbidity. One skilled in the
art will recognize that dosage will depend upon a variety of
factors including the condition (health) of the subject, the body
weight of the subject, kind of concurrent treatment, if any,
frequency of treatment, therapeutic ratio, as well as the severity
and stage of the pathological condition.
[0217] Also disclosed are kits that comprise a compound disclosed
herein in one or more containers. The disclosed kits can optionally
include pharmaceutically acceptable carriers and/or diluents. In
one embodiment, a kit includes one or more other components,
adjuncts, or adjuvants as described herein. In another embodiment,
a kit includes one or more anti-cancer agents, such as those agents
described herein. In one embodiment, a kit includes instructions or
packaging materials that describe how to administer a compound or
composition of the kit. Containers of the kit can be of any
suitable material, e.g., glass, plastic, metal, etc., and of any
suitable size, shape, or configuration. In one embodiment, a
compound and/or agent disclosed herein is provided in the kit as a
solid, such as a tablet, pill, or powder form. In another
embodiment, a compound and/or agent disclosed herein is provided in
the kit as a liquid or solution. In one embodiment, the kit
comprises an ampoule or syringe containing a compound and/or agent
disclosed herein in liquid or solution form.
[0218] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
EXAMPLES
[0219] The following examples are set forth to illustrate the
methods and results according to the disclosed subject matter.
These examples are not intended to be inclusive of all aspects of
the subject matter disclosed herein, but rather to illustrate
representative methods and results. These examples are not intended
to exclude equivalents and variations which are apparent to one
skilled in the art.
[0220] Efforts have been made to ensure accuracy with respect to
numbers (e.g., amounts, temperature, etc.) but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric. There
are numerous variations and combinations of reaction conditions,
e.g., component concentrations, temperatures, pressures and other
reaction ranges and conditions that can be used to optimize the
product purity and yield obtained from the described process. Only
reasonable and routine experimentation will be required to optimize
such process conditions.
Example 1
[0221] Cyclic heptapeptide cyclo(F.PHI.RRRRQ) (cF.PHI.R.sub.4,
where .PHI. is L-2-naphthylalanine) was found to be efficiently
internalized by mammalian cells. In this study, its mechanism of
internalization was investigated by perturbing various endocytic
events through the introduction of pharmacologic agents and genetic
mutations. The results show that cF.PHI.R.sub.4 can bind directly
to membrane phospholipids, can be internalized into human cancer
cells through endocytosis, and can escape from early endosomes into
the cytoplasm. Its cargo capacity was examined with a wide variety
of molecules including small-molecule dyes, linear and cyclic
peptides of various charged states, and proteins. Depending on the
nature of the cargos, they may be delivered by endocyclic
(insertion of cargo into the cF.PHI.R.sub.4 ring), exocyclic
(attachment of cargo to the Gln side chain), or bicyclic approaches
(fusion of cF.PHI.R.sub.4 and cyclic cargo rings). The overall
delivery efficiency (i.e., delivery of cargo into the cytoplasm and
nucleus) of cF.PHI.R.sub.4 was 4-12-fold higher than those of
nonaarginine (R.sub.9), HIV Tat derived peptide (Tat), or
penetratin (Antp). The higher delivery efficiency, coupled with
superior serum stability, minimal toxicity, and synthetic
accessibility, renders cF.PHI.R.sub.4 a useful transporter for
intracellular cargo delivery and a suitable system for
investigating the mechanism of endosomal escape.
Introduction
[0222] The plasma membrane presents a major challenge in drug
discovery, especially for biologics such as peptides, proteins and
nucleic acids. One potential strategy to subvert the membrane
barrier and deliver the biologics into cells is to attach them to
"cell-penetrating peptides (CPPs)". Since the initial observation
that HIV trans-activator of transcription, Tat, internalizes into
mammalian cells and activates viral replication in the late 1980s
(Frankel, A D and Pabo, C O. Cell, 1988, 55, 1189-1193; Green, M
and Loewenstein, P M. Cell, 1988, 55, 1179-1188) a large number of
CPPs consisting of 6-20 residues have been reported (Langel, U.
Cell-penetrating peptides: methods and protocols, Humana Press, New
York, 2011, p xv; Schmidt, N et al. FEBS Lett., 2010, 584,
1806-1813; Futaki, S. Adv. Drug Delivery Rev., 2005, 57, 547-558;
Stewart, K M et al. Org. Biomol. Chem., 2008, 6, 2242-2255;
Deshayes, S et al. Cell. Mol. Life Sci., 2005, 62, 1839-1849; Goun,
E A et al. ChemBioChem, 2005, 7, 1497-1515). CPPs have been used to
deliver small-molecule drugs (Rothbard, J B et al. Nat. Med., 2000,
6, 1253-1257; Nori, A et al. Bioconjugate Chem., 2003, 14, 44-50),
DNA (Hoyer, J and Neundorf, I. Acc. Chem. Res., 2012, 45,
1048-1056; Eguchi, A et al. J. Biol. Chem., 2001, 276,
26204-26210), RNA (Nakase, I et al. Acc. Chem. Res., 2012, 45,
1132-1139; Andaloussi, S E et al. Nucleic Acids Res., 2011, 39,
3972-3987; Jeong, J H et al. Bioconjugate Chem., 2009, 20, 5-14;
Muratovska, A and Eccles, M R. FEBS Lett., 2004, 558, 63-68),
proteins (Wadia, J S and Dowdy, S F. Adv. Drug Delivery Rev., 2005,
57, 579-596; Pooga, M et al. FASEB J., 2001, 15, 1451-1453;
Schwarze, S R et al. Science, 1999, 285, 1569-1572), and
nanoparticles (Josephson, L et al. Bioconjugate Chem., 1999, 10,
186-191; Gupta, B et al. Adv. Drug Delivery Rev., 2005, 57,
637-651; Liu, J et al. Biomacromolecules, 2001, 2, 362-8), into
mammalian cells and tissues through either covalent attachment or
electrostatic association. Many CPPs display minimal toxicity and
immunogenicity at physiologically relevant concentrations (Saar, K
et al. Anal. Biochem., 2005, 345, 55-65; Suhorutsenko, J et al.
Bioconjugate Chem., 2011, 22, 2255-2262) and the incorporation of
specific unnatural amino acids (Rueping, M et al. ChemBioChem,
2002, 3, 257-259) and other chemical moieties (Cooley, C B et al. J
Am. Chem. Soc., 2009, 131, 16401-16403; Pham, W et al. Chembiochem,
2004, 5, 1148-1151) have been found to increase stability and
cytosolic delivery.
[0223] Despite three decades of investigation, the fundamental
basis for CPP activity remains elusive. Two distinct and
non-mutually exclusive mechanisms have been proposed for the CPPs
whose primary sequences are characterized by having multiple
arginine residues. In the first mechanism (direct membrane
translocation), the arginine guanidinium groups interact with
phospholipids of the plasma membrane to generate neutral ion pairs
that passively diffuse across the membrane (Herce, H D and Garcia,
AE. Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 20805-20810; Hirose,
H et al. Mol. Ther., 2012, 20, 984-993) or promote the formation of
transient pores that permit the CPPs to traverse the lipid bilayer
(Herce, H D et al. Biophys. J., 2009, 97, 1917-1925; Palm-Apergi, C
et al. FASEB J., 2009, 23, 214-223). In the second mechanism, CPPs
associate with cell surface glycoproteins and membrane
phospholipids, internalize into cells through endocytosis (Richard,
J P et al. J Biol. Chem., 2005, 280, 15300-15306; Ferrari, A et al.
Mol. Ther., 2003, 8, 284-294; Fittipaldi, A et al. J Biol. Chem.,
2003, 278, 34141-34149; Kaplan, I M et al. J Controlled Release,
2005, 102, 247-253; Nakase, I et al. Biochemistry, 2007, 46,
492-501) and subsequently exit from endosomes into the cytoplasm.
Taken together, the majority of data show that at low CPP
concentrations, cellular uptake occurs mostly through endocytosis,
whereas direct membrane translocation becomes prevalent at
concentrations above 10 .mu.M (Duchardt, F et al. Traffic, 2007, 8,
848-866). However, the mechanism(s) of entry and the efficiency of
uptake may vary with the CPP identity, cargo, cell type, and other
factors (Mueller, J et al. Bioconjugate Chem., 2008, 19, 2363-2374;
Maiolo, J R et al. Biochim. Biophys. Acta., 2005, 1712,
161-172).
[0224] CPPs that enter cells via endocytosis must exit from
endocytic vesicles in order to reach the cytosol. Unfortunately,
the endosomal membrane has proven to be a significant barrier
towards cytoplasmic delivery by these CPPs; often a negligible
fraction of the peptides escapes into the cell interior (El-Sayed,
A et al. AAPSJ., 2009, 11, 13-22; Varkouhi, A K et al. J.
Controlled Release, 2011, 151, 220-228; Appelbaum, J S et al. Chem.
Biol., 2012, 19, 819-830). For example, even in the presence of the
fusogenic hemagglutinin peptide HA2, which has been demonstrated to
enhance endosomal cargo release, >99% of a Tat-Cre fusion
protein remains entrapped in macropinosomes 24 h after initial
uptake (Kaplan, I M et al. J Controlled Release, 2005, 102,
247-253). Recently, two new types of CPPs with improved endosomal
escape efficiencies have been discovered. Appelbaum et al. showed
that folded miniature proteins containing a discrete penta-arginine
motif were able to effectively overcome endosomal entrapment and
reach the cytosol of mammalian cells (Appelbaum, J S et al. Chem.
Biol., 2012, 19, 819-830). This motif consists of five arginines
across three turns of an .alpha.-helix, and proteins containing
this motif were released from early (Rab5.sup.+) endosomes into the
cell interior. It has also been found that cyclization of certain
arginine-rich CPPs enhances their cellular uptake (Qian, Z et al.
ACS Chem. Biol., 2013, 8, 423-431; Lattig-Tunnemann, G et al. Nat.
Commun., 2011, 2, 453; Mandal, D et al. Angew. Chem. Int. Ed.,
2011, 50, 9633-9637; Zhao, K et al. Soft Matter, 2012, 8,
6430-6433). Small amphipathic cyclic peptides such as
cyclo(F.PHI.RRRRQ) (cF.PHI.R.sub.4, where .PHI. is
L-2-naphthylalanine) are internalized by mammalian cells in an
energy-dependent manner, and enter the cytoplasm and nucleus with
efficiencies 2-5-fold higher than that of nonaarginine (R.sub.9)
(Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431). Moreover,
membrane impermeable cargos such as phosphopeptides can be inserted
into the cF.PHI.R.sub.4 ring resulting in their delivery into the
cytoplasm of target cells. However, insertion of a cargo into the
cyclic peptide ring, which is referred to herein as the
"endocyclic" delivery method (FIG. 1A), is limited to relatively
short peptides (.ltoreq.7 amino acids), as large rings display poor
internalization efficiency (Qian, Z et al. ACS Chem. Biol., 2013,
8, 423-431).
[0225] To gain insight into the cF.PHI.R.sub.4 mechanism of action
and potentially design cyclic CPPs of still higher efficiency,
herein the internalization mechanism of cF.PHI.R.sub.4 was
investigated through the use of artificial membranes and
pharmacologic agents as well as genetic mutations that perturb
various endocytic events. The data show that cF.PHI.R.sub.4 can
bind directly to the plasma membrane phospholipids and can enter
cells through endocytosis. Like the miniature proteins displaying
the penta-arginine motif (Appelbaum, J S et al. Chem. Biol., 2012,
19, 819-830), cF.PHI.R.sub.4 can escape from the early endosomes
into the cytosol. The ability of cF.PHI.R.sub.4 to deliver a wide
range of cargo molecules, including linear peptides of varying
charges, cyclic peptides, and large proteins, into the cytoplasm of
mammalian cells by exocyclic (attachment of cargo to the Gln side
chain; FIG. 1B) or bicyclic delivery methods (fusion of the
cF.PHI.R.sub.4 and cyclic cargo rings; FIG. 1C) was also examined.
It was found that cF.PHI.R.sub.4 is tolerant to the size and nature
of cargos and efficiently transported all of the cargos tested into
the cytoplasm and nucleus of mammalian cells. In addition,
cF.PHI.R.sub.4 exhibits superior stability against proteolysis over
linear CPPs but minimal cytotoxicity. cF.PHI.R.sub.4 therefore
provides a practically useful transporter for cytosolic cargo
delivery as well as a system for investigating the mechanism of
early endosomal cargo release.
[0226] Materials. Reagents for peptide synthesis were purchased
from Advanced ChemTech (Louisville, Ky.), NovaBiochem (La Jolla,
Calif.), or Anaspec (San Jose, Calif.). 2,2'-Dipyridyl disulfide,
Lissamine rhodamine B sulfonyl chloride, fluorescein isothiocyanate
(FITC), dexamethasone (Dex), coenzyme A trilithium salt,
FITC-labeled dextran (dextran.sup.FITC) and human serum were
purchased from Sigma-Aldrich (St. Louis, Mo.). Cell culture media,
fetal bovine serum (FBS), penicillin-streptomycin, 0.25%
trypsin-EDTA, Hoescht 33342, Alexa488-labeled dextran
(dextran.sup.Alexa488), Dulbecco's phosphate-buffered saline (DPBS)
(2.67 mM potassium chloride, 1.47 mM potassium phosphate monobasic,
137 mM sodium chloride, 8.06 mM sodium phosphate dibasic), and
Lipofectamine 2000 were purchased from Invitrogen (Carlsbad,
Calif.). PD-10 desalting columns were purchased from GE-Healthcare
(Piscataway, N.J.). Nuclear staining dye DRAQ5.TM. was purchased
from Thermo Scientific (Rockford, Ill.), while cell proliferation
kit (MTT) was purchased from Roche (Indianapolis, Ind.).
Anti-phosphotyrosine (pY) antibody (clone 4G10) was purchased from
Millipore (Temecula, Calif.).
[0227] Rink resin LS (100-200 mesh, 0.2 mmol/g) was purchased from
Advanced ChemTech. LC-SMCC (succinimidyl-4-[N-maleimidomethyl]
cyclohexane-1-carboxy-[6-amidocaproate]) was purchased from Thermo
Scientific (Rockford, Ill.), while
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium
salt) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phophoethanolamine
(POPE), sphingomyelin (Brain, Porcine), and cholesterol were
purchased from Avanti Polar Lipids (Alabaster, Ala.). Heparan
sulfate (HO-03103, Lot #HO-10697) was obtained from Celcus
Laboratories (Cincinnati, Ohio).
[0228] Peptide Synthesis and Labeling. Peptides were synthesized on
Rink Resin LS (0.2 mmol/g) using standard Fmoc chemistry. The
typical coupling reaction contained 5 equiv of Fmoc-amino acid, 5
equiv of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and 10 equiv of diisopropylethylamine
(DIPEA) and was allowed to proceed with mixing for 75 min. After
the addition of the last (N-terminal) residue, the allyl group on
the C-terminal Glu residue was removed by treatment with
Pd(PPh.sub.3).sub.4 and phenylsilane (0.1 and 10 equiv,
respectively) in anhydrous DCM (3.times.15 min). The N-terminal
Fmoc group was removed by treatment with 20% piperidine in DMF and
the peptide was cyclized by treatment with
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP)/HOBt/DIPEA (5, 5, and 10 equiv) in DMF
for 3 h. The peptides were deprotected and released from the resin
by treatment with 82.5:5:5:5:2.5 (v/v)
TFA/thioanisole/water/phenol/ethanedithiol for 2 h. The peptides
were triturated with cold ethyl ether (3.times.) and purified by
reversed-phase HPLC on a Cis column. The authenticity of each
peptide was confirmed by MALDI-TOF mass spectrometry.
[0229] Peptide labeling with FITC was performed by dissolving the
purified peptide (.about.1 mg) in 300 .mu.L of 1:1:1 (vol/vol)
DMSO/DMF/150 mM sodium bicarbonate (pH 8.5) and mixing with 10
.mu.L of FITC in DMSO (100 mg/mL). After 20 min at room
temperature, the reaction mixture was subjected to reversed-phase
HPLC on a Cis column to isolate the FITC-labeled peptide. To
generate rhodamine- and Dex-labeled peptides (FIG. 2), an
N.sup.8-4-methoxytrityl-L-lysine was added to the C-terminus. After
the solid phase peptide synthesis, the lysine side chain was
selectively deprotected using 1% (v/v) trifluoroacetic acid in
CH.sub.2Cl.sub.2. The resin was incubated with Lissamine rhodamine
B sulfonyl chloride/DIPEA (5 equiv each) in DMF overnight. The
peptides were fully deprotected, triturated with diethyl ether, and
purified by HPLC. The Dex-labeled peptide was produced by
incubating the resin with dexamethasone-21-thiopropionic
acid/HBTU/DIPEA (5, 5, and 10 equiv) in DMF for 3 h (Appelbaum, J S
et al. Chem. Biol., 2012, 19, 819-830). The peptide was then
deprotected, triturated, and purified by HPLC. Bicyclic peptides,
phosphocoumaryl aminopropionic acid (pCAP), and pCAP-containing
peptides (PCPs) were synthesized as previously described (Lian, W
et al. J. Am. Chem. Soc., 2013, 135, 11990-11995; Mitra, S and
Barrios, A M. Bioorg. Med. Chem. Lett., 2005, 15, 5124-5145;
Stanford, S M et al. Proc. Natl. Acad. Sci. U.S.A., 2012, 109,
13972-13977). The authenticity of each peptide was confirmed by
MALDI-TOF mass spectrometry.
[0230] Preparation of cF.PHI.R.sub.4--Protein Conjugates. The gene
coding for the catalytic domain of PTP1B (amino acids 1-321) was
amplified by the polymerase chain reaction using PTP1B cDNA as
template and oligonucleotides
5'-ggaattccatatggagatggaaaaggagttcgagcag-3' and
5'-gggatccgtcgacattgtgtggctccaggattcgtttgg-3' as primers. The
resulting DNA fragment was digested with endonucleases Nde I and
Sal I and inserted into prokaryotic vector pET-22b(+)-ybbR (Yin, J
et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15815-15820). This
cloning procedure resulted in the addition of a ybbR tag
(VLDSLEFIASKL) to the N-terminus of PTP1B. Expression and
purification of the ybbR tagged PTP1B were carried out as
previously described (Ren, L et al. Biochemistry, 2011, 50,
2339-2356).
[0231] Peptide cF.PHI.R.sub.4 containing a C-terminal cysteine
(cF.PHI.R.sub.4-SH, .about.10 .mu.mol; FIG. 3) was dissolved in 1
mL of degassed DPBS and mixed with 2,2'-dipyridyl disulfide (5
equiv) dissolved in acetone (0.5 mL). After 2 h at room
temperature, the reaction product cF.PHI.R.sub.4-SS-Py was purified
by reversed-phase HPLC. The product was incubated with coenzyme A
(2 equiv) in DPBS for 2 h. The resulting cF.PHI.R.sub.4-SS-CoA
adduct was purified again by reversed-phase HPLC. Green fluorescent
protein (GFP) containing an N-terminal ybbR tag (VLDSLEFIASKL) and
a C-terminal six-histidine tag was expressed in Escherichia coli
and purified as previously described (Yin, J et al. Proc. Natl.
Acad. Sci. U.S.A., 2005, 102, 15815-15820). Next, ybbR-GFP (30
.mu.M), cF.PHI.R.sub.4-SS-CoA (30 .mu.M), and phosphopantetheinyl
transferase Sfp (0.5 .mu.M) were mixed in 50 mM HEPES (pH 7.4), 10
mM MgCl.sub.2 (total volume 1.5 mL) and incubated at 37.degree. C.
for 15 min. The labeled protein, cF.PHI.R.sub.4-S-S-GFP (FIG. 3),
was separated from unreacted cF.PHI.R.sub.4-SS-CoA by passing the
reaction mixture through a PD-10 desalting column. GFP conjugated
to Tat (Tat-S-S-GFP) and cF.PHI.R.sub.4-conjugated PTP1B
(cF.PHI.R.sub.4-PTP1B) were prepared in a similar fashion (FIG.
4).
[0232] Peptide containing a C-terminal lysine (cF.PHI.R.sub.4-Lys,
.about.10 .mu.mol; FIG. 4) was synthesized on the solid phase,
deprotected and released from the support, dissolved in degassed
DPBS (pH 7.4, 1 mL), and mixed with bifunctional linker LC-SMCC (5
equiv) dissolved in DMSO (0.2 mL). After incubation at room
temperature for 2 h, the reaction product cF.PHI.R.sub.4-SMCC (FIG.
4) was purified by reversed-phase HPLC equipped with a Cis column.
The product was then mixed with coenzyme A (2 equiv) in DPBS and
incubated for 2 h. The resulting cF.PHI.R.sub.4-SMCC-CoA adduct was
purified again by reversed-phase HPLC. Next, ybbR-tagged PTP1B (30
.mu.M), cF.PHI.R.sub.4-SMCC-CoA (30 .mu.M), and phosphopantetheinyl
transferase Sfp (0.5 .mu.M) were mixed in 50 mM HEPES (pH 7.4), 10
mM MgCl.sub.2 (total volume of 1.5 mL) and incubated at 37.degree.
C. for 15 min. The labeled protein (cF.PHI.R.sub.4-PTP1B; FIG. 4)
was separated from unreacted cF.PHI.R.sub.4-SMCC-CoA by passing the
reaction mixture through a PD-10 desalting column eluted with
DPBS.
[0233] Cell Culture and Transfection. HEK293, HeLa, MCF-7, NIH 3T3
and A549 cells were maintained in medium consisting of DMEM, 10%
FBS and 1% penicillin/streptomycin. Jurkat, H1650, and H1299 cells
were grown in RPMI-1640 supplemented with 10% FBS and 1%
penicillin/streptomycin. Cells were cultured in a humidified
incubator at 37.degree. C. with 5% CO.sub.2. For HeLa cells
transfection, cells were seeded onto 96-well plate at a density of
10,000 cells/well. Following attachment, cells were transfected
with plasmids encoding Rab5-green fluorescent protein fusion
(Rab5-GFP), Rab7-GFP (Addgene plasmid #28047), glucocorticoid
receptor (C638G)-GFP fusion (GR-GFP) (Holub, J M et al.
Biochemistry, 2013, 50, 9036-6046), DsRed-Rab5 WT (Addgene plasmid
#13050) or DsRed-Rab5.sup.Q79L (Addgene plasmid #29688) following
Lipofectamine 2000 manufacturer protocols.
[0234] Preparation of Small Unilamellar Vesicles (SUVs). SUVs were
prepared by modifying a previously reported procedure (Magzoub, M
et al. Biochim. Biophys. Acta, 2002, 1563, 53-63). A proper lipid
mixture was dissolved in chloroform in a test tube. The lipid
mixture was dried gently by blowing argon over the solution, and
kept in a desiccator overnight. The dried lipids were rehydrated in
DPBS to final total lipid concentration of 10 mM. The suspension
was rigorously mixed by vortexing and sonication on ice until it
became clear. A typical preparation yields a homogeneous solution
containing vesicles with average diameter of .about.80 nm and
polydispersity (PdI) index of <0.15 as determined by dynamic
light scattering measurements using Zeta Sizer Nano Series
(Malvern, Brookhaven, Conn.). The SUV solution was stored at
4.degree. C. and used for FP experiments on the same day.
[0235] Fluorescence Polarization. A typical experiment was
performed by incubating 100 nM FITC-labeled peptide with varying
concentrations of heparan sulfate (0-5,000 nM) in DPBS for 2 h at
room temperature. The FP values were measured on a Molecular
Devices Spectramax M5 spectrofluorimeter, with excitation and
emission wavelengths at 485 and 525 nm, respectively. EC.sub.50
were determined by plotting the FP values as a function of heparan
sulfate concentrations and fitted to a four-parameter logistic
curve with GraphPad PRISM ver.6 software.
[0236] To obtain the EC.sub.50 value of CPP with lipid membranes,
the FP experiment was similarly conducted using 100 nM FITC-labeled
peptide with increasing concentrations of SUV solutions (0-10 mM)
in DPBS. The FP values were similarly measured, plotted, and
analyzed.
