U.S. patent application number 12/102086 was filed with the patent office on 2009-07-09 for reagents and methods for smooth muscle therapies.
This patent application is currently assigned to Arizona Board of Regents. Invention is credited to Colleen Brophy, Padmini Komalavilas, Joshi Lokesh, Alyssa Panitch, Brandon Seal.
Application Number | 20090176695 12/102086 |
Document ID | / |
Family ID | 23220338 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090176695 |
Kind Code |
A1 |
Brophy; Colleen ; et
al. |
July 9, 2009 |
Reagents and Methods for Smooth Muscle Therapies
Abstract
The present invention provides novel polypeptides comprising
heat shock protein 20 (HSP20)-derived polypeptides to treat or
inhibit smooth muscle vasospasm, as well to treat and inhibit
smooth muscle cell proliferation and migration.
Inventors: |
Brophy; Colleen;
(Scottsdale, AZ) ; Komalavilas; Padmini; (Tempe,
AZ) ; Panitch; Alyssa; (Higley, AZ) ; Seal;
Brandon; (Mesa, AZ) ; Lokesh; Joshi; (Tempe,
AZ) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Arizona Board of Regents
|
Family ID: |
23220338 |
Appl. No.: |
12/102086 |
Filed: |
April 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11078256 |
Mar 11, 2005 |
7381699 |
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12102086 |
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10226956 |
Aug 23, 2002 |
7135453 |
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11078256 |
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60314535 |
Aug 23, 2001 |
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Current U.S.
Class: |
514/1.1 ;
435/320.1; 435/325; 435/375; 530/324; 530/326; 530/327; 530/329;
536/23.1 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101; A61P 15/06 20180101; C07K 5/0819 20130101;
A61P 9/10 20180101; C07K 7/08 20130101; A61P 1/04 20180101; A61P
21/00 20180101; A61P 15/00 20180101; A61P 25/06 20180101; A61P 9/00
20180101; A61P 9/08 20180101; C07K 5/0806 20130101; C07K 5/0808
20130101; C07K 5/1024 20130101; C07K 7/06 20130101; A61P 13/12
20180101; A61P 9/12 20180101; A61P 11/06 20180101; A61P 15/10
20180101; A61P 11/00 20180101; A61P 35/00 20180101; A61P 43/00
20180101; A61P 39/02 20180101; A61P 21/02 20180101; A61P 9/14
20180101 |
Class at
Publication: |
514/7 ; 530/324;
530/326; 530/327; 530/329; 536/23.1; 435/320.1; 435/325;
435/375 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 7/08 20060101 C07K007/08; C07K 7/06 20060101
C07K007/06; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12N 5/06 20060101 C12N005/06 |
Goverment Interests
STATEMENT OF GOVERNMENT FUNDING
[0002] The U.S. Government through the National Institute of
Health, provided financial assistance for this project under Grant
No. RO1 HL58027-06. Therefore, the United States Government may own
certain rights to this invention.
Claims
1. A polypeptide consisting of a sequence according to general
formula I: X1-X2-[X3-A(X4)APLP-X5-].sub.u-X6 wherein X1 is absent
or is one or more molecules comprising one or more aromatic ring;
X2 is absent or comprises a transduction domain; X3 is 0, 1, 2, 3,
or 4 amino acids of the sequence WLRR (SEQ ID NO:1); X4 is selected
from the group consisting of S, T, Y, D, E, phosphoserine analogs
and phosphotyrosine analogs; X5 is 0, 1, 2, or 3 amino acids of a
sequence of genus Z1-Z2-Z3, wherein Z1 is selected from the group
consisting of G and D; Z2 is selected from the group consisting of
L and K; and Z3 is selected from the group consisting of S and T;
and X6 is absent or comprises a transduction domain; and wherein u
is 1-5.
2. The polypeptide of claim 1 wherein either X2 or X6 comprises a
transduction domain.
3. The polypeptide of claim 1 wherein X4 is phosphorylated.
4. The polypeptide of claim 1 wherein X1 is a molecule comprising
an aromatic ring.
5. The polypeptide of claim 4 wherein X1 is selected from the group
consisting of F, Y, W; and compounds comprising
9-fluoroenylmethyl.
6. A polypeptide comprising a sequence according to general formula
II: X1-X2-[X3-A(X4)APLP-X5].sub.u-X6 wherein X1 is absent or is one
or more molecules comprising one or more aromatic ring; X2 is
absent or comprises a cell transduction domain; X3 is 0-14 amino
acids of the sequence of heat shock protein 20 between residues 1
and 14 of SEQ ID NO:297; X4 is selected from the group consisting
of S, T, Y, D, E, phosphoserine analogs and phosphotyrosine
analogs; X5 is 0-140 amino acids of heat shock protein 20 between
residues 21 and 160 of SEQ ID NO:297; X6 is absent or comprises a
cell transduction domain; and wherein at least one of X2 and X6
comprise a transduction domain.
6. The polypeptide of claim 5 wherein X4 is phosphorylated.
7. The polypeptide of claim 5 wherein X1 is a molecule comprising
an aromatic ring.
8. The polypeptide of claim 7 wherein X1 is selected from the group
consisting of F, Y, W; and compounds comprising
9-fluoroenylmethyl.
9. A pharmaceutical composition, comprising one or more
polypeptides according to claim 1, and a pharmaceutically
acceptable carrier.
10. A pharmaceutical composition, comprising one or more
polypeptides according to claim 5, and a pharmaceutically
acceptable carrier.
11. An isolated nucleic acid sequence encoding the polypeptide of
claim 1.
12. An isolated nucleic acid sequence encoding the polypeptide of
claim 5.
13. An expression vector comprising the nucleic acid of claim
11.
14. An expression vector comprising the nucleic acid of claim
12.
15. A host cell comprising the expression vector of claim 13.
16. A host cell comprising the expression vector of claim 14.
17. An improved biomedical device, wherein the biomedical device
comprises one or more polypeptides according to claim 1 disposed on
or in the biomedical device.
18. An improved biomedical device, wherein the biomedical device
comprises one or more polypeptides according to claim 5 disposed on
or in the biomedical device.
19. A method for inhibiting smooth muscle cell proliferation and/or
migration, comprising contacting the smooth muscle cells with an
amount effective to inhibit smooth muscle cell proliferation and/or
migration of one or more polypeptides according to claim 1.
20. A method for inhibiting smooth muscle cell proliferation and/or
migration, comprising contacting the smooth muscle cells with an
amount effective to inhibit smooth muscle cell proliferation and/or
migration of one or more polypeptides according to claim 5.
21. A method for inhibiting smooth muscle cell proliferation and/or
migration, comprising contacting the smooth muscle cells with an
amount effective to inhibit smooth muscle cell proliferation and/or
migration of HSP20, or a functional equivalent thereof.
22. A method for treating or inhibiting a disorder selected from
the group consisting of intimal hyperplasia, stenosis, restenosis,
and/or atherosclerosis, comprising contacting a subject in need
thereof with an amount effective to treat or inhibit intimal
hyperplasia, stenosis, restenosis, and/or atherosclerosis of one or
more polypeptides according to claim 1.
23. A method for treating or inhibiting a disorder selected from
the group consisting of intimal hyperplasia, stenosis, restenosis,
and/or atherosclerosis, comprising contacting a subject in need
thereof with an amount effective to treat or inhibit intimal
hyperplasia, stenosis, restenosis, and/or atherosclerosis of one or
more polypeptides according to claim 5.
24. A method for treating or inhibiting a disorder selected from
the group consisting of intimal hyperplasia, stenosis, restenosis,
and/or atherosclerosis, comprising contacting a patient in need
thereof with an amount effective to treat or inhibit intimal
hyperplasia, stenosis, restenosis, and/or atherosclerosis of HSP20,
or a functional equivalent thereof.
25. A method for treating or inhibiting smooth muscle spasm,
comprising contacting a subject in need thereof with an amount
effective to inhibit vasoconstriction of one or more polypeptides
according to claim 1.
26. A method for treating or inhibiting smooth muscle spasm,
comprising contacting a subject in need thereof with an amount
effective to inhibit vasoconstriction of one or more polypeptides
according to claim 5.
27. A method for treating or inhibiting smooth muscle spasm,
comprising contacting a subject in need thereof with an amount
effective to inhibit vasoconstriction of HSP20, or a functional
equivalent thereof.
28. A composition comprising: (a) a polypeptide according to claim
1; and (b) an inhibitor of HSP27.
29. A composition comprising: (a) a polypeptide according to claim
5; and (b) an inhibitor of HSP27.
30. A method for inhibiting smooth muscle spasm, comprising
contacting a graft with an amount effective to inhibit
vasoconstriction of one or more polypeptides of claim 1.
31. A method for inhibiting smooth muscle spasm, comprising
contacting a graft with an amount effective to inhibit
vasoconstriction of one or more polypeptides of claim 5.
32. A method for inhibiting smooth muscle spasm, comprising
contacting a graft with an amount effective to inhibit
vasoconstriction of HSP20, or a functional equivalent thereof.
Description
CROSS REFERENCE
[0001] This application claims priority from U.S. Provisional
Patent Application Ser. No. 60/314,535 filed Aug. 23, 2001, the
disclosure of which is incorporated by reference herein in its
entirety.
FIELD OF INVENTION
[0003] This invention relates generally to the fields of cell
biology, molecular biology, pharmaceuticals, and smooth muscle
biology.
BACKGROUND
[0004] There are three types of muscles: cardiac, skeletal, and
smooth. Smooth muscles are found in the walls of blood vessels,
airways, the gastrointestinal tract, and the genitourinary tract.
The caliber of tubes lined by these muscles is dependent on a
dynamic balance between the state of contraction and the state of
relaxation of the muscles in these organs. Contraction and
relaxation of smooth muscles are mediated by different signaling
pathways inside the muscles. Pathways which induce relaxation also
inhibit contraction. Sustained contraction of muscle is a "spasm"
of the muscle. This spasm can be prevented by activating pathways
or systems which induce relaxation, or in other words, inhibit
contraction.
[0005] For the most part, smooth muscles are unique in that they
lack the ordered structure of cardiac and skeletal muscles and that
they are able to maintain tonic contractions with minimal oxygen
use. Pathologic tonic contraction is a state in which the muscles
are in spasm.
[0006] Many pathological conditions are associated with spasm of
vascular smooth muscle ("vasospasm"), the smooth muscle that lines
blood vessels. Vasospasm, of the vessel causes narrowing of the
vessel lumen, limiting blood flow. Spasm of any vessel leads to
ischemia to the organ that the vessel supplies blood to. Ischemia
is reversible lack of blood flow and oxygen supply to the tissues.
In the case of spasm of the vessels in the heart it leads to
cardiac ischemia and/or infarction; spasm of vessels in the brain
leads to stroke; spasm of the vessels that supply the intestines
leads to mesenteric ischemia, a lack of relaxation of the vessels
in the penis leads to impotence, since erection requires
vasodilation of the corpra cavernosal (penile) blood vessels; and
spasm of the intracranial blood vessels leads to migraines.
[0007] Excessive vasoconstriction (or inadequate vasodilation)
occur in other disease states as well. Hypertension (high blood
pressure) is caused by excessive vasoconstriction, as well as
thickening, of the vessel wall, particularly in the smaller vessels
of the circulation. This process may affect the lung vessels as
well and cause pulmonary (lung) hypertension and asthma
(bronchospasm). Other disorders known to be associated with
excessive constriction, or inadequate dilation of smooth muscles
include toxemia of pregnancy, pre-term labor,
pre-eclampsia/eclampsia, Raynaud's disease or phenomenon, anal
fissure, achalasia, hemolytic-uremia, and Prinzmetal's angina, a
form of coronary spasm that causes angina. Spasm in the coronary
arteries also occurs during mechanical manipulation of coronary
arteries, such as during angioplasty and stenting. This spasm can
lead to ischemia and infarction.
[0008] Surgical procedures involving the vasculature are also
complicated by vasospasm of smooth muscle, which may result in both
short term and long term complications including restenosis and
vascular occlusion. There is a general pattern in which vasospasm,
if persistent, leads to constrictive remodeling/intimal
hyperplasia, and ultimately vascular occlusion. Corrective surgical
procedures, such as stenting of a blood vessel, angioplasty, and
implanting prosthetic devices such as dialysis access fistulas and
shunts, are accompanied by damage to the smooth muscle. This leads
to smooth muscle cell proliferation and migration. This ultimately
leads to constrictive remodeling and intimal hyperplasia. This
process leads to restenosis, prosthetic graft failure, stent and
stent graft failure, microvascular graft failure, atherosclerosis,
and transplant vasculopathy.
[0009] While incompletely understood, intimal hyperplasia is
mediated by a sequence of events that include endothelial cell
injury and vascular smooth muscle proliferation and migration from
the media to the intima. This is associated with a phenotypic
modulation of the smooth muscle cells from a contractile to a
synthetic phenotype. The "synthetic" smooth muscle cells secrete
extracellular matrix proteins, which leads to pathologic vascular
occlusion, as described above. Furthermore, increased proliferation
and migration of smooth muscle cells can also lead to smooth muscle
cell tumors, such as leiomyosarcomas and leiomyomas.
[0010] Thus, it would be of great benefit to identify new methods
and therapeutics to treat or inhibit smooth muscle vasospasm,
promote smooth muscle relaxation, improve other therapies involving
smooth muscle, and to treat and inhibit smooth muscle cell
proliferation and migration.
SUMMARY OF THE INVENTION
[0011] The present invention provides new methods and therapeutics
to treat or inhibit smooth muscle vasospasm, promote smooth muscle
relaxation, improve other therapies involving smooth muscle, and to
treat and inhibit smooth muscle cell proliferation and
migration.
[0012] In one aspect, the present invention provides polypeptides
consisting of an amino acid sequence according to general formula
I:
X1-X2-[X3-A(X4)APLP-X5-].sub.u-X6
[0013] wherein X1 is absent or is one or more molecules comprising
one or more aromatic ring;
[0014] X2 is absent or comprises a transduction domain;
[0015] X3 is 0, 1, 2, 3, or 4 amino acids of the sequence WLRR (SEQ
ID NO:1);
[0016] X4 is selected from the group consisting of S, T, Y, D, E,
hydroxylysine, hydroxyproline, phosphoserine analogs and
phosphotyrosine analogs;
[0017] X5 is 0, 1, 2, or 3 amino acids of a sequence of genus
Z1-Z2-Z3,
[0018] wherein Z1 is selected from the group consisting of G and
D;
[0019] Z2 is selected from the group consisting of L and K; and
[0020] Z3 is selected from the group consisting of S and T;
[0021] X6 is absent or comprises a transduction domain; and
[0022] wherein u is 1-5.
[0023] In a preferred embodiment, X4 is phosphorylated. In a
further preferred embodiment, at least one of X2 and X6 comprises a
transduction domain.
[0024] In another aspect, the invention provides polypeptides
consisting of an amino acid sequence according to the general
formula II:
X1-X2-[X3-A(X4)APLP-X5].sub.u-X6
[0025] wherein X1 is absent or is one or more molecules comprising
one or more aromatic ring;
[0026] X2 is absent or comprises a cell transduction domain;
[0027] X3 is 0-14 amino acids of the sequence of heat shock protein
20 between residues 1 and 14 of SEQ ID NO:297;
[0028] X4 is selected from the group consisting of S, T, Y, D, E,
hydroxylysine, hydroxyproline, phosphoserine analogs and
phosphotyrosine analogs;
[0029] X5 is 0-140 amino acids of heat shock protein 20 between
residues 21 and 160 of SEQ ID NO:297;
[0030] X6 is absent or comprises a cell transduction domain;
and
[0031] wherein at least one of X2 and X6 comprise a transduction
domain.
[0032] In a preferred embodiment, X4 is phosphorylated.
[0033] In another aspect, the present invention provides
pharmaceutical compositions, comprising one or more polypeptides of
the present invention and a pharmaceutically acceptable
carrier.
[0034] In another aspect, the present invention provides isolated
nucleic acid sequences encoding a polypeptide of the present
invention. In further aspects, the present invention provides
recombinant expression vectors comprising the nucleic acid
sequences of the present invention, and host cells transfected with
the recombinant expression vectors of the present invention.
[0035] In another aspect, the invention provides improved
biomedical devices, wherein the biomedical devices comprise one or
more polypeptides of the present invention disposed on or in the
biomedical device. In various embodiments, such biomedical devices
include stents, grafts, shunts, stent grafts, angioplasty devices,
balloon catheters, fistulas, and any implantable drug delivery
device.
[0036] In another aspect, the invention provides methods for
inhibiting smooth muscle cell proliferation and/or migration,
comprising contacting the smooth muscle cells with an amount
effective to inhibit smooth muscle cell proliferation and/or
migration of one or more polypeptide of the present invention. In
various preferred embodiments of this aspect of the invention, the
method is used to treat or prevent a disorder selected from the
group consisting of intimal hyperplasia, stenosis, restenosis, and
atherosclerosis. In various other preferred embodiments of this
aspect of the invention, the method is performed on a subject who
has undergone, is undergoing, or will undergo a procedure selected
from the group consisting of angioplasty, vascular stent placement,
endarterectomy, atherectomy, bypass surgery, vascular grafting,
organ transplant, prosthetic implant, microvascular
reconstructions, plastic surgical flap reconstruction, and catheter
emplacement. In a further embodiment of this aspect of the
invention, the method is used to treat smooth muscle cell
tumors.
[0037] In a further aspect, the present invention comprises methods
for treating or inhibiting a disorder selected from the group
consisting of intimal hyperplasia, stenosis, restenosis, and/or
atherosclerosis, comprising contacting a subject in need thereof
with an amount effective to treat or inhibit intimal hyperplasia,
stenosis, restenosis, and/or atherosclerosis of HSP20, or a
functional equivalent thereof.
[0038] In a further aspect, the present invention comprises methods
for treating smooth muscle cell tumors comprising contacting a
subject in need thereof with an amount effective to treat smooth
muscle tumors of HSP20, or a functional equivalent thereof.
[0039] In a further aspect, the present invention provides a method
for treating or preventing smooth muscle spasm, comprising
contacting a subject in need thereof with an amount effective to
inhibit smooth muscle spasm of one or more polypeptides of the
present invention. In various preferred embodiments of this aspect
of the invention, the muscle cell spasm is associated with a
disorder or condition selected from the group consisting of angina,
Prinzmetal's angina (coronary vasospasm), ischemia, stroke,
bradycardia, hypertension, pulmonary (lung) hypertension, asthma
(bronchospasm), toxemia of pregnancy, pre-term labor,
pre-eclampsia/eclampsia, Raynaud's disease or phenomenon,
hemolytic-uremia, non-occlusive mesenteric ischemia, anal fissure,
achalasia, impotence, migraine, ischemic muscle injury associated
with smooth muscle spasm, and vasculopathy, such as transplant
vasculopathy.
[0040] In a further aspect, the present invention provides methods
for promoting smooth muscle relaxation, comprising contacting
smooth muscle with an amount effect effective to promote smooth
muscle relaxation with one or more of the polypeptide of the
present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1. Mesangial cells were transfected with vectors
containing green fluorescent protein (GFP) alone, GFP fused to the
5' end of the wild type cDNA for HSP20 (WT), or GFP fused to an
HSP20 construct in which the PKA phosphorylation site was mutated
to an alanine (S16A-HSP20)(MUT) and the number of wrinkles under
the cells was determined after treatment with dibutyryl cAMP (10
.PHI.M) for 0 minutes, 30 minutes, 60 minutes, or 90 minutes.
[0042] FIG. 2. Mesangial cells were transduced with FITC-TAT-HSP20
and the number of wrinkles under the cells was determined at the
time points indicated using phase contrast microscopy (n=10,
*=p<0.05 compared to time 0).
[0043] FIG. 3. Transverse strips of bovine carotid artery smooth
muscle, denuded of endothelium, were pre-contracted with serotonin
(1 .mu.M for 10 minutes), cumulative doses of
FITC-phospho-HSP20-TAT, FITC-scrambled phosphoHSP20-TAT
(FITC-NH.sub.2-.beta.AGGGGYGRKKRRQRRRPRKS*LWALGRPLA-COOH, open
circles) (SEQ ID NO:305), or FITC-TAT
(FITC-NH.sub.2-.beta.AGGGGYGRKKRRQRRR, closed triangles) (SEQ ID
NO:306) were added every 10 minutes, and the percent contraction
was calculated. The force is depicted as a percentage of the
maximal serotonin contraction (n=5, *=p<0.05 compared to 0
peptide added).
[0044] FIG. 4. Rings of porcine coronary artery in which the
endothelium was not denuded, were pre-contracted with serotonin (1
.mu.M for 10 minutes), cumulative doses of PTD-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH, closed circles)
(SEQ ID NO:307) or PTD-scrambled-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH open circles) (SEQ
ID NO:308) were added every 10 minutes, and the percentage of
relaxation was calculated as a percentage of the maximal serotonin
contraction (n=5, *=p<0.05 compared to 0 peptide added). The
concentrations of peptide used are depicted on the x axis.
[0045] FIG. 5. Homogenates of mesangial cells (lane 1), rat aortic
smooth muscle cells (lane 2), and PKG transfected rat aortic smooth
muscle cells (lane 3) were immunoblotted for PKG (panel A) or HSP20
(panel B). In a separate experiment, mesangial cells were untreated
(panel C) or treated with dibutyryl cAMP (10 .mu.M, 15 minutes,
panel D). The proteins were separated by 2-dimensional
electrophoresis, transferred to immobilon and probed with
anti-HSP20 antibodies. Increases in the phosphorylation of HSP20
leads to a shift in the electrophoretic mobility of the protein to
a more acidic isoform (arrow).
[0046] FIG. 6. Transfected mesangial cells were fixed, and the
actin filaments were stained with fluorescent-labeled phalloidin.
Mesangial cells were transfected with EGFP alone (EGFP), S16A-HSP20
(MUT-EGFP), or wild type HSP20 (WT-EGFP). The cells were plated on
a glass slides, and not treated (CONT) or treated with dibutyryl
cAMP (10 .mu.M, for 30 minutes, db-cAMP). The cells were fixed and
stained with rhodamine phalloidin. Dibutyryl cAMP led to a loss of
central actin stress fibers in EGFP but not S16A-HSP20 cells. In
the cells overexpressing HSP20 the actin fibers were peripherally
localized.
[0047] FIG. 7. Bovine aortic endothelial cells were plated on glass
coverslips (80K-100K cells) in DMEM plus 10% FBS over night (24
wells plate). The cells were serum starved (no serum) for one hour
and incubated in the presence of the peptide analogues of HSP20
[NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH-pHSP20 (10 uM)
(SEQ ID NO:307) or scrambled analogues of HSP20
[NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH-scHSP20 (10 uM)]
(SEQ ID NO:308) for 30 minutes. The cells were fixed with 3%
glutaraldehyde and the number of focal adhesions was detected with
interference reflection microscopy. The Hep I peptide was used as a
positive control.
[0048] FIG. 8. Confluent A10 cells were serum starved (0.5% fetal
bovine serum, FBS) for 48 hours. A linear wound was made in the
smooth muscle cell monolayer using a rubber scraper and the
scratched edges were marked using metal pins. The cells were
changed to 10% FBS media containing PTD-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307),
or PTD-scrambled-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ ID NO:308)
(50 .mu.M) and incubated for 24 hours. The cells were fixed and
stained with hematoxylin. The number of cells migrating into a 1
cm.sup.2 scratched area were counted as an index for migration. In
additional experiments, the migration of A10 cells was determined
in a Boyden chamber assay.
[0049] FIG. 9. A10 cells were serum starved for 3 days. The cells
were then treated with media containing 10% fetal bovine serum,
PTD-pHSP20 (NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID
NO:307), or PTD-scrambled-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ ID NO:308)
(50 .mu.M). After 24 hours cell counts were performed.
DETAILED DESCRIPTION OF THE INVENTION
[0050] Within this application, unless otherwise stated, the
techniques utilized may be found in any of several well-known
references such as: Molecular Cloning: A Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene
Expression Technology (Methods in Enzymology, Vol. 185, edited by
D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to
Protein Purification" in Methods in Enzymology (M. P. Deutshcer,
ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to
Methods and Applications (Innis, et al. 1990. Academic Press, San
Diego, Calif.), Culture of Animal Cells: A Manual of Basic
Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York,
N.Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed.
E. J. Murray, The Humana Press Inc., Clifton, N.J.)
[0051] In one aspect, the present invention provides polypeptides
consisting of an amino acid sequence according to general formula
I:
X1-X2-[X3-A(X4)APLP-X5-].sub.u-X6
[0052] wherein X1 is absent or is one or more molecules comprising
one or more aromatic ring;
[0053] X2 is absent or comprises a transduction domain;
[0054] X3 is 0, 1, 2, 3, or 4 amino acids of the sequence WLRR (SEQ
ID NO:1);
[0055] X4 is selected from the group consisting of S, T, Y, D, E,
hydroxylysine, hydroxyproline, phosphoserine analogs and
phosphotyrosine analogs;
[0056] X5 is 0, 1, 2, or 3 amino acids of a sequence of genus
Z1-Z2-Z3,
[0057] wherein Z1 is selected from the group consisting of G and
D;
[0058] Z2 is selected from the group consisting of L and K; and
[0059] Z3 is selected from the group consisting of S and T;
[0060] X6 is absent or comprises a transduction domain; and
[0061] wherein u is 1-5.
[0062] Both single letter and three letter amino acid abbreviations
are used within the application. As used herein, "norL" means
norleucine and "Orn" means ornithine.
[0063] The term "polypeptide" is used in its broadest sense to
refer to a sequence of subunit amino acids, amino acid analogs, or
peptidomimetics. The subunits are linked by peptide bonds, except
where noted (including when the X2 position is a non-amino acid
molecule that contains an aromatic ring). The polypeptides
described herein may be chemically synthesized or recombinantly
expressed.
[0064] Preferably, the polypeptides of the present invention are
chemically synthesized. Synthetic polypeptides, prepared using the
well known techniques of solid phase, liquid phase, or peptide
condensation techniques, or any combination thereof, can include
natural and unnatural amino acids. Amino acids used for peptide
synthesis may be standard Boc (N.alpha.-amino protected
N.alpha.-t-butyloxycarbonyl) amino acid resin with the standard
deprotecting, neutralization, coupling and wash protocols of the
original solid phase procedure of Merrifield (1963, J. Am. Chem.
Soc. 85:2149-2154), or the base-labile N.alpha.-amino protected
9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by
Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and
Boc N.alpha.-amino protected amino acids can be obtained from
Sigma, Cambridge Research Biochemical, or other chemical companies
familiar to those skilled in the art. In addition, the polypeptides
can be synthesized with other N.alpha.-protecting groups that are
familiar to those skilled in this art.
[0065] Solid phase peptide synthesis may be accomplished by
techniques familiar to those in the art and provided, for example,
in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition,
Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int.
J. Pept. Protein Res. 35:161-214, or using automated synthesizers.
The polypeptides of the invention may comprise D-amino acids (which
are resistant to L-amino acid-specific proteases in vivo), a
combination of D- and L-amino acids, and various "designer" amino
acids (e.g., .beta.-methyl amino acids, C.alpha.-methyl amino
acids, and N.alpha.-methyl amino acids, etc.) to convey special
properties. Synthetic amino acids include ornithine for lysine, and
norleucine for leucine or isoleucine.
[0066] In addition, the polypeptides can have peptidomimetic bonds,
such as ester bonds, to prepare peptides with novel properties. For
example, a peptide may be generated that incorporates a reduced
peptide bond, i.e., R.sub.1--CH.sub.2--NH--R.sub.2, where R.sub.1
and R.sub.2 are amino acid residues or sequences. A reduced peptide
bond may be introduced as a dipeptide subunit. Such a polypeptide
would be resistant to protease activity, and would possess an
extended half-live in vivo.
[0067] According to various embodiments of the polypeptides of
general formula I, the region [X3-A(X4)APLP-X5-].sub.u may be
present in 1, 2, 3, 4, or 5 copies. In a preferred embodiment, it
is present in 1 copy. In other embodiments, it is present in
multiple copies to provide increased efficacy for use of the
polypeptides for inhibiting one or more of smooth muscle cell
proliferation, smooth muscle cell migration, and smooth muscle
spasm, and/or also for promoting smooth muscle vasorelaxation.
[0068] According to various embodiments of the polypeptides of
general formula I, X4 is S, T, Y, D E, a phosphoserine mimic, or a
phosphotyrosine mimic. It is more preferred that X4 is S, T, or Y;
more preferred that X4 is S or T, and most preferred that X4 is S.
In these embodiments where X4 is S, T, or Y, it is most preferred
that X4 is phosphorylated. When X4 is D or E, these residues have a
negative charge that mimics the phosphorylated state. The
polypeptides of the invention are optimally effective in the
methods of the invention when X4 is phosphorylated, is a
phosphoserine or phosphotyrosine mimic, or is another mimic of a
phosphorylated amino acid residue, such as a D or E residue.
Examples of phosphoserine mimics include, but are not limited to,
sulfoserine, amino acid mimics containing a methylene substitution
for the phosphate oxygen, 4-phosphono(difluoromethyl)phenylanaline,
and L-2-amino-4-(phosphono)-4,4-difluorobutanoic acid. Other
phosphoserine mimics can be made by those of skill in the art; for
example, see Otaka et al., Tetrahedron Letters 36:927-930 (1995).
Examples of phosphotyrosine mimics include, but are not limited to,
phosphonomethylphenylalanine, difluorophosphonomethylphenylalanine,
fluoro-O-malonyltyrosine and O-malonyltyrosine. (See, for example,
Akamatsu et. al., Bioorg Med Chem 1997 January; 5(1):157-63).
[0069] In another preferred embodiment, X1 is one or more molecules
comprising an aromatic ring. In one preferred embodiment, the one
or molecules comprising an aromatic ring are amino acids, and X1 is
(F/Y/W).sub.z, wherein "z" is 1-5 amino acids. Thus, for example,
X1 can be 1 or 2 amino acid residues of any combination of F, Y,
and W, such as F, FF, Y, YY, W, WW, FY, FW, YF, YW, WY, and WF.
Alternatively, X1 can be a 3, 4, or 5 amino acid combination of F,
Y, and W. In another preferred embodiment, the molecule comprising
an aromatic ring is selected from the group of molecules comprising
one or more aromatic rings which can optionally be substituted with
halogen, lower alkyl, lower alkylthio, trifluoromethyl, lower
acyloxy, aryl, and heteroaryl. In a most preferred embodiment, the
one or more molecule comprising one or more aromatic ring comprise
9-fluorenylmethyl (Fm). Examples of such molecules include, but are
not limited to 9-fluorenylmethylcarbonyl,
9-fluorenylmethylcarbamates, 9-fluorenylmethylcarbonates,
9-fluorenylmethyl esters, 9-fluorenylmethylphosphates, and
S-9-fluorenylmethyl thioethers. In embodiments wherein the molecule
comprising an aromatic ring is not an amino acid, it can be
attached to the polypeptide by methods known in the art, including
but not limited to, standard Fmoc protection chemistry employed in
peptide synthesis.
[0070] According to various embodiments of the polypeptides of
general formula I, X3 is 0, 1, 2, 3, or 4 amino acids of the
sequence WLRR (SEQ ID NO:1). If X3 consists of only one amino acid
of the sequence, an "R" is present, since it is the
carboxy-terminal amino acid of the sequence and it would be present
at the amino terminus of the rest of the A(X4)APLP (SEQ ID NO: 2)
sequence. If X3 consists of two amino acids of WLRR (SEQ ID NO:1),
then the two amino acids added will be "RR". Other variations will
be apparent to one of skill in the art based on the teachings
herein.
[0071] Similarly, variations in the residues that can make up X5
will be apparent to one of skill in the art based on the teachings
herein.
[0072] Thus, according to these various aspects, a representative
sample of polypeptides according to general formula I include, but
are not limited to the following: (ASAPLP).sub.u (SEQ ID NO:3);
(ATAPLP).sub.u (SEQ ID NO:4); (RASAPLP).sub.u (SEQ ID NO:5);
(RATAPLP).sub.u (SEQ ID NO:6); (AYAPLP).sub.u (SEQ ID NO:7);
(RAYAPLP).sub.u (SEQ ID NO:8); (RRASAPLP).sub.u (SEQ ID NO:9);
(LRRASAPLP).sub.u (SEQ ID NO:10); (WLRRASAPLP).sub.u; (SEQ ID
NO:11) (RRATAPLP).sub.u (SEQ ID NO:12); (LRRATAPLP).sub.u (SEQ ID
NO:13); (WLRRATAPLP).sub.u (SEQ ID NO:14); (RRAYAPLP).sub.u (SEQ ID
NO:15); (LRRAYAPLP).sub.u (SEQ ID NO:16); (WLRRAYAPLP).sub.u (SEQ
ID NO:17); (RRASAPLPG).sub.u (SEQ ID NO:18); (RRASAPLPD).sub.u (SEQ
ID NO:19); (RRASAPLPGL).sub.u (SEQ ID NO:20); (RRASAPLPGK).sub.u
(SEQ ID NO:21); (RRASAPLPDL).sub.u (SEQ ID NO:22);
(RRASAPLPDK).sub.u (SEQ ID NO:23); (RRASAPLPGLS).sub.u (SEQ ID
NO:24); (RRASAPLPGLT).sub.u (SEQ ID NO:25); (RRASAPLPGKS).sub.u
(SEQ ID NO:26); (RRASAPLPGKT).sub.u (SEQ ID NO:27);
(RRASAPLPDLS).sub.u (SEQ ID NO:28); RRASAPLPDLT).sub.u (SEQ ID
NO:29); (RRASAPLPDKS).sub.u (SEQ ID NO:30); (RRASAPLPDKT).sub.u
(SEQ ID NO:31); (LRRASAPLPG).sub.u (SEQ ID NO:32);
(LRRASAPLPD).sub.u (SEQ ID NO:33); (LRRASAPLPGL).sub.u (SEQ ID
NO:34); (LRRASAPLPGK).sub.u (SEQ ID NO:35); (LRRASAPLPDL).sub.u
(SEQ ID NO:36); (LRRASAPLPDK).sub.u (SEQ ID NO:37);
(LRRASAPLPGLS).sub.u (SEQ ID NO:38); (LRRASAPLPGLT).sub.u (SEQ ID
NO:39); (LRRASAPLPGKS).sub.u (SEQ ID NO:40); (LRRASAPLPGKT).sub.u
(SEQ ID NO:41); (LRRASAPLPDLS).sub.u (SEQ ID NO:42);
(LRRASAPLPDLT).sub.u (SEQ ID NO:43); (LRRASAPLPDKS).sub.u (SEQ ID
NO:44); (LRRASAPLPDKT).sub.u (SEQ ID NO:45); (WLRRASAPLPG).sub.u
(SEQ ID NO:46); (WLRRASAPLPD).sub.u (SEQ ID NO:47);
(WLRRASAPLPGL).sub.u (SEQ ID NO:48); (WLRRASAPLPGK).sub.u (SEQ ID
NO:49); (WLRRASAPLPDL).sub.u (SEQ ID NO:50); (WLRRASAPLPDK).sub.u
(SEQ ID NO:51); (WLRRASAPLPGLS).sub.u (SEQ ID NO:52);
(WLRRASAPLPGLT).sub.u (SEQ ID NO:53); (WLRRASAPLPGKS).sub.u (SEQ ID
NO:54); (WLRRASAPLPGKT).sub.u (SEQ ID NO:55); (WLRRASAPLPDLS).sub.u