[0237] Image Analysis. Raw images were uniformly modified using
imageJ. Pearson's correlation coefficient (R) was obtained from
endosomal regions using Just Another Colocalization Plugin (JACoP)
(Bolte, S and Cordelieres, F P. J Microsc., 2006, 224, 213-232).
For GR-GFP translocation assay, individual GFP and Hoescht images
were loaded into a customized CellProfiler pipeline and colored to
grey (Carpenter, A E et al. Genome Biol., 2006, 7, R100). Nuclei
were distinguished from the Hoescht image via Otsu automatic
three-class thresholding, with pixels of the middle intensity class
assigned to background. Clumped objects were identified using
Laplacian of Gaussian modeling and separated by shape. The nuclear
region was defined as the diameter of the Hoescht objects shrunken
by 1 .mu.m, while the cytosolic ring region was defined as the
region between the nuclear diameter and the nuclear diameter
expanded 2 .mu.m. The translocation ratio was defined as the mean
GFP signal inside the nuclear region divided by the mean GFP signal
within the cytosolic region measured per cell, and 30-70 cells from
15-30 images were captured for each condition tested.
[0238] Confocal Microscopy. To examine the co-localization between
rhodamine-labeled cyclic peptide (cF.PHI.R.sub.4.sup.Rho) and
Rab5.sup.+ or Rab7.sup.+ endosomes, HeLa cells transfected with
Rab5-GFP or Rab7-GFP were plated (200 .mu.L, 10.sup.4 cells/well,
96-well glass bottom MatriPlates) the day prior to the experiment.
On the day of experiment, HeLa cells were treated with 1 .mu.M
cF.PHI.R.sub.4.sup.Rho in DMEM media supplemented with 300 nM
Hoescht 33342 for 30 min. After that, the cells were washed with
HKR buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2 mM KCl, 1 mM
CaCl.sub.2), 1 mM MgCl.sub.2) and imaged using a PerkinElmer
LiveView spinning disk confocal microscope.
[0239] For GR translocation assay, HeLa cells transfected with
GR-GFP were plated as described above (Holub, J M et al.
Biochemistry, 2013, 50, 9036-6046). The cells were treated for 30
min with DMEM media containing 1 .mu.M Dex or Dex-peptide conjugate
and 300 nM Hoescht 33342 and imaged using a Zeiss Axiovert 200M
epifluorescence microscope outfitted with Ziess Axiocam mRM camera
and an EXFO-Excite series 120 Hg arc lamp. To examine the effect of
endocytosis inhibitors, transfected HeLa cells were pretreated for
30 min with clear DMEM containing the inhibitors before incubation
with Dex or Dex-peptide conjugates. To test whether Rab5 activity
is required for endosomal escape, HeLa cells were transfected with
GR-GFP and DsRed-Rab5 WT or DsRed-Rab5.sup.Q79L before treatment
with Dex or Dex-peptide conjugate and imaged as described above
(Appelbaum, J S et al. Chem. Biol., 2012, 19, 819-830).
[0240] To examine the internalization of rhodamine-labeled
peptides, 5.times.10.sup.4 HEK293 cells were plated in a 35 mm
glass-bottomed microwell dish (MatTek). On the day of experiment,
the cells were incubated with the peptide solution (5 .mu.M) and
0.5 mg/mL dextran.sup.FITC at 37.degree. C. for 2 h. The cells were
gently washed with DPBS twice and imaged on a Visitech Infinity 3
Hawk 2D-array live cell imaging confocal microscope. To detect the
internalization of pCAP-containing peptides, HEK293 cells were
similarly plated and incubated with the peptide solution (5 .mu.M)
at 37.degree. C. for 60 min. After removal of the medium, the cells
were gently washed with DPBS containing sodium pervanadate (1 mM)
twice and incubated for 10 min in DPBS containing 5 .mu.M nuclear
staining dye DRAQ5. The resulting cells were washed with DPBS twice
and imaged on a spinning disk confocal microscope (UltraView Vox
CSUX1 system). To monitor GFP internalization, 5.times.10.sup.4
HEK293 cells were seeded in a 35 mm glass-bottomed microwell dish
and cultured overnight. Cells were treated with
cF.PHI.R.sub.4-S-S-GFP (1 .mu.M) at 37.degree. C. for 2 h. After
removal of the medium, the cells were incubated in DPBS containing
5 .mu.M DRAQ5 for 10 min. The cells were washed with DPBS twice and
imaged on a Visitech Infinity 3 Hawk 2D-array live cell imaging
confocal microscope.
[0241] Flow Cytometry. To quantify the delivery efficiencies of
pCAP-containing peptides, HeLa cells were cultured in six-well
plates (5.times.10.sup.5 cells per well) for 24 h. On the day of
experiment, the cells were incubated with 10 .mu.M pCAP-containing
peptide in clear DMEM with 1% FBS at 37.degree. C. for 2 h. The
cells were washed with DPBS containing 1 mM sodium pervanadate,
detached from plate with 0.25% trypsin, suspended in DPBS
containing 1% bovine serum albumin, and analyzed on a BD FACS Aria
flow cytometer with excitation at 355 nm. Data were analyzed with
Flowjo software (Tree Star).
[0242] To estimate the effect of cF.PHI.R.sub.4 on endocytosis,
HeLa cells were seeded in six-well plates (5.times.10.sup.5 cells
per well) and allowed to adhere overnight. Following adherence,
cells were treated with clear DMEM containing no supplement, 1
.mu.M cF.PHI.R.sub.4 peptide, 100 .mu.M dextran.sup.Alexa488 (Life
Technologies, D-22910), or both 1 .mu.M cyclic peptide and 100
.mu.M dextran.sup.Alexa488 for 30 min under standard cell culture
conditions. The cells were washed with DPBS twice, removed from the
plate with 0.25% trypsin, diluted into clear DMEM containing 10%
FBS, pelleted at 300 g for 5 min, washed once with DPBS and
resuspended in 200 .mu.L of DPBS. Whole-cell dextran uptake was
analyzed on a BD Accuri C6 flow cytometer using the manufacturer
FL1 laser and filter set.
[0243] Immunoblotting. NIH 3T3 cells were cultured in full growth
media to reach 80% confluence. The cells were starved in serum free
media for 3 h and treated with different concentrations of
cF.PHI.R.sub.4-PTP1B or untagged PTP1B for 2 h, followed by 30 min
incubation in media supplemented with 1 mM sodium pervanadate. The
solutions were removed and the cells were washed with cold DPBS
twice. The cells were detached and lysed in 50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1% NP-40, 10 mM sodium pyrophosphate, 5 mM iodoacetic
acid, 10 mM NaF, 1 mM EDTA, 2 mM sodium pervanadate, 0.1 mg/mL
phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 0.1 mg/mL
trypsin inhibitor. After 30 min incubation on ice, the cell lysate
was centrifuged at 15,000 rpm for 25 min in a microcentrifuge. The
total cellular proteins were separated by SDS-PAGE and transferred
electrophoretically to PVDF membrane, which was immunoblotted using
anti-pY antibody 4G10.
[0244] Serum Stability Test. The stability tests were carried by
modifying a previously reported procedure (Nguyen, L T et al. PLoS
One, 2010, 5, e12684). Diluted human serum (25%) was centrifuged at
15,000 rpm for 10 min, and the supernatant was collected. A peptide
stock solution was diluted into the supernatant to a final
concentration of 5 .mu.M for cF.PHI.R.sub.4 and Antp and 50 .mu.M
for peptides R.sub.9 and Tat and incubated at 37.degree. C. At
various time points (0-6 h), 200-.mu.L aliquots were withdrawn and
mixed with 50 .mu.L of 15% trichloroacetic acid and incubated at
4.degree. C. overnight. The final mixture was centrifuged at 15,000
rpm for 10 min in a microcentrifuge, and the supernatant was
analyzed by reversed-phase HPLC equipped with a Cis column
(Waters). The amount of remaining peptide (%) was determined by
integrating the area underneath the peptide peak (monitored at 214
nm) and compared with that of the control reaction (no serum).
[0245] Cytotoxicity Assay. MTT assays were performed to evaluate
cyclic peptide's cytotoxicity against several mammalian cell lines
(Mosmann, T. J. Immunol. Methods, 1983, 65, 55-63). One hundred
.mu.L of MCF-7, HEK293, H1299, H1650, A549 (1.times.10.sup.5
cells/mL) cells were placed in each well of a 96-well culture plate
and allowed to grow overnight. Varying concentrations of the
peptide (5 or 50 .mu.M) were added to the each well and the cells
were incubated at 37.degree. C. with 5% CO.sub.2 for 24 to 72 h.
Ten .mu.L of MTT stock solution was added into each well. Addition
of 10 .mu.L of the solution to the growth medium (no cell) was used
as a negative control. The plate was incubated at 37.degree. C. for
4 h. Then 100 .mu.L of SDS-HCl solubilizing buffer was added into
each well, and the resulting solution was mixed thoroughly. The
plate was incubated at 37.degree. C. for another 4 h. The
absorbance of the formazan product was measured at 570 nm using a
Molecular Devices Spectramax M5 plate reader. Each experiment was
performed in triplicates and the cells without any peptide added
were treated as control.
[0246] cF.PHI.R.sub.4Binds to Membrane Phospholipids. It was
previously observed that incubation of 1 .mu.M FITC-labeled cyclic
peptide cF.PHI.R.sub.4.sup.FITC with vesicles containing negatively
charged phospholipids (90% phosphatidylcholine (PC) and 10%
phosphatidylglycerol (PG)) resulted in quenching of the peptide
fluorescence, consistent with direct binding of cF.PHI.R.sub.4 to
phospholipids (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431).
To test the potential role of membrane binding during endocytic
uptake of CPPs, SUVs that mimic the outer membrane of mammalian
cells (45% PC, 20% phosphatidylethanolamine, 20% sphingomyelin, and
15% cholesterol) were prepared and tested for binding to
FITC-labeled cF.PHI.R.sub.4, R.sub.9, and Tat (each at 100 nM) by a
fluorescence polarization (FP) assay. cF.PHI.R.sub.4 bound to the
neutral SUVs with an EC.sub.50 value (lipid concentration at which
half of cF.PHI.R.sub.4.sup.FITC is bound) of 2.1 i 0.1 mM (FIG.
5A). R.sub.9 showed much weaker binding to the artificial membrane
(EC.sub.50>10 mM), whereas Tat did not bind at all. Next, the
CPPs were tested for binding to heparan sulfate, which was
previously proposed to be the primary binding target of cationic
CPPs (Nakase, I et al. Biochemistry, 2007, 46, 492-501; Rusnati, M
et al. J. Biol. Chem., 1999, 274, 28198-28205; Tyagi, M et al. J.
Biol. Chem., 2001, 276, 3254-3261; Ziegler, A and Seelig, J.
Biophys. J., 2004, 86, 254-263; Goncalves, E et al. Biochemistry,
2005, 44, 2692-2702; Ziegler, A. Adv. Drug Delivery Rev., 2008, 60,
580-597). R.sub.9 and Tat both bound to heparan sulfate with high
affinity, having EC.sub.50 values of 144 and 304 nM, respectively
(FIG. 5B). Under the same condition, cF.PHI.R.sub.4 showed no
detectable binding to heparan sulfate. These results are in
agreement with the previous observations that non-amphipathic
cationic CPPs (e.g., Tat and R.sub.9) bind tightly with cell
surface proteoglycans (e.g. heparan sulfate) but only weakly with
membrane lipids (Ziegler, A. Adv. Drug Delivery Rev., 2008, 60,
580-597). The insufficient number of positive charges of
cF.PHI.R.sub.4 is likely responsible for its lack of strong
electrostatic interaction with heparan sulfate. On the other hand,
the amphipathic nature and the more rigid cyclic structure of
cF.PHI.R.sub.4 should facilitate its binding to neutral lipid
membranes. These data, together with the inhibition pattern by
various endocytic inhibitors described above, suggest that
cF.PHI.R.sub.4 can bind directly to the plasma membrane
phospholipids and can be internalized by all of the endocytic
mechanisms in a piggyback manner.
[0247] Intracellular Delivery of Peptidyl Cargos. Since endocyclic
delivery by cF.PHI.R.sub.4 is limited to a heptapeptide or smaller
cargos (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431), in this
study the ability of cF.PHI.R.sub.4 to deliver cargos of varying
sizes and physicochemical properties attached to the Gln side chain
(FIG. 1B, exocyclic delivery) was tested. First, positively charged
(RRRRR), neutral (AAAAA), hydrophobic (FFFF), and negatively
charged peptides [DE(pCAP)LI] were covalently attached to
cF.PHI.R.sub.4. The first three peptides were labeled with
rhodamine B at a C-terminal lysine side chain (FIG. 2), and their
internalization into HEK293 cells was examined by live-cell
confocal microscopy. Cells incubated for 2 h with 5 .mu.M peptide
cF.PHI.R.sub.4-A.sub.5 (FIG. 6A) or cF.PHI.R.sub.4-R.sub.5 (FIG.
6B) showed evidence of both punctate and diffuse fluorescence, with
the latter distributed almost uniformly throughout the cell. In
contrast, the fluid phase endocytic marker dextran.sup.FITC
displayed predominantly punctate fluorescence, indicative of
endosomal localization. The diffuse rhodamine fluorescence suggests
that a fraction of the peptides reached the cytosol and nucleus of
the cells. Co-incubation of cells with cF.PHI.R.sub.4 (1 .mu.M) and
dextran.sup.Alexa488 increased the internalization of the endocytic
marker by 15% (FIG. 7), suggesting that cF.PHI.R.sub.4 can activate
endocytosis in cultured cells. cF.PHI.R.sub.4-F.sub.4 was not
tested due to its poor aqueous solubility.
[0248] Peptide cF.PHI.R.sub.4-DE(pCAP)LI (cF.PHI.R.sub.4-PCP; FIG.
2) was designed to test the ability of cF.PHI.R.sub.4 to deliver
negatively charged cargos as well as to compare the cytoplasmic
delivery efficiency of cF.PHI.R.sub.4 with those of other widely
used CPPs such as R.sub.9, Tat, and penetratin (Antp). Thus,
untagged PCP [Ac-DE(pCap)LI-NH2] and PCP conjugated to R.sub.9
(R.sub.9-PCP), Tat (Tat-PCP), or Antp (Antp-PCP) were also
prepared. Note that cF.PHI.R.sub.4-PCP carries a net charge of zero
at physiological pH. pCAP is non-fluorescent, but upon entering the
cell interior, should be rapidly dephosphorylated by endogenous
protein tyrosine phosphatases (PTPs) to produce a fluorescent
product, coumaryl aminopropionic acid (CAP, excitation 355 nm;
emission 450 nm) (Mitra, S and Barrios, A M. Bioorg. Med. Chem.
Lett., 2005, 15, 5124-5145; Stanford, S M et al. Proc. Natl. Acad.
Sci. U.S.A., 2012, 109, 13972-13977). When assayed against a PTP
panel in vitro, all four CPP-PCP conjugates were efficiently
dephosphorylated (Table 8). This assay detects only the CPP-cargo
inside the cytoplasm and nucleus, where the catalytic domains of
all known mammalian PTPs are localized (Alonso, A et al. Cell,
2004, 117, 699-711). Further, CAP is fluorescent only in its
deprotonated state (pKa=7.8); even if some dephosphorylation occurs
inside the endosome (pH 6.5-4.5) or lysosome (pH 4.5), it would
contribute little to the total fluorescence (FIG. 8). Treatment of
HEK293 cells with 5 .mu.M cF.PHI.R.sub.4-PCP for 60 min resulted in
diffuse blue fluorescence throughout the cell, suggesting that
cF.PHI.R.sub.4-PCP reached the cell interior, whereas the untagged
PCP failed to enter cells under the same condition (FIG. 9A). When
HEK293 cells were pretreated with the PTP inhibitor sodium
pervanadate for 1 h prior to incubation with cF.PHI.R.sub.4-PCP (5
.mu.M), the CAP fluorescence in the cells diminished to background
levels. HEK293 cells treated with R.sub.9-PCP, Antp-PCP, or Tat-PCP
under identical conditions showed weak fluorescence, consistent
with the poor ability of these peptides to access the cell interior
(FIG. 9A). To quantify the relative intracellular PCP delivery
efficiency, HeLa cells were treated with each peptide and analyzed
by fluorescence activated cell sorting (FIG. 9B).
cF.PHI.R.sub.4-PCP was most efficiently internalized by the HeLa
cells, with a mean fluorescence intensity (MFI) of 3510 arbitrary
units (AU), whereas R.sub.9-PCP, Antp-PCP, Tat-PCP, and untagged
PCP produced MFI values of 960, 400, 290, and 30 AU, respectively
(FIG. 9C). Again, when cells were treated with cF.PHI.R.sub.4-PCP
in the presence of sodium pervanadate, the amount of CAP
fluorescence was reduced to near background levels (70 AU). Thus,
cF.PHI.R.sub.4 is capable of delivering peptidyl cargos of varying
physicochemical properties into the cytoplasm with efficiencies
3.7-12-fold higher than R.sub.9, Antp, and Tat.
TABLE-US-00008 TABLE 8 Kinetic Activities (k.sub.cat/K.sub.M,
M.sup.-1 s.sup.-1) of Recombinant PTPs against pCAP-Containing
Peptides.sup.a PTP cF.PHI.R.sub.4-PCP Tat-PCP R.sub.9-PCP Antp-PCP
PTP1B 37100 13800 14700 17400 TCPTP 2780 560 457 970 SHP2 7400 2290
248 2210 CD45 35100 21800 2940 22300 VHR 2460 1460 6240 2030
.sup.ak.sub.cat/K.sub.M was measured as previously described (Ren,
L et al. Biochemistry, 2011, 50, 2339-2356).
[0249] Intracellular Delivery of Cyclic Peptides. In recent years,
there has been much interest in cyclic peptides as therapeutic
agents and biomedical research tools (Driggers, E M et al. Nat.
Rev. Drug Discov., 2008, 7, 608-624; Marsault, E and Peterson, M L.
J Med. Chem., 2011, 54, 1961-2004). For example, cyclic peptides
are effective for inhibition of protein-protein interactions (Lian,
W et al. J. Am. Chem. Soc., 2013, 135, 11990-11995; Liu, T et al.
ACS Comb. Sci., 2011, 13, 537-546; Dewan, V et al. ACS Chem. Biol.,
2012, 7, 761-769; Wu, X et al. Med. Chem. Commun., 2013, 4,
378-382), which are challenging targets for conventional small
molecules. A major obstacle in developing cyclic peptide
therapeutics is that they are generally impermeable to the cell
membrane (Kwon, Y U and Kodadek, T. Chem. Biol., 2007, 14, 671-677;
Rezai, T et al. J Am. Chem. Soc., 2006, 128, 2510-2511; Chatterjee,
J et al. Acc. Chem. Res., 2008, 41, 1331-1342). The attempt to
deliver cyclic peptides by cF.PHI.R.sub.4 by the endocyclic method
had only limited success; increase in the cargo size from 1 to 7
residues led to progressively poorer cellular uptake, likely
because the larger, more flexible rings bind more poorly to the
cell membrane (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431).
To overcome this limitation, a bicyclic peptide system was
explored, in which one ring contains a CPP motif (e.g.,
F.PHI.R.sub.4) while the other ring consists of peptide sequences
specific for the desired targets (FIG. 1C). The bicyclic system
should in principle be able to accommodate cargos of any size,
because the cargo does not change the structure of the CPP ring and
should have less impact on its delivery efficiency. The additional
rigidity of a bicyclic structure should also improve its metabolic
stability as well as the target-binding affinity and specificity.
The bicyclic peptides were readily synthesized by forming three
amide bonds between a trimesoyl scaffold and three amino groups on
the corresponding linear peptide (i.e., the N-terminal amine, the
side chain of a C-terminal diaminopropionic acid (Dap), and the
side chain of a lysine (or ornithine, Dap) imbedded in between the
CPP and target-binding motifs) (Lian, W et al. J Am. Chem. Soc.,
2013, 135, 11990-11995). To test the validity of this approach,
F.PHI.R.sub.4 was chosen in the C-terminal ring as the CPP moiety
and peptides of different lengths and charges (AAAAA, AAAAAAA,
RARAR, or DADAD) were chosen as cargo (Table 8, compounds 13-16).
For comparison, two monocyclic peptides containing F.PHI.R.sub.4 as
transporter and peptides A.sub.5 and A.sub.7 as cargos (Table 8,
compounds 17 and 18) were also prepared. All of the peptides were
labeled at a C-terminal lysine side chain with rhodamine B (FIG. 2)
and their internalization into HEK293 cells was examined by
live-cell confocal microscopy. Treatment of cells with 5 .mu.M
peptide for 2 h resulted in efficient internalization of all six
peptides (FIG. 10), although FACS analysis indicated that the
uptake of bicyclo(F.PHI.R.sub.4-A.sub.5).sup.Rho was .about.3-fold
more efficient than the corresponding monocyclic peptide (compound
17). The intracellular distribution of the internalized peptides
was quite different between the bicyclic and monocyclic peptides.
While the four bicyclic peptides showed evidence for their presence
in both the cytoplasm/nucleus (as indicated by the diffuse
rhodamine fluorescence) and the endosomes (as indicated by the
fluorescence puncta), the monocyclic peptides exhibited
predominantly punctate fluorescence that overlapped with that of
the endocytic marker dextran.sup.FITC. In all cases, the endocytic
marker displayed only punctate fluorescence, indicating that the
endosomes were intact in the cells treated with the peptides. These
results indicate that the increased structural rigidity of the
bicyclic peptides facilitates both the initial uptake by
endocytosis and endosomal release, presumably because of their
improved binding to the plasma and endosomal membranes. The
bicyclic system may provide a general strategy for intracellular
delivery of cyclic and bicyclic peptides.
[0250] Intracellular Delivery of Protein Cargos. To test whether
cF.PHI.R.sub.4 is capable of transporting full-length proteins into
mammalian cells, GFP was attached to the N-terminus of
cF.PHI.R.sub.4 through a disulfide bond (FIG. 11A and FIG. 3). GFP
was chosen because of its intrinsic fluorescence. The disulfide
exchange reaction is highly specific, efficient, and reversible;
upon entering the cytoplasm, the CPP-S-S-protein conjugate can be
rapidly reduced to release the native protein. Although
cF.PHI.R.sub.4 can be directly attached to a native or engineered
surface cysteine residue(s) on a cargo protein, a GFP variant
containing a 12-amino acid ybbR tag at its N-terminus was used and
phosphopantetheinyl transferase Sfp was used to enzymatically
attach cF.PHI.R.sub.4 to the ybbR tag (Yin, J et al. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 15815-15820). This permitted the
attachment of a single cF.PHI.R.sub.4 unit to GFP in a
site-specific manner. For comparison, a Tat-S-S-GFP conjugate was
generated in the same manner. Incubation of HEK293 cells in the
presence of 1 .mu.M cF.PHI.R.sub.4-S-S-GFP resulted in accumulation
of green fluorescence inside the cells (FIG. 11B). The fluorescence
signal was diffuse and present throughout the entire cell volume,
but with higher concentrations in the nucleus. Some of the cells
contained small spots of intense green fluorescence (indicated by
arrows in FIG. 11B), which may represent endosomally sequestered
cF.PHI.R.sub.4-S-S-GFP or aggregated GFP inside the cell. The
untagged GFP was unable to enter cells, whereas Tat-S-S-GFP entered
cells less efficiently than cF.PHI.R.sub.4-S-S-GFP (FIG. 11B); FACS
analysis of HaLa cells treated with 1 .mu.M protein revealed a
5.5-fold higher total intracellular fluorescence for the latter.
The fluorescence puncta in the cell periphery as well as lack of
any detectable fluorescence in the nuclear region of Tat-S-S-GFP
treated cells indicate that Tat-S-S-GFP is mostly entrapped in the
endosomes, in agreement with previous reports (Kaplan, I M et al.
J. Controlled Release, 2005, 102, 247-253). Thus, with a protein as
cargo, cF.PHI.R.sub.4 also has higher efficiency than Tat with
regard to both initial uptake and endosomal escape.
[0251] To demonstrate the generality of cF.PHI.R.sub.4 for protein
delivery, a functional enzyme, the catalytic domain of PTP1B (amino
acids 1-321), was chosen to be delivered into the cell interior. To
show that a non-cleavable linkage is also compatible with the
delivery method, cF.PHI.R.sub.4 was conjugated to ybbR-tagged PTP1B
via a thioether bond (cF.PHI.R.sub.4-PTP1B) (FIG. 4). In vitro
assay using p-nitrophenyl phosphate as substrate showed that
addition of the cF.PHI.R.sub.4 tag does not affect the catalytic
activity of PTP1B (Table 9). NIH 3T3 cells were incubated for 2 h
in the presence of untagged PTP1B or cF.PHI.R.sub.4-PTP1B and their
global pY protein levels were analyzed by anti-pY western blotting
(FIG. 12A). Treatment of the cells with cF.PHI.R.sub.4-PTP1B, but
not untagged PTP1B, resulted in concentration-dependent decrease in
pY levels of most, but not all, proteins. The total cellular
protein levels, as detected by Coomassie blue staining, were
unchanged (FIG. 12B), indicating that the observed decrease in pY
levels was due to dephosphorylation of the pY proteins by
cF.PHI.R.sub.4-PTP1B and/or secondary effects caused by the
introduction of cF.PHI.R.sub.4-PTP1B (e.g., inactivation of
cellular protein tyrosine kinases). Interestingly, different
proteins exhibited varying dephosphorylation kinetics. Several
proteins in the 150-200 kD range were completely dephosphorylated
upon the addition of 62 nM cF.PHI.R.sub.4-PTP1B, whereas proteins
of .about.80 kD remained phosphorylated at 500 nM
cF.PHI.R.sub.4-PTP1B. The changes in the pY pattern are consistent
with the broad substrate specificity of PTP1B (Ren, L et al.
Biochemistry, 2011, 50, 2339-2356) and very similar to that caused
by overexpression of PTP1B inside the cytosol of mammalian cells
(LaMontagne Jr., K R et al. Proc. Nat. Acad. Sci. U.S.A., 1998, 95,
14094-14099). These results indicate that cF.PHI.R.sub.4 can
deliver PTP1B into the interior of NIH 3T3 cells in the
catalytically active form and to sufficient levels to perturb the
cell signaling process. cF.PHI.R.sub.4 thus provides a tool for
introducing other functional proteins, especially proteins that
cannot be genetically expressed (e.g., toxic and chemically
modified proteins), into mammalian cells in order to study their
cellular functions.
TABLE-US-00009 TABLE 9 Kinetic Activities (k.sub.cat/K.sub.M,
M.sup.-1 s.sup.-1) of PTP1B and cF.PHI.R.sub.4-PTP1B against
pNPP.sup.a enzyme k.sub.cat/K.sub.M (M.sup.-1 s.sup.-1) PTP1B 1340
cF.PHI.R.sub.4-PTP1B 1600 .sup.apNPP = p-nitrophenyl phosphate;
k.sub.cat/K.sub.M was measured as previously described (Ren, L et
al. Biochemistry, 2011, 50, 2339-2356).
[0252] Stability and Cytotoxicity of cF.PHI.R.sub.4. The relative
stability of cF.PHI.R.sub.4, R.sub.9, Tat, and Antp (Table 8,
compounds 19-22) against proteolytic degradation was determined by
incubating the CPPs in 25% human serum at 37.degree. C. and
following the disappearance of the full-length peptides by
reversed-phase HPLC. The cationic tryptophan-containing peptide,
Antp, was least stable among the four CPPs; it was degraded at a
half-life of <20 min and was completely digested after 2 h (FIG.
13A). R.sub.9 and Tat were slightly more stable than Antp, having
half-lives of .about.30 min. In contrast, cF.PHI.R.sub.4 was
remarkably stable against serum proteases. There was less than 10%
degradation after 6 h of incubation; after 24 h of incubation in
the serum, >70% of cF.PHI.R.sub.4 remained intact. Numerous
other studies have also demonstrated that cyclization of peptides
increases their proteolytic stabilities (Nguyen, L T et al. PLoS
One, 2010, 5, e12684). The potential cytotoxicity of cF.PHI.R.sub.4
was assessed by MTT assays with five different human cell lines
(HEK293, MCF-7, A549, H1650, and H1299). After 24 or 48 h of
incubation with up to 50 .mu.M cF.PHI.R.sub.4, there was no
significant growth inhibition for any of the cell lines (FIG. 13B
and FIG. 14). After 72 h, a slight growth inhibition (up to 20%)
was observed at 50 .mu.M (FIG. 14). Thus, cF.PHI.R.sub.4 is
relatively nontoxic to mammalian cells.
[0253] In this study, it was demonstrated that cF.PHI.R.sub.4 can
be effective for exocyclic delivery of small-molecule, peptide, and
protein cargos into the cytoplasm and nucleus of mammalian cells.
By using a pCAP-containing peptide as cargo/reporter, it was shown
that cF.PHI.R.sub.4 can be 3.7-12-fold more efficient than R.sub.9,
Tat, and Antp for cytoplasmic cargo delivery, making cF.PHI.R.sub.4
one of the most active CPPs known to date. Although modification of
polybasic CPPs such as addition of hydrophobic acyl groups has
previously been reported to enhance cellular uptake by a similar
magnitude (Pham, W et al. Chembiochem, 2004, 5, 1148-1151), these
previous studies have not established whether the enhanced uptake
translates into a similar increase in the cytoplasmic CPP
concentration. The pCAP-based reporter system described herein can
provide a simple, robust method to quantitatively assess the
cytoplasmic delivery efficiency of other CPPs. Several lines of
evidence indicate that cF.PHI.R.sub.4 can enter cells through
multiple endocytic mechanisms, including its failure to enter cells
at 4.degree. C. or in the presence of sodium azide, partial overlap
between the fluorescence puncta of cF.PHI.R.sub.4.sup.Rho and the
fluid phase endocytic marker dextran.sup.FITC, colocalization of
cF.PHI.R.sub.4.sup.Rho and endosomal proteins Rab5 and Rab7, and
decreased cF.PHI.R.sub.4.sup.Dex uptake upon administration of
endocytic inhibitors. The minimal effect of the PI3K inhibitor
wortmannin and the Rab5 Q79L mutation on the cytoplasmic delivery
of cF.PHI.R.sub.4, in addition to the strong colocalization
observed between cF.PHI.R.sub.4 and Rab5.sup.+ endosomes, suggest
that cF.PHI.R.sub.4 can escape from early endosomes (FIG. 15). In
comparison, Tat has been demonstrated to enter cells through
endocytosis and release from late endosomes, while R.sub.9 escapes
endosomes prior to Rab7 recruitment (Appelbaum, J S et al. Chem.
Biol., 2012, 19, 819-830). Early endosomal release can offer
advantages, especially for peptide and protein cargos, since it can
minimize cargo degradation by late endosomal and lysosomal
proteases and denaturation caused by acidification during endosomal
maturation. Indeed, both GFP and PTP1B delivered into the cytoplasm
by cF.PHI.R.sub.4 were in their folded, active forms, as evidenced
by the green fluorescence and the ability to dephosphorylate
intracellular pY proteins, respectively. Additionally, due to its
more rigid structure, cF.PHI.R.sub.4 can be more stable against
proteolytic degradation than linear peptides, and due to its
smaller size, cF.PHI.R.sub.4 can be less expensive to synthesize
and potentially less likely to interfere with the cargo function.
These properties can make cF.PHI.R.sub.4 a useful transporter for
cytosolic delivery of small-molecule to protein cargos. Direct
protein delivery can provide a useful research tool, e.g., for
studying the cellular function of a protein, as it can offer
improved temporal control over DNA transfection and subsequent gene
expression and can allow delivery of chemically modified proteins
and proteins whose overexpression can cause toxicity. The ability
of cF.PHI.R.sub.4 to escape from early endosomes and its simple
structure can also provide an excellent system for elucidating the
mechanism of endosomal escape and the factors that influence the
escape efficiency.
Example 2
[0254] Cyclization of peptide ligands can be effective for
improving their stability against proteolytic degradation and in
some cases their cell permeability. However, this strategy is not
compatible with proteins that recognize peptide ligands in the
extended conformations (e.g., .beta.-strand and .alpha.-helix). In
this work, a general strategy for intracellular delivery of linear
peptide ligands was developed, by fusing them with an amphipathic
sequence motif (e.g., RRRR.PHI.F, where .PHI. is L-naphthylalanine)
and cyclizing the resulting conjugate through a disulfide bond. The
cyclized peptides can have enhanced proteolytic stability and
membrane permeability; upon entering the cytoplasm/nucleus of a
cell, the disulfide bond can be cleaved by the reducing
intracellular environment to release the linear, biologically
active peptide. This strategy was applied to generate cell
permeable peptides as caspase substrates and inhibitors against the
CAL PDZ domain for potential treatment of cystic fibrosis.
[0255] The applicability of linear peptides as drugs is often
limited by their susceptibility to proteolytic cleavage and poor
membrane permeability. Cyclization of peptides can be effective for
improving their proteolytic stability (Nguyen, L T et al. PLoS One,
2010, 5, e12684). Moreover, it was recently reported that
cyclization of certain amphipathic peptides (e.g., F.PHI.RRRR,
where .PHI. is L-2-naphthylalanine) can render them cell permeable
through an active transport mechanism (Qian, Z et al. ACS Chem.
Biol. 2013, 8, 423). Biologically active cyclic peptides can be
delivered into the cytoplasm and nucleus of mammalian cells by
incorporating into them these short sequence motifs (Qian, Z et al.
ACS Chem. Biol. 2013, 8, 423). However, in many circumstances,
binding to a molecular target (e.g., PDZ (Doyle, D A et al. Cell
1996, 85, 1067; Morais Cabral, J H et al., Nature 1996, 382, 649)
and BIR domains (Wu, G et al. Nature 2000, 408, 1008)) can require
that the peptidyl ligand exist in its extended conformation (e.g.,
.alpha.-helix and .beta.-strand) and cyclization may interfere with
target binding. Herein, a potentially general strategy for
delivering linear peptide ligands into mammalian cells through
reversible, disulfide bond-mediated cyclization is examined. When
present in the oxidizing extracellular environment, the peptides
can exist as macrocycles, which can have enhanced stability against
proteolysis and cell permeability. Upon entering the cell (i.e.,
cytoplasm and/or nucleus), the disulfide bond can be reduced by the
intracellular thiols to produce the linear, biologically active
peptides (FIG. 16) (Cascales, L et al. J Biol Chem. 2011, 286,
36932; Jha, D et al. Bioconj Chem. 2011, 22, 319).
[0256] Materials. Reagents for peptide synthesis were purchased
from Advanced ChemTech (Louisville, Ky.), NovaBiochem (La Jolla,
Calif.), or Anaspec (San Jose, Calif.). Rink resin LS (100-200
mesh, 0.2 mmol/g) was purchased from Advanced ChemTech.
Dextrane.sup.Rho, trypsin and .alpha.-chymotrypsin were purchased
from Sigma-Aldrich (St. Louis, Mo.). Cell culture media, fetal
bovine serum, penicillin-streptomycin, 0.25% trypsin-EDTA, and DPBS
were purchased from Invitrogen (Carlsbad, Calif.). Nuclear staining
dye DRAQ5.TM. was purchased from Thermo Scientific (Rockford,
Ill.). Caspase-3, Human, recombinant protein was purchased from EMD
Chemicals (San Diego, Calif.).
[0257] Peptide Synthesis. Most peptides were synthesized on Rink
Resin LS (0.2 mmol/g) using standard Fmoc chemistry. The typical
coupling reaction contained 5 equiv of Fmoc-amino acid, 5 equiv of
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and 10 equiv of diisopropylethylamine
(DIPEA) and was allowed to proceed with mixing for 75 min. The
peptides were deprotected and released from the resin by treatment
with 92.5:2.5:2.5:2.5 (v/v) trifluoroacetic acid
(TFA)/water/phenol/triisopropylsilane (TIPS) for 2 h. The peptides
were triturated with cold ethyl ether (3.times.) and purified by
reversed-phase HPLC equipped with a Cis column. Peptide labeling
with fluorescein isothiocyanate (FITC) was performed by dissolving
the purified peptides (.about.1 mg each) in 300 .mu.L of 1:1:1
DMSO/DMF/150 mM sodium bicarbonate (pH 8.5) and mixing with 10
.mu.L of FITC in DMSO (100 mg/mL). After 20 min at room
temperature, the reaction mixture was subjected to reversed-phase
HPLC on a Cis column to isolate the FITC-labeled peptide.
[0258] To generate disulfide bond mediated cyclic peptides, the
3,3'-dithiodipropionic acid (10 equiv) were coupled on the
N-terminal using 10 equiv N,N'-Diisopropylcarbodiimide (DIC) and
0.1 equiv 4-(dimethylamino)pyridine (DMAP) in anhydrous DCM for 2 h
after the removal of the N-terminal Fmoc protection group by
treatment with 20% (v/v) piperidine in DMF. The resin then was
incubated in 20% .beta.-mercaptoethanol in DMF for 2 h twice to
expose the free thiol. Triturated crude linear peptides were
incubated in 5% DMSO in pH 7.4 PBS buffer overnight (Tam, J P et
al. J. Am. Chem. Soc. 1991, 113, 6657), followed by trituration and
HPLC purification as described above (Tam, J P et al. J Am. Chem.
Soc. 1991, 113, 6657).
[0259] To produce thioether mediated cyclic peptides,
4-bromobutyric acid (10 equiv) was coupled on the N-terminal using
10 equiv DIC and 0.1 equiv DMAP in anhydrous DCM for 2 h after the
removal of the N-terminal Fmoc protection group by treatment with
20% (v/v) piperidine in DMF. The 4-methoxytrityl (Mmt) protection
group on the L-cysteine side chain was selectively removed using 1%
trifluoroacetic acid (TFA) in DCM. Thioether formation was
conducted by incubating the resin in 1% DIPEA in DMF under nitrogen
protection overnight. The cyclized peptide was then triturated and
purified as described above (Roberts, K D et al. Tetrahedron Lett.
1998, 39, 8357).
[0260] Fmoc-Asp(Wang-resin)-AMC (AMC=7-amino-4-methylcoumarin)
(NovaBiochem) was used as a solid support to synthesize fluorogenic
caspase substrates. Standard Fmoc chemistry was employed to
synthesize the peptide on solid phase. These peptides were released
from the resin by the treatment with 95:2.5:2.5 (v/v)
TFA/phenol/water for 2 h (Maly, D J et al. J Org. Chem. 2002, 67,
910).
[0261] Cell Culture. HeLa cells were maintained in medium
consisting of DMEM, 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin. Jurkat cells were maintained in medium
consisting of RPMI-1640, 10% FBS and 1% penicillin/streptomycin.
The bronchial epithelial CFBE cell line, homozygous for the
.DELTA.F508-CFTR mutation, was maintained in DMEM containing
L-glutamine supplemented with 10% FBS and 1%
penicillin/streptomycin. The tissue culture plates were coated
using human fibronectin (1 mg/ml), collagen I bovine (3 mg/ml), and
bovine serum albumin (1 mg/ml) Cells were cultured in a humidified
incubator at 37.degree. C. with 5% CO.sub.2.
[0262] Confocal Microscopy. To detect peptide internalization, 1 mL
of HeLa cell suspension (5.times.10.sup.4 cells) was seeded in a 35
mm glass-bottomed microwell dish (MatTek) and cultured overnight.
Cells were gently washed with DPBS twice and treated with FITC
labeled peptides (5 .mu.M) and dextran.sup.Rho (0.5 mg mL.sup.-1)
in phenol-red free DMEM containing 1% serum at 37.degree. C. for 1
h in the presence of 5% CO.sub.2. After removal of the medium, the
cells were gently washed with DPBS twice and incubated with 5 .mu.M
DRAQ5 in DPBS for 10 min. The cells were again washed with DPBS
twice and imaged on a Visitech Infinity 3 Hawk 2D-array live cell
imaging confocal microscope. Images were captured under the same
parameters and adjusted under the same setting using MetaMorph
(Molecular Devices).
[0263] Flow Cytometry. HeLa cells were cultured in six-well plates
(5.times.10.sup.5 cells per well) for 24 h. On the day of
experiment, the cells were incubated with 5 .mu.M FITC labeled
peptide in clear DMEM with 1% FBS at 37.degree. C. for 2 h. The
cells were washed with DPBS, detached from plate with 0.25%
trypsin, diluted into clear DMEM containing 10% FBS, pelleted at
250 g for 5 min, washed once with DPBS and resuspended in DPBS
containing 1% bovine serum albumin, and analyzed on a BD FACS Aria
flow cytometer. Data were analyzed with Flowjo software (Tree
Star).
[0264] To quantify the delivery efficiencies of PCP-conjugated
peptides, HeLa cells were cultured in six-well plates
(5.times.10.sup.5 cells per well) for 24 h. On the day of
experiment, the cells were incubated with 5 .mu.M pCAP-containing
peptide in clear DMEM with 1% FBS at 37.degree. C. for 2 h. The
cells were washed with DPBS containing 1 mM sodium pervanadate,
detached from plate with 0.25% trypsin, suspended in DPBS
containing 1% bovine serum albumin, and analyzed on a BD FACS Aria
flow cytometer with excitation at 355 nm.
[0265] Peptide Proteolysis Stability Assay. The stability tests
were carried out by slightly modifying a previously reported
procedure (Frackenpohl, J et al. Chembiochem 2001, 2, 445). 24
.mu.L of 1.5 mM peptide solution was incubated at 37.degree. C.
with 30 .mu.L 50 .mu.M of .alpha.-chymotrypsin and 30 .mu.L 50
.mu.M of trypsin in 200 .mu.L of working buffer (50 mM Tris-HCl, pH
8.0, NaCl (100 mM), CaCl.sub.2) (10 mM)). At various time points
(0-12 h), 40 .mu.L aliquots were withdrawn and mixed with 40 .mu.L
of 15% trichloroacetic acid and incubated at 4.degree. C.
overnight. The final mixture was centrifuged at 15,000 rpm for 10
min in a microcentrifuge, and the supernatant was analyzed by
reversed-phase HPLC equipped with a Cis column (Waters). The amount
of remaining peptide (%) was determined by integrating the area
underneath the peptide peak (monitored at 214 nm) and compared with
that of control reaction (no proteases).
[0266] In Cellulo Fluorimetric Assay. 100 .mu.L of Jurkat cell
suspension (5.times.10.sup.5 cells/mL) was seeded in 96-well plate
one hour prior to the experiment. Ten .mu.L of staurosporine stock
solution (10 .mu.M) was added into half of the wells to induce
apoptosis, while 10 .mu.L of media was added to the other wells.
After 1 h incubation, caspase-3 fluorogenic substrates were added
to the cells to a final concentration of 5 .mu.M. The fluorescence
of the released coumarin was measured on the Spectramax M5 plate
reader with excitation and emission wavelengths at 360 and 440 nm
at various times points (0-6 h). The fluorescence unit (FU)
increases between induced and uninduced cells were plotted against
the time to present caspase-3 activities measured using various
fluorogenic substrates in living cell in real-time. Three
independent sets of experiments, each performed in triplicate, were
conducted.
[0267] In Vitro Fluorimetric Assay. 0.5 .mu.L (100 U/.mu.L)
caspase-3 enzyme was first incubated with 90 .mu.L of reaction
buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT) for 30 min in
96-well plate. Fluorogenic substrates (10 .mu.L, 100 .mu.M) were
mixed into the above solutions to start the reactions, and the
plate was measured on a Spectramax M5 plate reader (Ex=360 nm,
Em=440 nm) (Molecular Devices). Fluorescence units (FU) increase at
one-minute intervals was correlated to the release of Amc due to
protease activity. The AFU/min was calculated from the linear
portion of the reaction curve. Reported values are averages of
three trials with the standard deviation indicated.
[0268] Fluorescence anisotropy. The full fluorescence anisotropy
(FA) titration experiment was performed by incubating 100 nM
fluorophore-labeled peptidyl ligands with varying concentrations
(0-6 .mu.M) of CAL-PDZ (Cushing, P R et al. Biochemistry 2008, 47,
10084) in FA buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM
glutathione, 0.1% (w/v) bovine serum albumin) for 2 h at room
temperature. The FA values were measured on a Molecular Devices
Spectramax M5 spectrofluorimeter, with excitation and emission
wavelengths at 485 nm and 525 nm, respectively. Equilibrium
dissociation constants (K.sub.D) were determined by plotting the
fluorescence anisotropy values as a function of CAL-PDZ
concentration. The titration curves were fitted to the following
equation, which assumes a 1:1 binding stoichiometry
Y = ( A min + ( A max .times. Q b Q f - A min ) ( ( L + x + K D ) -
( ( L + x + K D ) 2 - 4 .times. L .times. x ) 2 .times. L ) ) ( 1 +
( Q b Q f - 1 ) .times. ( ( L + x + K D ) - ( ( L + x + K D ) 2 - 4
.times. L .times. x ) 2 .times. L ) ) ##EQU00001##
[0269] where Y is the measured anisotropy at a given CAL-PDZ
concentration x; L is the bicyclic peptide concentration;
Q.sub.b/Q.sub.f is the correction fact for dye-protein interaction;
A.sub.max is the maximum anisotropy when all the peptides are bound
to CAP-PDZ, while A.sub.min is the minimum anisotropy when all the
peptides are free.
[0270] Immunofluorescent Staining. Briefly, the bronchial
epithelial CFBE cells, homozygous for the DF508-CFTR mutation, were
treated with 10 mM Corr-4a in the presence and absence of 50 .mu.M
unlabeled peptide 8. After the treatments, cells were fixed in cold
methanol for 20 min. The slides were then incubated in 1% BSA/PBS
for 10 min, followed by incubation at 37.degree. C. for 1 h with
mouse anti-human monoclonal CFTR antibody (R&D Systems).
Thereafter, the slides were incubated at 37.degree. C. for 45 min
with Alexa Fluor.RTM. 488-conjugated anti-mouse IgG2a secondary
antibody. Cells were visualized on a Leica TCS SP2 AOBS confocal
laser scanning microscope. All measurements were conducted in a
double-blinded manner by two independent investigators.
[0271] SPQ intracellular chloride concentration assay. A SPQ
(6-Methoxy-N-(3-sulfopropyl)quinolinium) assay was utilized to
estimate the transport activity of .DELTA.F508-CFTR activity in
CFBE cells, as the fluorescence of SPQ is negatively correlated
with increasing concentration of intracellular chloride (Illsley, N
P and Verkman, A S. Biochemistry 1987, 26, 1215). CFBE cells were
grown on 96-well plate, which was pre-coated with 1 mg/ml human
fibronectin, 3 mg/ml collagen I bovine, and 1 mg/ml bovine serum
albumin, using DMEM media supplemented with L-glutamine and 10%
FBS. Cells were first treated in the presence or absence of 20
.mu.M CFTR corrector VX809 (Van Goor, F et al. Proc. Natl. Acad.
Sci. U.S.A. 2011, 108, 18843) for 24 h and 50 .mu.M CAL-PDZ domain
inhibitors for 1 h. Cells were then loaded with SPQ using hypotonic
shock at 37.degree. C. for 15 min with 10 mM SPQ containing 1:1
(v/v) Opti-MEM/water solution. The cells were then washed and
incubated twice for 10 min with fluorescence quenching NaI buffer
(130 mM NaI, 5 mM KNO.sub.3, 2.5 mM Ca(NO.sub.3).sub.2, 2.5 mM
Mg(NO.sub.3).sub.2, 10 mM D-glucose, 10 mM N-(2-hydroxyethyl)
piperazine-N'-(2-ethanesulfonic) acid (HEPES, pH 7.4)).
Subsequently, the cells were switched to a dequenching isotonic
NaNO.sub.3 buffer (identical to NaI buffer except that 130 mM NaI
was replaced with 130 mM NaNO.sub.3) with a CFTR activation
cocktail (10 .mu.M forskolin and 50 .mu.M genistein). Fluorescence
non-specific to CFTR-mediated iodide efflux was measured by
incubating the cells with the activation cocktail and the CFTR
specific inhibitor GlyH101 (10 .mu.M). The effects of CAL-PDZ
inhibitors were evaluated by the fluorescence increasing rate above
the basal level. The fluorescence of dequenched SPQ was measured
using the plate reader VICTOR X3 (Perkin Elmer) with excitation
wavelength at 350 nm and DAPI emission filter. The data was
presented as mean standard deviation from at least three individual
experiments.