(SEQ ID NO:56); (WLRRASAPLPDLT).sub.u (SEQ ID NO:57);
(WLRRASAPLPDKS).sub.u (SEQ ID NO:58); (WLRRASAPLPDKT).sub.u (SEQ ID
NO:59); (RRATAPLPG).sub.u (SEQ ID NO:60); (RRATAPLPD).sub.u (SEQ ID
NO:61); (RRATAPLPGL).sub.u (SEQ ID NO:62); (RRATAPLPGK).sub.u (SEQ
ID NO:63); (RRATAPLPDL).sub.u (SEQ ID NO:64); (RRATAPLPDK).sub.u
(SEQ ID NO:65); (RRATAPLPGLS).sub.u (SEQ ID NO:66);
(RRATAPLPGLT).sub.u (SEQ ID NO:67); (RRATAPLPGKS).sub.u (SEQ ID
NO:68); (RRATAPLPGKT).sub.u (SEQ ID NO:69); (RRATAPLPDLS).sub.u
(SEQ ID NO:70); (RRATAPLPDLT).sub.u (SEQ ID NO:71);
(RRATAPLPDKS).sub.u (SEQ ID NO:72); (RRATAPLPDKT).sub.u (SEQ ID
NO:73); (LRRATAPLPG).sub.u (SEQ ID NO:74); (LRRATAPLPD).sub.u (SEQ
ID NO:75); (LRRATAPLPGL).sub.u (SEQ ID NO:76); (LRRATAPLPGK).sub.u
(SEQ ID NO:77); (LRRATAPLPDL).sub.u (SEQ ID NO:78);
(LRRATAPLPDK).sub.u (SEQ ID NO:79); (LRRATAPLPGLS).sub.u (SEQ ID
NO:80); (LRRATAPLPGLT).sub.u (SEQ ID NO:81); (LRRATAPLPGKS).sub.u
(SEQ ID NO:82); (LRRATAPLPGKT).sub.u (SEQ ID NO:83);
(LRRATAPLPDLS).sub.u (SEQ ID NO:84); (LRRATAPLPDLT).sub.u (SEQ ID
NO:85); (LRRATAPLPDKS).sub.u (SEQ ID NO:86); (LRRATAPLPDKT).sub.u
(SEQ ID NO:87); (WLRRATAPLPG).sub.u (SEQ ID NO:88);
(WLRRATAPLPD).sub.u (SEQ ID NO:89); (WLRRATAPLPGL).sub.u (SEQ ID
NO:90); (WLRRATAPLPGK).sub.u (SEQ ID NO:91); (WLRRATAPLPDL).sub.u
(SEQ ID NO:92); (WLRRATAPLPDK).sub.u (SEQ ID NO:93);
(WLRRATAPLPGLS).sub.u (SEQ ID NO:94); (WLRRATAPLPGLT).sub.u (SEQ ID
NO:95); (WLRRATAPLPGKS).sub.u (SEQ ID NO:96); (WLRRATAPLPGKT).sub.u
(SEQ ID NO:97); (WLRRATAPLPDLS).sub.u (SEQ ID NO:98);
(WLRRATAPLPDLT).sub.u (SEQ ID NO:99); (WLRRATAPLPDKS).sub.u (SEQ ID
NO:100); (WLRRATAPLPDKT).sub.u (SEQ ID NO:101); (RRAYAPLPG).sub.u
(SEQ ID NO:102); (RRAYAPLPD).sub.u (SEQ ID NO:103);
(RRAYAPLPGL).sub.u (SEQ ID NO:104); (RRAYAPLPGK).sub.u (SEQ ID
NO:105); (RRAYAPLPDL).sub.u (SEQ ID NO:106); (RRAYAPLPDK).sub.u
(SEQ ID NO:107); (RRAYAPLPGLS).sub.u (SEQ ID NO:108);
(RRAYAPLPGLT).sub.u (SEQ ID NO:109); (RRAYAPLPGKS).sub.u (SEQ ID
NO:110; (RRAYAPLPGKT).sub.u (SEQ ID NO:111); (RRAYAPLPDLS).sub.u
(SEQ ID NO:112); (RRAYAPLPDLT).sub.u (SEQ ID NO:113);
(RRAYAPLPDKS).sub.u (SEQ ID NO:114); (RRAYAPLPDKT).sub.u (SEQ ID
NO:115); (LRRAYAPLPG).sub.u (SEQ ID NO:116); (LRRAYAPLPD).sub.u
(SEQ ID NO:117); (LRRAYAPLPGL).sub.u (SEQ ID NO:118);
(LRRAYAPLPGK).sub.u (SEQ ID NO:119); (LRRAYAPLPDL).sub.u (SEQ ID
NO:120); (LRRAYAPLPDK).sub.u (SEQ ID NO:121); (LRRAYAPLPGLS).sub.u
(SEQ ID NO:122); (LRRAYAPLPGLT).sub.u (SEQ ID NO:123);
(LRRAYAPLPGKS).sub.u (SEQ ID NO:124); (LRRAYAPLPGKT).sub.u (SEQ ID
NO:125); (LRRAYAPLPDLS).sub.u (SEQ ID NO:126); (LRRAYAPLPDLT).sub.u
(SEQ ID NO:127); (LRRAYAPLPDKS).sub.u (SEQ ID NO:128);
(LRRAYAPLPDKT).sub.u (SEQ ID NO:129); (WLRRAYAPLPG).sub.u (SEQ ID
NO:130); (WLRRAYAPLPD).sub.u (SEQ ID NO:131); (WLRRAYAPLPGL).sub.u
(SEQ ID NO:132); (WLRRAYAPLPGK).sub.u (SEQ ID NO:133);
(WLRRAYAPLPDL).sub.u (SEQ ID NO:134); (WLRRAYAPLPDK).sub.u (SEQ ID
NO:135); (WLRRAYAPLPGLS).sub.u (SEQ ID NO:136);
(WLRRAYAPLPGLT).sub.u (SEQ ID NO:137); (WLRRAYAPLPGKS).sub.u (SEQ
ID NO:138); (WLRRAYAPLPGKT).sub.u (SEQ ID NO:139);
(WLRRAYAPLPDLS).sub.u (SEQ ID NO:140); (WLRRAYAPLPDLT).sub.u (SEQ
ID NO:141); (WLRRAYAPLPDKS).sub.u (SEQ ID NO:142); and
(WLRRAYAPLPDKT).sub.u (SEQ ID NO:143); ((F/Y/W)RRASAPLP).sub.u (SEQ
ID NO:144); ((F/Y/W)LRRASAPLP).sub.u (SEQ ID NO:145);
((F/Y/W)WLRRASAPLP).sub.u; (SEQ ID NO:146) ((F/Y/W)RRATAPLP).sub.u,
(SEQ ID NO:147); ((F/Y/W)LRRATAPLP).sub.u (SEQ ID NO:148);
((F/Y/W)WLRRATAPLP).sub.u (SEQ ID NO:149); ((F/Y/W)RRAYAPLP).sub.u
(SEQ ID NO:150); ((F/Y/W)LRRAYAPLP).sub.u (SEQ ID NO:151);
((F/Y/W)WLRRAYAPLP).sub.u (SEQ ID NO:152); ((F/Y/W)RRASAPLPG).sub.u
(SEQ ID NO:153); ((F/Y/W)RRASAPLPD).sub.u (SEQ ID NO:154);
((F/Y/W)RRASAPLPGL).sub.u (SEQ ID NO:155);
((F/Y/W)RRASAPLPGK).sub.u (SEQ ID NO:156);
((F/Y/W)RRASAPLPDL).sub.u (SEQ ID NO:157);
((F/Y/W)RRASAPLPDK).sub.u (SEQ ID NO:158);
((F/Y/W)RRASAPLPGLS).sub.u (SEQ ID NO:159);
((F/Y/W)RRASAPLPGLT).sub.u (SEQ ID NO:160);
((F/Y/W)RRASAPLPGKS).sub.u; (SEQ ID NO:161);
((F/Y/W)RRASAPLPGKT).sub.u (SEQ ID NO:162);
((F/Y/W)RRASAPLPDLS).sub.u (SEQ ID NO:163);
((F/Y/W)RRASAPLPDLT).sub.u (SEQ ID NO:164);
((F/Y/W)RRASAPLPDKS).sub.u (SEQ ID NO:165);
((F/Y/W)RRASAPLPDKT).sub.u (SEQ ID NO:166);
((F/Y/W)LRRASAPLPG).sub.u (SEQ ID NO:167);
((F/Y/W)LRRASAPLPD).sub.u (SEQ ID NO:168);
((F/Y/W))LRRASAPLPGL).sub.u (SEQ ID NO:169);
((F/Y/W)LRRASAPLPGK).sub.u (SEQ ID NO:170);
((F/Y/W)LRRASAPLPDL).sub.u (SEQ ID NO:171);
((F/Y/W)LRRASAPLPDK).sub.u (SEQ ID NO:172);
((F/Y/W)LRRASAPLPGLS).sub.u (SEQ ID NO:173);
((F/Y/W)LRRASAPLPGLT).sub.u (SEQ ID NO:174);
((F/Y/W)LRRASAPLPGKS).sub.u (SEQ ID NO:175);
((F/Y/W)LRRASAPLPGKT).sub.u (SEQ ID NO:176);
((F/Y/W)LRRASAPLPDLS).sub.u (SEQ ID NO:177);
((F/Y/W)LRRASAPLPDLT).sub.u (SEQ ID NO:178);
((F/Y/W)LRRASAPLPDKS).sub.u (SEQ ID NO:179);
((F/Y/W)LRRASAPLPDKT).sub.u (SEQ ID NO:180);
((F/Y/W)WLRRASAPLPG).sub.u (SEQ ID NO:181);
((F/Y/W)WLRRASAPLPD).sub.u (SEQ ID NO:182);
((F/Y/W)WLRRASAPLPGL).sub.u (SEQ ID NO:183);
((F/Y/W)WLRRASAPLPGK).sub.u (SEQ ID NO:184);
((F/Y/W)WLRRASAPLPDL).sub.u (SEQ ID NO:185);
((F/Y/W)WLRRASAPLPDK).sub.u (SEQ ID NO:186);
((F/Y/W)WLRRASAPLPGLS).sub.u (SEQ ID NO:187);
((F/Y/W)WLRRASAPLPGLT).sub.u (SEQ ID NO:188);
((F/Y/W)WLRRASAPLPGKS).sub.u (SEQ ID NO:189);
((F/Y/W)WLRRASAPLPGKT).sub.u (SEQ ID NO:190);
((F/Y/W)WLRRASAPLPDLS).sub.u (SEQ ID NO:191);
((F/Y/W)WLRRASAPLPDLT).sub.u (SEQ ID NO:192);
((F/Y/W)WLRRASAPLPDKS).sub.u (SEQ ID NO:193);
((F/Y/W)WLRRASAPLPDKT).sub.u (SEQ ID NO:194);
((F/Y/W)RRATAPLPG).sub.u (SEQ ID NO:195); ((F/Y/W)RRATAPLPD).sub.u
(SEQ ID NO:196); ((F/Y/W)RRATAPLPGL).sub.u (SEQ ID NO:197);
((F/Y/W)RRATAPLPGK).sub.u (SEQ ID NO:198);
((F/Y/W)RRATAPLPDL).sub.u (SEQ ID NO:199);
((F/Y/W)RRATAPLPDK).sub.u (SEQ ID NO:200);
((F/Y/W)RRATAPLPGLS).sub.u (SEQ ID NO:201);
((F/Y/W)RRATAPLPGLT).sub.u (SEQ ID NO:202);
((F/Y/W)RRATAPLPGKS).sub.u (SEQ ID NO:203);
((F/Y/W)RRATAPLPGKT).sub.u (SEQ ID NO:204);
((F/Y/W)RRATAPLPDLS).sub.u (SEQ ID NO:205);
((F/Y/W)RRATAPLPDLT).sub.u (SEQ ID NO:206);
((F/Y/W)RRATAPLPDKS).sub.u (SEQ ID NO:207);
((F/Y/W)RRATAPLPDKT).sub.u (SEQ ID NO:208);
((F/Y/W)LRRATAPLPG).sub.u (SEQ ID NO:209);
((F/Y/W)LRRATAPLPD).sub.u (SEQ ID NO:210);
((F/Y/W)LRRATAPLPGL).sub.u (SEQ ID NO:211);
((F/Y/W)LRRATAPLPGK).sub.u (SEQ ID NO:212);
((F/Y/W)LRRATAPLPDL).sub.u (SEQ ID NO:213);
((F/Y/W)LRRATAPLPDK).sub.u (SEQ ID NO:214); ((F/Y/W)LRRATAPLPGLS
).sub.u (SEQ ID NO:215); ((F/Y/W)LRRATAPLPGLT).sub.u (SEQ ID
NO:216); ((F/Y/W)LRRATAPLPGKS).sub.u (SEQ ID NO:217);
((F/Y/W)LRRATAPLPGKT).sub.u (SEQ ID NO:218);
((F/Y/W)LRRATAPLPDLS).sub.u (SEQ ID NO:219);
((F/Y/W)LRRATAPLPDLT).sub.u (SEQ ID NO:220);
((F/Y/W)LRRATAPLPDKS).sub.u (SEQ ID NO:221);
((F/Y/W)LRRATAPLPDKT).sub.u (SEQ ID NO:222);
((F/Y/W)WLRRATAPLPG).sub.u (SEQ ID NO:223);
((F/Y/W)WLRRATAPLPD).sub.u (SEQ ID NO:224);
((F/Y/W)WLRRATAPLPGL).sub.u (SEQ ID NO:225);
((F/Y/W)WLRRATAPLPGK).sub.u (SEQ ID NO:226);
((F/Y/W)WLRRATAPLPDL).sub.u (SEQ ID NO:227);
((F/Y/W)WLRRATAPLPDK).sub.u (SEQ ID NO:228);
((F/Y/W)WLRRATAPLPGLS).sub.u (SEQ ID NO:229);
((F/Y/W)WLRRATAPLPGLT).sub.u (SEQ ID NO:230);
((F/Y/W)WLRRATAPLPGKS).sub.u (SEQ ID NO:231);
((F/Y/W)WLRRATAPLPGKT).sub.u (SEQ ID NO:232);
((F/Y/W)WLRRATAPLPDLS).sub.u (SEQ ID NO:233);
((F/Y/W)WLRRATAPLPDLT).sub.u (SEQ ID NO:234);
((F/Y/W)WLRRATAPLPDKS).sub.u (SEQ ID NO:235);
((F/Y/W)WLRRATAPLPDKT).sub.u (SEQ ID NO:236);
((F/Y/W)RRAYAPLPG).sub.u (SEQ ID NO:237); ((F/Y/W)RRAYAPLPD).sub.u
(SEQ ID NO:238); ((F/Y/W)RRAYAPLPGL).sub.u (SEQ ID NO:239);
((F/Y/W)RRAYAPLPGK).sub.u (SEQ ID NO:240);
((F/Y/W)RRAYAPLPDL).sub.u (SEQ ID NO:241);
((F/Y/W)RRAYAPLPDK).sub.u (SEQ ID NO:242);
((F/Y/W)RRAYAPLPGLS).sub.u (SEQ ID NO:243);
((F/Y/W)RRAYAPLPGLT).sub.u (SEQ ID NO:244);
((F/Y/W)RRAYAPLPGKS).sub.u (SEQ ID NO:245);
((F/Y/W)RRAYAPLPGKT).sub.u (SEQ ID NO:246);
((F/Y/W)RRAYAPLPDLS).sub.u (SEQ ID NO:247);
((F/Y/W)RRAYAPLPDLT).sub.u (SEQ ID NO:248);
((F/Y/W)RRAYAPLPDKS).sub.u (SEQ ID NO:249);
((F/Y/W)RRAYAPLPDKT).sub.u (SEQ ID NO:250);
((F/Y/W)LRRAYAPLPG).sub.u (SEQ ID NO:251);
((F/Y/W)LRRAYAPLPD).sub.u (SEQ ID NO:252);
((F/Y/W)LRRAYAPLPGL).sub.u (SEQ ID NO:253);
((F/Y/W)LRRAYAPLPGK).sub.u (SEQ ID NO:254);
((F/Y/W)LRRAYAPLPDL).sub.u (SEQ ID NO:255);
((F/Y/W)LRRAYAPLPDK).sub.u (SEQ ID NO:256);
((F/Y/W)LRRAYAPLPGLS).sub.u (SEQ ID NO:257);
((F/Y/W)LRRAYAPLPGLT).sub.u (SEQ ID NO:258);
((F/Y/W)LRRAYAPLPGKS).sub.u (SEQ ID NO:259);
((F/Y/W)LRRAYAPLPGKT).sub.u (SEQ ID NO:260);
((F/Y/W)LRRAYAPLPDLS).sub.u (SEQ ID NO:261);
((F/Y/W)LRRAYAPLPDLT).sub.u (SEQ ID NO:262);
((F/Y/W)LRRAYAPLPDKS).sub.u (SEQ ID NO:263);
((F/Y/W)LRRAYAPLPDKT).sub.u (SEQ ID NO:264);
((F/Y/W)WLRRAYAPLPG).sub.u (SEQ ID NO:265);
((F/Y/W)WLRRAYAPLPD).sub.u (SEQ ID NO:266);
((F/Y/W)WLRRAYAPLPGL).sub.u (SEQ ID NO:267);
((F/Y/W)WLRRAYAPLPGK).sub.u (SEQ ID NO:268);
((F/Y/W)WLRRAYAPLPDL).sub.u (SEQ ID NO:269);
((F/Y/W)WLRRAYAPLPDK).sub.u (SEQ ID NO:270);
((F/Y/W)WLRRAYAPLPGLS).sub.u (SEQ ID NO:271);
((F/Y/W)WLRRAYAPLPGLT).sub.u (SEQ ID NO:272);
((F/Y/W)WLRRAYAPLPGKS).sub.u (SEQ ID NO:273);
((F/Y/W)WLRRAYAPLPGKT).sub.u (SEQ ID NO:274);
((F/Y/W)WLRRAYAPLPDLS).sub.u (SEQ ID NO:275);
((F/Y/W)WLRRAYAPLPDLT).sub.u (SEQ ID NO:276);
((F/Y/W)WLRRAYAPLPDKS).sub.u (SEQ ID NO:277); and
((F/Y/W)WLRRAYAPLPDKT).sub.u (SEQ ID NO:278) wherein "u" is as
defined above, and (F/Y/W) means that the residue is selected from
F, Y, and W. Other specific polypeptides falling within the scope
of general formula I will be readily apparent to one of skill in
the art based on the teachings herein.
[0073] In a further embodiment, the polypeptides of the present
invention consist of a combination of different sequences from the
region [X3-A(X4)APLP-X5-].sub.u. In this embodiment, for example,
the polypeptide can consist of 1 copy of SEQ ID NO:9 and 1 copy of
SEQ ID NO:143. In a different example, the polypeptide could
consist of 2 copies of SEQ ID NO:200 and 3 copies of SEQ ID NO:62.
It will be apparent to one of skill in the art that many such
combinations are possible based on the teachings of the present
invention.
[0074] In a preferred embodiment, at least one of X2 and X6
comprises a transduction domain. As used herein, the term
"transduction domain" means one or more amino acid sequence or any
other molecule that can carry the active domain across cell
membranes. These domains can be linked to other polypeptides to
direct movement of the linked polypeptide across cell membranes. In
some cases the transducing molecules do not need to be covalently
linked to the active polypeptide (for example, see sequence ID
291). In a preferred embodiment, the transduction domain is linked
to the rest of the polypeptide via peptide bonding. (See, for
example, Cell 55: 1179-1188, 1988; Cell 55: 1189-1193, 1988; Proc
Natl Acad Sci USA 91: 664-668, 1994; Science 285: 1569-1572, 1999;
J Biol Chem 276: 3254-3261, 2001; and Cancer Res 61: 474-477, 2001)
In this embodiment, any of the polypeptides as described above
would include at least one transduction domain. In a further
embodiment, both X2 and X6 comprise transduction domains. In a
further preferred embodiment, the transduction domain(s) is/are
selected from the group consisting of (R).sub.4-9 (SEQ ID NO:279);
GRKKRRQRRRPPQ (SEQ ID NO:280); AYARAAARQARA (SEQ ID NO:281);
DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:282);
GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:283); PLSSIFSRIGDP (SEQ ID
NO:284); AAVALLPAVLLALLAP (SEQ ID NO:285); AAVLLPVLLAAP (SEQ ID
NO:286); VTVLALGALAGVGVG (SEQ ID NO:287); GALFLGWLGAAGSTMGAWSQP
(SEQ ID NO:288); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:289);
KLALKLALKALKAALKLA (SEQ ID NO:290); KETWWETWWTEWSQPKKKRKV (SEQ ID
NO:291); KAFAKLAARLYRKAGC (SEQ ID NO:292); KAFAKLAARLYRAAGC (SEQ ID
NO:293); AAFAKLAARLYRKAGC (SEQ ID NO:294); KAFAALAARLYRKAGC (SEQ ID
NO:295); KAFAKLAAQLYRKAGC (SEQ ID NO:296), and AGGGGYGRKKRRQRRR
(SEQ ID NO:306).
[0075] In another embodiment, the present invention provides a
polypeptide comprising a sequence according to general formula
II:
X1-X2-[X3-A(X4)APLP-X5].sub.u-X6
[0076] wherein X1 is absent or is one or more molecules comprising
one or more aromatic ring;
[0077] X2 is absent or comprises a cell transduction domain;
[0078] X3 is 0-14 amino acids of the sequence of heat shock protein
20 between residues 1 and 14 of SEQ ID NO:297;
[0079] X4 is selected from the group consisting of S, T, Y, D, E,
hydroxylysine, hydroxyproline, phosphoserine analogs and
phosphotyrosine analogs;
[0080] X5 is 0-140 amino acids of heat shock protein 20 between
residues 21 and 160 of SEQ ID NO:297;
[0081] X6 is absent or comprises a cell transduction domain;
and
[0082] wherein at least one of X2 and X6 comprise a transduction
domain.
[0083] Thus, in various preferred embodiments of the polypeptide of
general formula II, X4 is S, T, Y, D, E, a phosphoserine analog, or
a phosphotyrosine analog. In a preferred embodiment, X4 is S, T, or
Y. In a more preferred embodiment, X4 is S or T. In a most
preferred embodiment, X4 is S.
[0084] In these embodiments where X4 is S, T, or Y, it is most
preferred that X4 is phosphorylated. When X4 is D or E, these
residues have a negative charge that mimics the phosphorylated
state. The polypeptides of the invention are optimally effective in
the methods of the invention when X4 is phosphorylated, is a
phosphoserine or phosphotyrosine mimic, or is another mimic of a
phosphorylated amino acid residue, such as a D or E residue.
[0085] In a further preferred embodiment, X1 is one or more
molecules comprising one or more aromatic ring, as disclosed above,
with preferred embodiments as disclosed above.
[0086] According to these embodiments, the polypeptide comprises at
least one transduction domain. In a further embodiment, both X2 and
X6 comprise a transduction domain. Preferred embodiments of such
transduction domains are as described above.
[0087] One preferred embodiment of the polypeptide of general
formula II comprises full length HSP20 (X1-X2-SEQ ID
NO:297-X6).
TABLE-US-00001 (SEQ ID NO:297) Met Glu Ile Pro Val Pro Val Gln Pro
Ser Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Ser Ala Pro Gly
Arg Leu Phe Asp Gln Arg Phe Gly Glu Gly Leu Leu Glu Ala Glu Leu Ala
Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr Tyr Leu Arg Ala Pro Ser Val
Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro Gly His Phe Ser Val Leu
Leu Asp Val Lys His Phe Ser Pro Glu Glu Ile Ala Val Lys Val Val Gly
Glu His Val Glu Val His Ala Arg His Glu Glu Arg Pro Asp Glu His Gly
Phe Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro Gly Val Asp
Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val Leu Ser Ile Gln
Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys.