[0272] A homodectic amphipathic cyclic peptide, cyclo(F.PHI.RRRRQ)
(cF.PHI.R.sub.4), has been reported as a highly active
cell-penetrating peptide (CPP) which can enter the cytoplasm of
mammalian cells through endocytosis and endosomal escape (Qian, Z
et al. ACS Chem. Biol. 2013, 8, 423). To test the validity of the
reversible cyclization strategy, a
N-3-mercaptopropionyl-F.PHI.RRRRCK-NH2 peptide was synthesized and
then cyclized by forming an intramolecular disulfide bond (FIG. 17;
Table 10, peptide 1). A linear peptide of the same sequence (Table
10, peptide 2) was also synthesized by replacing the N-terminal
3-mercaptopropionyl group with a butyryl group and the C-terminal
cysteine with 2-aminobutyric acid (Abu or U). Both peptides were
labeled at a C-terminal lysine residue with fluorescein
isothiocyanate (FITC) and their cellular uptake was assessed by
live-cell confocal microscopy and flow cytometry. HeLa cells
treated with the cyclic peptide (5 .mu.M) showed strong, diffuse
green fluorescence throughout the entire cell volume, whereas the
endocytosis marker, rhodamine-labeled dextran (dextran.sup.Rho),
exhibited only punctate fluorescence in the cytoplasmic region
(FIG. 18A). The nearly uniform distribution of FITC fluorescence in
both cytoplasmic and nuclear regions suggests that the cyclic
peptide was efficiently internalized by HeLa cells and like the
parent cyclic peptide, cF.PHI.R.sub.4, was able to efficiently
escape from the endosome. In contrast, cells treated with the
linear control peptide showed much weaker intracellular
fluorescence under the same imaging condition. Quantitation of the
total intracellular fluorescence by fluorescence-activated cell
sorting (FACS) gave mean fluorescence intensity (MFI) of 27,100,
5530, and 1200 arbitrary units (AU), for cells treated with the
disulfide cyclized peptide, linear peptide, and FITC alone,
respectively (FIG. 18B). A highly negatively charged pentapeptide,
Asp-Glu-pCAP-Leu-Ile (PCP, where pCAP is phosphocoumaryl
aminopropionic acid), was also used as cargo and attached to
peptides 1 and 2 through a polyethyleneglycol linker (FIG. 17).
pCAP is non-fluorescent but, when delivered into the mammalian
cytoplasm, undergoes rapid dephosphorylation to generate a
fluorescent product, coumaryl aminopropionic acid (CAP) (Stanford,
S M et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 13972). The
pCAP assay therefore provides a quantitative assessment of the
cytoplasmic/nuclear concentrations of different CPPs (Qian, Z et
al. ACS Chem. Biol. 2013, 8, 423). FACS analysis of HeLa cells
treated with 5 .mu.M peptide 1-PCP and peptide 2-PCP gave MFI
values of 3020 and 700, respectively (FIG. 19). Thus, the above
results indicate that cyclization of F.PHI.RRRR through a disulfide
bond can have a similar effect to the N-to-C cyclization and can
increase its cellular uptake efficiency by .about.5-fold (Qian, Z
et al. ACS Chem. Biol. 2013, 8, 423). In addition, cyclization by
disulfide bond formation can enhance the proteolytic resistance of
the peptide. Incubation of peptide 1 with a protease cocktail for
12 h resulted in <50% degradation, whereas the linear peptide 2
was degraded with a half-life of .about.20 min under the same
condition (FIG. 20).
TABLE-US-00010 TABLE 10 Sequences of peptides. SEQ Pep- ID tide NO
ID Peptide Sequence.sup.a 123 1 ##STR00046## 124 2
CH.sub.3CH.sub.2CH.sub.2CO-F.PHI.RRRRUK(FITC)-NH.sub.2 125 3
Ac-DMUD-Amc 126 4 ##STR00047## 127 5 ##STR00048## 128 6
CH.sub.3CH.sub.2CH.sub.2CO-RRRR.PHI.FD.OMEGA.UD-Amc 129 7
Ac-RRRRRRRRRD.OMEGA.UD-Amc 130 8 ##STR00049## 131 9
FITC-URRRRFWQUTRV-OH 132 11 ##STR00050## .sup.aAmc,
7-amino-4-methylcourmarin; FITC, fluorescein isothiocyanate; .PHI.,
L-2-naphthylalanine; .OMEGA., norleucine; U, 2-aminobutyric
acid.
[0273] To illustrate the utility of the reversible cyclization
strategy, it was used to deliver specific caspase substrates into
cells and monitor intracellular caspase activities in real time
(Riedl, S J and Shi, Y. Nat. Rev. Mol. Cell Biol. 2004, 5, 897).
Although peptidyl coumarin derivatives have been widely used to
detect caspase activities in vitro (Maly, D J et al. Chembiochem
2002, 3, 16), they are generally not suitable for in vivo
applications due to impermeability to the mammalian cell membrane.
To generate a cell permeable caspase substrate, a caspase 3/7
substrate, Ac-Asp-Nle-Abu-Asp-Amc (Thornberry, N A et al. J. Biol.
Chem. 1997, 272, 17907) (Table 10, peptide 3, where Amc is
7-amino-4-methylcoumarin and Ne is norleucine), was fused with the
CPP motif RRRR.PHI.F. The fusion peptide was subsequently cyclized
by the addition of a 3-mercaptopropionyl group to its N-terminus,
replacement of the C-terminal Abu with a cysteine, and formation of
an intramolecular disulfide bond, to give cyclic peptide 4 (Table
10). For comparison, an isosteric but irreversibly cyclized peptide
(Table 10, peptide 5) was synthesized by forming a thioether bond
between an N-terminal bromobutyryl moiety and the C-terminal
cysteine (FIG. 17). A linear control peptide of the same sequence
was also prepared as described above (Table 10 peptide 6). Finally,
the caspase 3/7 substrate was conjugated to nonaarginine (R.sub.9)
to generate a positive control peptide (Table 10, peptide 7). In
vitro kinetic analysis revealed that fusion of the caspase 3/7
substrate to RRRR.PHI.F and R.sub.9 decreased its activity by 53%
and 72%, respectively, relative to peptide 3, whereas cyclization
by thioether formation rendered the peptide inactive toward
recombinant caspase 3 (Table 11). The activity of peptide 4 toward
caspase 3 could not be reliably determined because the caspase
assay required a reducing environment, which would cleave the
disulfide bond. Given the structural similarity between peptides 4
and 5, it can be assumed that peptide 4 in the cyclic form is also
inactive toward caspases, but has similar activity to peptide 6
after reductive cleavage of the disulfide bond.
TABLE-US-00011 TABLE 11 In vitro activity of various fluorogenic
substrates against recombinant caspase-3 enzyme. Peptide ID
.DELTA.FU/min 3 159 .+-. 19 5 No detectable activity 6 74.7 .+-.
5.5 7 45.3 .+-. 6.5
[0274] Jurkat cells were pretreated with the kinase inhibitor
staurosporin to induce caspase activities and thus apoptosis
(Belmokhtar, C A et al. Biochem. J 1996, 315, 21). These cells were
then incubated with peptides 3-7 and the amount of Amc released was
monitored at various time points (0-10 h). The impermeable caspase
substrate (peptide 3) produced little fluorescence increase over
the 10-h period (FIG. 21). Peptide 4 produced the fastest
fluorescence increase, reaching 459 fluorescence units (FU),
followed by peptides 7 and 6. Peptide 5, which is inactive toward
caspase 3, also produced AMC in a time-dependent manner, albeit at
a much slower rate (99 FU). This slow rate of AMC release can be
attributed to hydrolysis by other intracellular proteases and
peptidases. Consistent with this interpretation, pretreatment of
Jurkat cells with a pancaspase inhibitor Z-VAD(OMe)-FMK (Slee, E A
et al. Biochem J 1996, 315, 21) followed by incubation with peptide
4 released AMC at a rate that was similar to that of peptide 5
alone. One explanation of the above observations is that both
peptides 4 and 5 can enter the cell interior efficiently, but only
peptide 4 can be converted into the linear caspase substrate inside
the cells.
[0275] Many protein-protein interactions (PPIs) are mediated by
protein domains binding short peptides in their extended
conformations (e.g., .alpha.-helix and .beta.-strand) (Pawson, T
and Nash, P. Science 2003, 300, 445). For example, the PDZ domain
is a common structural domain of 80-90 amino acids found in the
signaling proteins of bacteria to man (Doyle, D A et al. Cell 1996,
85, 1067; Morais Cabral, J H et al., Nature 1996, 382, 649; Lee, H
J and Zheng, J J. Cell Commun. Signal. 2010, 8, 8). PDZ domains
recognize specific sequences at the C-termini of their binding
partners and the bound peptide ligands are in their extended
.beta.-strand conformation (Doyle, D A et al. Cell 1996, 85, 1067;
Songyang, Z et al. Science 1997, 275, 73). It was recently reported
that the activity of cystic fibrosis membrane conductance regulator
(CFTR), a chloride ion channel protein mutated in cystic fibrosis
(CF) patients, is negatively regulated by CFTR-associated ligand
(CAL) through its PDZ domain (CAL-PDZ) (Wolde, M et al. J Biol.
Chem. 2007, 282, 8099). Inhibition of the CFTR/CAL-PDZ interaction
was shown to improve the activity of .DELTA.Phe508-CFTR, the most
common form of CFTR mutation (Cheng, S H et al. Cell 1990, 63, 827;
Kerem, B S et al. Science 1989, 245, 1073), by reducing its
proteasome-mediated degradation (Cushing, P R et al. Angew. Chem.
Int. Ed. 2010, 49, 9907). Previous library screening and rational
design have identified several peptidyl inhibitors of the CAL-PDZ
domain of moderate potencies (K.sub.D values in the high nM to low
.mu.M range) (Cushing, P R et al. Angew. Chem. Int. Ed. 2010, 49,
9907; Roberts, K E et al. PLos Comput. Biol. 2008, 8, e1002477;
Kundu, R et al. Angew. Chem. Int. Ed. 2012, 51, 7217-7220).
However, none of the peptide inhibitors were cell permeable,
limiting their therapeutic potential.
[0276] Starting with a hexapeptide ligand for the CAL-PDZ domain,
WQVTRV (Roberts, K E et al. PLos Comput. Biol. 2008, 8, e1002477),
a disulfide-mediated cyclic peptide was designed by adding the
sequence CRRRRF to its N-terminus and replacing the Val at the -3
position with a cysteine (Table 10, peptide 8). Thus, in peptide 8,
the tryptophan residue at the -5 position was designed to serve the
dual function of PDZ binding and membrane translocation. To
facilitate affinity measurements and quantitation of its cellular
uptake, a FITC group was added to the N-terminus of peptide 8. FA
analysis showed that in the absence of a reducing agent, peptide 8
showed no detectable binding to CAL-PDZ domain (FIG. 22A). In the
presence of 2 mM tris(carboxylethyl)phosphine, which can reduce the
disulfide bond, peptide 8 bound to the CAL-PDZ domain with a
K.sub.D value of 489 nM. Peptide 8 was readily cell permeable;
incubation of HeLa cells with 5 .mu.M peptide 8 for 2 h resulted in
intense and diffuse fluorescence throughout the entire cell (FIG.
22B).
[0277] As expected, peptide 8 is readily cell permeable (FIG. 25C).
Bronchial epithelial CFBE cells, which are homozygous for the
.DELTA.F508-CFTR mutation, were treated with 10 .mu.M Corr-4a in
the presence and absence of 50 .mu.M unlabeled peptide 8. Peptide
8, by inhibiting the function of CAL-PDZ domain, is expected to
increase the amount of .DELTA.F508-CFTR protein transferred to the
plasma membrane, whereas Corr-4a is a small molecule that helps
folding of .DELTA.F508-CFTR protein delivered to the plasma
membrane. Immunostaining of untreated cells (FIG. 25D, panel I)
showed that most of the expressed .DELTA.F508-CFTR was in the
endoplasmic reticulum surrounding the cell nucleus. In contrast,
treatment of cells with Corr-4a and peptide 8 resulted in much
greater amounts of the protein at the cell surface (FIG. 25D panel
II). Quantitation of the cell population revealed that a small but
significant percentage of cells have wild-type like distribution of
.DELTA.F508-CFTR at the cell surface (FIG. 25D). Finally, an SPQ
assay was utilized to quantitate the ion channel activity of
.DELTA.F508-CFTR CFBE cells untreated or treated with CTFR folding
corrector VX809 and peptide 8. Again, VX809 and peptide 8 acted
synergistically to improve the function of the channel activity of
.DELTA.F508-CFTR (FIG. 25E).
Example 3
[0278] Cyclic peptides have great potential as therapeutic agents
and research tools but are generally impermeable to the cell
membrane. Fusion of the cyclic peptides with a cyclic
cell-penetrating peptide can produce bicyclic peptides that can be
cell permeable and can retain the ability to recognize specific
intracellular targets. Application of this strategy to protein
tyrosine phosphatase 1B and peptidyl prolyl cis-trans isomerase
Pin1 resulted in potent, selective, proteolytically stable, and
biologically active inhibitors against the enzymes.
[0279] Cyclic peptides (and depsipeptides) exhibit a wide range of
biological activities (Pomilio, A B et al. Curr. Org. Chem. 2006,
10, 2075-2121). Several innovative methodologies have recently been
developed to synthesize cyclic peptides, either individually
(Meutermans, W D F et al. J Am. Chem. Soc. 1999, 121, 9790-9796;
Schafmeister, C E et al. J Am. Chem. Soc. 2000, 122, 5891-5892;
Sun, Y et al. Org. Lett. 2001, 3, 1681-1684; Kohli, R M et al.
Nature 2002, 418, 658-661; Qin, C et al. J Comb. Chem. 2004, 6,
398-406; Turner, R A et al. Org. Lett. 2007, 9, 5011-5014; Hili, R
et al. J Am. Chem. Soc. 2010, 132, 2889-2891; Lee, J et al. J Am.
Chem. Soc. 2009, 131, 2122-2124; Frost, J R et al. ChemBioChem
2013, 14, 147-160) or combinatorially (Eichler, J et al. Mol.
Divers. 1996, 1, 233-240; Giebel, L B et al. Biochemistry 1995, 34,
15430-15435; Scott, C P et al. Proc. Natd. Acad. Sci. USA 1999, 96,
13638-13643; Millward, S W et al. J. Am. Chem. Soc. 2005, 127,
14142-14143; Sako, Y et al. J. Am. Chem. Soc. 2008, 130,
7232-7234.; Li, S et al. Chem. Commun. 2005, 581-583.; Joo, S H et
al. J. Am. Chem. Soc. 2006, 128, 13000-13009; Heinis, C et al. Nat.
Chem. Biol. 2009, 5, 502-507; Tse, B N et al. J. Am. Chem. Soc.
2008, 130, 15611-15626), and screen them for biological activity. A
particularly exciting application of cyclic peptides is the
inhibition of protein-protein interactions (PPIs) (Leduc, A M et
al. Proc. Natd. Acad. Sic. USA 2003, 100, 11273-11278; Millward, S
W et al. ACS Chem Biol 2007, 2, 625-634; Tavassoli, A et al. ACS
Chem. Biol. 2008, 3, 757-764.; Wu, X et al. Med. Chem. Commun.
2013, 4, 378-382; Birts, C N et al. Chem. Sci. 2013, 4, 3046-3057;
Kawakami, T et al. ACS Chem. Biol. 2013, 8, 1205-1214; Lian, W et
al. J Am. Chem. Soc.2013, 135, 11990-11995), which remain
challenging targets for conventional small molecules. However, a
major limitation of cyclic peptides is that they are generally
impermeable to the cell membrane, precluding any application
against intracellular targets, which include most of the
therapeutically relevant PPIs. Although formation of intramolecular
hydrogen bonds (Rezai, T et al. J. Am. Chem. Soc.2006, 128,
14073-14080) or N.sup..alpha.-methylation of the peptide backbone
(Chatterjee, J et al. Acc. Chem. Res. 2008, 41, 1331-1342; White, T
R et al. Nat. Chem. Biol. 2011, 7, 810-817) can improve the
membrane permeability of certain cyclic peptides, alternative
strategies to increase the cell permeability of cyclic peptides are
clearly needed.
[0280] Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical
member of the PTP superfamily and plays numerous roles during
eukaryotic cell signaling. Because of its role in negatively
regulating insulin and leptin receptor signaling, PTP1B is a valid
target for treatment of type II diabetes and obesity (Elchelby, M
et al. Science 1999, 283, 1544-1548; Zabolotny, J M et al. Dev Cell
2002, 2, 489-495). A large number of PTP1B inhibitors have been
reported (He, R et al. in New Therapeutic Strategies for Type 2
Diabetes: Small Molecule Approaches. Ed. R. M. Jones, RSC
Publishing 2012, pp 1142), however, none of them have succeeded in
the clinic. Designing PTP inhibitors is challenging because most of
the phosphotyrosine (pY) isosteres, such as difluorophosphonomethyl
phenylalanine (F.sub.2Pmp) (Burke Jr., T R et al. Biochem. Biophys.
Res. Commun. 1994, 204, 129-134), are impermeable to the cell
membrane. Additionally, because all PTPs share a similar active
site, achieving selectivity for a single PTP has been difficult.
Herein, a potentially general approach to designing cell-permeable
cyclic peptidyl inhibitors against intracellular proteins such as
PTP1B is reported.
[0281] Materials. Fmoc-protected amino acids were purchased from
Advanced ChemTech (Louisville, Ky.), Peptides International
(Louisville, Ky.), or Aapptec (Louisville, Ky.). Fmoc-F.sub.2Pmp-OH
was purchased from EMD Millipore (Darmstadt, Germany).
Aminomethyl-ChemMatrix resin (0.66 mmol/g) was from SJPC (Quebec,
Canada). Rink resin LS (100-200 mesh, 0.2 mmol/g) and
N-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) were
purchased from Advanced ChemTech.
O-Benzotriazole-N,N,N,N'-tetramethyluronium hexafluorophosphate
(HBTU), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU), 1-hydroxybenzotriazole hydrate (HOBt)
were purchased from Aapptec. Phenyl isothiocyanate in 1-mL sealed
ampoules, fluorescein isothiocyanate (FITC), rhodamine B-labeled
dextran (dextran.sup.Rho) were purchased from Sigma-Aldrich. Cell
culture media, fetal bovine serum (FBS), penicillin-streptomycin,
0.25% trypsin-EDTA, Dulbecco's phosphate-buffered saline (DPBS)
(2.67 mM potassium chloride, 1.47 mM potassium phosphate monobasic,
137 mM sodium chloride, 8.06 mM sodium phosphate dibasic.), and
anti-phospho-IR/IGF1R antibody were purchased from Invitrogen
(Carlsbad, Calif.). Nuclear staining dye DRAQ5' and
anti-.beta.-actin antibody were purchased from Thermo Scientific
(Rockford, Ill.). Antibody 4G10 was purchased from Millipore
(Temecula, Calif.). All solvents and other chemical reagents were
obtained from Sigma-Aldrich (St. Louis, Mo.) and were used without
further purification unless noted otherwise.
[0282] Cell Culture. A549, HEK293, and HepG2 cells were maintained
in Dulbecco's modified Eagle medium (DMEM) supplemented with 10%
FBS in a humidified incubator at 37.degree. C. with 5%
CO.sub.2.
[0283] Protein Expression, Purification and Labeling. The gene
coding for the catalytic domain of PTP1B (amino acids 1-321) was
amplified by the polymerase chain reaction using PTP1B cDNA as
template and oligonucleotides
5'-ggaattccatatggagatggaaaaggagttcgagcag-3' and
5'-gggatccgtcgacattgtgtggctccaggattcgtttgg-3' as primers. The
resulting DNA fragment was digested with endonucleases Nde I and
Sal I and inserted into prokaryotic vector pET-22b(+)-ybbR (Yin, J
et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15815-15820). This
cloning procedure resulted in the addition of a ybbR tag
(VLDSLEFIASKL) to the N-terminus of PTP1B. Expression and
purification of the ybbR-tagged PTP1B were carried out as
previously described (Ren, L et al. Biochemistry 2011, 50,
2339-2356). Texas Red labeling of PTP1B was carried out by treating
the ybbR-tagged PTP1B protein (80 .mu.M) in 50 mM HEPES, pH 7.4, 10
mM MgCl.sub.2 with Sfp phosphopantetheinyl transferase (1 .mu.M)
and Texas Red-CoA (100 .mu.M) for 30 min at room temperature (Yin,
J et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15815-15820). The
reaction mixture was passed through a G-25 fast-desalting column
equilibrated in 30 mM HEPES, pH 7.4, 150 mM NaCl to remove any free
dye molecules. The full-length human S16A/Y23A mutant Pin1 was
expressed and purified from E. coli as previously described (Liu, T
et al. J. Med. Chem. 2010, 53, 2494-2501).
[0284] Library Synthesis. The cyclic peptide library was
synthesized on 1.35 g of aminomethyl-ChemMatrix resin (0.57
mmol/g). The library synthesis was performed at room temperature
unless otherwise noted. The linker sequence (BBM) was synthesized
using standard Fmoc chemistry. The typical coupling reaction
contained 5 equiv of Fmoc-amino acid, 5 equiv of HBTU and 10 equiv
of diisopropylethylamine (DIPEA) and was allowed to proceed with
mixing for 2 h. The Fmoc group was removed by treatment twice with
20% (v/v) piperidine in DMF (5+15 min), and the beads were
exhaustively washed with DMF (6.times.). To spatially segregate the
beads into outer and inner layers, the resin (after removal of
N-terminal Fmoc group) was washed with DMF and water, and soaked in
water overnight. The resin was quickly drained and suspended in a
solution of Fmoc-Glu(.delta.-NHS)-OAll (0.10 equiv), Boc-Met-OSu
(0.4 equiv) and N-methylmorpholine (2 equiv) in 20 mL of 1:1 (v/v)
DCM/diethyl ether (Joo, S H et al. J. Am. Chem. Soc. 2006, 128,
13000-13009). The mixture was incubated on a carousel shaker for 30
min. The beads were washed with 1:1 DCM/diethyl ether (3.times.)
and DMF (8.times.). Next, the Fmoc group was removed by piperidine
treatment. Then, Fmoc-Arg(Pbf)-OH (4.times.), Fmoc-Nal-OH, and
Fmoc-Phe-OH were sequentially coupled by standard Fmoc chemistry to
half of the resin. The other half was coupled with the same amino
acids in the reverse sequence. The resin was combined and the
random sequence was synthesized by the split-and-pool method using
5 equiv of Fmoc-amino acids, 5 equiv HATU and 10 equiv DIPEA as the
coupling agent. The coupling reaction was repeated once to ensure
complete coupling at each step. For random positions, a 24-amino
acid set was selected based on their structural diversity,
metabolic stability, and commercial availability, including 10
proteinogenic .alpha.-L-amino acids (Ala, Asp, Gln, Gly, His, Ile,
Ser, Trp, Pro, and Tyr), 5 nonproteinogenic .alpha.-L-amino acids
(L-4-fluorophenylalanine (Fpa), L-homoproline (Pip), L-norleucine
(Nle), L-phenylglycine (Phg) and
L-4-(phosphonodifluoromethyl)phenylalanine (F.sub.2Pmp)), and nine
.alpha.-D-amino acids (D-2-naphthylalanine (D-Nal), D-Ala, D-Asn,
D-Glu, D-Leu, D-Phe, D-Pro, D-Thr, and D-Val). To differentiate
isobaric amino acids during PED-MS analysis, 4% (mol/mol) of
CD.sub.3CO.sub.2D was added to the coupling reactions of D-Ala,
D-Leu, and D-Pro, while 4% CH.sub.3CD.sub.2CO.sub.2D was added to
the Nle reactions. Fmoc-F.sub.2Pmp-OH (0.06 equiv) and Fmoc-Tyr-OH
(0.54 equiv) was placed in the middle of the random positions using
HATU/DIPEA. After the entire sequence was synthesized, the allyl
group on the C-terminal Glu residue was removed by treatment with a
DCM solution containing tetrakis(triphenylphosphine)palladium
[Pd(PPh.sub.3).sub.4, 0.25 equiv] and phenylsilane (5 equiv) for 15
min (3.times.). The beads were sequentially washed with 0.5% (v/v)
DIPEAin DMF, 0.5% (w/v) sodium dimethyldithiocarbamate hydrate in
DMF, DMF (3.times.), DCM (3.times.), and DMF (3.times.). The Fmoc
group on the N-terminal random residue was removed by piperidine as
described above. The beads were washed with DMF (6.times.), DCM
(3.times.), and 1 M HOBt in DMF (3.times.). For peptide
cyclization, a solution of PyBOP/HOBt/DIPEA (5, 5, 10 equiv,
respectively) in DMF was mixed with the resin and the mixture was
incubated on a carousel shaker for 3 h. The resin was washed with
DMF (3.times.) and DCM (3.times.) and dried under vacuum for >1
h. Side-chain deprotection was carried out with a modified reagent
K 78.5:7.5:5:5:2.5:1:1 (v/v)
TFA/phenol/water/thioanisole/ethanedithiol/anisole/triisopropylsilane)
for 3 h. The resin was washed with TFA and DCM and dried under
vacuum before storage at -20.degree. C.
[0285] Library Screening and Peptide Sequencing. Library resin (100
mg, .about.300,000 beads) was swollen in DCM, washed extensively
with DMF, doubly distilled H.sub.2O, and incubated in 1 mL of
blocking buffer (PBS, pH 7.4, 150 mM NaCl, 0.05% Tween 20 and 0.1%
gelatin) containing 20 nM Texas red-labeled PTP1B at 4.degree. C.
for 3 h. The beads were examined under an Olympus SZX12 microscope
equipped with a fluorescence illuminator (Olympus America, Center
Valley, Pa.) and the most intensely fluorescent beads were manually
collected as positive hits. Beads containing encoding linear
peptides were individually sequenced by partial Edman
degradation-mass spectrometry (PED-MS) (Liu, T et al. J Med. Chem.
2010, 53, 2494-2501).
[0286] Individual Peptide Synthesis and Labeling. Monocyclic and
bicyclic peptides were synthesized on Rink Resin LS (0.2 mmol/g)
using standard Fmoc chemistry. For monocyclic peptides, after the
last (N-terminal) residue was coupled, the allyl group on the
C-terminal Glu residue was removed by treatment with
Pd(PPh.sub.3).sub.4 and phenylsilane (0.1 and 10 equiv,
respectively) in anhydrous DCM (3.times.15 min). The N-terminal
Fmoc group was removed by treatment with 20% (v/v) piperidine in
DMF and the peptide was cyclized by treatment with PyBOP/HOBt/DIPEA
(5, 5, and 10 equiv) in DMF for 3 h. For bicyclic peptides, the
N-terminal Fmoc group was removed with piperidine and a trimesic
acid was coupled on the N-terminal amine using HBTU as a coupling
agent. The allyloxycarbonyl groups on the side chains of two Dap
residues were removed by treatment with Pd(PPh.sub.3).sub.4 and
phenylsilane (0.1 and 10 equiv, respectively) in anhydrous DCM for
2 h. The resulting peptide was cyclized with PyBOP as described
above. The peptides were deprotected and released from the resin by
treatment with 82.5:5:5:5:2.5 (v/v)
TFA/thioanisole/water/phenol/ethanedithiol for 2 h. The peptides
were triturated with cold ethyl ether (3.times.) and purified by
reversed-phase HPLC on a Cis column. The authenticity of each
peptide was confirmed by MALDI-TOF mass spectrometry. Peptide
labeling with FITC was performed by dissolving the purified peptide
(.about.1 mg) in 300 .mu.L of 1:1:1 (vol/vol) DMSO/DMF/150 mM
sodium bicarbonate (pH 8.5) and mixing with 10 .mu.L of FITC in
DMSO (100 mg/mL). After 20 min at room temperature, the reaction
mixture was subjected to reversed-phase HPLC on a Cis column to
isolate the FITC-labeled peptide.
[0287] PTP Inhibition Assay. PTP assays were performed in a quartz
microcuvette (total volume 150 .mu.L). The reaction mixture
contains 100 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2 mM EDTA, 1 mM TCEP,
0-1 .mu.M of PTP inhibitor, and 500 .mu.M para-nitrophenyl
phosphate (pNPP). The enzymatic reaction was initiated by the
addition of PTP (final concentration 15-75 nM) and monitored
continuously at 405 nm on a UV-VIS spectrophotometer. Initial rates
were calculated from the reaction progress curves (typically <60
s). The half-maximal inhibition constant (IC.sub.50) was defined as
the concentration of an inhibitor that reduced the enzyme activity
to 50% and was obtained by plotting the rates (V) against the
inhibitor concentration [1] and fitting the data against the
equation
V = V 0 ( 1 + [ I ] IC 5 .times. 0 ) ##EQU00002##
where V.sub.0 is the enzymatic reaction rate in the absence of
inhibitor. The inhibition constant (K.sub.i) was determined by
measuring the initial rates at fixed enzyme concentration (15 nM)
but varying concentrations of pNPP (0-24 mM) and inhibitor (0-112
nM). The reaction rate (V) was plotted against the pNPP
concentration ([S]) and fitted against the equation
1 V = K .times. 1 [ S ] + 1 V max ##EQU00003##
to obtain the Michaelis constant K. The K.sub.i value was obtained
by plotting the K values against the inhibitor concentration [1]
and fitted to equation
K K 0 = 1 + [ I ] K i ##EQU00004##
where K.sub.0 is the Michaelis constant in the absence of inhibitor
([I]=0).
[0288] Confocal Microscopy. Approximately 5.times.10.sup.4 A549
cells were seeded in 35-mm glass-bottomed microwell dish (MatTek)
containing 1 mL of media and cultured for one day. A549 cells were
gently washed with DPBS once and treated with the FITC-labeled
PTP1B inhibitors (5 .mu.M), dextran.sup.Rho (1 mg mL.sup.-1) in
growth media for 2 h at 37.degree. C. in the presence of 5%
CO.sub.2. The peptide-containing media was removed and the cells
were washed with DPBS three times and incubated for 10 min in 1 mL
of DPBS containing 5 .mu.M DRAQ5. The cells were again washed with
DPBS twice. Then the cells were imaged on a Visitech Infinity 3
Hawk 2D-array live cell imaging confocal microscope (with a
60.times. oil immersion lens) at 37.degree. C. in the presence of
5% CO.sub.2. Live-cell confocal microscopic imaging of HEK293 cells
after treatment with FITC-labeled Pin1 inhibitors were similarly
conducted.
[0289] Immunoblotting. A549 cells were cultured in full growth
media to reach 80% confluence. The cells were starved in serum free
media for 3 h and treated with varying concentrations of PTP1B
inhibitors for 2 h, followed by 30 min incubation in media
supplemented with 1 mM sodium pervanadate. The solutions were
removed and the cells were washed with cold DPBS twice. The cells
were detached and lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%
NP-40, 10 mM sodium pyrophosphate, 5 mM iodoacetic acid, 10 mM NaF,
1 mM EDTA, 2 mM sodium pervanadate, 0.1 mg/mL phenylmethanesulfonyl
fluoride, 1 mM benzamidine, and 0.1 mg/mL trypsin inhibitor. After
30 min incubation on ice, the cell lysate was centrifuged at 15,000
rpm for 25 min in a microcentrifuge. The total cellular proteins
were separated by SDS-PAGE and transferred electrophoretically to a
PVDF membrane, which was immunoblotted using anti-phosphotyrosine
antibody 4G10. The same samples were analyzed on a separate
SDS-PAGE gel and stained by Coomassie brilliant blue to ascertain
equal sample loading in all lanes.
[0290] To test the inhibitor's effect on insulin signaling pathway,
HepG2 cells were cultured to reach 80% confluence. The cells were
starved for 4 h in serum free DMEM media before being treated with
PTP1B inhibitor (2 h), followed by stimulation with 100 nM insulin
for 5 min. The samples were analyzed by SDS-PAGE as described above
and immunoblotted using anti-phospho-IR/IGF1R antibody. The PVDF
membrane was also probed by anti-.beta.-actin antibody as the
loading control.
[0291] Serum Stability Test. The stability tests were carried by
modifying a previously reported procedure (Nguyen, L T et al. PLoS
One 2010, 5, e12684). Diluted human serum (25%) was centrifuged at
15,000 rpm for 10 min, and the supernatant was collected. A peptide
stock solution was diluted into the supernatant to a final
concentration of 5 .mu.M and incubated at 37.degree. C. At various
time points (0-24 h), 200-.mu.L aliquots were withdrawn and mixed
with 50 .mu.L of 15% trichloroacetic acid and incubated at
4.degree. C. overnight. The final mixture was centrifuged at 15,000
rpm for 10 min in a microcentrifuge, and the supernatant was
analyzed by reversed-phase HPLC equipped with a Cis column. The
amount of remaining peptide (%) was determined by integrating the
area underneath the peptide peak (monitored at 214 nm) and
comparing with that of the control reaction (no serum).
[0292] Fluorescence Anisotropy. FA experiments were carried out by
incubating 100 nM FITC-labeled peptide with varying concentrations
of protein in 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM magnesium
acetate, and 0.1% bovine serum albumin (BSA) for 2 h at room
temperature. The FA values were measured on a Molecular Devices
Spectramax M5 plate reader, with excitation and emission
wavelengths at 485 and 525 nm, respectively. Equilibrium
dissociation constants (K.sub.D) were determined by plotting the FA
values as a function of protein concentration and fitting the curve
to the following equation:
Y = ( A min + ( A max .times. Q b Q f - A min ) ( ( L + x + K D ) -
( ( L + x + K D ) 2 - 4 .times. L .times. x ) 2 .times. L ) ) ( 1 +
( Q b Q f - 1 ) .times. ( ( L + x + K D ) - ( ( L + x + K D ) 2 - 4
.times. L .times. x ) 2 .times. L ) ) ##EQU00005##
where Y is the FA value at a given protein concentration x, L is
the peptide concentration, Q.sub.b/Q.sub.f is the correction factor
for fluorophore-protein interaction, A.sub.max is the maximum FA
value when all of the peptides are bound to protein, while
A.sub.min is the minimum FA value when all of the peptides are
free. FA competition assay was performed by incubating 100 nM
FITC-labeled Pin1 inhibitor 5 with 1 .mu.M Pin1, followed by the
addition of 0-5 .mu.M unlabeled inhibitor. The FA values were
measured similarly on a pate reader. IC.sub.50 values were obtained
by plotting the FA values against the competitor concentration and
curve fitting using the four-parameter dose-response inhibition
equation (Prism 6, GraphPad).
[0293] A class of cell-penetrating peptides (CPPs),
cyclo(Phe-Nal-Arg-Arg-Arg-Arg-Gln) (cF.PHI.R.sub.4, where .PHI. or
Nal is L-naphthylalanine), were recently discovered (Qian, Z et al.
ACS Chem. Biol. 2013, 8, 423-431). Unlike previous CPPs, which are
typically linear peptides and predominantly entrapped in the
endosome, cF.PHI.R.sub.4 can efficiently escape from the endosome
into the cytoplasm. Short peptide cargos (1-7 aa) could be
delivered into mammalian cells by directly incorporating them into
the cF.PHI.R.sub.4 ring. The possibility of developing bifunctional
cyclic peptides containing both cell-penetrating and target-binding
sequences as cell-permeable inhibitors against intracellular
proteins was examined. To generate specific inhibitors against
PTP1B, a one-bead-two-compound library was synthesized on spatially
segregated ChemMatrix resin (Liu, R et al. J Am. Chem. Soc. 2002,
124, 7678-7680), in which each bead displayed a bifunctional cyclic
peptide on its surface and contained the corresponding linear
peptide in its interior as an encoding tag (FIG. 23 and FIG. 24).
The bifunctional cyclic peptides all featured the amphipathic CPP
motif F.PHI.R.sub.4 (or its inverse sequence RRRR.PHI.F) on one
side and a random pentapeptide sequence
(X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5) on the other side, where
X.sup.2 represents a 9:1 (mol/mol) mixture of Tyr and F2Pmp while
X.sup.1 and X.sup.3--X.sup.5 are any of the 24 amino acids that
included 10 proteinogenic L-amino acids (Ala, Asp, Gln, Gly, His,
Ile, Pro, Ser, Tyr, Trp), 5 unnatural .alpha.-L-amino acids (F2Pmp,
L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-phenylglycine
(Phg), L-pipecolic acid (Pip)), and 9.alpha.-D-amino acids (D-Ala,
D-Asn, D-Glu, D-Leu, L-O-naphthylalanine (D-Nal), D-Phe, D-Pro,
D-Thr, and D-Val). The use of 9:1 Tyr/F2Pmp ratio at the X.sup.2
position, together with a 5-fold reduction of the surface peptide
loading, reduced the amount of F2Pmp-containing peptides at the
bead surface by 50-fold, increasing the stringency and minimizing
nonspecific binding during library screening (Chen, X et al. J.
Comb. Chem. 2009, 11, 604-611). Screening of the library
(theoretical diversity 6.6.times.10.sup.5) against Texas
red-labeled PTP1B resulted in 65 positive beads, which were
individually sequenced by partial Edman degradation-mass
spectrometry (PED-MS) (Thakkar, A et al. Anal. Chem. 2006, 78,
5935-5939) to give 42 complete sequences (Table 12). Interestingly,
most of the selected PTP1B inhibitors contained the inverse CPP
motif (RRRR.PHI.F).
TABLE-US-00012 TABLE 12 Peptide Sequences Selected from Cyclic
Peptide Library against PTP1B.sup.a. SEQ ID Bead NO. No. Sequence
136 1 Pro-Pip-Gly-F.sub.2Pmp-Tyr-Arg 137 2
Ser-Pip-Ile-F.sub.2Pmp-F.sub.2Pmp-Arg 138 3
Ile-His-Ile-F.sub.2Pmp-Ile-Arg 139 4
Ala-D-Ala-Ile-F.sub.2Pmp-Pip-Arg 140 5
Fpa-Ser-Pip-F.sub.2Pmp-D-Val-Arg 141 6
Pip-D-Asn-Pro-F.sub.2Pmp-Ala-Arg 142 7
Tyr-Phg-Ala-F.sub.2Pmp-Gly-Arg 143 8
Ala-His-Ile-F.sub.2Pmp-D-Ala-Arg 144 9
Gly-D-Asn-Gly-F.sub.2Pmp-D-Pro-Arg 145 10
D-Phe-Gln-Pip-F.sub.2Pmp-Ile-Arg 146 11
Ser-Pro-Gly-F.sub.2Pmp-His-Arg 147 12
Pip-Tyr-Ile-F.sub.2Pmp-His-Arg 148 13*
Ser-D-Val-Pro-F.sub.2Pmp-His-Arg 149 14
Ala-Ile-Pro-F.sub.2Pmp-D-Asn-Arg 150 15
Fpa-Ser-Ile-F.sub.2Pmp-Gln-Phe 151 16
Ala-D-Ala-Phg-F.sub.2Pmp-D-Phe-Arg 152 17
D-Asn-D-Thr-Phg-F.sub.2Pmp-Phg-Arg 153 18*
Ile-Pro-Phg-F.sub.2Pmp-Nle-Arg 154 19
Gln-Pip-Fpa-F.sub.2Pmp-Pip-Arg 155 20
D-Asn-Ala-Fpa-F.sub.2Pmp-Gly-Arg 156 21
D-Asn-D-Thr-Tyr-F.sub.2Pmp-Ala-Arg 157 22
D-Glu-Ala-Phg-F.sub.2Pmp-D-Val-Arg 158 23
Ile-D-Val-Phg-F.sub.2Pmp-Ala-Arg 159 24
Tyr-D-Thr-Phg-F.sub.2Pmp-Ala-Arg 160 25
D-Asn-Pip-Phg-F.sub.2Pmp-Ile-Arg 161 26
Pip-D-Asn-Trp-F.sub.2Pmp-His-Arg 162 27
Tyr-Pip-D-Val-F.sub.2Pmp-Ile-Arg 163 28
D-Asn-Ser-D-Ala-F.sub.2Pmp-Gly-Arg 164 29*
D-Thr-D-Asn-D-Val-F.sub.2Pmp-D-Ala- Arg 165 30
D-Asn-D-Thr-D-Val-F.sub.2Pmp-D-Thr- Arg 166 31
Ser-Ile-D-Thr-F.sub.2Pmp-Tyr-Arg 167 32
D-Asn-Fpa-D-Asn-F.sub.2Pmp-D-Leu- Arg 168 33
Tyr-D-Asn-D-Asn-F.sub.2Pmp-Nle-Arg 169 34
D-Asn-Tyr-D-Asn-F.sub.2Pmp-Gly-Arg 170 35
Ala-Trp-D-Asn-F.sub.2Pmp-Ala-Arg 171 36
D-Val-D-Thr-His-F.sub.2Pmp-Tyr-Arg 172 37
Pro-Phg-His-F.sub.2Pmp-Pip-Arg 173 38
D-Asn-Phg-His-F.sub.2Pmp-Gly-Arg 174 39
Pro-Ala-His-F.sub.2Pmp-Gly-Arg 175 40
Ala-Tyr-His-F.sub.2Pmp-Ile-Arg 176 41
D-Asn-Pip-D-Glu-F.sub.2Pmp-Tyr-Arg 177 42
D-Val-Ser-Ser-F.sub.2Pmp-D-Thr-Arg .sup.aFpa,
L-4-fluorophenylalanine; Pip, L-homoproline; Nle, L-norleucine;
Phg, L-phenylglycine; F.sub.2Pmp,
L-4-(phosphonodifluoromethyl)phenylalanine. *Sequences subjected to
further analysis.
[0294] Three hit sequences
(D-Thr-D-Asn-D-Val-F2Pmp-D-Ala-Arg-Arg-Arg-Arg-Nal-Phe-Gln
(inhibitor 1), Ser-D-Val-Pro-F2Pmp-His-Arg-Arg-Arg-Arg-Nal-Phe-Gln
(inhibitor 2), and
Ile-Pro-Phg-F2Pmp-Nle-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 3))
were resynthesized and purified by HPLC. All three peptides are
competitive PTP1B inhibitors (Table 13), with peptide 2 being most
potent (K.sub.1=54 nM) (FIG. 25). Confocal microscopic analysis of
human cells treated with fluorescein isothiocyanate (FITC)-labeled
inhibitor 2 indicated poor cellular uptake of the peptide (FIG.
26a). It has previously been shown that as the size of the cargo
inserted into the cF.PHI.R.sub.4 ring increases, the cellular
uptake efficiency of the cyclic peptides decreases (Qian, Z et al.
ACS Chem. Biol. 2013, 8, 423-431). Larger rings can be more
conformationally flexible and may bind less tightly to the cell
surface receptors (e.g., membrane phospholipids) during
endocytosis. The negatively charged F2Pmp may also interact
intramolecularly with the F.PHI.R.sub.4 motif and interfere with
its CPP function.
TABLE-US-00013 TABLE 13 Potency of Selected Monocyclic Peptide
Inhibitors against PTP1B SEQ ID Monocyclic IC.sub.50 NO Inhibitor
Sequence (nM) 178 1
cyclo(D-Thr-D-Asn-D-Val-F.sub.2Pmp-D-Ala-Arg-Arg- ~100
Arg-Arg-Nal-Phe-Gln) 179 2
cyclo(Ser-D-Val-Pro-F.sub.2Pmp-His-Arg-Arg-Arg-Arg- ~30
Nal-Phe-Gln) 180 3
cyclo(Ile-Pro-Phg-F.sub.2Pmp-Nle-Arg-Arg-Arg-Arg-Nal- ~200
Phe-Gln)
[0295] To improve the cell permeability of inhibitor 2, a bicyclic
system in which the CPP motif is placed in one ring whereas the
target-binding sequence constitutes the other ring (FIG. 23) was
explored. The bicyclic system keeps the CPP ring to a minimal size
which, according to the previously observed trend (Qian, Z et al.
ACS Chem. Biol. 2013, 8, 423-431), can result in more efficient
cellular uptake. The bicyclic system should be able to accommodate
cargos of any size, because incorporation of the latter does not
change the size of CPP ring and, therefore, should not affect the
delivery efficiency of the cyclic CPP. The use of a rigid scaffold
(e.g., trimesic acid) may also help keep the CPP and cargo motifs
away from each other and minimize any mutual interference. The
smaller rings of a bicyclic peptide, compared to its monocyclic
counterpart, can result in greater structural rigidity and improved
metabolic stability.
[0296] To convert the monocyclic PTP1B inhibitor 2 into a bicyclic
peptide, the Gln residue (used for attachment to the solid support
and peptide cyclization) was replaced with (S)-2,3-diaminopropionic
acid (Dap) and a second Dap residue was inserted at the junction of
CPP and PTP1B-binding sequences (C-terminal to His) (FIG. 23).
Synthesis of the bicycle was accomplished by the formation of three
amide bonds between a trimesic acid and the N-terminal amine and
the side chains of the two Dap residues (FIG. 27) (Lian, W et al.
J. Am. Chem. Soc.2013, 135, 11990-11995). Briefly, the linear
peptide was synthesized on Rink amide resin using the standard Fmoc
chemistry and NR-alloxycarbonyl (Alloc)-protected Dap. After
removal of the N-terminal Fmoc group, the exposed amine was
acylated with trimesic acid. Removal of the Alloc groups with
Pd(PPh.sub.3).sub.4 followed by treatment with PyBOP afforded the
desired bicyclic structure. To facilitate labeling with fluorescent
probes, a lysine was added to the C-terminus. The bicyclic peptide
(peptide 4) was deprotected by TFA and purified to homogeneity by
HPLC.
[0297] Bicyclic peptide 4 can act as a competitive inhibitor of
PTP1B, with a K.sub.1 value of 37 nM (FIG. 26b). It can be highly
selective for PTP1B. When assayed against p-nitrophenyl phosphate
as a substrate (500 .mu.M), inhibitor 4 had IC.sub.50 values of 30
and 500 nM for PTP1B and TCPTP, respectively (FIG. 26c and Table
14). It exhibited minimal inhibition of any of the other PTPs
tested (.ltoreq.10% inhibition of HePTP, SHP-1, PTPRC, PTPH1, or
PTPRO at 1 .mu.M inhibitor concentration). Inhibitor 4 has improved
cell permeability over peptide 2, as detected by live-cell confocal
microscopy of A549 cells treated with FITC-labeled inhibitor 4
(FIG. 26a). The treated cells showed both diffuse fluorescence
throughout the cytoplasm and nucleus as well as fluorescence
puncta, indicating that a fraction of the inhibitors reached the
cytoplasm and nucleus while the rest was likely entrapped in the
endosomes. Incubation of inhibitor 4 in human serum for 24 h at
37.degree. C. resulted in .about.10% degradation, whereas 91% of
inhibitor 2 was degraded under the same condition (FIG. 28).
Overall, inhibitor 4 compares favorably with the small-molecule
PTP1B inhibitors reported to date (Qian, Z et al. ACS Chem. Biol.
2013, 8, 423-431) with respect to potency, selectivity over the
highly similar TCPTP (17-fold), cell permeability, and
stability.
TABLE-US-00014 TABLE 14 Selectivity of Bicyclic Inhibitor 4 against
Various PTPs.sup.a PTP PTP1B TCPTP HePTP PTPRC SHP1 PTPRO PTPH1
IC.sub.50 30 .+-. 4 500 .+-. NA NA NA NA NA (nM) 250 .sup.aNA, no
significant inhibition at 1 .mu.M inhibitor.
[0298] Inhibitor 4 was next tested for its ability to perturb PTP1B
function during cell signaling. Treatment of A549 cells with
inhibitor 4 (0-5 .mu.M) resulted in dose-dependent increases in the
phosphotyrosine (pY) levels of a large number of proteins,
consistent with the broad substrate specificity of PTP1B (Ren, L et
al. Biochemistry 2011, 50, 2339) (FIG. 29a). Analysis of the same
samples by Coomassie blue staining showed similar amounts of
proteins in all samples (FIG. 29b), indicating that the increased
pY levels reflected increased phosphorylation (or decreased PTP
reaction) instead of changes in the total protein levels.