[0088] Another preferred embodiment of the polypeptide of general
formula TI comprises full length HSP20 with the serine at position
16 substitute with aspartic acid (X1-X2-SEQ ID NO:298-X6):
TABLE-US-00002 (SEQ ID NO:298) Met Glu Ile Pro Val Pro Val Gln Pro
Ser Trp Leu Arg Arg Ala Asp Ala Pro Leu Pro Gly Leu Ser Ala Pro Gly
Arg Leu Phe Asp Gln Arg Phe Gly Glu Gly Leu Leu Glu Ala Glu Leu Ala
Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr Tyr Leu Arg Ala Pro Ser Val
Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro Gly His Phe Ser Val Leu
Leu Asp Val Lys His Phe Ser Pro Glu Glu Ila Ala Val Lys Val Val Gly
Glu His Val Glu Val His Ala Arg His Glu Glu Arg Pro Asp Glu His Gly
Phe Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro Gly Val Asp
Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val Leu Ser Ile Gln
Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys.
[0089] Another preferred embodiment of the polypeptide of general
formula II comprises full length HSP20 with the serine at position
16 substitute with glutamic acid (X1-X2-SEQ ID NO:299-X6):
TABLE-US-00003 (SEQ ID NO:299) Met Glu Ile Pro Val Pro Val Gln Pro
Ser Trp Leu Arg Arg Ala Glu Ala Pro Leu Pro Gly Leu Ser Ala Pro Gly
Arg Leu Phe Asp Gln Arg Phe Gly Glu Gly Leu Leu Glu Ala Glu Leu Ala
Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr Tyr Leu Arg Ala Pro Ser Val
Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro Gly Phe Ser Val Leu Leu
Asp Val Lys His Phe Ser Pro Glu Glu Ile Ala Val Lys Val Val Gly Glu
His Val Glu Val His Ala Arg His Glu Glu Arg Pro Asp Glu His Gly Phe
Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro Gly Val Asp Pro
Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val Leu Ser Ile Gln Ala
Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys.
[0090] Other preferred embodiments according to general formula II
are the peptides disclosed above as embodiments of general formula
I with the required transduction domain at either X2 or X6, or
both. Still further preferred embodiments according to general
formula II are the following:
TABLE-US-00004 X1-X2-SEQ ID NO:300-X6, wherein (SEQ ID NO: 300) is
Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Lys; X1-X2-SEQ ID
NO:301-X6, wherein (SEQ ID NO: 301) is Trp Leu Arg Arg Ala Asp Ala
Pro Leu Pro Gly Leu Lys; and X1-X2-SEQ ID NO:302-X6, wherein (SEQ
ID NO: 302) is Trp Leu Arg Arg Ala Glu Ala Pro Leu Pro Gly Leu
Lys.
[0091] In these embodiments of the polypeptides according to
general formula II, it is preferred that the polypeptides are
phosphorylated, most preferably at residue 16, or contain
phosphorylation mimics at the position of amino acid residue
16.
[0092] In a further aspect, the present invention provides a
composition, comprising one or more polypeptides of the present
invention, and an inhibitor of HSP27. HSP27 is closely related to
HSP20; the two proteins often co-exist in macromolecular
aggregates, and both are actin-associated proteins. Increases in
the phosphorylation of HSP27 are associated with smooth muscle
contraction, and transfection of cells with dominant active
phosphorylated mutants of HSP27 leads to stress fiber formation
(Mol Cell Biol 15: 505-516, 1995). Furthermore, increases in the
phosphorylation of HSP27 are associated with smooth muscle cell
migration. HSP20, in contrast, promotes vasorelaxation, and the
data presented herein demonstrates that phosphorylated analogues of
HSP20 lead to a loss of stress fiber formation, and inhibit smooth
muscle cell proliferation and migration (See the examples below).
Thus, the data indicate that HSP20 and HSP27 have opposing
functions. Therefore, the combined use of one or more polypeptides
of the invention and an inhibitor of HSP27 will have enhanced
efficacy in carrying out the methods of the invention for
inhibiting smooth muscle cell proliferation and/or migration, for
promoting smooth muscle relaxation, and for inhibiting smooth
muscle spasm (see below).
[0093] As used herein, an "inhibitor" of HSP27 includes HSP27
antibodies, anti-sense HSP27 nucleic acids, or small molecule
inhibitors of the phosphorylation of HSP27, such as SB203580
(available from SmithKline Beecham).
[0094] The polypeptides may be subjected to conventional
pharmaceutical operations such as sterilization and/or may contain
conventional adjuvants, such as preservatives, stabilizers, wetting
agents, emulsifiers, buffers etc.
[0095] In another aspect, the present invention provides
pharmaceutical compositions, comprising one or more of the
polypeptides disclosed herein, and a pharmaceutically acceptable
carrier. Such pharmaceutical compositions are especially useful for
carrying out the methods of the invention described below.
[0096] For administration, the polypeptides are ordinarily combined
with one or more adjuvants appropriate for the indicated route of
administration. The compounds may be admixed with lactose, sucrose,
starch powder, cellulose esters of alkanoic acids, stearic acid,
talc, magnesium stearate, magnesium oxide, sodium and calcium salts
of phosphoric and sulphuric acids, acacia, gelatin, sodium
alginate, polyvinylpyrrolidine, dextran sulfate, heparin-containing
gels, and/or polyvinyl alcohol, and tableted or encapsulated for
conventional administration. Alternatively, the compounds of this
invention may be dissolved in saline, water, polyethylene glycol,
propylene glycol, carboxymethyl cellulose colloidal solutions,
ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,
tragacanth gum, and/or various buffers. Other adjuvants and modes
of administration are well known in the pharmaceutical art. The
carrier or diluent may include time delay material, such as
glyceryl monostearate or glyceryl distearate alone or with a wax,
or other materials well known in the art.
[0097] The polypeptides or pharmaceutical compositions thereof may
be administered by any suitable route, including orally,
parentally, by inhalation spray, rectally, or topically in dosage
unit formulations containing conventional pharmaceutically
acceptable carriers, adjuvants, and vehicles. The term parenteral
as used herein includes, subcutaneous, intravenous, intra-arterial,
intramuscular, intrasternal, intratendinous, intraspinal,
intracranial, intrathoracic, infusion techniques or
intraperitoneally. Preferred embodiments for administration vary
with respect to the condition being treated, and are described in
detail below.
[0098] The polypeptides may be made up in a solid form (including
granules, powders or suppositories) or in a liquid form (e.g.,
solutions, suspensions, or emulsions). The polypeptides of the
invention may be applied in a variety of solutions. Suitable
solutions for use in accordance with the invention are sterile,
dissolve sufficient amounts of the polypeptides, and are not
harmful for the proposed application.
[0099] In another aspect, the present invention provides an
isolated nucleic acid sequence encoding a polypeptide of the
present invention. Appropriate nucleic acid sequences according to
this aspect of the invention will be apparent to one of skill in
the art based on the disclosure provided herein and the general
level of skill in the art. One example of such a nucleic acid
sequence is provided as SEQ ID NO:320.
[0100] In another aspect, the present invention provides an
expression vector comprising DNA control sequences operably linked
to the isolated nucleic acid molecules of the present invention, as
disclosed above. "Control sequences" operably linked to the nucleic
acid sequences of the invention are nucleic acid sequences capable
of effecting the expression of the nucleic acid molecules. The
control sequences need not be contiguous with the nucleic acid
sequences, so long as they function to direct the expression
thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the nucleic acid sequences and the promoter sequence can still
be considered "operably linked" to the coding sequence. Other such
control sequences include, but are not limited to, polyadenylation
signals, termination signals, and ribosome binding sites.
[0101] Such expression vectors can be of any type known in the art,
including but not limited plasmid and viral-based expression
vectors.
[0102] In a further aspect, the present invention provides
genetically engineered host cells comprising the expression vectors
of the invention. Such host cells can be prokaryotic cells or
eukaryotic cells, and can be either transiently or stably
transfected, or can be transduced with viral vectors.
[0103] In another aspect, the invention provides improved
biomedical devices, wherein the biomedical devices comprise one or
more of the polypeptides of the present invention disposed on or in
the biomedical device. In a preferred embodiment, the one or more
polypeptides are phosphorylated, as discussed above.
[0104] As used herein, a "biomedical device" refers to a device to
be implanted into a subject, for example, a human being, in order
to bring about a desired result. Particularly preferred biomedical
devices according to this aspect of the invention include, but are
not limited to, stents, grafts, shunts, stent grafts, fistulas,
angioplasty devices, balloon catheters and any implantable drug
delivery device.
[0105] As used herein, the term "grafts" refers to both natural and
prosthetic grafts and implants. In a most preferred embodiment, the
graft is a vascular graft.
[0106] As used herein, the term "stent" includes the stent itself,
as well as any sleeve or other component that may be used to
facilitate stent placement.
[0107] As used herein, "disposed on or in" means that the one or
more polypeptides can be either directly or indirectly in contact
with an outer surface, an inner surface, or embedded within the
biomedical device. "Direct" contact refers to disposition of the
polypeptides directly on or in the device, including but not
limited to soaking a biomedical device in a solution containing the
one or more polypeptides, spin coating or spraying a solution
containing the one or more polypeptides onto the device, implanting
any device that would deliver the polypeptide, and administering
the polypeptide through a catheter directly on to the surface or
into any organ.
[0108] "Indirect" contact means that the one or more polypeptides
do not directly contact the biomedical device. For example, the one
or more polypeptides may be disposed in a matrix, such as a gel
matrix or a viscous fluid, which is disposed on the biomedical
device. Such matrices can be prepared to, for example, modify the
binding and release properties of the one or more polypeptides as
required.
[0109] In a further embodiment, the biomedical device further
comprises an inhibitor of the small heat shock protein HSP27
disposed on or in the biomedical device. In a preferred embodiment,
such inhibitors are selected from HSP27 antibodies, anti-sense
HSP27 nucleic acids, or small molecule inhibitors of the
phosphorylation of HSP27, such as SB203580.
[0110] In another aspect, the invention provides methods for
inhibiting smooth muscle cell proliferation and/or migration,
comprising contacting the smooth muscle cells with an amount
effective to inhibit smooth muscle cell proliferation and/or
migration of HSP20, or functional equivalents thereof, such as one
or more polypeptide according to general formula I or II. In a most
preferred embodiment, the one or more polypeptides are
phosphorylated as disclosed above. In a further embodiment, the
method further comprises contacting the smooth muscle cells with an
amount effective to inhibit smooth muscle cell proliferation and/or
migration of an inhibitor of the small heat shock protein HSP27. In
a further embodiment, the method further comprises contacting the
cells with an amount of PKG sufficient to stimulate HSP20
phosphorylation, wherein the contacting comprises transfecting the
cells with an expression vector that is capable of directing the
expression of PKG, or by transducing the cells with a
PKG-transduction domain chimera.
[0111] Intimal hyperplasia is a complex process that leads to graft
failure, and is the most common cause of failure of arterial bypass
grafts. While incompletely understood, intimal hyperplasia is
mediated by a sequence of events that include endothelial cell
injury and subsequent vascular smooth muscle proliferation and
migration from the media to the intima. This process is associated
with a phenotypic modulation of the smooth muscle cells from a
contractile to a synthetic phenotype. The "synthetic" smooth muscle
cells secrete extracellular matrix proteins, which leads to
pathologic narrowing of the vessel lumen leading to graft stenoses
and ultimately graft failure. Such endothelial cell injury and
subsequent smooth muscle cell proliferation and migration into the
intima also characterizes restenosis, most commonly after
angioplasty to clear an obstructed blood vessel. As discussed
below, HSP20, and functional equivalents thereof, such as the
polypeptides of general formula I and II, inhibit smooth muscle
cell proliferation and migration.
[0112] In this aspect, the method can be in vitro or in vivo. In
one embodiment, the method is in vitro, wherein a vein or arterial
graft is contacted with HSP20 or a functional equivalent(s)
thereof, prior to grafting in a patient, in order to inhibit smooth
muscle cell proliferation and/or migration, and thus to inhibit
subsequent intimal hyperplasia and stenosis after placement of the
graft, which could lead to graft failure. This can be accomplished,
for example, by delivering the recombinant expression vectors (most
preferably a viral vector, such as an adenoviral vector) of the
invention to the site, and transfecting the smooth muscle cells.
More preferably, delivery into smooth muscle cells is accomplished
by using the polypeptides of general formula I or II that include
at least one transduction domain to facilitate entry into the
smooth muscle cells. The examples below demonstrate the ability of
the polypeptides of the invention that contain at least one
transduction domain to be delivered into smooth muscle cells.
[0113] In a more preferred in vitro embodiment, the method
comprises contacting the graft with one or more of the polypeptides
of the invention that include at least one transduction domain.
Upon placement of the graft, it is preferred that the subject
receiving be treated systemically with heparin, as heparin has been
shown to bind to protein transduction domains and prevent them from
transducing into cells. This approach will lead to localized
protein transduction of the graft alone, and not into peripheral
tissues.
[0114] In various other preferred embodiments of this aspect, the
method is performed in vivo, and is used to treat or prevent a
disorder selected from the group consisting of intimal hyperplasia,
stenosis, restenosis, and atherosclerosis. In these embodiments,
the contacting may be direct, by contacting a blood vessel in a
subject being treated with HSP20 or a functional equivalent(s)
thereof, such as the polypeptides according to general formula I or
II. For example, a liquid preparation of HSP20 or a functional
equivalent(s) thereof, such as the polypeptides according to
general formula I or II, can be forced through a porous catheter,
or otherwise injected through a catheter to the injured site, or a
gel or viscous liquid containing the one or more polypeptides could
be spread on the injured site. In these embodiment of direct
delivery, it is most preferred that the HSP20 or a functional
equivalent(s) thereof, such as the polypeptides according to
general formula I or II be delivered into smooth muscle cells at
the site of injury or intervention. This can be accomplished, for
example, by delivering the recombinant expression vectors (most
preferably a viral vector, such as an adenoviral vector) of the
invention to the site. More preferably, delivery into smooth muscle
cells is accomplished by using the polypeptides of general formula
I or II that include at least one transduction domain to facilitate
entry into the smooth muscle cells. The examples below demonstrate
the ability of the polypeptides of the invention that contain at
least one transduction domain to be delivered into smooth muscle
cells.
[0115] In various other preferred embodiments of this aspect of the
invention, the method is performed on a subject who has undergone,
is undergoing, or will undergo a procedure selected from the group
consisting of angioplasty, vascular stent placement,
endarterectomy, atherectomy, bypass surgery (such as coronary
artery bypass surgery; peripheral vascular bypass surgeries),
vascular grafting, organ transplant, prosthetic device implanting,
microvascular reconstructions, plastic surgical flap construction,
and catheter emplacement.
[0116] In a further embodiment of this aspect of the invention, the
method is used to treat smooth muscle cell tumors. In a preferred
embodiment, the tumor is a leiomyosarcoma, which is defined as a
malignant neoplasm that arises from muscle. Since leiomyosarcomas
can arise from the walls of both small and large blood vessels,
they can occur anywhere in the body, but peritoneal, uterine, and
gastro-intestinal (particularly esophageal) leiomyosarcomas are
more common. Alternatively, the smooth muscle tumor can be a
leiomyoma, a non-malignant smooth muscle neoplasm. In a most
preferred embodiment, the one or more polypeptides are
phosphorylated as disclosed above. In a further embodiment, the
method further comprises contacting the smooth muscle cells with an
amount effective to inhibit smooth muscle cell proliferation and/or
migration of an inhibitor of the small heat shock protein
HSP27.
[0117] As further discussed in the examples below, HSP20, and
functional equivalents thereof, such as the polypeptides of general
formula I and II, also disrupt actin stress fiber formation and
adhesion plaques, each of which have been implicated in intimal
hyperplasia. The data further demonstrate a direct inhibitory
effect of the polypeptides of the present invention on intimal
hyperplasia. Thus, in another aspect, the present invention further
provides methods for treating or inhibiting one or more disorder
selected from the group consisting of intimal hyperplasia,
stenosis, restenosis, and atherosclerosis, comprising contacting a
subject in need thereof with an amount effective to treat or
inhibit intimal hyperplasia, stenosis, restenosis, and/or
atherosclerosis of HSP20, or a functional equivalent thereof, such
as one or more polypeptides according to general formula I or II.
Delivery of the HSP20, or a functional equivalent thereof, such as
one or more polypeptides according to general formula I or II, in
this aspect are as disclosed above. In a most preferred embodiment,
the one or more polypeptides are phosphorylated as disclosed above.
In a further embodiment, the method further comprises contacting
the smooth muscle cells with an amount effective to inhibit smooth
muscle cell proliferation and/or migration of an inhibitor of the
small heat shock protein HSP27.
[0118] In various other preferred embodiments of this aspect of the
invention, the method is performed on a subject who has undergone,
is undergoing, or will undergo a procedure selected from the group
consisting of angioplasty, vascular stent placement,
endarterectomy, atherectomy, bypass surgery, vascular grafting,
microvascular reconstructions, plastic surgical flap construction,
organ transplant, and catheter emplacement.
[0119] In a further aspect, the present invention provides methods
to treat smooth muscle cell tumors, comprising administering to a
subject in need thereof of an amount effective of HSP20, or a
functional equivalent thereof, such as one or more polypeptides
according to general formula I or II, to inhibit smooth muscle
tumor growth and/or metastasis. In a preferred embodiment, the
tumor is a leiomyosarcomas. Alternatively, the smooth muscle tumor
can be a leiomyoma. In a further embodiment, the method further
comprises contacting the smooth muscle cells with an amount
effective to inhibit smooth muscle cell proliferation and/or
migration of an inhibitor of the small heat shock protein
HSP27.
[0120] In a further aspect, the present invention provides a method
for treating or preventing smooth muscle spasm, comprising
contacting a subject or graft in need thereof with an amount
effective to inhibit smooth muscle spasm of HSP20, or a functional
equivalent thereof, such as one or more polypeptides according to
general formula I or II. In a most preferred embodiment, the one or
more polypeptides are phosphorylated as disclosed above. In a
further embodiment, the method further comprises contacting the
smooth muscle with an amount effective to inhibit smooth muscle
cell proliferation and/or migration of an inhibitor of the small
heat shock protein HSP27. In a further embodiment, the method
further comprises contacting the smooth muscle with an amount
effective of PKG to stimulate HSP20 phosphorylation, as described
above.
[0121] The examples below demonstrate that HSP20, and equivalents
thereof, such as the polypeptides according to general formula I
and II, are effective at inhibiting smooth muscle spasm, such as
vasospasm. While not being limited by a specific mechanism of
action, it is believed that HSP20, and equivalents thereof, such as
the polypeptides according to general formula I and II, exert their
anti-smooth muscle spasm effect by promoting smooth muscle
vasorelaxation and inhibiting contraction.
[0122] Smooth muscles are found in the walls of blood vessels,
airways, the gastrointestinal tract, and the genitourinary tract.
Pathologic tonic contraction of smooth muscle constitutes spasm.
Many pathological conditions are associated with spasm of vascular
smooth muscle ("vasospasm"), the smooth muscle that lines blood
vessels. This can cause symptoms such as angina and ischemia (if a
heart artery is involved), or stroke as in the case of subarachnoid
hemorrhage induced vasospasm if a brain vessel is involved.
Hypertension (high blood pressure) is caused by excessive
vasoconstriction, as well as thickening, of the vessel wall,
particularly in the smaller vessels of the circulation.
[0123] Thus, in one embodiment of this aspect of the invention, the
muscle cell spasm comprises a vasospasm, and the method is used to
treat or inhibit vasospasm. Preferred embodiments of the method
include, but are not limited to, methods to treat or inhibit
angina, coronary vasospasm, Prinzmetal's angina (episodic focal
spasm of an epicardial coronary artery), ischemia, stroke,
bradycardia, and hypertension.
[0124] In another embodiment of this aspect of the invention,
smooth muscle spasm is inhibited by treatment of a graft, such as a
vein or arterial graft, with HSP20, or a functional equivalent
thereof, such as one or more polypeptides according to general
formula I or II, as described above. One of the ideal conduits for
peripheral vascular and coronary reconstruction is the greater
saphenous vein. However, the surgical manipulation during harvest
of the conduit often leads to vasospasm. The exact etiology of
vasospasm is complex and most likely multifactorial. Most
investigations have suggested that vasospasm is either due to
enhanced constriction or impaired relaxation of the vascular smooth
muscle in the media of the vein. Numerous vasoconstricting agents
such as endothelin-1 and thromboxane are increased during surgery
and result in vascular smooth muscle contraction. Other
vasoconstrictors such as norepinephrine, 5-hydroxytryptamine,
acetylcholine, histamine, angiotensin II, and phenylephrine have
been implicated in vein graft spasm. Papaverine is a smooth muscle
vasodilator that has been used. In circumstances where spasm occurs
even in the presence of papaverine, surgeons use intraluminal
mechanical distension to break the spasm. This leads to injury to
the vein graft wall and subsequent intimal hyperplasia. Intimal
hyperplasia is the leading cause of graft failure.
[0125] Thus, in this embodiment, the graft can be contacted with
HSP20 or a functional equivalent(s) thereof, during harvest from
the graft donor, subsequent to harvest (before implantation),
and/or during implantation into the graft recipient. This can be
accomplished, for example, by delivering the recombinant expression
vectors (most preferably a viral vector, such as an adenoviral
vector) of the invention to the site, and transfecting the smooth
muscle cells. More preferably, delivery into smooth muscle is
accomplished by using the polypeptides of general formula I or II
that include at least one transduction domain to facilitate entry
into the smooth muscle cells. The examples below demonstrate the
ability of the polypeptides of the invention that contain at least
one transduction domain to be delivered into smooth muscle cells.
During graft implantation, it is preferred that the subject
receiving be treated systemically with heparin, as heparin has been
shown to bind to protein transduction domains and prevent them from
transducing into cells. This approach will lead to localized
protein transduction of the graft alone, and not into peripheral
tissues. The methods of this embodiment of the invention inhibit
vein graft spasm during harvest and/or implantation of the graft,
and thus improve both short and long term graft success.
[0126] In various other embodiments, the muscle cell spasm is
associated with a disorder including, but not limited to pulmonary
(lung) hypertension, asthma (bronchospasm), toxemia of pregnancy,
pre-term labor, pre-eclampsia/eclampsia, Raynaud's disease or
phenomenon, hemolytic-uremia, non-occlusive mesenteric ischemia
(ischemia of the intestines that is caused by inadequate blood flow
to the intestines), anal fissure (which is caused by persistent
spasm of the internal anal sphincter), achalasia (which is caused
by persistent spasm of the lower esophageal sphincter), impotence
(which is caused by a lack of relaxation of the vessels in the
penis, erection requires vasodilation of the corpra cavernosal
(penile) blood vessels), migraine (which is caused by spasm of the
intracranial blood vessels), ischemic muscle injury associated with
smooth muscle spasm, and vasculopathy, such as transplant
vasculopathy (a reaction in the transplanted vessels which is
similar to atherosclerosis, it involves constrictive remodeling and
ultimately obliteration of the transplanted blood vessels, this is
the leading cause of heart transplant failure).
[0127] Preferred routes of delivery for these various indications
of the different aspects of the invention vary. Topical
administration is preferred for methods involving treatment or
inhibition of vein graft spasm, intimal hyperplasia, restenosis,
prosthetic graft failure due to intimal hyperplasia, stent, stent
graft failure due to intimal hyperplasia/constrictive remodeling,
microvascular graft failure due to vasospasm, transplant
vasculopathy, and male and female sexual dysfunction. As used
herein, "topical administration" refers to delivering the
polypeptide onto the surface of the organ.
[0128] Intrathecal administration, defined as delivering the
polypeptide into the cerebrospinal fluid is the preferred route of
delivery for treating or inhibiting stroke and subarachnoid
hemorrhage induced vasospasm. Intraperitoneal administration,
defined as delivering the polypeptide into the peritoneal cavity,
is the preferred route of delivery for treating or inhibiting
non-occlusive mesenteric ischemia. Oral administration is the
preferred route of delivery for treating or inhibiting achalasia.
Intravenous administration is the preferred route of delivery for
treating or inhibiting hypertension and bradycardia. Administration
via suppository is preferred for treating or inhibiting anal
fissure. Aerosol delivery is preferred for treating or inhibiting
asthma (ie: bronchospasm). Intrauterine administration is preferred
for treating or inhibiting pre-term labor and
pre-eclampsia/eclampsia.
[0129] In practicing these various aspects of the invention, the
amount or dosage range of the polypeptides or pharmaceutical
compositions employed is one that effectively teats or inhibits one
or more of smooth muscle cell proliferation, smooth muscle cell
migration, smooth muscle spasm; and/or that promotes smooth muscle
relaxation. Such an inhibiting (or promoting in the case of smooth
muscle relaxation) amount of the polypeptides that can be employed
ranges generally between about 0.01 .mu.g/kg body weight and about
10 mg/kg body weight, preferably ranging between about 0.05
.mu.g/kg and about 5 mg/kg body weight.
[0130] The present invention may be better understood with
reference to the accompanying examples that are intended for
purposes of illustration only and should not be construed to limit
the scope of the invention, as defined by the claims appended
hereto.
EXAMPLES
Example 1
[0131] This Example illustrates a study of cyclic
nucleotide-dependent phosphorylation of HSP20 in mesangial cells.
The contractile phenotype and expression of PKG is lost as smooth
muscle cells are passaged in culture. Mesangial cells have been
shown to maintain a contractile phenotype in culture. To determine
if mesangial cells in culture continue to express PKG and HSP20,
multiply passaged mesangial cells were compared to multiply
passaged vascular smooth muscle cells and smooth muscle cells that
had been stably transfected with PKG. The cells were homogenized
and immunoblots were performed using rabbit polyclonal antibodies
against PKG and HSP20. Multiply passaged vascular smooth muscle
cells did not express PKG or HSP20. However, smooth muscle cells
that had been stably transfected with PKG express PKG and HSP20.
Cultured mesangial cells expressed similar amounts of both PKG and
HSP20 as the PKG transfected vascular smooth muscle cells. These
data suggest that the expression of PKG and the PKG substrate
protein, HSP20, are coordinately regulated and that the expression
of these proteins may be important for maintaining the cells in a
contractile phenotype. Since phenotypic modulation from a
contractile to a synthetic phenotype has been implicated in the
response to injury model of atherogenesis and in the development of
intimal hyperplasia, we propose that introducing HSP20 either via
protein transduction or by gene transfection will provide a novel
therapeutic approach to maintain cells in a contractile phenotype
and prevent intimal hyperplasia.
Example 2
[0132] This Example illustrates the production of the HSP20 S16A
mutant wherein the phosphorylation site (serine 16) was mutated to
an alanine. The cDNA for HSP20 was cloned into pEGFP-C2 expression
vector (commercially available from Clontech, Inc.) For production
of the HSP20 S16A mutant, a single nucleotide mutation was
introduced in the HSP20 cDNA sequence using a two complimentary
oligonucleotide strategy with Pfu polumerase (commercially
available from Stratagene, La Jolla, Calif.). All sequences were
confirmed for orientation, the presence of the appropriate
mutations, and the absence of other mutations, using a 377
Perkin-Elmer ABI Prism DNA sequencer (Foster City, Calif.). Similar
techniques can be used to mutate the serine 16 for an aspartic or
glutamic acid.
Example 3
[0133] This experiment illustrates that genetic manipulation of
muscle like cells can alter their ability to contract.
Specifically, engineering the cells to overexpress HSP20 prevents
them from contracting (going into a state of spasm). If the cells
overexpress a mutated form of HSP20 that cannot be phosphorylated,
they remain contracted (in spasm) even when treated with potent
agents which cause relaxation. This experiment demonstrates that
the phosphorylation of HSP20 is the seminal event required for
muscles to relax.
[0134] The experiment was performed in the following fashion:
Mesangial cells were transfected with vectors containing green
fluorescent protein (GFP) alone, GFP fused to the 5' end of the
wild type cDNA for HSP20 (WT), or GFP fused to an HSP20 construct
in which the PKA phosphorylation site was mutated to an alanine
(S16A-HSP20) (MUT). The cells were plated on a silicone rubber
substrata in the presence of serum for 48 hours. The plates were
then placed on the stage of a Zeiss Inverted Fluorescence
Microscope with DeltaVision image acquisition and deconvolution
software (Applied Precision, Issaquah, Wash.). The DeltaVision
software was configured to eight different cells (x and y axis) on
each plate with 7 z-axis images taken at 2 nm intervals.
Fluorescent and phase contrast images were obtained such that the
cells and silicone membrane were imaged in close succession. During
the scanning process, the z-axis of each cell was monitored to
assure that the imaging stacks were maintained at the appropriate
level as the cells relaxed on the silicone membrane. Baseline
images were acquired for one hour. The cells were then treated with
the cells were treated with dibutyryl cAMP (10 .PHI.M) for 0
minutes, 30 minutes, 60 minutes, or 90 minutes, and the results are
illustrated in FIG. 1.
[0135] The control cells expressing the green fluorescent protein
(GFP) in which the HSP20 was not changed relaxed over time (the
wrinkles under the cells disappeared) when treated with dibutyryl
cAMP. The cells over expressing HSP20 tagged to GFP (WT) did not
form wrinkles; they were unable to contract or go into spasm. The
cells expressing the mutated form of HSP20 that could not be
phosphorylated (S16A-HSP20) (MUT) formed abundant wrinkles
(contracted) but did not relax (remained in spasm) in response to
dibutyryl cAMP. This figure is representative of 6 separate
transfections in which at least 12 cells were imaged with each
construct and the aggregate data is illustrated graphically
(*=p<0.05 compared to the initial number of wrinkles). Similar
findings were observed when cyclic nucleotide-dependent signaling
pathways were activated with sodium nitroprusside (10 .mu.M),
dibutyryl cGMP (10 .mu.M), or forskolin (10 .mu.M) (data not
shown). There were no changes in the wrinkles in the substrata in
untreated cells imaged for 90 minutes (data not shown). These data
demonstrate that over expression of wild type HSP20 inhibits
contraction of the cells and expression of the S16A-HSP20 mutated
protein inhibits relaxation. Thus, these data show that the
phosphorylation of HSP20 is necessary and sufficient for the
relaxation of smooth muscles, and suggests that phosphorylation of
HSP20 represents a point in which the cyclic nucleotide signaling
pathways converge to prevent contraction or cause relaxation.
Example 4
[0136] This experiment demonstrates that transduction of peptide
analogues of phosphorylated HSP20 relaxes muscle-like cells.
Phosphopeptide analogues of HSP20 were synthesized containing the
TAT sequence (NH.sub.2-.beta.AGGGGYGRKKRRQRRRWLRRAS*APLPGLK-COOH)
(referred to as FITC-TAT-HSP20) (SEQ ID NO:304) (the asterisk
indicates that the "S" residue is phosphorylated). Mesangial cells
were plated on a silicone substrata in the presence of serum and
after 48 hours the cells were treated with the FITC-TAT-HSP20
phosphopeptide (50 .mu.M). The number of wrinkles under the cells
was determined at the time points indicated using phase contrast
microscopy (n=10, *=p<0.05 compared to time 0). Results of this
Experiment are illustrated in FIG. 2. Treatment of the cells with
the phosphopeptide analogue of HSP20 led to a time dependent loss
of wrinkles (relaxation of the cells). This experiment demonstrates
that transduction of phosphopeptide analogues of HSP20 also relaxes
the cells.
Example 5
[0137] This experiment demonstrates that transduction of
phosphopeptide analogues of HSP20 relax and prevent spasm in intact
strips of vascular smooth muscle. Transverse strips of bovine
carotid artery smooth muscle, denuded of endothelium, were
suspended in a muscle bath containing bicarbonate buffer (120 mM
NaCl, 4.7 mM KCl, 1.0 mM MgSO.sub.4, 1.0 mM NaH.sub.2PO.sub.4, 10
mM glucose, 1.5 mM CaCl.sub.2, and 25 mM Na.sub.2HCO.sub.3, pH
7.4), equilibrated with 95% O.sub.2/5% CO.sub.2, at 37.degree. C.
at one gram of tension for 2 hours. The muscles were pre-contracted
with serotonin (1 .mu.M for 10 minutes) and cumulative doses of
FITC-phosphoHSP20-TAT, FITC-scrambled phosphoHSP20-TAT
(FITC-NH.sub.2-.beta.AGGGGYGRKKRRQRRRPRKS*LWALGRPLA-COOH, open
circles) (SEQ ID NO:305), or FITC-TAT
(FITC-NH.sub.2-.beta.AGGGGYGRKKRRQRRR, closed triangles) (SEQ ID
NO:306) were added every 10 minutes. The force is depicted as a
percentage of the maximal serotonin contraction (n=5, *=p<0.05
compared to 0 peptide added). Representative strips were fixed in
4% paraformaldehyde and examined under fluorescence microscopy
(40.times. magnification). The internal elastic lamina (IEL)
autofluoresced, and the media and adventitia (ADV) also displayed
fluorescence (not shown).
[0138] The results of this Experiment are illustrated in FIG. 3.
Transduction of pre-contracted strips of intact bovine corotid
artery smooth muscle with the FITC-TAT-HSP20 phosphopeptide led to
a dose dependent decrease in serotonin pre-contracted muscles.
Peptides containing the scrambled sequence or FITC-TAT alone had no
significant effect on contractile force. This shows that the
phosphopeptide analogues relax and prevent spasm in intact vascular
smooth muscles. There was a diffuse fluorescence staining pattern
of the muscle strips after transduction with the FITC-TAT-HSP20
phosphopeptide which shows that the peptides enter the muscles.
Example 6
[0139] This experiment shows that a different transducing peptide
can introduce the HSP20 phosphopeptide analogues. In addition, it
demonstrates that phosphopeptide analogues of HSP20 relax and
prevent spasm in smooth muscles from a different vascular bed in a
different species. Finally, it shows that transduction of HSP20
analogues relax and prevent spasm in muscles in which an intact
endothelium is present.
[0140] Rings of porcine coronary artery in which the endothelium
was not denuded, were suspended in a muscle bath containing
bicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO.sub.4, 1.0
mM NaH.sub.2PO.sub.4, 10 mM glucose, 1.5 mM CaCl.sub.2, and 25 mM
Na.sub.2HCO.sub.3, pH 7.4), equilibrated with 95% O.sub.2/5%
CO.sub.2, at 37.degree. C. at one gram of tension for 2 hours. The
muscles were pre-contracted with serotonin (1 .mu.M for 10 minutes)
and cumulative doses of PTD-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH, closed circles)
(SEQ ID NO:307) or PTD-scrambled-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH open circles) (SEQ
ID NO:308) were added every 10 minutes (FIG. 4). The percentage of
relaxation is depicted as a percentage of the maximal serotonin
contraction (n=5, *=p<0.05 compared to 0 peptide added). The
concentrations of peptide used are depicted on the x axis.
Representative rings were treated with peptides (1 mM final
concentration) linked to FITC (15 minutes at 37.degree. C.), fixed
in 4% paraformaldehyde and examined under fluorescence microscopy
(40.times. magnification).
[0141] The results of this experiment are illustrated in FIG. 4.
This shows that phosphopeptide analogues of HSP20 relax and prevent
spasm in muscles from a different species and different vascular
bed. There was marked fluorescence in the strips treated with
protein transduction analogues. This demonstrates that a protein
transduction peptide that is different than TAT can transduce the
phosphopeptide analogue and relax and prevent spasm in muscles.
Example 7
[0142] This experiment shows that protein transduction of
phosphopeptide analogues of HSP20 can relax and prevent spasm in
non-vascular smooth muscles.
[0143] The internal anal sphincter was obtained from a human
pathology specimen after an abdominal perineal resection for
cancer. The smooth muscles were equilibrated in a muscle bath as
described in example 6. The muscles developed tonic sustained
contractions when warmed in the bicarbonate buffer. These
contractions relaxed with the addition of the guanylate cyclase
activator, sodium nitroprusside (SNP). When the sodium
nitroprusside was washed out of the bath, the muscles again
contracted spontaneously. The muscles were then treated with the
PTD-phosphopeptide analogues of HSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH, 1 mM final
concentration) (SEQ ID NO:307). The muscles relaxed and spasm was
prevented in response to the phosphopeptide analogues and the
relaxation was sustained.
[0144] Intestinal smooth muscle was obtained from the tinea coli of
a pig. These muscles were equilibrated in a muscle bath as
described in example 6. The muscles produced transient contractions
in response to high extracellular potassium chloride (KCl 110 mM)
and in response to carbachol (10.sup.-6 M). The muscles were then
treated with a dextran gel containing the PTD-phosphopeptide
analogues of HSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH, 1 mM final
concentration) (SEQ ID NO:307) and treated with carbachol.
Treatment with the phosphopeptide analogues significantly
attenuated the contractile response to carbachol.
[0145] Tracheal and corpra cavernosal smooth muscles were obtained
from a New Zealand White rabbits after sacrifice. The muscles were
equilibrated in a muscle bath as described in example 6. The
tracheal muscles were pre-contracted with carbachol and the corpra
cavernosal smooth muscles were pre-contracted with norepinephrine.
The muscles were then treated with the PTD-phosphopeptide analogues
of HSP20 (NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH) (SEQ ID
NO:307). Both the tracheal and the corpra cavernosal muscles
relaxed and spasm was prevented in response to the phosphopeptide
analogues.
[0146] The results of these experiments show that the
phosphopeptide analogues of HSP20 relax and prevent spasm in human
anal sphincter smooth muscles, porcine intestinal smooth muscle,
rabbit tracheal smooth muscles, and rabbit corpra cavernosal smooth
muscles.
Example 8
[0147] This experiment shows that protein transduction of
phosphopeptide analogues of HSP20 can relax and prevent spasm in
human saphenous vein smooth muscles.
[0148] Human saphenous vein was obtained from remnants that were
discarded after vascular bypass operations. Rings of the saphenous
vein were equilibrated in a muscle bath as described in experiment
4. The rings were treated with a 7.5% dextran gel alone, or a 7.5%
dextran gel containing the phosphopeptide analogues of HSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH) (SEQ ID NO:307).
The rings were then treated with serotonin (1 uM). The rings
treated with the 7.5% dextran gel alone contracted in response to
serotonin. However, the rings treated with the 7.5% dextran gel
that contained the HSP20 phosphopeptide analogues did not contract
in response to serotonin.
[0149] These results show that the phosphopeptide analogues of
HSP20 prevent spasm (contraction) of human saphenous vein smooth
muscles.
Example 9
[0150] This experiment was performed to demonstrate that smaller
peptide analogues of phosphorylated HSP20 relax and prevent spasm
of smooth muscles even more effectively than the larger
analogues.
[0151] Rings of rabbit aorta in which the endothelium was not
denuded, were suspended in a muscle bath containing bicarbonate
buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO.sub.4, 1.0 mM
NaH.sub.2PO.sub.4, 10 mM glucose, 1.5 mM CaCl.sub.2, and 25 mM
Na.sub.2HCO.sub.3, pH 7.4), equilibrated with 95% O.sub.2/5%
CO.sub.2, at 37.degree. C. at one gram of tension for 2 hours. The
rings were pre-contracted with norepinephrine (10.sup.-7 M) and
treated with RRRRRRApSAPLP (SEQ ID NO:309) or RRRRWLRRApSAPLP (SEQ
ID NO:310). Both peptides caused rapid and complete relaxation and
inhibition of spasm of the muscles, and the relaxation was faster
and the muscles remained in a relaxed state for longer than when
the longer peptides were used. The peptides used were prepared by
Fmoc-based peptide synthesis, and the peptides retained the Fmoc
moiety at the amino terminus of the peptide.