Remarkably, the increase in tyrosine phosphorylation was already
apparent at 8 nM inhibitor 4. Interestingly, further increase in
inhibitor concentration beyond 1 .mu.M reversed the effect on
tyrosine phosphorylation, an observation that was also made
previously by Zhang and co-workers with a different PTP1B inhibitor
(Xie, L et al. Biochemistry 2003, 42, 12792-12804). To obtain
further evidence that the intracellular PTP1B was inhibited by
peptide 4, the pY level of insulin receptor (IR), a
well-established PTP1B substrate in vivo (Elchelby, M et al.
Science 1999, 283, 1544-1548; Zabolotny, J M et al. Dev. Cell 2002,
2, 489-495), was monitored by immunoblotting with specific
antibodies against the pY.sup.1162pY.sup.1163 site. Again,
treatment with inhibitor 4 caused dose-dependent increase in
insulin receptor phosphorylation up to 1 .mu.M inhibitor and the
effect leveled off at higher concentrations (FIG. 29c,d). Taken
together, these data indicate that bicyclic inhibitor 4 can
efficiently enter mammalian cells and can inhibit PTP1B in vivo.
The decreased phosphorylation at higher inhibitor concentrations
may be caused by nonspecific inhibition of other PTPs (which may in
turn down regulate protein tyrosine kinases). It may also reflect
the pleiotropic roles played by PTP1B, which can both negatively
and positively regulate the activities of different protein kinases
(Lessard, L et al. Biochim. Biophys. Acta 2010, 1804, 613).
[0299] To test the generality of the bicyclic approach, it was
applied to design cell permeable inhibitors against peptidyl prolyl
cis-trans isomerase Pin1, a potential target for treatment of a
variety of human diseases including cancer (Lu, K P and Zhou, X Z.
Nat. Rev. Mol. Cell Biol. 2007, 8, 904-916), for which potent,
selective, and biologically active inhibitors are still lacking
(More, J D and Potter, A. Bioorg. Med. Chem. Lett. 2013, 23,
4283-91). Thus, a previously reported monocyclic peptide (5), which
is a potent inhibitor against Pin1 in vitro (K.sub.D 258 nM) but
membrane impermeable (Liu, T et al. J. Med. Chem. 2010, 53,
2494-2501), was fused with cF.PHI.R.sub.4 (FIG. 30). In addition,
the L-Tyr at the pThr+3 position was replaced with an Arg to
improve the aqueous solubility. The resulting bicyclic peptide 6
bound Pin1 with a K.sub.D value of 131 nM (Table 15 and FIG. 31).
Insertion of a D-Ala at the pThr+5 position to increase the
separation between the Pin1-binding and cell-penetrating motifs
improved the inhibitor potency by .about.2-fold (K.sub.D=72 nM for
inhibitor 7). Inhibitor 7 competed with FITC-labeled inhibitor 5
for binding to Pin1 (FIG. 32), indicating that they both can bind
to the Pin1 active site. Substitution of D-Thr for D-pThr of
inhibitor 7 reduced its potency by .about.10-fold (K.sub.D=620 nM
for inhibitor 8, Table 16), whereas further replacement of the
pipecolyl residue with D-Ala abolished Pin1 inhibitory activity
(peptide 9). The bicyclic inhibitors 7-9 were cell permeable (FIG.
33). Treatment of HeLa cells with inhibitor 7 resulted in time- and
dose-dependent inhibition of cell growth (45% inhibition after
3-day treatment at 20 .mu.M inhibitor 7), whereas the monocyclic
inhibitor 5 and inactive peptide 9 had no effect (FIG. 34). Peptide
8 also inhibited cell growth, but to a lesser extent than inhibitor
7.
TABLE-US-00015 TABLE 15 Dissociation Constants of Monocyclic and
Bicyclic Peptides against Pin1 as Determined by FA Analysis SEQ ID
Pin1 NO Inhibitor Sequence.sup.a K.sub.D (nM) 181 5
cyclo(D-Ala-Sar-D-pThr-Pip-Nal-Tyr-Gln)-Lys-NH.sub.2 258 .+-. 65
182 6 bicyclo[Tm(D-Ala-Sar-D-pThr-Pip-Nal-Arg-Ala)-Dap- 131 .+-. 44
(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys-NH.sub.2 183 7
bicyclo[Tm(D-Ala-Sar-D-pThr-Pip-Nal-Arg-Ala-D-Ala)- 72 .+-. 21
Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys-NH.sub.2 184 8
bicyclo[Tm(D-Ala-Sar-D-Thr-Pip-Nal-Arg-Ala-D-Ala)- 620 .+-. 120
Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys-NH.sub.2 185 9
bicyclo[Tm(D-Ala-Sar-D-Thr-D-Ala-Nal-Arg-Ala-D-Ala)- >>6000
Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys-NH.sub.2 .sup.aDap,
L-2,3-diaminopropionic acid; Nal, L-.beta.-naphthylalanine; Pip,
L-pipecolic acid; Sar, sarcosine; Tm, trimesic acid. For FA
analysis, all peptides were labeled at the C-terminal lysine
side-chain with FITC.
[0300] In conclusion, a potentially general approach to designing
cell-permeable bicyclic peptides against intracellular targets was
developed. These preliminary studies show that replacement of the
PTP1B-binding motif with other peptide sequences of different
physicochemical properties also resulted in their efficient
delivery into cultured mammalian cells. The availability of a
general intracellular delivery method should greatly expand the
utility of cyclic peptides in drug discovery and biomedical
research.
Example 4
[0301] Also discussed herein are the CPP sequences in Table 16. All
uptake/delivery efficiencies are in Table 17 are relative to that
of cF.PHI.R.sub.4 (290-1F, 100%). SUV1 are small unilamellar
vesicles that mimic the neutral outer membrane of mammalian cells
[45% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE),
20% sphingomyelin (SM), and 15% cholesterol (CHO)]. SUV2 are small
unilamellar vesicles that mimic the negatively charged endosomal
membrane of mammalian cells [50% PC, 20% PE, 10%
phosphatidylinositol (PI), and 20%
bis(monoacylglycerol)phosphate].
[0302] Measurements were carried out fluorescence polarization
using FITC-labeled cyclic peptides against increasing
concentrations of vesicles. Experiments were performed at pH 7.4
and 5.5 (pH inside late endosomes).
[0303] The overall delivery efficiency appears to correlate with
the CPPs' binding affinity to the endosomal membrane at pH 7.4.
i.e., tighter binding leads to higher delivery efficiency.
TABLE-US-00016 TABLE 16 Cyclic CPPs and their cellular uptake and
membrane binding properties. Uptake Membrane Binding K.sub.D (mM)
SEQ CPP Efficiency SUV1 SUV1 SUV2 SUV2 ID NO Sequence (%) pH 7.4 pH
5.5 pH 7.4 pH 5.5 290-1F 186 c(F.PHI.RRRRQ) 100 4 1.1 0.66 0.63
290-12F 187 c(Ff.PHI.RrRrQ) 681 0.8 0.026 0.004 290-9F 188
c(f.PHI.RrRrQ) 602 1.2 0.81 0.033 0.012 290-11F 189 c(f.PHI.RrRrRQ)
542 2.7 0.092 0.019 290-18F 190 c(F.PHI.rRrRq) 205 0.75 0.04 0.022
290-13F 191 c(F.PHI.rRrRQ) 200 0.68 0.28 0.04 290-6F 192
c(F.PHI.RRRRRQ) 184 2.2 0.12 0.019 290-3F 193 c(RRFR.PHI.RQ) 163
0.22 0.4 0.26 290-7F 194 c(FF.PHI.RRRRQ) 134 1.65 0.11 0.007 290-8F
195 c(RFRFR.PHI.RQ) 98 0.4 0.39 0.082 290-5F 196 c(F.PHI.RRRQ) 97
10.1 2.4 0.066 290-4F 197 c(FRRRR.PHI.Q) 59 7.24 0.54 0.11 290-10F
198 c(rRFR.PHI.RQ) 52 1.2 0.87 0.17 290-2F 199 c(RR.PHI.FRRQ) 47
1.95 0.69 0.025 Tat 32 too weak too weak 3.3 4.5 R.sub.9 35 too
weak too weak 0.47 0.03 .PHI. = L-naphthylalanine; .PHI. =
D-naphthylalanine; f = D-phenylalanine; r = D-arginine; q =
D-glutamine
Example 5
[0304] Cardiomyocytes are in general difficult to transfect with
DNA and delivering proteins into them by using previous CPPs have
not been successful. There is therefore an unmet need for
delivering therapeutic proteins into heart tissues.
[0305] The disclosed cyclic CPPs are very effective in delivering
proteins into cardiomyocytes. Fluorescein isothiocyanate
(FITC)-labeled cyclic CPPs [c(F.PHI.RRRRQ)-K(FITC)-NH.sub.2 and
c(f.PHI.RrRrQ)-K(FITC)-NH.sub.2] were synthesized and their
internalization into mouse ventricular cardiac myocytes was tested
by treating the cells with 5 .mu.M FITC-labeled peptide for 3 h.
After washing away the extracellular peptides, the internalization
of CPPs was examined by fluorescent live-cell confocal microscopy.
Both peptides exhibited significant and predominantly diffused
fluorescence throughout the cells, indicating efficient
internalization of the CPPs into cardiac muscle cells (FIGS. 35a
and 35b). Whether the cyclic CPPs are capable of transporting
full-length proteins into cardiac muscle cells was tested.
Calmodulin (with an engineered Thr5Cys), a multifunctional
calcium-binding messenger protein, was conjugated to
c(F.PHI.RRRRQ)-C--NH.sub.2 at the Cys residue near N-terminus
through a disulfide bond. The disulfide exchange reaction is highly
specific, efficient, and reversible. Further, upon entering the
cytosol of cells, the disulfide linkage is expected to be reduced
to release the native protein (FIG. 35c). The CPP-protein conjugate
was chemically labeled on amino-groups with cyanine3, which permits
visualization of the internalized calmodulin. Mouse ventricular
cardiac myocytes were incubated with 6 .mu.M of the CPP-calmodulin
conjugate for 3 h, and examined by live-cell confocal microscopy.
The intracellular fluorescence signal was present throughout the
entire cell volume and displayed a sarcomeric pattern (FIG. 35d),
indicating the internalized calmodulin was properly integrated into
the cellular machinery. These data indicate that the disclosed
cyclic CPPs such as c(F.PHI.RRRRQ) are uniquely capable of
delivering small molecules as well as proteins (likely in their
native form) into cardiomyocytes with high efficiency, opening the
door to future therapeutic applications.
Example 6
[0306] Pin1 is a phosphorylation-dependent peptidyl-prolyl
cis/trans isomerase (PPIase). It contains an N-terminal WW domain
and a C-terminal catalytic domain, both of which recognize specific
phosphoserine (pSer)/phos-phothreonine (pThr)-Pro motifs in their
protein substrates. Through cis-trans isomerization of specific
pSer/pThr-Pro bonds, Pin1 regulates the levels, activities, as well
as intracellular localization of a wide variety of phosphoproteins.
For example, Pin1 controls the in vivo stability of cyclin D1 and
cyclin E and switches c-Jun, c-Fos, and NF-.kappa.B between their
inactive unstable forms and active stable forms. Isomerization by
Pin1 also regulates the catalytic activity of numerous cell-cycle
signaling proteins such as phosphatase CDC25C and kinase Wee1.
Finally, Pin1-catalyzed conformational changes in .beta.-catenin
and NF-.kappa.B lead to subcellular translocation.
[0307] Given its critical roles in cell-cycle regulation and
increased expression levels and activity in human cancers, Pin1 has
been proposed as a potential target for the development of
anticancer drugs. Pin1 is also implicated in neural degenerative
diseases such as Alzheimer's disease. Therefore, there have been
significant interests in developing specific inhibitors against
Pin1. Small-molecule inhibitors such as Juglone, PiB,
dipentamenthylene thiauram monosulfide and halogenated
phenyl-isothiazolone (TME-001) generally lack sufficient potency
and/or specificity. A number of potent peptidyl Pin1 inhibitors
have been reported and are more selective than the small-molecule
inhibitors. However, peptidyl inhibitors are generally impermeable
to the cell membrane and therefore have limited utility as
therapeutics or in vivo probes. A cell-permeable bicyclic peptidyl
inhibitor against Pin1, in which one ring (A ring) featured a
Pin1-binding phosphopeptide motif [D-pThr-Pip-Nal, where Pip and
Nal are (R)-piperidine-2-carboxylic acid and L-naphthylalanine,
respectively] while the second ring (B ring) contained a
cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg is shown in FIG.
36, peptide 1. Although the bicyclic peptidyl inhibitor is potent
(K.sub.D=72 nM) and active in cellular assays, its D-pThr moiety
might be metabolically labile due to hydrolysis by nonspecific
phosphatases. The negative charges of the phosphate group might
also impede the cellular entry of the inhibitor. Here a
non-phosphorylated bicyclic peptidyl inhibitor against Pin1 was
prepared by screening a peptide library and hit optimization. The
resulting bicyclic peptidyl inhibitor is potent and selective
against Pin1 in vitro, cell-permeable, and metabolically stable in
biological assays.
[0308] Although removal of the phosphoryl group of peptide 1
significantly reduced its potency against Pin1, the
nonphosphorylated peptide (FIG. 36, peptide 2) was still a
relatively potent Pin1 inhibitor (K.sub.D=0.62 .mu.M). The potency
of peptide 2 might be further improved by optimizing the sequences
flanking the D-Thr-Pip-Nal motif. So a second-generation bicyclic
peptide library,
bicyclo[Tm-(X.sup.1X.sup.2X.sup.3-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
-Arg-Arg-Arg-Dap)]-.beta.-Ala-.beta.-Ala-Pra-.beta.-Ala-Hmb-.beta.-Ala-.be-
ta.-Ala-Met-resin (FIG. 35, where Tm was trimesic acid, Dap was
2,3-diaminopropionic acid, .beta.-Ala was .beta.-alanine, Pra was
L-propargylglycine, and Hmb was 4-hydroxymethyl benzoic acid), by
randomizing the three N-terminal residues of peptide 2. X.sup.1 and
X.sup.2 represented any of the 27 amino acid building blocks that
included 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His,
Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5 nonproteinogenic
.alpha.-L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine
(Nle), L-ornithine (Om), L-phenylglycine (Phg), and L-Nal],
6.alpha.-D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and
D-Val], and 4 N.sup..alpha.-methylated L-amino acids
[L-N.sup..alpha.-methylalanine (Mal), L-N.sup..alpha.-methyleucine
(Mle), L-N.sup..alpha.-methylphenylalanine (Mpa), and sarcosine
(Sar)], while X.sup.3 was Asp, Glu, D-Asp, D-Glu, or D-Thr.
Incorporation of these nonproteinogenic amino acids was expected to
increase both the structural diversity and the proteolytic
stability of the library peptides. The library had a theoretical
diversity of 5.times.27.times.27 or 3645 different bicyclic
peptides, most (if not all) of which were expected to be
cell-permeable. The library was synthesized on 500 mg of TentaGel
microbeads (130 .mu.m, .about.7.8.times.10.sup.5 beads/g,
.about.350 pmol peptides/bead). Peptide cyclization was achieved by
forming three amide bonds between Tm and the N-terminal amine and
the sidechain amines of the two Dap residues.
TABLE-US-00017 TABLE 17 Hit Sequences from Peptide Library
Screening.sup.a hit X.sup.1 X.sup.2 X.sup.3 1 Pro Sar D-Asp 2 Pro
Sar D-Asp 3 D-Phe Fpa D-Thr 4 His Phg D-Thr 5 Mpa Ile D-Glu 6 Phg
His D-Glu 7 Mpa Gly D-Thr
[0309] The .beta.-Ala provides a flexible linker, while Pra serves
as a handle for on-bead labeling of the bicyclic peptides with
fluorescent probes through click chemistry. The ester linkage of
Hmb enables selective release of the bicyclic peptides from the
resin for solution-phase binding analysis. Finally, the C-terminal
Met allows peptide release from the resin by CNBr cleavage prior to
MS analysis. .sup.aHits 1-3 were selected from 1.sup.st-round
screening, whereas hits 4-7 were selected after 2.sup.nd-round
screening.
[0310] The library (100 mg of resin) was screened against a
S16A/Y23A mutant Pin1, which has a defective WW domain. The mutant
Pin1 was produced as a maltose-binding protein (MBP) fusion at the
N-terminus. During the first round of screening, Texas red-labeled
MBP-Pin1 was incubated with the peptide library and fluorescent
beads were removed from the library under a microscope. Three
positive beads had substantially greater fluorescence intensities
than the rest of hits and were directly subjected to peptide
sequencing by partial Edman degradation mass spectroscopy (PED-MS)
(Table 17). The other 13 fluorescent beads were subjected to a
second round of screening, during which the bicyclic peptide on
each bead was labeled with tetramethylrhodamine (TMR) azide at the
Pra residue and released from the bead by treatment with a NaOH
solution.
TABLE-US-00018 TABLE 18 Sequences and Pin1 Binding Affinities of
Peptides Used SEQ ID K.sub.D NO Peptide Peptide sequence (.mu.M)
200 1
bicyclo[Tm-(D-Ala-Sar-D-pThr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-
0.072 .+-. 0.021 Arg-Arg-Arg-Arg-Dap)]-Lys 201 2
bicyclo[Tm-(D-Ala-Sar-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
0.62 .+-. 0.12 Arg-Arg-Arg-Dap)]-Lys 202 3
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
0.87 .+-. 0.17 Arg-Arg-Arg-Dap)]-Lys 203 4
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-
0.67 .+-. 0.12 Arg-Arg-Arg-Arg-Dap)]-Lys 204 5
bicyclo[Tm-(Mpa-Gly-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
1.08 .+-. 0.12 Arg-Arg-Arg-Dap)]-Lys 205 6
bicyclo[Tm-(Phg-His-D-Glu-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
1.47 .+-. 0.19 Arg-Arg-Arg-Dap)]-Lys 206 7
bicyclo[Tm-(Mpa-Ile-D-Glu-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
1.25 .+-. 0.20 Arg-Arg-Arg-Dap)]-Lys 207 8
bicyclo[Tm-(His-Phg-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-
1.40 .+-. 0.24 Arg-Arg-Arg-Dap)]-Lys 208 9
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-
2.59 .+-. 0.37 Arg-Dap)]-Lys 209 10
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg)-Dap-(Phe-Nal-Arg-Arg-Arg-
3.42 .+-. 0.61 Arg-Dap)]-Lys 210 11
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala)-Dap-(Phe-Nal-Arg-Arg-
0.90 .+-. 0.25 Arg-Arg-Dap)]-Lys 211 12
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg)-Dap-(Phe-Nal-Arg-Arg-Arg-
2.36 .+-. 0.48 Arg-Dap)]-Lys 212 13
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala-.beta.-Ala)-Dap-(Phe-Nal--
Arg- 2.08 .+-. 0.31 Arg-Arg-Arg-Dap)]-Lys 213 14
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-.beta.-Ala-D-Ala)-Dap-(Phe-Na-
l-Arg- 1.75 .+-. 0.18 Arg-Arg-Arg-Dap)]-Lys 214 15
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-.beta.-Ala-.beta.-Ala)-Dap-(P-
he-Nal-Arg- 4.83 .+-. 0.96 Arg-Arg-Arg-Dap)]-Lys 215 16
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala-D-Ala-D-Ala)-Dap-(Phe-Nal-
- 2.49 .+-. 0.57 Arg-Arg-Arg-Arg-Dap)]-Lys 216 17
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Tyr-D-Ala)-Dap-(Phe-Nal-Arg-
2.17 .+-. 0.55 Arg-Arg-Arg-Dap)]-Lys 217 18
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Val-D-Ala)-Dap-(Phe-Nal-Arg-
1.75 .+-. 0.24 Arg-Arg-Arg-Dap)]-Lys 218 19
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Ala)-Dap-(Phe-Nal-Arg-
0.72 .+-. 0.09 Arg-Arg-Arg-Dap)]-Lys 219 20
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Asp-D-Ala)-Dap-(Phe-Nal-Arg-
3.19 .+-. 0.50 Arg-Arg-Arg-Dap)]-Lys 220 21
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ser-D-Phe)-Dap-(Phe-Nal-
0.57 .+-. 0.11 Arg-Arg-Arg-Arg-Dap)]-Lys 221 22
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Phe)-Dap-(Phe-Nal-Arg-
0.48 .+-. 0.07 Arg-Arg-Arg-Dap)]-Lys 222 23
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Val)-Dap-(Phe-Nal-Arg-
1.92 .+-. 0.19 Arg-Arg-Arg-Dap)]-Lys 223 24
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Arg)-Dap-(Phe-Nal-Arg-
1.31 .+-. 0.10 Arg-Arg-Arg-Dap)]-Lys 224 25
bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Asp)-Dap-(Phe-Nal-Arg-
4.60 .+-. 1.42 Arg-Arg-Arg-Dap)]-Lys 225 26
bicyclo[Tm-(D-Phe-4-Fpa-D-Thr-Pip-Nal-Arg-Gly-D-Ala)-Dap-(Phe-Nal-
0.74 .+-. 0.11 Arg-Arg-Arg-Arg-Dap)]-Lys 226 27
bicyclo[Tm-(D-Phe-4-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
0.27 .+-. 0.08 Arg-Arg-Arg-Arg-Dap)]-Lys 227 28
bicyclo[Tm-(D-Phe-Phe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
1.26 .+-. 0.28 Arg-Arg-Arg-Arg-Dap)]-Lys 228 29
bicyclo[Tm-(D-Phe-3,4-diFPhe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-
0.41 .+-. 0.10 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 229 30
bicyclo[Tm-(D-Phe-4-ClPhe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-
0.78 .+-. 0.05 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 230 31
bicyclo[Tm-(D-Phe-His-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
1.68 .+-. 0.17 Arg-Arg-Arg-Arg-Dap)]-Lys 231 32
bicyclo[Tm-(D-Phe-4-BrPhe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-
1.78 .+-. 0.42 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 232 33
bicyclo[Tm-(D-Ala-4-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-
1.49 .+-. 0.11 Arg-Arg-Arg-Arg-Dap)]-Lys 233 34
bicyclo[Tm-(D-Val-4-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-
1.07 .+-. 0.16 Arg-Arg-Arg-Arg-Dap)]-Lys 234 35
bicyclo[Tm-(D-2-Fpa-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
0.59 .+-. 0.05 Arg-Arg-Arg-Arg-Dap)]-Lys 235 36
bicyclo[Tm-(D-3-Fpa-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
0.39 .+-. 0.05 Arg-Arg-Arg-Arg-Dap)]-Lys 236 37
bicyclo[Tm-(D-4-Fpa-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
0.12 .+-. 0.03 Arg-Arg-Arg-Arg-Dap)]-Lys 237 38
bicyclo[Tm-(D-4-CyanoPhe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-
0.35 .+-. 0.04 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 238 39
bicyclo[Tm-(D-4-Phe-Fpa-D-Ile-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
0.46 .+-. 0.17 Arg-Arg-Arg-Arg-Dap)]-Lys 239 40
bicyclo[Tm-(D-4-Phe-Fpa-D-Nle-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-
0.71 .+-. 0.12 Arg-Arg-Arg-Arg-Dap)]-Lys 240 41
bicyclo[Tm-(D-4-Phe-Fpa-D-homoGlu-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-
0.87 .+-. 0.08 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 241 42
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Arg-Arg-
0.98 .+-. 0.18 Arg-Arg-Nal-Phe-Dap)]-Lys 242 43
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Arg-Arg-
1.38 .+-. 0.16 Nal-Phe-Arg-Arg-Dap)]-Lys 243 44
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Arg-Nal-
0.45 .+-. 0.05 Arg-Phe-Arg-Arg-Dap)]-Lys 244 45
bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(D-Arg-Arg-
3.10 .+-. 0.38 D-Arg-Arg-Nal-D-Phe-Dap)]-Lys 245 46
cyclo(D-Ala-Sar-D-pThr-Pip-Nal-Tyr-Gln)-Lys 0.24 .+-. 0.04 246 47
bicyclo[Tm-(D-Ala-Sar-D-Thr-D-Ala-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-
No Arg-Arg-Arg-Arg-Dap)]-Lys binding
[0311] The released peptides were incubated with 5 .mu.M MBP-Pin1
and the increase of fluorescence anisotropy (FA) was measured. For
bicyclic peptides that showed .gtoreq.50% FA increase (relative to
the no-protein control), the corresponding beads (5 beads, which
still contained the linear encoding peptides) were sequenced by
PED-MS to give 4 additional complete sequences (Table 1). All 7 hit
sequences contained a D-amino acid at the X.sup.3 position,
consistent with the previous observation that Pin1 prefers D-pThr
over pThr at this position. There is a strong preference for
hydrophobic especially aromatic hydrophobic residues at the X.sup.1
position, but no obvious selectivity at the X.sup.2 position.