[0152] These data show that poly arginine sequences can transduce
the HSP20 analogues and induce relaxation and that the smaller
sequence ApSAPLP (SEQ ID NO:3) (where "p' indicates that the S
residue is phosphorylated) causes rapid and complete relaxation and
inhibition of spasm.
Example 10
[0153] This experiment illustrates that HSP20 is expressed in
mesangial cells and in rat aortic smooth muscle cells that have
been stably transfected with PKG. It also illustrates that
relaxation of mesangial cells is associated with increases in the
phosphorylation of HSP20.
[0154] Homogenates of mesangial cells (FIG. 5, lane 1), rat aortic
smooth muscle cells (FIG. 5, lane 2), and PKG transfected rat
aortic smooth muscle cells (FIG. 5, lane 3) were immunoblotted for
PKG (FIG. 5, panel A) or HSP20 (FIG. 5, panel B). In a separate
experiment, mesangial cells were untreated (FIG. 5, panel C) or
treated with dibutyryl cAMP (10 .mu.M, 15 minutes, FIG. 5, panel
D). The proteins were separated by 2-dimensional electrophoresis,
transferred to immobilon and probed with anti-HSP20 antibodies.
Increases in the phosphorylation of HSP20 lead to a shift in the
electrophoretic mobility of the protein to a more acidic isoform
(arrow).
[0155] These data show that activation of cyclic
nucleotide-dependent signaling pathways in mesangial cells (as
shown in FIG. 2) is associated with phosphorylation of HSP20.
Example 11
[0156] This experiment illustrates that cells expressing HSP20
(stably PKG transfected cells) are able to contract.
[0157] Rat aortic smooth muscle cells that are multiply passaged or
that stably express PKG were cultured on a silicone substrata in
the presence of serum. The cells were imaged with phase contrast
microscopy (10.times. magnification). The multiply passaged cells
did not form wrinkles on the substrata whereas the PKG transfected
cells formed wrinkles. To determine if the wrinkles were
reversible, PKG transfected cells were treated with dibutyryl cAMP
(10 uM) for 30 minutes. Dibutyryl cAMP led to a decrease in wrinkle
formation.
[0158] Taken together with example 10, these results show that the
expression of HSP20 is associated with a contractile phenotype.
Vascular smooth muscle cells exist in widely divergent phenotypes.
In the normal vessel wall, the smooth muscle cells are in a well
differentiated contractile phenotype and are capable of generating
force. In response to injury, or cell culture conditions, the cells
modulate to a synthetic or secretory phenotype. These cells
proliferate and secrete matrix proteins contributing to intimal
hyperplasia. Phenotypic modulation is associated with changes in
gene expression, protein expression, morphology, and physiologic
responses. This leads to pathologic narrowing of the vessel lumen
which occurs in atherosclerosis and intimal hyperplasia. This leads
to stenotic lesions and ultimately occlusion of the vessel. Thus,
maintaining expression of HSP20 is important for the maintenance of
the contractile phenotype.
Example 12
[0159] Cellular processes such as cell adhesion, cytokinesis, cell
motility, migration, and contraction all require dynamic
reorganization of the actin cytoskeleton These experiments show
that the phosphorylation of HSP20 modulates changes in these actin
filaments.
[0160] Transfected mesangial cells were fixed and the actin
filaments were stained with fluorescent-labeled phalloidin.
Mesangial cells were transfected with EGFP alone (EGFP), S16A-HSP20
(MUT-EGFP, or wild type HSP20 (WT-EGFP). The cells were plated on a
glass slides, and not treated (CONT) or treated with dibutyryl cAMP
(10 .mu.M, for 30 minutes, db-cAMP). The cells were fixed and
stained with rhodamine phalloidin. Dibutyryl cAMP led to a loss of
central actin stress fibers in EGFP but not S16A-HSP20 cells. In
the cells overexpressing HSP20 the actin fibers were peripherally
localized. The results of this experiment are illustrated in FIG.
6.
[0161] These experiments show that activation of cyclic
nucleotide-dependent signaling pathways, which lead to increases in
the phosphorylation of HSP20, are associated with a loss of central
actin stress fibers. Over-expression of HSP20 was also associated
with a loss of actin stress fibers. In cells overexpressing a
mutated form of HSP20 in which the serine has been replaced with an
alanine (S16A-HSP20) and cannot be phosphorylated, there is no loss
of these central actin fibers with activation of cyclic
nucleotide-dependent signaling pathways. These studies demonstrate
that phosphorylation of HSP20 is associated with changes in actin
fiber formation.
Example 13
[0162] This experiment shows that protein transduction of smooth
muscle cells with phosphopeptide analogues of HSP20 also leads to
changes in actin fiber formation.
[0163] Rat aortic smooth muscle cells were treated with
lyophosphatidic acid (LPA) in the presence and absence of
FITC-TAT-pHSP20
(FITC-NH.sub.2-.beta.AGGGGYGRKKRRQRRRWLRRAS*APLPGLK-COOH, 50 uM)
(SEQ ID NO:307) Lysophosphatidic acid (LPA) is a substance which
promotes actin fiber formation. Inhibition of the actions of LPA
have been shown to inhibit intimal hyperplasia.
[0164] The cells were fixed and stained with phalloidin and images
were obtained with confocal microscopy. LPA led to robust actin
stress fiber formation, whereas there was a loss of central actin
stress fibers in the cells treated with LPA in the presence of the
FITC-TAT-pHSP20 peptide. These studies show that protein
transduction with the phosphopeptide analogues of HSP20 inhibit
LPA-induced actin fiber formation. These studies confirm that HSP20
has a direct role in modulating actin fiber formation.
Example 14
[0165] Cell adhesion formation involves the interaction between
integrins and extracellular matrix substrates. This induces
integrin clustering. The cells then form actin microfilaments and
the cells spread. If the appropriate signals are provided by the
matrix, the cells proceed to organize their cytoskeleton as
characterized by the formation of focal adhesions and
actin-containing stress fibers. Cell adhesion is a dynamic
reversible process integral to cell migration. Activation of cGMP
leads to focal adhesion disassembly. These studies show that
phosphopeptide analogues of HSP20 mediate focal adhesion
disassembly.
[0166] Bovine aortic endothelial cells were plated on glass
coverslips (80K-100K cells) in DMEM plus 10% FBS over night (24
wells plate). The cells were serum starved (no serum) for one hour
and incubated in the presence of the peptide analogues of HSP20
[NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH-pHSP20 (10 uM)
(SEQ ID NO:307) or scrambled analogues of HSP20
[NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH-scHSP20 (10 uM)]
(SEQ ID NO:308) for 30 minutes. The cells were fixed with 3%
glutaraldehyde and the number of focal adhesions was detected with
interference reflection microscopy. The Hep I peptide was used as a
positive control. This peptide binds to the heparin binding site of
thrombospondin in induces focal adhesion disassembly. Both Hep I
and pHSP20 led to focal adhesion disassembly. The results are
illustrated in FIG. 7.
[0167] Treatment with PTD-pHSP20 led to a disassociation of focal
adhesions similar to the disassociation induced by a peptide from
the amino-terminal heparin-binding domain of thrombospondin 1 (Hep
1). The scrambled peptide had no significant effect on focal
adhesion disassembly. These studies show that phosphorylated HSP20
mediates focal adhesion disassembly. This weakens cell attachment
and prevents the formation of the attachments necessary for cell
migration.
Example 15
[0168] This Experiment shows that the phosphorylated peptide
analogues of HSP20 directly inhibit cell migration.
[0169] Confluent A10 cells were serum starved (0.5% fetal bovine
serum, FBS) for 48 hours. A linear wound was made in the smooth
muscle cell monolayer using a rubber scraper and the scratched
edges were marked using metal pins. The cells were changed to 10%
FBS media containing PTD-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307),
or PTD-scrambled-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ ID NO:308)
(50 .mu.M) and incubated for 24 hours. The cells were fixed and
stained with hematoxylin. The number of cells migrating into a 1
cm.sup.2 scratched area were counted as an index for migration. In
additional experiments, the migration of A10 cells was determined
in a Boyden chamber assay. In both cases the phosphopeptide
analogue of HSP20 led to inhibition of migration. FIG. 8
illustrates the results of these experiments.
[0170] These results show that transduction of phosphopeptide
analogues of HSP20 inhibits serum-induced migration of smooth
muscle cells.
Example 16
[0171] This experiment shows that transduction of phosphopeptide
analogues of HSP20 inhibits serum-induced proliferation of smooth
muscle cells.
[0172] A10 cells were serum starved for 3 days. The cells were then
treated with media containing 10% fetal bovine serum, PTD-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307),
or PTD-scrambled-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ ID NO:308)
(50 .mu.M). After 24 hours cell counts were performed. The number
of cells per well in the serum starved plates averaged 109
cells/well (+/-7.4) compared to 276+/-6.1 in wells containing 10%
fetal bovine serum (FBS). In the presence of FBS, the
phosphopeptide analogues of HSP20 containing a transduction domain
inhibited of smooth muscle proliferation, 149+/-14.6 compared to
transduction of the scrambled phosphopeptide analogue of HSP20
(242.3+/-15.3 cells/well). The results of these experiments are
illustrated in FIG. 9.
[0173] The results from these examples demonstrate that HSP20 is
associated with a contractile phenotype and that transduction of
phosphopeptide analogues of HSP20 inhibit actin fiber formation,
focal adhesion formation, smooth muscle cell migration and smooth
muscle proliferation. These are cellular processes that lead to
intimal hyperplasia, and other disorders as discussed throughout
the application.
Example 17
[0174] These experiments show that HSP20 inhibits intimal
hyperplasia in human saphenous vein grafts.
[0175] Segments of human saphenous vein were cultured in media
containing 30% fetal bovine serum. The segments were treated for 14
days with media containing serum alone, serum and the
phosphopeptide analogue of HSP20 (PTD-pHSP20
(NH.sub.2-.beta.AYARRAAARQARAWLRRAS*APLPGLK-COOH, 10:M) (SEQ ID
NO:307), or the PTD-scrambled phosphopeptide analogue
(NH.sub.2-.beta.AYARRAAARQARAPRKS*LWALGRPLA-COOH, 10:.mu.M). (SEQ
ID NO:308) The rings were fixed in formalin, stained with
hematoxylin and eosin, and the intimal area was measured
morphometrically. There was a significant reduction in intimal area
in the rings transduced with the phosphopeptide analogues of HSP20
compared to the rings treated with serum alone or serum and
transduction of the scrambled analogue.
These results show that intimal hyperplasia can be inhibited in
human saphenous vein segments by transduction of the phosphopeptide
analogues of HSP20.
Example 18
[0176] This Experiment illustrates that plant cells can be
engineered to produce recombinant HSP20.
[0177] Tobacco BY-2 cells were transformed with vector alone
(vector transformed) or with His tagged HSP20 constructs
(6.times.his-HSP20 transformed). Western blots were performed on
cells lysates using anti-6.times.his monoclonal antibodies. There
is immunoreactivity of a 20 kDa polypeptide in the HSP20 lysates
but not the empty vector transformed lysates.
[0178] Optical sections from confocal immunofluorescence images of
processed tobacco cells transiently-expressing either myc-epitope
tagged HSP20, probed with anti-myc antibodies, HSP20, probed with
anti-HSP20 antibodies, TAT-HSP20, probed with anti-HSP20
antibodies, or HISTAT-HSP20, probed with anti-his antibodies.
Expression is present with all 4 constructs (bar=100 .mu.m).
[0179] These data show that plants can be engineered to produce
proteins that contain a protein transduction sequence and the HSP20
molecule. This represents an alternative source of production of
HSP20.
Sequence CWU 1
1
32014PRTArtificial sequenceSynthetic peptide 1Trp Leu Arg
Arg126PRTArtificial sequenceSynthetic peptide 2Ala Xaa Ala Pro Leu
Pro1 536PRTArtificial sequenceSynthetic peptide 3Ala Ser Ala Pro
Leu Pro1 546PRTArtificial sequenceSynthetic peptide 4Ala Thr Ala
Pro Leu Pro1 557PRTArtificial sequenceSynthetic peptide 5Arg Ala
Ser Ala Pro Leu Pro1 567PRTArtificial sequenceSynthetic peptide
6Arg Ala Thr Ala Pro Leu Pro1 576PRTArtificial sequenceSynthetic
peptide 7Ala Tyr Ala Pro Leu Pro1 587PRTArtificial
sequenceSynthetic peptide 8Arg Ala Tyr Ala Pro Leu Pro1
598PRTArtificial sequenceSynthetic peptide 9Arg Arg Ala Ser Ala Pro
Leu Pro1 5109PRTArtificial sequenceSynthetic peptide 10Leu Arg Arg
Ala Ser Ala Pro Leu Pro1 51110PRTArtificial sequenceSynthetic
peptide 11Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro1 5
10128PRTArtificial sequenceSynthetic peptide 12Arg Arg Ala Thr Ala
Pro Leu Pro1 5139PRTArtificial sequenceSynthetic peptide 13Leu Arg
Arg Ala Thr Ala Pro Leu Pro1 51410PRTArtificial sequenceSynthetic
peptide 14Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro1 5
10158PRTArtificial sequenceSynthetic peptide 15Arg Arg Ala Tyr Ala
Pro Leu Pro1 5169PRTArtificial sequenceSynthetic peptide 16Leu Arg
Arg Ala Tyr Ala Pro Leu Pro1 51710PRTArtificial sequenceSynthetic
peptide 17Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro1 5
10189PRTArtificial sequenceSynthetic peptide 18Arg Arg Ala Ser Ala
Pro Leu Pro Gly1 5199PRTArtificial sequenceSynthetic peptide 19Arg
Arg Ala Ser Ala Pro Leu Pro Asp1 52010PRTArtificial
sequenceSynthetic peptide 20Arg Arg Ala Ser Ala Pro Leu Pro Gly
Leu1 5 102110PRTArtificial sequenceSynthetic peptide 21Arg Arg Ala
Ser Ala Pro Leu Pro Gly Lys1 5 102210PRTArtificial
sequenceSynthetic peptide 22Arg Arg Ala Ser Ala Pro Leu Pro Asp
Leu1 5 102310PRTArtificial sequenceSynthetic peptide 23Arg Arg Ala
Ser Ala Pro Leu Pro Asp Lys1 5 102411PRTArtificial
sequenceSynthetic peptide 24Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu
Ser1 5 102511PRTArtificial sequenceSynthetic peptide 25Arg Arg Ala
Ser Ala Pro Leu Pro Gly Leu Thr1 5 102611PRTArtificial
sequenceSynthetic peptide 26Arg Arg Ala Ser Ala Pro Leu Pro Gly Lys
Ser1 5 102711PRTArtificial sequenceSynthetic peptide 27Arg Arg Ala
Ser Ala Pro Leu Pro Gly Lys Thr1 5 102811PRTArtificial
sequenceSynthetic peptide 28Arg Arg Ala Ser Ala Pro Leu Pro Asp Leu
Ser1 5 102911PRTArtificial sequenceSynthetic peptide 29Arg Arg Ala
Ser Ala Pro Leu Pro Asp Leu Thr1 5 103011PRTArtificial
sequenceSynthetic peptide 30Arg Arg Ala Ser Ala Pro Leu Pro Asp Lys
Ser1 5 103111PRTArtificial sequenceSynthetic peptide 31Arg Arg Ala
Ser Ala Pro Leu Pro Asp Lys Thr1 5 103210PRTArtificial
sequenceSynthetic peptide 32Leu Arg Arg Ala Ser Ala Pro Leu Pro
Gly1 5 103310PRTArtificial sequenceSynthetic peptide 33Leu Arg Arg
Ala Ser Ala Pro Leu Pro Asp1 5 103411PRTArtificial
sequenceSynthetic peptide 34Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly
Leu1 5 103511PRTArtificial sequenceSynthetic peptide 35Leu Arg Arg
Ala Ser Ala Pro Leu Pro Gly Lys1 5 103611PRTArtificial
sequenceSynthetic peptide 36Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp
Leu1 5 103711PRTArtificial sequenceSynthetic peptide 37Leu Arg Arg
Ala Ser Ala Pro Leu Pro Asp Lys1 5 103812PRTArtificial
sequenceSynthetic peptide 38Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly
Leu Ser1 5 103912PRTArtificial sequenceSynthetic peptide 39Leu Arg
Arg Ala Ser Ala Pro Leu Pro Gly Leu Thr1 5 104012PRTArtificial
sequenceSynthetic peptide 40Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly
Lys Ser1 5 104112PRTArtificial sequenceSynthetic peptide 41Leu Arg
Arg Ala Ser Ala Pro Leu Pro Gly Lys Thr1 5 104212PRTArtificial
sequenceSynthetic peptide 42Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp
Leu Ser1 5 104312PRTArtificial sequenceSynthetic peptide 43Leu Arg
Arg Ala Ser Ala Pro Leu Pro Asp Leu Thr1 5 104412PRTArtificial
sequenceSynthetic peptide 44Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp
Lys Ser1 5 104512PRTArtificial sequenceSynthetic peptide 45Leu Arg
Arg Ala Ser Ala Pro Leu Pro Asp Lys Thr1 5 104611PRTArtificial
sequenceSynthetic peptide 46Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro
Gly1 5 104711PRTArtificial sequenceSynthetic peptide 47Trp Leu Arg
Arg Ala Ser Ala Pro Leu Pro Asp1 5 104812PRTArtificial
sequenceSynthetic peptide 48Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro
Gly Leu1 5 104912PRTArtificial sequenceSynthetic peptide 49Trp Leu
Arg Arg Ala Ser Ala Pro Leu Pro Gly Lys1 5 105012PRTArtificial
sequenceSynthetic peptide 50Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro
Asp Leu1 5 105112PRTArtificial sequenceSynthetic peptide 51Trp Leu
Arg Arg Ala Ser Ala Pro Leu Pro Asp Lys1 5 105213PRTArtificial
sequenceSynthetic peptide 52Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro
Gly Leu Ser1 5 105313PRTArtificial sequenceSynthetic peptide 53Trp
Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Thr1 5
105413PRTArtificial sequenceSynthetic peptide 54Trp Leu Arg Arg Ala
Ser Ala Pro Leu Pro Gly Lys Ser1 5 105513PRTArtificial
sequenceSynthetic peptide 55Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro
Gly Lys Thr1 5 105613PRTArtificial sequenceSynthetic peptide 56Trp
Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp Leu Ser1 5
105713PRTArtificial sequenceSynthetic peptide 57Trp Leu Arg Arg Ala
Ser Ala Pro Leu Pro Asp Leu Thr1 5 105813PRTArtificial
sequenceSynthetic peptide 58Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro
Asp Lys Ser1 5 105913PRTArtificial sequenceSynthetic peptide 59Trp
Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp Lys Thr1 5
10609PRTArtificial sequenceSynthetic peptide 60Arg Arg Ala Thr Ala
Pro Leu Pro Gly1 5619PRTArtificial sequenceSynthetic peptide 61Arg
Arg Ala Thr Ala Pro Leu Pro Asp1 56210PRTArtificial
sequenceSynthetic peptide 62Arg Arg Ala Thr Ala Pro Leu Pro Gly
Leu1 5 106310PRTArtificial sequenceSynthetic peptide 63Arg Arg Ala
Thr Ala Pro Leu Pro Gly Lys1 5 106410PRTArtificial
sequenceSynthetic peptide 64Arg Arg Ala Thr Ala Pro Leu Pro Asp
Leu1 5 106510PRTArtificial sequenceSynthetic peptide 65Arg Arg Ala
Thr Ala Pro Leu Pro Asp Lys1 5 106611PRTArtificial
sequenceSynthetic peptide 66Arg Arg Ala Thr Ala Pro Leu Pro Gly Leu
Ser1 5 106711PRTArtificial sequenceSynthetic peptide 67Arg Arg Ala
Thr Ala Pro Leu Pro Gly Leu Thr1 5 106811PRTArtificial
sequenceSynthetic peptide 68Arg Arg Ala Thr Ala Pro Leu Pro Gly Lys
Ser1 5 106911PRTArtificial sequenceSynthetic peptide 69Arg Arg Ala
Thr Ala Pro Leu Pro Gly Lys Thr1 5 107011PRTArtificial
sequenceSynthetic peptide 70Arg Arg Ala Thr Ala Pro Leu Pro Asp Leu
Ser1 5 107111PRTArtificial sequenceSynthetic peptide 71Arg Arg Ala
Thr Ala Pro Leu Pro Asp Leu Thr1 5 107211PRTArtificial
sequenceSynthetic peptide 72Arg Arg Ala Thr Ala Pro Leu Pro Asp Lys
Ser1 5 107311PRTArtificial sequenceSynthetic peptide 73Arg Arg Ala
Thr Ala Pro Leu Pro Asp Lys Thr1 5 107410PRTArtificial
sequenceSynthetic peptide 74Leu Arg Arg Ala Thr Ala Pro Leu Pro
Gly1 5 107510PRTArtificial sequenceSynthetic peptide 75Leu Arg Arg
Ala Thr Ala Pro Leu Pro Asp1 5 107611PRTArtificial
sequenceSynthetic peptide 76Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly
Leu1 5 107711PRTArtificial sequenceSynthetic peptide 77Leu Arg Arg
Ala Thr Ala Pro Leu Pro Gly Lys1 5 107811PRTArtificial
sequenceSynthetic peptide 78Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp
Leu1 5 107911PRTArtificial sequenceSynthetic peptide 79Leu Arg Arg
Ala Thr Ala Pro Leu Pro Asp Lys1 5 108012PRTArtificial
sequenceSynthetic peptide 80Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly
Leu Ser1 5 108112PRTArtificial sequenceSynthetic peptide 81Leu Arg
Arg Ala Thr Ala Pro Leu Pro Gly Leu Thr1 5 108212PRTArtificial
sequenceSynthetic peptide 82Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly
Lys Ser1 5 108312PRTArtificial sequenceSynthetic peptide 83Leu Arg
Arg Ala Thr Ala Pro Leu Pro Gly Lys Thr1 5 108412PRTArtificial
sequenceSynthetic peptide 84Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp
Leu Ser1 5 108512PRTArtificial sequenceSynthetic peptide 85Leu Arg
Arg Ala Thr Ala Pro Leu Pro Asp Leu Thr1 5 108612PRTArtificial
sequenceSynthetic peptide 86Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp
Lys Ser1 5 108712PRTArtificial sequenceSynthetic peptide 87Leu Arg
Arg Ala Thr Ala Pro Leu Pro Asp Lys Thr1 5 108811PRTArtificial
sequenceSynthetic peptide 88Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro
Gly1 5 108911PRTArtificial sequenceSynthetic peptide 89Trp Leu Arg
Arg Ala Thr Ala Pro Leu Pro Asp1 5 109012PRTArtificial
sequenceSynthetic peptide 90Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro
Gly Leu1 5 109112PRTArtificial sequenceSynthetic peptide 91Trp Leu
Arg Arg Ala Thr Ala Pro Leu Pro Gly Lys1 5 109212PRTArtificial
sequenceSynthetic peptide 92Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro
Asp Leu1 5 109312PRTArtificial sequenceSynthetic peptide 93Trp Leu
Arg Arg Ala Thr Ala Pro Leu Pro Asp Lys1 5 109413PRTArtificial
sequenceSynthetic peptide 94Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro
Gly Leu Ser1 5 109513PRTArtificial sequenceSynthetic peptide 95Trp
Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly Leu Thr1 5
109613PRTArtificial sequenceSynthetic peptide 96Trp Leu Arg Arg Ala
Thr Ala Pro Leu Pro Gly Lys Ser1 5 109713PRTArtificial
sequenceSynthetic peptide 97Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro
Gly Lys Thr1 5 109813PRTArtificial sequenceSynthetic peptide 98Trp
Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp Leu Ser1 5
109913PRTArtificial sequenceSynthetic peptide 99Trp Leu Arg Arg Ala
Thr Ala Pro Leu Pro Asp Leu Thr1 5 1010013PRTArtificial
sequenceSynthetic peptide 100Trp Leu Arg Arg Ala Thr Ala Pro Leu
Pro Asp Lys Ser1 5 1010113PRTArtificial sequenceSynthetic peptide
101Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp Lys Thr1 5
101029PRTArtificial sequenceSynthetic peptide 102Arg Arg Ala Tyr
Ala Pro Leu Pro Gly1 51039PRTArtificial sequenceSynthetic peptide
103Arg Arg Ala Tyr Ala Pro Leu Pro Asp1 510410PRTArtificial
sequenceSynthetic peptide 104Arg Arg Ala Tyr Ala Pro Leu Pro Gly
Leu1 5 1010510PRTArtificial sequenceSynthetic peptide 105Arg Arg
Ala Tyr Ala Pro Leu Pro Gly Lys1 5 1010610PRTArtificial
sequenceSynthetic peptide 106Arg Arg Ala Tyr Ala Pro Leu Pro Asp
Leu1 5 1010710PRTArtificial sequenceSynthetic peptide 107Arg Arg
Ala Tyr Ala Pro Leu Pro Asp Lys1 5 1010811PRTArtificial
sequenceSynthetic peptide 108Arg Arg Ala Tyr Ala Pro Leu Pro Gly
Leu Ser1 5 1010911PRTArtificial sequenceSynthetic peptide 109Arg
Arg Ala Tyr Ala Pro Leu Pro Gly Leu Thr1 5 1011011PRTArtificial
sequenceSynthetic peptide 110Arg Arg Ala Tyr Ala Pro Leu Pro Gly
Lys Ser1 5 1011111PRTArtificial sequenceSynthetic peptide 111Arg
Arg Ala Tyr Ala Pro Leu Pro Gly Lys Thr1 5 1011211PRTArtificial
sequenceSynthetic peptide 112Arg Arg Ala Tyr Ala Pro Leu Pro Asp
Leu Ser1 5 1011311PRTArtificial sequenceSynthetic peptide 113Arg
Arg Ala Tyr Ala Pro Leu Pro Asp Leu Thr1 5 1011411PRTArtificial
sequenceSynthetic peptide 114Arg Arg Ala Tyr Ala Pro Leu Pro Asp
Lys Ser1 5 1011511PRTArtificial sequenceSynthetic peptide 115Arg
Arg Ala Tyr Ala Pro Leu Pro Asp Lys Thr1 5 1011610PRTArtificial
sequenceSynthetic peptide 116Leu Arg Arg Ala Tyr Ala Pro Leu Pro
Gly1 5 1011710PRTArtificial sequenceSynthetic peptide 117Leu Arg
Arg Ala Tyr Ala Pro Leu Pro Asp1 5 1011811PRTArtificial
sequenceSynthetic peptide 118Leu Arg Arg Ala Tyr Ala Pro Leu Pro
Gly Leu1 5 1011911PRTArtificial sequenceSynthetic peptide 119Leu
Arg Arg Ala Tyr Ala Pro Leu Pro Gly Lys1 5 1012011PRTArtificial
sequenceSynthetic peptide 120Leu Arg Arg Ala Tyr Ala Pro Leu Pro
Asp Leu1 5 1012111PRTArtificial sequenceSynthetic peptide 121Leu
Arg Arg Ala Tyr Ala Pro Leu Pro Asp Lys1 5 1012212PRTArtificial
sequenceSynthetic peptide 122Leu Arg Arg Ala Tyr Ala Pro Leu Pro
Gly Leu Ser1 5 1012312PRTArtificial sequenceSynthetic peptide
123Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Leu Thr1 5
1012412PRTArtificial sequenceSynthetic peptide 124Leu Arg Arg Ala
Tyr Ala Pro Leu Pro Gly Lys Ser1 5 1012512PRTArtificial
sequenceSynthetic peptide 125Leu Arg Arg Ala Tyr Ala Pro Leu Pro
Gly Lys Thr1 5 1012612PRTArtificial sequenceSynthetic peptide
126Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Leu Ser1 5
1012712PRTArtificial sequenceSynthetic peptide 127Leu Arg Arg Ala
Tyr Ala Pro Leu Pro Asp Leu Thr1 5 1012812PRTArtificial
sequenceSynthetic peptide 128Leu Arg Arg Ala Tyr Ala Pro Leu Pro
Asp Lys Ser1 5 1012912PRTArtificial sequenceSynthetic peptide
129Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Lys Thr1 5
1013011PRTArtificial sequenceSynthetic peptide 130Trp Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Gly1 5 1013111PRTArtificial
sequenceSynthetic peptide 131Trp Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Asp1 5 1013212PRTArtificial sequenceSynthetic peptide 132Trp
Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Leu1 5 1013312PRTArtificial
sequenceSynthetic peptide 133Trp Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Gly Lys1 5 1013412PRTArtificial sequenceSynthetic peptide
134Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Leu1 5
1013512PRTArtificial sequenceSynthetic peptide 135Trp Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Asp Lys1 5 1013613PRTArtificial
sequenceSynthetic peptide 136Trp Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Gly Leu Ser1 5 1013713PRTArtificial sequenceSynthetic peptide
137Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Leu Thr1 5
1013813PRTArtificial sequenceSynthetic peptide 138Trp Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Gly Lys Ser1 5 1013913PRTArtificial
sequenceSynthetic peptide 139Trp Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Gly Lys Thr1 5 1014013PRTArtificial sequenceSynthetic peptide
140Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Leu Ser1 5
1014113PRTArtificial sequenceSynthetic peptide 141Trp Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Asp Leu Thr1 5 1014213PRTArtificial
sequenceSynthetic peptide 142Trp Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Asp Lys Ser1 5 1014313PRTArtificial sequenceSynthetic peptide
143Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Lys Thr1 5
101449PRTArtificial sequenceSynthetic peptide 144Xaa Arg Arg Ala
Ser Ala Pro Leu Pro1 514510PRTArtificial sequenceSynthetic peptide
145Xaa Leu Arg Arg Ala Ser Ala Pro Leu Pro1 5 1014611PRTArtificial
sequenceSynthetic peptide 146Xaa Trp Leu Arg Arg Ala Ser Ala Pro
Leu Pro1 5 101479PRTArtificial sequenceSynthetic peptide 147Xaa Arg
Arg Ala Thr Ala Pro Leu Pro1 514810PRTArtificial sequenceSynthetic
peptide 148Xaa Leu Arg Arg Ala Thr Ala Pro Leu Pro1 5
1014911PRTArtificial sequenceSynthetic peptide 149Xaa Trp Leu Arg
Arg Ala Thr Ala Pro Leu Pro1 5 101509PRTArtificial
sequenceSynthetic peptide 150Xaa Arg Arg Ala Tyr Ala Pro Leu Pro1
515110PRTArtificial sequenceSynthetic peptide 151Xaa Leu Arg Arg
Ala Tyr Ala Pro Leu Pro1 5 1015211PRTArtificial sequenceSynthetic
peptide 152Xaa Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro1 5
1015310PRTArtificial sequenceSynthetic peptide 153Xaa Arg Arg Ala
Ser Ala Pro Leu Pro Gly1 5 1015410PRTArtificial sequenceSynthetic
peptide 154Xaa Arg Arg Ala Ser Ala Pro Leu Pro Asp1 5
1015511PRTArtificial sequenceSynthetic peptide 155Xaa Arg Arg Ala
Ser Ala Pro Leu Pro Gly Leu1 5 1015611PRTArtificial
sequenceSynthetic peptide 156Xaa Arg Arg Ala Ser Ala Pro Leu Pro
Gly Lys1 5 1015711PRTArtificial sequenceSynthetic peptide 157Xaa
Arg Arg Ala Ser Ala Pro Leu Pro Asp Leu1 5 1015811PRTArtificial
sequenceSynthetic peptide 158Xaa Arg Arg Ala Ser Ala Pro Leu Pro
Asp Lys1 5 1015912PRTArtificial sequenceSynthetic peptide 159Xaa
Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Ser1 5 1016012PRTArtificial
sequenceSynthetic peptide 160Xaa Arg Arg Ala Ser Ala Pro Leu Pro
Gly Leu Thr1 5 1016112PRTArtificial sequenceSynthetic peptide
161Xaa Arg Arg Ala Ser Ala Pro Leu Pro Gly Lys Ser1 5
1016212PRTArtificial sequenceSynthetic peptide 162Xaa Arg Arg Ala
Ser Ala Pro Leu Pro Gly Lys Thr1 5 1016312PRTArtificial
sequenceSynthetic peptide 163Xaa Arg Arg Ala Ser Ala Pro Leu Pro
Asp Leu Ser1 5 1016412PRTArtificial sequenceSynthetic peptide
164Xaa Arg Arg Ala Ser Ala Pro Leu Pro Asp Leu Thr1 5
1016512PRTArtificial sequenceSynthetic peptide 165Xaa Arg Arg Ala
Ser Ala Pro Leu Pro Asp Lys Ser1 5 1016612PRTArtificial
sequenceSynthetic peptide 166Xaa Arg Arg Ala Ser Ala Pro Leu Pro
Asp Lys Thr1 5 1016711PRTArtificial sequenceSynthetic peptide
167Xaa Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly1 5
1016811PRTArtificial sequenceSynthetic peptide 168Xaa Leu Arg Arg
Ala Ser Ala Pro Leu Pro Asp1 5 1016912PRTArtificial
sequenceSynthetic peptide 169Xaa Leu Arg Arg Ala Ser Ala Pro Leu
Pro Gly Leu1 5 1017012PRTArtificial sequenceSynthetic peptide
170Xaa Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Lys1 5
1017112PRTArtificial sequenceSynthetic peptide 171Xaa Leu Arg Arg
Ala Ser Ala Pro Leu Pro Asp Leu1 5 1017212PRTArtificial
sequenceSynthetic peptide 172Xaa Leu Arg Arg Ala Ser Ala Pro Leu
Pro Asp Lys1 5 1017313PRTArtificial sequenceSynthetic peptide
173Xaa Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Ser1 5
1017413PRTArtificial sequenceSynthetic peptide 174Xaa Leu Arg Arg
Ala Ser Ala Pro Leu Pro Gly Leu Thr1 5 1017513PRTArtificial
sequenceSynthetic peptide 175Xaa Leu Arg Arg Ala Ser Ala Pro Leu
Pro Gly Lys Ser1 5 1017613PRTArtificial sequenceSynthetic peptide
176Xaa Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Lys Thr1 5
1017713PRTArtificial sequenceSynthetic peptide 177Xaa Leu Arg Arg
Ala Ser Ala Pro Leu Pro Asp Leu Ser1 5 1017813PRTArtificial
sequenceSynthetic peptide 178Xaa Leu Arg Arg Ala Ser Ala Pro Leu
Pro Asp Leu Thr1 5 1017913PRTArtificial sequenceSynthetic peptide
179Xaa Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp Lys Ser1 5
1018013PRTArtificial sequenceSynthetic peptide 180Xaa Leu Arg Arg
Ala Ser Ala Pro Leu Pro Asp Lys Thr1 5 1018112PRTArtificial
sequenceSynthetic peptide 181Xaa Trp Leu Arg Arg Ala Ser Ala Pro
Leu Pro Gly1 5 1018212PRTArtificial sequenceSynthetic peptide
182Xaa Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp1 5
1018313PRTArtificial sequenceSynthetic peptide 183Xaa Trp Leu Arg
Arg Ala Ser Ala Pro Leu Pro Gly Leu1 5 1018413PRTArtificial
sequenceSynthetic peptide 184Xaa Trp Leu Arg Arg Ala Ser Ala Pro
Leu Pro Gly Lys1 5 1018513PRTArtificial sequenceSynthetic peptide
185Xaa Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp Leu1 5
1018613PRTArtificial sequenceSynthetic peptide 186Xaa Trp Leu Arg
Arg Ala Ser Ala Pro Leu Pro Asp Lys1 5 1018714PRTArtificial
sequenceSynthetic peptide 187Xaa Trp Leu Arg Arg Ala Ser Ala Pro
Leu Pro Gly Leu Ser1 5 1018814PRTArtificial sequenceSynthetic
peptide 188Xaa Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Thr1
5 1018914PRTArtificial sequenceSynthetic peptide 189Xaa Trp Leu Arg
Arg Ala Ser Ala Pro Leu Pro Gly Lys Ser1 5 1019014PRTArtificial
sequenceSynthetic peptide 190Xaa Trp Leu Arg Arg Ala Ser Ala Pro
Leu Pro Gly Lys Thr1 5 1019114PRTArtificial sequenceSynthetic
peptide 191Xaa Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp Leu Ser1
5 1019214PRTArtificial sequenceSynthetic peptide 192Xaa Trp Leu Arg
Arg Ala Ser Ala Pro Leu Pro Asp Leu Thr1 5 1019314PRTArtificial
sequenceSynthetic peptide 193Xaa Trp Leu Arg Arg Ala Ser Ala Pro
Leu Pro Asp Lys Ser1 5 1019414PRTArtificial sequenceSynthetic
peptide 194Xaa Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Asp Lys Thr1
5 1019510PRTArtificial sequenceSynthetic peptide 195Xaa Arg Arg Ala
Thr Ala Pro Leu Pro Gly1 5 1019610PRTArtificial sequenceSynthetic
peptide 196Xaa Arg Arg Ala Thr Ala Pro Leu Pro Asp1 5
1019711PRTArtificial sequenceSynthetic peptide 197Xaa Arg Arg Ala
Thr Ala Pro Leu Pro Gly Leu1 5 1019811PRTArtificial
sequenceSynthetic peptide 198Xaa Arg Arg Ala Thr Ala Pro Leu Pro
Gly Lys1 5 1019911PRTArtificial sequenceSynthetic peptide 199Xaa
Arg Arg Ala Thr Ala Pro Leu Pro Asp Leu1 5 1020011PRTArtificial
sequenceSynthetic peptide 200Xaa Arg Arg Ala Thr Ala Pro Leu Pro