[0312] Hit Optimization. The 6 hit sequences (hits 1 and 2 have the
same sequence) were resynthesized with a Lys added to their
C-termini, labeled with fluorescein isothiocyanate (FITC), and
tested for binding to Pin1 by FA (Table 18, peptides 3-8). All six
peptides bound to Pin1 with moderate affinities (K.sub.D.about.1
.mu.M), but did not improve upon peptide 2 (K.sub.D=0.62 .mu.M).
Peptides 3 and 4 were used for structure-activity relationship
analysis and optimization. Either expanding or contracting the size
of the Pin1-binding ring (A ring) decreased the binding affinity
(Table 18, peptides 9-16). Replacement of the Ala residue of
peptide 3 with amino acids containing side chains of different
physicochemical properties including Arg, Asp, Ser, Tyr, and Val
also failed to significantly improve the binding affinity (Table
18, peptide 17-21). On the other hand, modification of the D-Ala
residue revealed that substitution of a D-Phe at this position
increases the Pin1 inhibitory activity by .about.2-fold
(K.sub.D=0.48 .mu.M for peptide 22).
[0313] Peptide 4 was subjected to similar SAR studies. As observed
for peptide 3, modification of the Ala residue of peptide 4 (into
Gly) had little effect (peptide 26), but replacement of the D-Ala
residue with D-Phe improved the binding affinity to Pin1 by
.about.2-fold (Table 18, K.sub.D=0.27 .mu.M for peptide 27).
Modifications of the Fpa residue at the X.sup.2 position (e.g.,
replacement with other halogenated phenylalanine analogs) all
decreased the inhibitor potency (peptides 28-32). Likewise, removal
of the aromatic side chain at the X.sup.1 position was detrimental
to Pin1 binding (peptides 33 and 34). However, substitution of
halogenated D-Phe analogs improved the Pin1 binding activity
(peptides 35-38). In particular, replacement of D-Phe with
D-4-fluorophenylalanine (D-Fpa)] resulted in the most potent Pin1
inhibitor of this series (K.sub.D=0.12 .mu.M for peptide 37) (FIGS.
36 and 37a). Further attempts to modify the D-Thr residue or the
CPP motif failed to improve the Pin1 activity (Table 18, peptides
39-45).
[0314] Biological Evaluation. To determine whether peptide 37 binds
to the catalytic site of Pin1, its ability to compete with peptide
1 for binding to Pin1 by FA analysis was examined. Peptide 1 had
previously been shown to bind to the Pin1 active site. As expected,
peptide 37 inhibited the binding of peptide 1 to Pin1 with an
IC.sub.50 value of 190 nM (FIG. 37b). Next, the catalytic activity
of Pin1 toward a peptide substrate, Suc-Ala-Glu-Pro-Phe-pNA, in the
presence of increasing concentrations of peptide 37 was monitored.
Peptide 37 inhibited the Pin1 activity in a concentration-dependent
manner, with an IC.sub.50 value of 170 nM (FIG. 37c). These results
demonstrate that peptide 37 binds at (or near) the active site of
Pin1.
[0315] The selectivity of peptide 37 was assessed by two different
tests. First, peptide 37 was tested for binding to a panel of
arbitrarily selected proteins including bovine serum albumin (BSA),
protein tyrosine phosphatases 1B, SHPT, and SHP2, the Grb2 SH2
domain, Ras, and tumor necrosis factor-.alpha.. Peptide 37 bound
weakly to BSA (K.sub.D.about.20 .mu.M), but not any of the other
six proteins. Peptide 37 was next tested for potential inhibition
of Pin4, FKBP12, and cyclophilin A, the three other common human
peptidyl-prolyl cis-trans isomerases. Although Pin4 is structurally
similar to Pin1 and has partially overlapping functions with Pin1,
peptide 37 only slightly inhibited Pin4 (.about.15% at 5 .mu.M
inhibitor), with an estimated IC.sub.50 value of .about.34 .mu.M
(FIG. 37c). Peptide 37 had no effect on the catalytic activity of
FKBP12 or cyclophilin A up to 5 .mu.M concentration. These data
suggest that peptide 37 is a highly specific inhibitor of Pin1.
[0316] The metabolic stability of peptide 37 was evaluated by
incubating it in human serum for varying periods of time and
analyzing the reaction mixtures by reversed-phase HPLC. The
pThr-containing Pin1 inhibitor 1 was used as a control. After 6 h
of incubation, 97% of peptide 37 remained intact, while .about.50%
of bicyclic peptide 1 was degraded after 3 h (FIG. 37d). Loss of
peptide 1 was accompanied by the concomitant appearance of a new
peak in HPLC. Mass spectrometric analysis of the new species
identified it as the dephosphorylation product of peptide 1
(peptide 2). This result is in agreement with our previous
observation that the structurally constrained bicyclic peptides are
highly resistant to proteolytic degradation. The D-pThr moiety
remains susceptible to hydrolysis by the nonspecific phosphatases
in human serum.
[0317] The cellular uptake efficiency of peptide 37, peptide 1, and
a previously reported membrane-impermeable monocyclic Pin1
inhibitor (Table 18, peptide 46) was assessed by incubating HeLa
cells with the FITC-labeled peptides (5 .mu.M) for 2 h and
quantifying the total intracellular fluorescence by flow cytometry
analysis. As expected, untreated cells and cells treated with
peptide 46 showed little cellular fluorescence, having mean
fluorescence intensity (MFI) values of 101 and 193, respectively
(FIG. 38a). By contrast, cells treated with peptides 1 and 37 gave
MFI values of 2562 and 8792, respectively. Thus, peptide 37 is
internalized by HeLa cells .about.4-fold more efficiently than
peptide 1. Presumably, the negative charged phosphate group of
peptide 1 interacted electrostatically with the positively charged
CPP motif and reduced the cellular uptake efficiency of the
latter.
[0318] Inhibition of Pin1 activity has previously been shown to
decrease cell proliferation. The effect of peptide 37 on the growth
of HeLa cells was examined by using the MTT cell viability assay.
The membrane impermeable peptide 46 and a cell-permeable but
inactive (defective in Pin1 binding) bicyclic peptide (Table 18,
peptide 47) were used as controls. Peptide 37 inhibited HeLa cell
growth in a concentration-dependent manner, with an IC.sub.50 value
of 1.0 .mu.M (FIG. 38b). As expected, neither peptide 46 nor 47 had
any effect on cell growth. A time-course study also showed
significant growth inhibition (>60%) after a 3-day treatment
with 5 .mu.M peptide 37, but not with peptide 46 or 47. The
phosphorylated bicyclic peptide 1 under similar testing conditions
had an IC.sub.50 value of 1.8 .mu.M.
[0319] Finally, to ascertain that Pin1 is the molecular target of
peptide 37 in vivo, the intracellular protein level of a
well-established Pin1 substrate, promyeloretinoic leukemia protein
(PML), was examined by western blot analysis. Pin1 negatively
regulates the PML level in a phosphorylation-dependent manner and
inhibition of Pin1 activity is expected to stabilize PML and
increase its intracellular level. Indeed, treatment of HeLa cells
with peptide 37 (0.2-5 .mu.M) resulted in concentration-dependent
increases in the PML level (FIG. 38c,d). The effect was already
significant at 0.2 .mu.M inhibitor (1.8-fold increase in the PML
level) and plateaued at .about.1 .mu.M (3.3-fold increase). Again,
bicyclic peptide 47 had no effect under the same conditions, while
peptide 1 (the positive control, at 5 .mu.M) increased the PML
level by 3.1-fold.
[0320] By screening a peptide library followed by conventional
medicinal chemistry approaches, the first potent, selective,
metabolically stable, and cell-permeable peptidyl inhibitor against
human Pin1 has been disclosed. Its high potency and selectivity
should make it a useful chemical probe for exploring the cellular
functions of Pin1.
[0321] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0322] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
25916PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 1Phe Xaa Arg Arg Arg Gln1
526PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 2Phe Xaa Arg Arg Arg Cys1
536PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanineSITE(6)..(6)selenocysteine
3Phe Xaa Arg Arg Arg Xaa1 546PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-napthylalanine 4Arg Arg Arg Xaa Phe Gln1
556PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-napthylalanine 5Arg Arg Arg Arg Xaa Phe1
566PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 6Phe Xaa Arg Arg Arg Arg1
577PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-napthylalanine 7Phe Xaa Arg Arg Arg Arg Gln1
587PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-napthylalanine 8Phe Xaa Arg Arg Arg Arg Gln1
597PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 9Phe Xaa Arg Arg Arg Arg Gln1
5107PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 10Phe Xaa Arg Arg Arg Arg
Gln1 5117PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-napthylalanine 11Arg Arg Phe Arg Xaa Arg
Gln1 5127PRTArtificial SequenceSynthetic
ConstructSITE(6)..(6)L-napthylalanine 12Phe Arg Arg Arg Arg Xaa
Gln1 5137PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-napthylalanine 13Arg Arg Phe Arg Xaa Arg
Gln1 5147PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-napthylalanine 14Arg Arg Xaa Phe Arg Arg
Gln1 5158PRTArtificial SequenceSynthetic Construct 15Cys Arg Arg
Arg Arg Phe Trp Gln1 5168PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-napthylalanine 16Phe Phe Xaa Arg Arg Arg Arg
Gln1 5178PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-napthylalanine 17Phe Phe Xaa Arg Arg Arg Arg
Gln1 5188PRTArtificial SequenceSynthetic
ConstructSITE(6)..(6)L-napthylalanine 18Arg Phe Arg Phe Arg Xaa Arg
Gln1 5198PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)selenocysteine 19Xaa Arg Arg Arg Arg Phe Trp
Gln1 5208PRTArtificial SequenceSynthetic Construct 20Cys Arg Arg
Arg Arg Phe Trp Gln1 5218PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 21Phe Xaa Arg Arg Arg Arg Gln
Lys1 5228PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 22Phe Xaa Arg Arg Arg Arg Gln
Cys1 5238PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 23Phe Xaa Arg Arg Arg Arg Arg
Gln1 5248PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-napthylalanine 24Phe Xaa Arg Arg Arg Arg Arg
Gln1 5259PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-napthylalanineSITE(8)..(8)L-norleucine 25Arg
Arg Arg Arg Xaa Phe Asp Xaa Cys1 5265PRTArtificial
SequenceSynthetic ConstructSITE(2)..(2)L-napthylalanine 26Phe Xaa
Arg Arg Arg1 5275PRTArtificial SequenceSynthetic Construct 27Phe
Trp Arg Arg Arg1 5285PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-napthylalanine 28Arg Arg Arg Xaa Phe1
5295PRTArtificial SequenceSynthetic Construct 29Arg Arg Arg Trp
Phe1 5305PRTArtificial SequenceSynthetic Construct 30Arg Arg Arg
Arg Arg1 5315PRTArtificial SequenceSynthetic Construct 31Ala Ala
Ala Ala Ala1 5324PRTArtificial SequenceSynthetic Construct 32Phe
Phe Phe Phe1335PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)phosphocoumaryl amino propionic acid 33Asp Glu
Xaa Leu Ile1 5347PRTArtificial SequenceSynthetic Construct 34Ala
Ala Ala Ala Ala Ala Ala1 5355PRTArtificial SequenceSynthetic
Construct 35Arg Ala Arg Ala Arg1 5365PRTArtificial
SequenceSynthetic Construct 36Asp Ala Asp Ala Asp1
5374PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)norleucineSITE(3)..(3)selenocysteine 37Asp Xaa
Xaa Asp1384PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)2-aminobutyric acid 38Xaa Thr Arg
Val1396PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 39Pro Xaa Gly Xaa Tyr Arg1 5406PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanineSITE(5)..(5)L-4-(phosphonodifluoromethyl)phenylalanine
40Ser Xaa Ile Xaa Xaa Arg1 5416PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
41Ile His Ile Xaa Ile Arg1 5426PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(5)..(-
5)L-homoproline 42Ala Ala Ile Xaa Xaa Arg1 5436PRTArtificial
SequenceSynthetic
ConstructSITE(1)..(1)L-4-fluorophenylalanineSITE(3)..(3)L-homoprolineSITE-
(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine 43Xaa Ser Xaa
Xaa Val Arg1 5446PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 44Xaa Asn Pro Xaa Ala Arg1 5456PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 45Thr Xaa Ala Xaa Gly Arg1 5466PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
46Ala His Ile Xaa Ala Arg1 5476PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
47Gly Asn Gly Xaa Pro Arg1 5486PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 48Phe Gln Xaa Xaa Ile Arg1 5496PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
49Ser Pro Gly Xaa His Arg1 5506PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 50Xaa Tyr Ile Xaa His Arg1 5516PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
51Ser Val Pro Xaa His Arg1 5526PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
52Ala Ile Pro Xaa Asn Arg1 5536PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-4-fluorophenylalanineSITE(4)..(4)L-4-(phosphonodif-
luoromethyl)phenylalanine 53Xaa Ser Ile Xaa Gln Phe1
5546PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 54Ala Ala Xaa Xaa Phe Arg1 5556PRTArtificial
SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-phenylglycine 55Asn Thr Xaa Xaa Xaa
Arg1 5566PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-norleucine 56Ile Pro Xaa Xaa Xaa
Arg1 5576PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(3)..(3)L-4-fluorophenylalanineSITE-
(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(5)..(5)L-homoprolin-
e 57Gln Xaa Xaa Xaa Xaa Arg1 5586PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-4-fluorophenylalanineSITE(4)..(4)L-4-(phosphonodif-
luoromethyl)phenylalanine 58Asn Ala Xaa Xaa Gly Arg1
5596PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
59Asn Thr Tyr Xaa Ala Arg1 5606PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 60Glu Ala Xaa Xaa Val Arg1 5616PRTArtificial
SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 61Ile Val Xaa Xaa Ala Arg1 5626PRTArtificial
SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 62Tyr Thr Xaa Xaa Ala Arg1 5636PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(3)..(3)L-phenylglycineSITE(4)..(4)-
L-4-(phosphonodifluoromethyl)phenylalanine 63Asn Xaa Xaa Xaa Ile
Arg1 5646PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 64Xaa Asn Trp Xaa His Arg1 5656PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 65Tyr Xaa Val Xaa Ile Arg1 5666PRTArtificial
SequenceSynthetic ConstructSITE(4)..(4)X4 =
L-4-(phosphonodifluoromethyl)phenylalanineSITE(4)..(4)L-4-(phosphonodiflu-
oromethyl)phenylalanine 66Asn Ser Ala Xaa Gly Arg1
5676PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
67Thr Asn Val Xaa Ala Arg1 5686PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
68Asn Thr Val Xaa Thr Arg1 5696PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
69Ser Ile Thr Xaa Tyr Arg1 5706PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-4-fluorophenylalanineSITE(4)..(4)L-4-(phosphonodif-
luoromethyl)phenylalanine 70Asn Xaa Asn Xaa Leu Arg1
5716PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(5)..(-
5)L-norleucine 71Tyr Asn Asn Xaa Xaa Arg1 5726PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
72Asn Tyr Asn Xaa Gly Arg1 5736PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
73Ala Trp Asn Xaa Ala Arg1 5746PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
74Val Thr His Xaa Tyr Arg1 5756PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-homoproline 75Pro Xaa His Xaa Xaa
Arg1 5766PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 76Asn Xaa His Xaa Gly Arg1 5776PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
77Pro Ala His Xaa Gly Arg1 5786PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
78Ala Tyr His Xaa Ile Arg1 5796PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 79Asn Xaa Glu Xaa Tyr Arg1 5806PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
80Val Ser Ser Xaa Thr Arg1 5818PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)sarcosineSITE(3)..(3)D-phosphothreonineSITE(4)..(4)L-
-pipecolic acidSITE(5)..(5)L-beta-naphthylalanine 81Ala Xaa Xaa Xaa
Xaa Tyr Asn Lys1 5829PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-pipe-
colic
acidSITE(6)..(6)L-beta-naphthylalanineSITE(9)..(9)L-2,3-diaminopropi-
onic acid 82Xaa Ala Xaa Xaa Xaa Xaa Arg Ala Xaa1 58310PRTArtificial
SequenceSynthetic ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-pipe-
colic
acidSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopro-
pionic acid 83Xaa Ala Xaa Xaa Xaa Xaa Arg Ala Ala Xaa1 5
108410PRTArtificial SequenceSynthetic ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-pipecolic
acidSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropioni-
c acid 84Xaa Ala Xaa Thr Xaa Xaa Arg Ala Ala Xaa1 5
108510PRTArtificial SequenceSynthetic ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)-
L-2,3-diaminopropionic acid 85Xaa Ala Xaa Thr Ala Xaa Arg Ala Ala
Xaa1 5 10868PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanineMOD_RES(8)..(8)Lys with
conjugated rhodamine B 86Phe Xaa Arg Arg Arg Arg Gln Lys1
5879PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanineSITE(9)..(9)dexamethasone
87Phe Xaa Arg Arg Arg Arg Gln Lys Xaa1 58816PRTArtificial
SequenceSynthetic ConstructSITE(2)..(2)dexamethasone 88Lys Xaa Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Tyr1 5 10
158912PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanine 89Phe Xaa Arg Arg Arg Arg
Gln Lys Phe Ile Thr Cys1 5 109013PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanineMOD_RES(13)..(13)Lys with
conjugated rhodamine B 90Phe Xaa Arg Arg Arg Arg Gln Arg Arg Arg
Arg Arg Lys1 5 109113PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanineMOD_RES(13)..(13)Lys with
conjugated rhodamine B 91Phe Xaa Arg Arg Arg Arg Gln Ala Ala Ala
Ala Ala Lys1 5 109212PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanineMOD_RES(12)..(12)Lys with
conjugated rhodamine B 92Phe Xaa Arg Arg Arg Arg Gln Phe Phe Phe
Phe Lys1 5 109313PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanineSITE(8)..(8)8-amino-3,6-dioxaocta-
noic acidSITE(11)..(11)phosphocoumaryl amino propionic acid 93Phe
Xaa Arg Arg Arg Arg Gln Xaa Asp Glu Xaa Leu Ile1 5
109415PRTArtificial SequenceSynthetic
ConstructSITE(10)..(10)8-amino-3,6-dioxaoctanoic
acidSITE(13)..(13)phosphocoumaryl amino propionic acid 94Arg Arg
Arg Arg Arg Arg Arg Arg Arg Xaa Asp Glu Xaa Leu Ile1 5 10
159515PRTArtificial SequenceSynthetic
ConstructSITE(10)..(10)8-amino-3,6-dioxaoctanoic
acidSITE(13)..(13)phosphocoumaryl amino propionic acid 95Arg Lys
Lys Arg Arg Gln Arg Arg Arg Xaa Asp Glu Xaa Leu Ile1 5 10
159622PRTArtificial SequenceSynthetic
ConstructSITE(17)..(17)8-amino-3,6-dioxaoctanoic
acidSITE(20)..(20)phosphocoumaryl amino propionic acid 96Arg Gln
Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 15Xaa
Asp Glu Xaa Leu Ile 209715PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(12)..(12)L-2-naphthylalanineSITE(14)..(14)L-2,3-diaminopropionic
acidMOD_RES(15)..(15)Lys with conjugated rhodamine B 97Xaa Ala Ala
Ala Ala Ala Lys Arg Arg Arg Arg Xaa Phe Xaa Lys1 5 10
159817PRTArtificial SequenceSynthetic ConstructSITE(1)..(1)trimesic
acidSITE(14)..(14)L-2-naphthylalanineSITE(16)..(16)L-2,3-diaminopropionic
acidMOD_RES(17)..(17)Lys with conjugated rhodamine B 98Xaa Ala Ala
Ala Ala Ala Ala Ala Lys Arg Arg Arg Arg Xaa Phe Xaa1 5 10
15Lys9914PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic acidSITE(11)..(11)Xaa can be any
naturally occurring amino
acidSITE(12)..(12)L-2-naphthylalanineSITE(13)..(13)Xaa can be any
naturally occurring amino
acidSITE(14)..(14)L-2,3-diaminopropionic acidSITE(15)..(15)Xaa can
be any naturally occurring amino acidMOD_RES(14)..(14)Lys with
conjugated rhodamine B 99Xaa Arg Ala Arg Ala Arg Arg Arg Arg Arg
Xaa Phe Xaa Lys1 5 1010015PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(12)..(12)L-2-naphthylalanineSITE(14)..(14)L-2,3-diaminopropionic
acidMOD_RES(15)..(15)Lys with conjugated rhodamine B 100Xaa Asp Ala
Asp Ala Asp Lys Arg Arg Arg Arg Xaa Phe Xaa Lys1 5 10
1510112PRTArtificial SequenceSynthetic
ConstructSITE(10)..(10)L-2-naphthylalanineMOD_RES(12)..(12)Lys with
conjugated rhodamine B 101Ala Ala Ala Ala Ala Arg Arg Arg Arg Xaa
Phe Lys1 5 1010214PRTArtificial SequenceSynthetic
ConstructSITE(12)..(12)L-2-naphthylalanineMOD_RES(14)..(14)Lys with
conjugated rhodamine B 102Ala Ala Ala Ala Ala Ala Ala Arg Arg Arg
Arg Xaa Phe Lys1 5 101038PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoproline 103Phe Xaa Arg Arg Arg Arg Cys
Lys1 51048PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(7)..(7)selenocysteine 104Phe
Xaa Arg Arg Arg Arg Xaa Lys1 51054PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-norleucineSITE(3)..(3)selenocysteineSITE(5)..(5)7--
amino-4-methylcourmarin 105Asp Xaa Xaa Asp110610PRTArtificial
SequenceSynthetic
ConstructSITE(5)..(5)L-homoprolineSITE(8)..(8)L-norleucine 106Arg
Arg Arg Arg Xaa Phe Asp Xaa Cys Asp1 5 1010710PRTArtificial
SequenceSynthetic
ConstructSITE(5)..(5)L-homoprolineSITE(8)..(8)L-norleucine 107Arg
Arg Arg Arg Xaa Phe Asp Xaa Cys Asp1 5 1010810PRTArtificial
SequenceSynthetic
ConstructSITE(5)..(5)L-homoprolineSITE(8)..(8)L-norleucineSITE(9)..(9)sel-
enocysteine 108Arg Arg Arg Arg Xaa Phe Asp Xaa Xaa Asp1 5
1010914PRTArtificial SequenceSynthetic
ConstructSITE(11)..(11)L-norleucineSITE(12)..(12)selenocysteineSITE(14)..-
(14)7-amino-4-methylcourmarin 109Arg Arg Arg Arg Arg Arg Arg Arg
Arg Asp Xaa Xaa Cys Xaa1 5 1011012PRTArtificial SequenceSynthetic
Construct 110Cys Arg Arg Arg Arg Phe Trp Gln Cys Thr Arg Val1 5
1011112PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)selenocysteineSITE(9)..(9)selenocysteine
111Xaa Arg Arg Arg Arg Phe Trp Gln Xaa Thr Arg Val1 5
101129PRTArtificial SequenceSynthetic Construct 112Cys Arg Arg Arg
Arg Phe Trp Gln Cys1 51139PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanine 113Phe Xaa Arg Arg Arg Arg
Pro Thr Pro1 51149PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-2-naphthylalanine 114Phe Xaa Arg Arg Arg Arg
Pro Cys Pro1 511512PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(10)..-
(10)L-beta-naphthylalanine 115Thr Asn Val Xaa Ala Arg Arg Arg Arg
Xaa Phe Gln1 5 1011612PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(10)..-
(10)L-beta-naphthylalanine 116Ser Val Pro Xaa His Arg Arg Arg Arg
Xaa Phe Gln1 5 1011712PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-norleucineSITE(10)..(10)L-beta-naphthylalan-
ine 117Ile Pro Xaa Xaa Xaa Arg Arg Arg Arg Xaa Phe Gln1 5
101188PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)sarcosineSITE(3)..(3)D-phosphothreonineSITE(4)..(4)L-
-pipecolic acidSITE(5)..(5)L-beta-naphthylalanine 118Ala Xaa Xaa
Xaa Xaa Tyr Gln Lys1 511917PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-pipe-
colic
acidSITE(6)..(6)L-beta-naphthylalanineSITE(9)..(9)L-2,3-diaminopropi-
onic acidSITE(11)..(11)Xaa can be any naturally occurring amino
acidSITE(16)..(16)L-2,3-diaminopropionic acid 119Xaa Ala Xaa Xaa
Xaa Xaa Arg Ala Xaa Phe Xaa Arg Arg Arg Arg Xaa1 5 10
15Lys12019PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-pipe-
colic
acidSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopro-
pionic
acidSITE(13)..(13)L-beta-naphthylalanineSITE(18)..(18)L-2,3-diamino-
propionic acid 120Xaa Ala Xaa Xaa Xaa Xaa Arg Ala Ala Xaa Phe Asn
Xaa Arg Arg Arg1 5 10 15Arg Xaa Lys12118PRTArtificial
SequenceSynthetic ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-pipecolic
acidSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropioni-
c
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropi-
onic acid 121Xaa Ala Xaa Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg
Arg Arg Arg1 5 10 15Xaa Lys12218PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)-
L-2,3-diaminopropionic acidSITE(12)..(12)Xaa can be any naturally
occurring amino acidSITE(17)..(17)L-2,3-diaminopropionic acid
122Xaa Ala Xaa Thr Ala Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg Arg Arg1