Asp Lys1 5 1020112PRTArtificial sequenceSynthetic peptide 201Xaa
Arg Arg Ala Thr Ala Pro Leu Pro Gly Leu Ser1 5 1020212PRTArtificial
sequenceSynthetic peptide 202Xaa Arg Arg Ala Thr Ala Pro Leu Pro
Gly Leu Thr1 5 1020312PRTArtificial sequenceSynthetic peptide
203Xaa Arg Arg Ala Thr Ala Pro Leu Pro Gly Lys Ser1 5
1020412PRTArtificial sequenceSynthetic peptide 204Xaa Arg Arg Ala
Thr Ala Pro Leu Pro Gly Lys Thr1 5 1020512PRTArtificial
sequenceSynthetic peptide 205Xaa Arg Arg Ala Thr Ala Pro Leu Pro
Asp Leu Ser1 5 1020612PRTArtificial sequenceSynthetic peptide
206Xaa Arg Arg Ala Thr Ala Pro Leu Pro Asp Leu Thr1 5
1020712PRTArtificial sequenceSynthetic peptide 207Xaa Arg Arg Ala
Thr Ala Pro Leu Pro Asp Lys Ser1 5 1020812PRTArtificial
sequenceSynthetic peptide 208Xaa Arg Arg Ala Thr Ala Pro Leu Pro
Asp Lys Thr1 5 1020911PRTArtificial sequenceSynthetic peptide
209Xaa Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly1 5
1021011PRTArtificial sequenceSynthetic peptide 210Xaa Leu Arg Arg
Ala Thr Ala Pro Leu Pro Asp1 5 1021112PRTArtificial
sequenceSynthetic peptide 211Xaa Leu Arg Arg Ala Thr Ala Pro Leu
Pro Gly Leu1 5 1021212PRTArtificial sequenceSynthetic peptide
212Xaa Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly Lys1 5
1021312PRTArtificial sequenceSynthetic peptide 213Xaa Leu Arg Arg
Ala Thr Ala Pro Leu Pro Asp Leu1 5 1021412PRTArtificial
sequenceSynthetic peptide 214Xaa Leu Arg Arg Ala Thr Ala Pro Leu
Pro Asp Lys1 5 1021513PRTArtificial sequenceSynthetic peptide
215Xaa Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly Leu Ser1 5
1021613PRTArtificial sequenceSynthetic peptide 216Xaa Leu Arg Arg
Ala Thr Ala Pro Leu Pro Gly Leu Thr1 5 1021713PRTArtificial
sequenceSynthetic peptide 217Xaa Leu Arg Arg Ala Thr Ala Pro Leu
Pro Gly Lys Ser1 5 1021813PRTArtificial sequenceSynthetic peptide
218Xaa Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly Lys Thr1 5
1021913PRTArtificial sequenceSynthetic peptide 219Xaa Leu Arg Arg
Ala Thr Ala Pro Leu Pro Asp Leu Ser1 5 1022013PRTArtificial
sequenceSynthetic peptide 220Xaa Leu Arg Arg Ala Thr Ala Pro Leu
Pro Asp Leu Thr1 5 1022113PRTArtificial sequenceSynthetic peptide
221Xaa Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp Lys Ser1 5
1022213PRTArtificial sequenceSynthetic peptide 222Xaa Leu Arg Arg
Ala Thr Ala Pro Leu Pro Asp Lys Thr1 5 1022312PRTArtificial
sequenceSynthetic peptide 223Xaa Trp Leu Arg Arg Ala Thr Ala Pro
Leu Pro Gly1 5 1022412PRTArtificial sequenceSynthetic peptide
224Xaa Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp1 5
1022513PRTArtificial sequenceSynthetic peptide 225Xaa Trp Leu Arg
Arg Ala Thr Ala Pro Leu Pro Gly Leu1 5 1022613PRTArtificial
sequenceSynthetic peptide 226Xaa Trp Leu Arg Arg Ala Thr Ala Pro
Leu Pro Gly Lys1 5 1022713PRTArtificial sequenceSynthetic peptide
227Xaa Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp Leu1 5
1022813PRTArtificial sequenceSynthetic peptide 228Xaa Trp Leu Arg
Arg Ala Thr Ala Pro Leu Pro Asp Lys1 5 1022914PRTArtificial
sequenceSynthetic peptide 229Xaa Trp Leu Arg Arg Ala Thr Ala Pro
Leu Pro Gly Leu Ser1 5 1023014PRTArtificial sequenceSynthetic
peptide 230Xaa Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro Gly Leu Thr1
5 1023114PRTArtificial sequenceSynthetic peptide 231Xaa Trp Leu Arg
Arg Ala Thr Ala Pro Leu Pro Gly Lys Ser1 5 1023214PRTArtificial
sequenceSynthetic peptide 232Xaa Trp Leu Arg Arg Ala Thr Ala Pro
Leu Pro Gly Lys Thr1 5 1023314PRTArtificial sequenceSynthetic
peptide 233Xaa Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp Leu Ser1
5 1023414PRTArtificial sequenceSynthetic peptide 234Xaa Trp Leu Arg
Arg Ala Thr Ala Pro Leu Pro Asp Leu Thr1 5 1023514PRTArtificial
sequenceSynthetic peptide 235Xaa Trp Leu Arg Arg Ala Thr Ala Pro
Leu Pro Asp Lys Ser1 5 1023614PRTArtificial sequenceSynthetic
peptide 236Xaa Trp Leu Arg Arg Ala Thr Ala Pro Leu Pro Asp Lys Thr1
5 1023710PRTArtificial sequenceSynthetic peptide 237Xaa Arg Arg Ala
Tyr Ala Pro Leu Pro Gly1 5 1023810PRTArtificial sequenceSynthetic
peptide 238Xaa Arg Arg Ala Tyr Ala Pro Leu Pro Asp1 5
1023911PRTArtificial sequenceSynthetic peptide 239Xaa Arg Arg Ala
Tyr Ala Pro Leu Pro Gly Leu1 5 1024011PRTArtificial
sequenceSynthetic peptide 240Xaa Arg Arg Ala Tyr Ala Pro Leu Pro
Gly Lys1 5 1024111PRTArtificial sequenceSynthetic peptide 241Xaa
Arg Arg Ala Tyr Ala Pro Leu Pro Asp Leu1 5 1024211PRTArtificial
sequenceSynthetic peptide 242Xaa Arg Arg Ala Tyr Ala Pro Leu Pro
Asp Lys1 5 1024312PRTArtificial sequenceSynthetic peptide 243Xaa
Arg Arg Ala Tyr Ala Pro Leu Pro Gly Leu Ser1 5 1024412PRTArtificial
sequenceSynthetic peptide 244Xaa Arg Arg Ala Tyr Ala Pro Leu Pro
Gly Leu Thr1 5 1024512PRTArtificial sequenceSynthetic peptide
245Xaa Arg Arg Ala Tyr Ala Pro Leu Pro Gly Lys Ser1 5
1024612PRTArtificial sequenceSynthetic peptide 246Xaa Arg Arg Ala
Tyr Ala Pro Leu Pro Gly Lys Thr1 5 1024712PRTArtificial
sequenceSynthetic peptide 247Xaa Arg Arg Ala Tyr Ala Pro Leu Pro
Asp Leu Ser1 5 1024812PRTArtificial sequenceSynthetic peptide
248Xaa Arg Arg Ala Tyr Ala Pro Leu Pro Asp Leu Thr1 5
1024912PRTArtificial sequenceSynthetic peptide 249Xaa Arg Arg Ala
Tyr Ala Pro Leu Pro Asp Lys Ser1 5 1025012PRTArtificial
sequenceSynthetic peptide 250Xaa Arg Arg Ala Tyr Ala Pro Leu Pro
Asp Lys Thr1 5 1025111PRTArtificial sequenceSynthetic peptide
251Xaa Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly1 5
1025211PRTArtificial sequenceSynthetic peptide 252Xaa Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Asp1 5 1025312PRTArtificial
sequenceSynthetic peptide 253Xaa Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Gly Leu1 5 1025412PRTArtificial sequenceSynthetic peptide
254Xaa Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Lys1 5
1025512PRTArtificial sequenceSynthetic peptide 255Xaa Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Asp Leu1 5 1025612PRTArtificial
sequenceSynthetic peptide 256Xaa Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Asp Lys1 5 1025713PRTArtificial sequenceSynthetic peptide
257Xaa Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Leu Ser1 5
1025813PRTArtificial sequenceSynthetic peptide 258Xaa Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Gly Leu Thr1 5 1025913PRTArtificial
sequenceSynthetic peptide 259Xaa Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Gly Lys Ser1 5 1026013PRTArtificial sequenceSynthetic peptide
260Xaa Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Lys Thr1 5
1026113PRTArtificial sequenceSynthetic peptide 261Xaa Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Asp Leu Ser1 5 1026213PRTArtificial
sequenceSynthetic peptide 262Xaa Leu Arg Arg Ala Tyr Ala Pro Leu
Pro Asp Leu Thr1 5 1026313PRTArtificial sequenceSynthetic peptide
263Xaa Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Lys Ser1 5
1026413PRTArtificial sequenceSynthetic peptide 264Xaa Leu Arg Arg
Ala Tyr Ala Pro Leu Pro Asp Lys Thr1 5 1026512PRTArtificial
sequenceSynthetic peptide 265Xaa Trp Leu Arg Arg Ala Tyr Ala Pro
Leu Pro Gly1 5 1026612PRTArtificial sequenceSynthetic peptide
266Xaa Trp Leu Arg Arg Ala
Tyr Ala Pro Leu Pro Asp1 5 1026713PRTArtificial sequenceSynthetic
peptide 267Xaa Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Leu1 5
1026813PRTArtificial sequenceSynthetic peptide 268Xaa Trp Leu Arg
Arg Ala Tyr Ala Pro Leu Pro Gly Lys1 5 1026913PRTArtificial
sequenceSynthetic peptide 269Xaa Trp Leu Arg Arg Ala Tyr Ala Pro
Leu Pro Asp Leu1 5 1027013PRTArtificial sequenceSynthetic peptide
270Xaa Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Lys1 5
1027114PRTArtificial sequenceSynthetic peptide 271Xaa Trp Leu Arg
Arg Ala Tyr Ala Pro Leu Pro Gly Leu Ser1 5 1027214PRTArtificial
sequenceSynthetic peptide 272Xaa Trp Leu Arg Arg Ala Tyr Ala Pro
Leu Pro Gly Leu Thr1 5 1027314PRTArtificial sequenceSynthetic
peptide 273Xaa Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Gly Lys Ser1
5 1027414PRTArtificial sequenceSynthetic peptide 274Xaa Trp Leu Arg
Arg Ala Tyr Ala Pro Leu Pro Gly Lys Thr1 5 1027514PRTArtificial
sequenceSynthetic peptide 275Xaa Trp Leu Arg Arg Ala Tyr Ala Pro
Leu Pro Asp Leu Ser1 5 1027614PRTArtificial sequenceSynthetic
peptide 276Xaa Trp Leu Arg Arg Ala Tyr Ala Pro Leu Pro Asp Leu Thr1
5 1027714PRTArtificial sequenceSynthetic peptide 277Xaa Trp Leu Arg
Arg Ala Tyr Ala Pro Leu Pro Asp Lys Ser1 5 1027814PRTArtificial
sequenceSynthetic peptide 278Xaa Trp Leu Arg Arg Ala Tyr Ala Pro
Leu Pro Asp Lys Thr1 5 102791PRTArtificial sequenceSynthetic
peptide 279Xaa128013PRTArtificial sequenceSynthetic peptide 280Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln1 5
1028112PRTArtificial sequenceSynthetic peptide 281Ala Tyr Ala Arg
Ala Ala Ala Arg Gln Ala Arg Ala1 5 1028234PRTArtificial
sequenceSynthetic peptide 282Asp Ala Ala Thr Ala Thr Arg Gly Arg
Ser Ala Ala Ser Arg Pro Thr1 5 10 15Glu Arg Pro Arg Ala Pro Ala Arg
Ser Ala Ser Arg Pro Arg Arg Pro20 25 30Val Glu28327PRTArtificial
sequenceSynthetic peptide 283Gly Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Leu Ile Asn Leu1 5 10 15Lys Ala Leu Ala Ala Leu Ala Lys
Lys Ile Leu20 2528412PRTArtificial sequenceSynthetic peptide 284Pro
Leu Ser Ser Ile Phe Ser Arg Ile Gly Asp Pro1 5 1028516PRTArtificial
sequenceSynthetic peptide 285Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Leu Leu Ala Pro1 5 10 1528612PRTArtificial
sequenceSynthetic peptide 286Ala Ala Val Leu Leu Pro Val Leu Leu
Ala Ala Pro1 5 1028715PRTArtificial sequenceSynthetic peptide
287Val Thr Val Leu Ala Leu Gly Ala Leu Ala Gly Val Gly Val Gly1 5
10 1528821PRTArtificial sequenceSynthetic peptide 288Gly Ala Leu
Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala Trp
Ser Gln Pro2028927PRTArtificial sequenceSynthetic peptide 289Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Leu Ile Asn Leu1 5 10
15Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu20
2529018PRTArtificial sequenceSynthetic peptide 290Lys Leu Ala Leu
Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys1 5 10 15Leu
Ala29121PRTArtificial sequenceSynthetic peptide 291Lys Glu Thr Trp
Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys1 5 10 15Lys Lys Arg
Lys Val2029216PRTArtificial sequenceSynthetic peptide 292Lys Ala
Phe Ala Lys Leu Ala Ala Arg Leu Tyr Arg Lys Ala Gly Cys1 5 10
1529316PRTArtificial sequenceSynthetic peptide 293Lys Ala Phe Ala
Lys Leu Ala Ala Arg Leu Tyr Arg Ala Ala Gly Cys1 5 10
1529416PRTArtificial sequenceSynthetic peptide 294Ala Ala Phe Ala
Lys Leu Ala Ala Arg Leu Tyr Arg Lys Ala Gly Cys1 5 10
1529516PRTArtificial sequenceSynthetic peptide 295Lys Ala Phe Ala
Ala Leu Ala Ala Arg Leu Tyr Arg Lys Ala Gly Cys1 5 10
1529616PRTArtificial sequenceSynthetic peptide 296Lys Ala Phe Ala
Lys Leu Ala Ala Gln Leu Tyr Arg Lys Ala Gly Cys1 5 10
15297160PRTArtificial sequenceSynthetic peptide 297Met Glu Ile Pro
Val Pro Val Gln Pro Ser Trp Leu Arg Arg Ala Ser1 5 10 15Ala Pro Leu
Pro Gly Leu Ser Ala Pro Gly Arg Leu Phe Asp Gln Arg20 25 30Phe Gly
Glu Gly Leu Leu Glu Ala Glu Leu Ala Ala Leu Cys Pro Thr35 40 45Thr
Leu Ala Pro Tyr Tyr Leu Arg Ala Pro Ser Val Ala Leu Pro Val50 55
60Ala Gln Val Pro Thr Asp Pro Gly His Phe Ser Val Leu Leu Asp Val65
70 75 80Lys His Phe Ser Pro Glu Glu Ile Ala Val Lys Val Val Gly Glu
His85 90 95Val Glu Val His Ala Arg His Glu Glu Arg Pro Asp Glu His
Gly Phe100 105 110Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro
Pro Gly Val Asp115 120 125Pro Ala Ala Val Thr Ser Ala Leu Ser Pro
Glu Gly Val Leu Ser Ile130 135 140Gln Ala Ala Pro Ala Ser Ala Gln
Ala Pro Pro Pro Ala Ala Ala Lys145 150 155 160298160PRTArtificial
sequenceSynthetic peptide 298Met Glu Ile Pro Val Pro Val Gln Pro
Ser Trp Leu Arg Arg Ala Asp1 5 10 15Ala Pro Leu Pro Gly Leu Ser Ala
Pro Gly Arg Leu Phe Asp Gln Arg20 25 30Phe Gly Glu Gly Leu Leu Glu
Ala Glu Leu Ala Ala Leu Cys Pro Thr35 40 45Thr Leu Ala Pro Tyr Tyr
Leu Arg Ala Pro Ser Val Ala Leu Pro Val50 55 60Ala Gln Val Pro Thr
Asp Pro Gly His Phe Ser Val Leu Leu Asp Val65 70 75 80Lys His Phe
Ser Pro Glu Glu Ile Ala Val Lys Val Val Gly Glu His85 90 95Val Glu
Val His Ala Arg His Glu Glu Arg Pro Asp Glu His Gly Phe100 105
110Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro Gly Val
Asp115 120 125Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val
Leu Ser Ile130 135 140Gln Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro
Pro Ala Ala Ala Lys145 150 155 160299160PRTArtificial
sequenceSynthetic peptide 299Met Glu Ile Pro Val Pro Val Gln Pro
Ser Trp Leu Arg Arg Ala Glu1 5 10 15Ala Pro Leu Pro Gly Leu Ser Ala
Pro Gly Arg Leu Phe Asp Gln Arg20 25 30Phe Gly Glu Gly Leu Leu Glu
Ala Glu Leu Ala Ala Leu Cys Pro Thr35 40 45Thr Leu Ala Pro Tyr Tyr
Leu Arg Ala Pro Ser Val Ala Leu Pro Val50 55 60Ala Gln Val Pro Thr
Asp Pro Gly His Phe Ser Val Leu Leu Asp Val65 70 75 80Lys His Phe
Ser Pro Glu Glu Ile Ala Val Lys Val Val Gly Glu His85 90 95Val Glu
Val His Ala Arg His Glu Glu Arg Pro Asp Glu His Gly Phe100 105
110Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro Gly Val
Asp115 120 125Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val
Leu Ser Ile130 135 140Gln Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro
Pro Ala Ala Ala Lys145 150 155 16030013PRTArtificial
sequenceSynthetic peptide 300Trp Leu Arg Arg Ala Ser Ala Pro Leu
Pro Gly Leu Lys1 5 1030113PRTArtificial sequenceSynthetic peptide
301Trp Leu Arg Arg Ala Asp Ala Pro Leu Pro Gly Leu Lys1 5
1030213PRTArtificial sequenceSynthetic peptide 302Trp Leu Arg Arg
Ala Glu Ala Pro Leu Pro Gly Leu Lys1 5 10303486DNAArtificial
sequenceSequence encoding rat HSP20 303atggagatac gcgtgccggt
acaacccagc tggctgcggc gtgcttccgc gccattacct 60ggcttcagta cccccggacg
attgtttgac cagaggtttg gggaaggttt acttgaggcg 120gaattagcaa
gtctatgtcc tgcagctata gcaccctact acctaagggc accatctgtc
180gcgctcccaa ctgcccaagt gcccacggat ccaggctatt tcagcgttct
gttagacgta 240aagcatttta gtccagaaga aatttcagta aaagtagtgg
gagaccatgt cgaggtacat 300gctagacacg aagagagacc tgatgaacac
ggtttcatcg ctcgagagtt tcaccggcgt 360tatcgcttgc cgccgggggt
tgatcccgcg gccgtcacat cagcactcag tccggaggga 420gttttatcca
tacaagccac accggcctct gctcaggcct cgcttccatc gcctcctgcg 480gcaaaa
48630429PRTArtificial sequenceSynthetic peptide 304Ala Gly Gly Gly
Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10 15Trp Leu Arg
Arg Ala Ser Ala Pro Leu Pro Gly Leu Lys20 2530529PRTArtificial
sequenceSynthetic peptide 305Ala Gly Gly Gly Gly Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg1 5 10 15Pro Arg Lys Ser Leu Trp Ala Leu
Gly Arg Pro Leu Ala20 2530616PRTArtificial sequenceSynthetic
peptide 306Ala Gly Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg1 5 10 1530726PRTArtificial sequenceSynthetic peptide 307Ala
Tyr Ala Arg Arg Ala Ala Ala Arg Gln Ala Arg Ala Trp Leu Arg1 5 10
15Arg Ala Ser Ala Pro Leu Pro Gly Leu Lys20 2530826PRTArtificial
sequenceSynthetic peptide 308Ala Tyr Ala Arg Arg Ala Ala Ala Arg
Gln Ala Arg Ala Pro Arg Lys1 5 10 15Ser Leu Trp Ala Leu Gly Arg Pro
Leu Ala20 2530912PRTArtificial sequenceSynthetic peptide 309Arg Arg
Arg Arg Arg Arg Ala Ser Ala Pro Leu Pro1 5 1031014PRTArtificial
sequenceSynthetic peptide 310Arg Arg Arg Arg Trp Leu Arg Arg Ala
Ser Ala Pro Leu Pro1 5 103113PRTArtificial sequenceSynthetic
peptide 311Leu Arg Arg13123PRTArtificial sequenceSynthetic peptide
312Gly Leu Ser13133PRTArtificial sequenceSynthetic peptide 313Gly
Leu Thr13143PRTArtificial sequenceSynthetic peptide 314Gly Lys
Ser13153PRTArtificial sequenceSynthetic peptide 315Gly Lys
Thr13163PRTArtificial sequenceSynthetic peptide 316Asp Leu
Ser13173PRTArtificial sequenceSynthetic peptide 317Asp Leu
Thr13183PRTArtificial sequenceSynthetic peptide 318Asp Lys
Ser13193PRTArtificial sequenceSynthetic peptide 319Asp Lys
Thr1320480DNAArtificial sequenceSequence encoding human HSP20
320atggaaattc ccgttccagt ccagcctagt tggctaagaa gagctagtgc
gcctttgccg 60ggtttgagtg cccccgggag gctatttgat caacgctttg gcgaggggtt
actggaggct 120gaattagcag cactttgtcc gaccacactc gcgccctatt
accttagagc gccgtctgta 180gccttaccag tcgctcaggt accaactgac
ccaggccact tctccgtttt attagacgtg 240aaacacttta gcccagaaga
gatagcagtc aaagttgtag gagagcatgt ggaagttcac 300gcgagacatg
aagagagacc agatgaacat ggtttcgtag cgagagaatt ccatcgacgg
360tatcgtctgc ccccaggagt cgatcctgca gctgtgacga gtgcattatc
gcctgaggga 420gtgctcagta tccaagcagc ccccgcgtca gcccaagccc
cgcctccggc tgctgccaag 480
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