5 10 15Xaa Lys1238PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoproline 123Phe Xaa Arg Arg Arg Arg Cys
Lys1 512416PRTArtificial SequenceSynthetic Construct 124Arg Gln Ile
Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
151255PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)2-aminobutyric
acidSITE(5)..(5)7-amino-4-methylcourmarin 125Asp Met Xaa Asp Xaa1
512610PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-homoprolineSITE(8)..(8)L-norleucine 126Arg
Arg Arg Arg Xaa Phe Asp Xaa Cys Asp1 5 1012710PRTArtificial
SequenceSynthetic
ConstructSITE(5)..(5)L-homoprolineSITE(8)..(8)L-norleucine 127Arg
Arg Arg Arg Xaa Phe Asp Xaa Cys Asp1 5 1012810PRTArtificial
SequenceSynthetic
ConstructSITE(5)..(5)L-homoprolineSITE(8)..(8)L-norleucineSITE(9)..(9)sel-
enocysteine 128Arg Arg Arg Arg Xaa Phe Asp Xaa Xaa Asp1 5
1012914PRTArtificial SequenceSynthetic
ConstructSITE(11)..(11)NleSITE(12)..(12)2-aminobutyric
acidSITE(14)..(14)7-amino-4-methylcourmarin 129Arg Arg Arg Arg Arg
Arg Arg Arg Arg Asp Xaa Xaa Asp Xaa1 5 1013012PRTArtificial
SequenceSynthetic Construct 130Cys Arg Arg Arg Arg Phe Trp Gln Cys
Thr Arg Val1 5 1013112PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)2-aminobutyric acidSITE(9)..(9)2-aminobutyric
acid 131Xaa Arg Arg Arg Arg Phe Trp Gln Xaa Thr Arg Val1 5
1013212PRTArtificial SequenceSynthetic Construct 132Cys Arg Arg Arg
Arg Phe Trp Gln Cys Thr Arg Val1 5 101339PRTArtificial
SequenceSynthetic Construct 133Arg Arg Arg Arg Arg Arg Arg Arg Arg1
513411PRTArtificial SequenceSynthetic Construct 134Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg1 5 1013516PRTArtificial
SequenceSynthetic Construct 135Arg Gln Ile Lys Ile Trp Phe Gln Asn
Arg Arg Met Lys Trp Lys Lys1 5 10 151366PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 136Pro Xaa Gly Xaa Tyr Arg1 51376PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanineSITE(5)..(5)L-4-(phosphonodifluoromethyl)phenylalanine
137Ser Xaa Ile Xaa Xaa Arg1 51386PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
138Ile His Ile Xaa Ile Arg1 51396PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(5)..(-
5)L-homoproline 139Ala Ala Ile Xaa Xaa Arg1 51406PRTArtificial
SequenceSynthetic
ConstructSITE(1)..(1)L-4-fluorophenylalanineSITE(3)..(3)L-homoprolineSITE-
(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine 140Xaa Ser Xaa
Xaa Val Arg1 51416PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 141Xaa Asn Pro Xaa Ala Arg1 51426PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 142Tyr Xaa Ala Xaa Gly Arg1 51436PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
143Ala His Ile Xaa Ala Arg1 51446PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
144Gly Asn Gly Xaa Pro Arg1 51456PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 145Phe Gln Xaa Xaa Ile Arg1 51466PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
146Ser Pro Gly Xaa His Arg1 51476PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 147Xaa Tyr Ile Xaa His Arg1 51486PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
148Ser Val Pro Xaa His Arg1 51496PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
149Ala Ile Pro Xaa Asn Arg1 51506PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-4-fluorophenylalanineSITE(4)..(4)L-4-(phosphonodif-
luoromethyl)phenylalanine 150Xaa Ser Ile Xaa Gln Phe1
51516PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 151Ala Ala Xaa Xaa Phe Arg1 51526PRTArtificial
SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-phenylglycine 152Asn Thr Xaa Xaa Xaa
Arg1 51536PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-norleucine 153Ile Pro Xaa Xaa Xaa
Arg1 51546PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(3)..(3)L-4-fluorophenylalanineSITE-
(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(5)..(5)L-homoprolin-
e 154Gln Xaa Xaa Xaa Xaa Arg1 51556PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-4-fluorophenylalanineSITE(4)..(4)L-4-(phosphonodif-
luoromethyl)phenylalanine 155Asn Ala Xaa Xaa Gly Arg1
51566PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
156Asn Thr Tyr Xaa Ala Arg1 51576PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 157Glu Ala Xaa Xaa Val Arg1 51586PRTArtificial
SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 158Ile Val Xaa Xaa Ala Arg1 51596PRTArtificial
SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 159Tyr Thr Xaa Xaa Ala Arg1 51606PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(3)..(3)L-phenylglycineSITE(4)..(4)-
L-4-(phosphonodifluoromethyl)phenylalanine 160Asn Xaa Xaa Xaa Ile
Arg1 51616PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 161Xaa Asn Trp Xaa His Arg1 51626PRTArtificial
SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 162Tyr Xaa Val Xaa Ile Arg1 51636PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
163Asn Ser Ala Xaa Gly Arg1 51646PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
164Thr Asn Val Xaa Ala Arg1 51656PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
165Asn Thr Val Xaa Thr Arg1 51666PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
166Ser Ile Thr Xaa Tyr Arg1 51676PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-4-fluorophenylalanineSITE(4)..(4)L-4-(phosphonodif-
luoromethyl)phenylalanine 167Asn Xaa Asn Xaa Leu Arg1
51686PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(5)..(-
5)L-norleucine 168Tyr Asn Asn Xaa Xaa Arg1 51696PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
169Asn Tyr Asn Xaa Gly Arg1 51706PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
170Ala Trp Asn Xaa Ala Arg1 51716PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
171Val Thr His Xaa Tyr Arg1 51726PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-homoproline 172Pro Xaa His Xaa Xaa
Arg1 51736PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanine 173Asn Xaa His Xaa Gly Arg1 51746PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
174Pro Ala His Xaa Gly Arg1 51756PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
175Ala Tyr His Xaa Ile Arg1 51766PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-homoprolineSITE(4)..(4)L-4-(phosphonodifluoromethy-
l)phenylalanine 176Asn Xaa Glu Xaa Tyr Arg1 51776PRTArtificial
SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanine
177Val Ser Ser Xaa Thr Arg1 517812PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(10)..-
(10)L-beta-naphthylalanine 178Thr Asn Val Xaa Ala Arg Arg Arg Arg
Xaa Phe Gln1 5 1017912PRTArtificial SequenceSynthetic
ConstructSITE(4)..(4)L-4-(phosphonodifluoromethyl)phenylalanineSITE(10)..-
(10)L-beta-naphthylalanine 179Ser Val Pro Xaa His Arg Arg Arg Arg
Xaa Phe Gln1 5 1018012PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-phenylglycineSITE(4)..(4)L-4-(phosphonodifluoromet-
hyl)phenylalanineSITE(5)..(5)L-norleucineSITE(10)..(10)L-beta-naphthylalan-
ine 180Ile Pro Xaa Xaa Xaa Arg Arg Arg Arg Xaa Phe Gln1 5
101818PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)sarcosineSITE(3)..(3)D-phosphothreonineSITE(4)..(4)L-
-homoprolineSITE(5)..(5)L-beta-naphthylalanine 181Ala Xaa Xaa Xaa
Xaa Tyr Gln Lys1 518216PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-homo-
prolineSITE(6)..(6)L-beta-naphthylalanineSITE(9)..(9)L-2,3-diaminopropioni-
c
acidSITE(11)..(11)L-beta-naphthylalanineSITE(16)..(16)L-2,3-diaminopropi-
onic acid 182Xaa Ala Xaa Xaa Xaa Xaa Arg Ala Xaa Phe Xaa Arg Arg
Arg Arg
Lys1 5 10 1518318PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-homo-
prolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropio-
nic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopro-
pionic acid 183Xaa Ala Xaa Xaa Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg
Arg Arg Arg1 5 10 15Xaa Lys18418PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 184Xaa Ala Xaa Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys18518PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)-
L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 185Xaa Ala Xaa Thr Ala Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys1867PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-naphthylalanine 186Phe Xaa Arg Arg Arg Arg
Gln1 51878PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-naphthylalanine 187Phe Phe Xaa Arg Arg Arg
Arg Gln1 51887PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-naphthylalanine 188Phe Xaa Arg Arg Arg Arg
Gln1 51898PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-naphthylalanine 189Phe Xaa Arg Arg Arg Arg
Arg Gln1 51908PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-naphthylalanineSITE(3)..(3)Xaa can be any
naturally occurring amino acid 190Cys Phe Xaa Arg Arg Arg Arg Gln1
51917PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-naphthylalanine 191Phe Xaa Arg Arg Arg Arg
Gln1 51928PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-naphthylalanine 192Phe Xaa Arg Arg Arg Arg
Arg Gln1 51937PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-naphthylalanine 193Arg Arg Phe Arg Xaa Arg
Gln1 51948PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-naphthylalanine 194Phe Phe Xaa Arg Arg Arg
Arg Gln1 51958PRTArtificial SequenceSynthetic
ConstructSITE(6)..(6)L-naphthylalanine 195Arg Phe Arg Phe Arg Xaa
Arg Gln1 51966PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)L-naphthylalanine 196Phe Xaa Arg Arg Arg Gln1
51977PRTArtificial SequenceSynthetic
ConstructSITE(6)..(6)L-naphthylalanine 197Phe Arg Arg Arg Arg Xaa
Gln1 51987PRTArtificial SequenceSynthetic
ConstructSITE(5)..(5)L-naphthylalanine 198Arg Arg Phe Arg Xaa Arg
Gln1 51997PRTArtificial SequenceSynthetic
ConstructSITE(3)..(3)L-naphthylalanine 199Arg Arg Xaa Phe Arg Arg
Gln1 520018PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(4)..(4)D-phosphothreonineSITE(5)..(5)L-homo-
prolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropio-
nic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopro-
pionic acid 200Xaa Ala Xaa Xaa Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg
Arg Arg Arg1 5 10 15Xaa Lys20118PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 201Xaa Ala Xaa Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys20217PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 202Xaa Pro Xaa Asp Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Lys20318PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 203Xaa Phe Xaa Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys20417PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)L-N(alpha)-methylphenylalanineSITE(5)..(5)L-homoprolineSI-
TE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 204Xaa Xaa Gly Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Lys20518PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)L-phenylglycineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-bet-
a-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 205Xaa Xaa His Glu Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys20618PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)L-N(alpha)-methylphenylalanineSITE(5)..(5)L-homoprolineSI-
TE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 206Xaa Xaa Ile Glu Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys20718PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-phenylglycineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-bet-
a-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 207Xaa His Xaa Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys20817PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic acidSITE(3)..(3)Xaa can be any
naturally occurring amino
acidSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(9)..(-
9)L-2,3-diaminopropionic
acidSITE(11)..(11)L-beta-naphthylalanineSITE(16)..(16)L-2,3-diaminopropio-
nic acid 208Xaa Pro Xaa Asp Xaa Xaa Arg Ala Xaa Phe Xaa Arg Arg Arg
Arg Xaa1 5 10 15Lys20916PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(8)..(8)L-2,3-diaminopropionic
acidSITE(10)..(10)L-beta-naphthylalanineSITE(15)..(15)L-2,3-diaminopropio-
nic acid 209Xaa Pro Xaa Asp Xaa Xaa Arg Xaa Phe Xaa Arg Arg Arg Arg
Xaa Lys1 5 10 1521017PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(9)..(9)L-2,3-diaminopropionic
acidSITE(11)..(11)Xaa can be any naturally occurring amino
acidSITE(16)..(16)L-beta-naphthylalanine 210Xaa Phe Xaa Thr Xaa Xaa
Arg Ala Xaa Phe Xaa Arg Arg Arg Arg Xaa1 5 10
15Lys21116PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(8)..(8)L-2,3-diaminopropionic
acidSITE(10)..(10)L-beta-naphthylalanineSITE(15)..(15)L-2,3-diaminopropio-
nic acid 211Xaa Phe Xaa Thr Xaa Xaa Arg Xaa Phe Xaa Arg Arg Arg Arg
Xaa Lys1 5 10 1521217PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(9)..(9)Xaa can be any naturally occurring amino
acidSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(11)..(11)L-beta-naphthylalanineSITE(16)..(16)L-2,3-diaminopropio-
nic acid 212Xaa Pro Xaa Asp Xaa Xaa Ala Ala Xaa Phe Xaa Arg Arg Arg
Arg Xaa1 5 10 15Lys21318PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 213Xaa Pro Xaa Asp Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys21418PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 214Xaa Pro Xaa Asp Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys21519PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(11)..(11)L-2,3-diaminopropionic
acidSITE(13)..(13)L-beta-naphthylalanineSITE(18)..(18)L-2,3-diaminopropio-
nic acid 215Xaa Pro Xaa Asp Xaa Xaa Arg Ala Ala Ala Xaa Phe Xaa Arg
Arg Arg1 5 10 15Arg Xaa Lys21618PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 216Xaa Pro Xaa Asp Xaa Xaa Arg Tyr Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys21718PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 217Xaa Pro Xaa Asp Xaa Xaa Arg Val Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys21818PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 218Xaa Pro Xaa Asp Xaa Xaa Arg Arg Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys21918PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 219Xaa Pro Xaa Asp Xaa Xaa Arg Asp Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22018PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 220Xaa Phe Xaa Thr Xaa Xaa Arg Ser Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22118PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 221Xaa Pro Xaa Asp Xaa Xaa Arg Arg Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22218PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 222Xaa Pro Xaa Asp Xaa Xaa Arg Arg Val Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22318PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 223Xaa Pro Xaa Asp Xaa Xaa Arg Arg Arg Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22418PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naph-
thylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 224Xaa Pro Xaa Asp Xaa Xaa Arg Arg Asp Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22518PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)4-L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)-
..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 225Xaa Phe Xaa Thr Xaa Xaa Arg Gly Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22618PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)4-L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)-
..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 226Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22718PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..-
(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 227Xaa Phe Phe Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22818PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)3,4-difluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)-
..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 228Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys22918PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)4-chlorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..(6-
)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 229Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23018PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..-
(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 230Xaa Phe His Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23118PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)4-bromophenylalaninSITE(5)..(5)L-homoprolineSITE(6)..(6)L-
-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 231Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23218PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)4-L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)-
..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 232Xaa Ala Xaa Thr Xaa Xaa Thr Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23318PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)4-L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)-
..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 233Xaa Val Xaa Thr Xaa Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23418PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-2-L-4-fluorophenylalanineSITE(3)..(3)L-4-fluorophenylal-
anineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..-
(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 234Xaa Xaa Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23518PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-3-L-4-fluorophenylalanineSITE(3)..(3)L-4-fluorophenylal-
anineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..-
(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 235Xaa Xaa Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23618PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-4-L-4-fluorophenylalanineSITE(3)..(3)L-4-fluorophenylal-
anineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..-
(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 236Xaa Xaa Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23718PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-4-CyanophenylalanineSITE(3)..(3)L-4-fluorophenylalanine-
SITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-
-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 237Xaa Xaa Xaa Thr Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23818PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-4-phenylalanineSITE(3)..(3)L-4-fluorophenylalanineSITE(-
5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3--
diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 238Xaa Xaa Xaa Ile Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys23919PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-4-phenylalanineSITE(3)..(3)L-4-fluorophenylalanineSITE(-
5)..(5)L-beta-naphthylalanineSITE(6)..(6)L-homoprolineSITE(7)..(7)L-beta-n-
aphthylalanineSITE(11)..(11)L-2,3-diaminopropionic
acidSITE(13)..(13)L-beta-naphthylalanineSITE(18)..(18)L-2,3-diaminopropio-
nic acid 239Xaa Xaa Xaa Asp Xaa Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg
Arg Arg1 5 10 15Arg Xaa Lys24018PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(2)..(2)D-4-phenylalanineSITE(3)..(3)L-4-fluorophenylalanineSITE(-
4)..(4)D-homogluatamineSITE(5)..(5)L-homoprolineSITE(6)..(6)L-beta-naphthy-
lalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 240Xaa Xaa Xaa Xaa Xaa Xaa Arg Ala Phe Xaa Phe Xaa Arg Arg
Arg Arg1 5 10 15Xaa Lys24118PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(15)..(15)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 241Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Arg Arg Arg Arg
Xaa Phe1 5 10 15Xaa Lys24218PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(13)..(13)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 242Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Arg Arg Xaa Phe
Arg Arg1 5 10 15Xaa Lys24318PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(12)..(12)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 243Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Arg Xaa Arg Phe
Arg Arg1 5 10 15Xaa Lys24418PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)L-4-fluorophenylalanineSITE(5)..(5)L-homoprolineSITE(6)..-
(6)L-beta-naphthylalanineSITE(10)..(10)L-2,3-diaminopropionic
acidSITE(15)..(15)L-beta-naphthylalanineSITE(17)..(17)L-2,3-diaminopropio-
nic acid 244Xaa Phe Xaa Thr Xaa Xaa Arg Ala Phe Xaa Arg Arg Arg Arg
Xaa Phe1 5 10 15Xaa Lys2458PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)sarcosineSITE(3)..(3)D-phosphothreonineSITE(4)..(4)L-
-homoprolineSITE(5)..(5)L-beta-naphthylalanine 245Ala Xaa Xaa Xaa
Xaa Tyr Gln Lys1 524617PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)trimesic
acidSITE(3)..(3)sarcosineSITE(5)..(5)L-beta-naphthylalanineSITE(9)..(9)L--
2,3-diaminopropionic
acidSITE(11)..(11)L-beta-naphthylalanineSITE(16)..(16)L-2,3-diaminopropio-
nic acid 246Xaa Ala Xaa Ala Xaa Arg Ala Ala Xaa Phe Xaa Arg Arg Arg
Arg Xaa1 5 10 15Lys2479PRTArtificial SequenceSynthetic
ConstructSITE(1)..(9)Any residue may be a D-amino
acidSITE(2)..(2)Xaa is L-naphthylalanine or PheSITE(3)..(3)Xaa is
Arg or L-naphthylalanineSITE(7)..(9)Xaa is any amino
acidSITE(7)..(9)Xaa may be present or absent 247Phe Xaa Xaa Arg Arg
Arg Xaa Xaa Xaa1 52489PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)D-PheSITE(2)..(2)Xaa is
L-naphthylalanineSITE(4)..(4)D-ArgSITE(6)..(6)D-ArgSITE(7)..(9)Xaa
is any amino acidSITE(7)..(9)Xaa may be present or absent 248Phe
Xaa Arg Arg Arg Arg Xaa Xaa Xaa1 52497PRTArtificial
SequenceSynthetic ConstructSITE(1)..(1)D-PheSITE(2)..(2)Xaa is
L-naphthylalanineSITE(4)..(4)D-ArgSITE(6)..(6)D-Arg 249Phe Xaa Arg
Arg Arg Arg Gln1 52508PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)D-PheSITE(2)..(2)Xaa is
L-naphthylalanineSITE(4)..(4)D-ArgSITE(6)..(6)D-Arg 250Phe Xaa Arg
Arg Arg Arg Arg Gln1 52519PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-PheSITE(3)..(3)Xaa is
L-naphthylalanineSITE(5)..(5)D-ArgSITE(7)..(9)Xaa is any amino
acidSITE(7)..(9)Xaa may be present or absent 251Phe Phe Xaa Arg Arg
Arg Xaa Xaa Xaa1 52528PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-PheSITE(3)..(3)Xaa is
L-naphthylalanineSITE(5)..(5)D-ArgSITE(7)..(7)D-Arg 252Phe Phe Xaa
Arg Arg Arg Arg Gln1 52539PRTArtificial SequenceSynthetic
ConstructSITE(1)..(9)Any residue may be a D-amino
acidSITE(2)..(2)Xaa is L-naphthylalanine or PheSITE(3)..(3)Xaa is
Arg or L-naphthylalanineSITE(7)..(9)Xaa is Arg, Phe or
GlnSITE(7)..(9)Xaa may be present or absent 253Phe Xaa Xaa Arg Arg
Arg Xaa Xaa Xaa1 52549PRTArtificial SequenceSynthetic
ConstructSITE(1)..(9)Any residue may be a D-amino
acidSITE(2)..(2)Xaa is L-naphthylalanine or PheSITE(3)..(3)Xaa is
Arg or L-naphthylalanineSITE(7)..(9)Xaa is any amino
acidSITE(7)..(9)Xaa may be present or absentSITE(9)..(9)therapeutic
moiety attached 254Phe Xaa Xaa Arg Arg Arg Xaa Xaa Xaa1
52559PRTArtificial SequenceSynthetic ConstructSITE(1)..(1)D-phe or
PheSITE(2)..(2)Xaa is L-naphthylalanine or D-PheSITE(3)..(3)Xaa is
Arg or L-naphthylalanineSITE(4)..(6)D-Arg or ArgSITE(7)..(9)Xaa is
any amino acidSITE(7)..(9)Xaa may be present or
absentSITE(9)..(9)therapeutic moiety attached 255Phe Xaa Xaa Arg
Arg Arg Xaa Xaa Xaa1 52567PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)D-pheSITE(2)..(2)Xaa is
L-naphthylalanineSITE(4)..(4)D-ArgSITE(6)..(6)D-ArgSITE(7)..(7)therapeuti-
c moiety attached 256Phe Xaa Arg Arg Arg Arg Gln1
52578PRTArtificial SequenceSynthetic
ConstructSITE(1)..(1)D-pheSITE(2)..(2)Xaa is
L-naphthylalanineSITE(4)..(4)D-ArgSITE(6)..(6)D-ArgSITE(8)..(8)therapeuti-
c moiety attached 257Phe Xaa Arg Arg Arg Arg Arg Gln1
52588PRTArtificial SequenceSynthetic
ConstructSITE(2)..(2)D-PheSITE(3)..(3)Xaa is
L-naphthylalanineSITE(5)..(5)D-ArgSITE(7)..(7)D-ArgSITE(8)..(8)therapeuti-
c moiety attached 258Phe Phe Xaa Arg Arg Arg Arg Gln1
52599PRTArtificial SequenceSynthetic ConstructSITE(1)..(9)Any
residue may be a D-amino acidSITE(2)..(2)Xaa is L-naphthylalanine
or PheSITE(3)..(3)Xaa is Arg or L-naphthylalanineSITE(7)..(9)Xaa is
Arg or PheSITE(7)..(9)Xaa may be present or
absentSITE(9)..(9)therapeutic moiety attached 259Phe Xaa Xaa Arg
Arg Arg Xaa Xaa Xaa1 5
* * * * *