U.S. patent application number 16/481128 was filed with the patent office on 2021-10-28 for fused in sarcoma (fus) nuclear translocation inhibitors for preventing fibrosis.
The applicant listed for this patent is The United States as Represented by The Department of Veterans Affairs, Vanderbilt University. Invention is credited to Manuel CHIUSA, Jack J. HAWIGER, Ambra A. POZZI, Jozef ZIENKIEWICZ.
Application Number | 20210332093 16/481128 |
Document ID | / |
Family ID | 1000005895399 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210332093 |
Kind Code |
A9 |
POZZI; Ambra A. ; et
al. |
October 28, 2021 |
FUSED IN SARCOMA (FUS) NUCLEAR TRANSLOCATION INHIBITORS FOR
PREVENTING FIBROSIS
Abstract
Disclosed herein are compositions and methods for inhibiting
collagen production mediated by the Fused in Sarcoma (FUS)
ribonucleoprotein. As disclosed herein, the C terminal domain of
FUS contains an uncommon nuclear localization sequence (NLS) motif
called PY-NLS that binds the nuclear import receptor transportin.
Phosphorylation of FUS leads to its association with transportin
and nuclear translocation with consequent increased in collagen
production. Therefore, disclosed herein is an isolated peptide
having a transportin-binding moiety, which inhibits FUS from
binding transportin, linked to a membrane translocating motif.
These compositions and methods can be used to inhibit FUS-mediated
collagen production, and treat fibrotic disease involving
FUS-mediated collagen accumulation in kidneys and other organs
displaying fibrotic diseases.
Inventors: |
POZZI; Ambra A.; (Nashville,
TN) ; CHIUSA; Manuel; (Nashville, TN) ;
HAWIGER; Jack J.; (Nashville, TN) ; ZIENKIEWICZ;
Jozef; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vanderbilt University
The United States as Represented by The Department of Veterans
Affairs |
Nashville
Washington |
TN
DC |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20190352355 A1 |
November 21, 2019 |
|
|
Family ID: |
1000005895399 |
Appl. No.: |
16/481128 |
Filed: |
January 29, 2018 |
PCT Filed: |
January 29, 2018 |
PCT NO: |
PCT/US2018/015702 PCKC 00 |
371 Date: |
July 26, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15297996 |
Oct 19, 2016 |
10568928 |
|
|
16481128 |
|
|
|
|
14251135 |
Apr 11, 2014 |
9492544 |
|
|
15297996 |
|
|
|
|
61810939 |
Apr 11, 2013 |
|
|
|
62451636 |
Jan 27, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1777 20130101;
C07K 2319/10 20130101; C07K 14/70557 20130101; C07K 14/4703
20130101; A61K 38/1825 20130101; C07K 2319/02 20130101; C07K 14/50
20130101; A61P 13/12 20180101; A61K 38/1709 20130101; C07K 2319/30
20130101; A61K 45/06 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C07K 14/705 20060101 C07K014/705; C07K 14/50 20060101
C07K014/50; A61P 13/12 20060101 A61P013/12; A61K 45/06 20060101
A61K045/06; A61K 38/17 20060101 A61K038/17; A61K 38/18 20060101
A61K038/18 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government Support under Grant
No. DK095761 awarded by the National Institutes of Health and Grant
No. BX002025 from the Department of Veterans Affairs. The
Government has certain rights in the invention.
Claims
1. An isolated peptide comprising a transportin-binding moiety
linked to a membrane translocating motif, wherein the
transportin-binding moiety inhibits Fused in Sarcoma (FUS)
ribonucleoprotein from binding transportin.
2. The peptide of claim 1, wherein the transportin-binding moiety
comprises a C-terminal fragment of a FUS ribonucleoprotein.
3. The peptide of claim 2, wherein the transportin-binding moiety
comprises the amino acid sequence SRGEHRQDRRERPY (SEQ ID NO:1), or
a conservative variant thereof.
4. The peptide of claim 1, wherein the membrane translocating motif
comprises a signal sequence hydrophobic region (SSHR).
5. The peptide of claim 4, wherein the SSHR is derived from a
fibroblast growth factor 4 (FGF4) protein.
6. (canceled)
7. The peptide of claim 4, wherein the membrane translocating motif
comprises the amino acid sequence XXXXLLPXXLLALLAP (SEQ ID NO:2) or
XXXXLLPXXLLAVLAP (SEQ ID NO:3), wherein X is any amino acid or
absent.
8. The peptide of claim 7, wherein the membrane translocating motif
comprises the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4) or
AAVALLPAVLLAVLAP (SEQ ID NO:5).
9. The peptide of claim 1, wherein the SSHR is derived from an
integrin .beta..sub.3 protein.
10. (canceled)
11. The peptide of claim 1, wherein the polypeptide comprises the
amino acid sequence AAVALLPAVLLALLAP--SRGEHRQDRRERPY (SEQ ID NO:6)
or AAVALLPAVLLAVLAP--SRGEHRQDRRERPY (SEQ ID NO:7), wherein "--" is
a linker or peptide bond.
12. The peptide of claim 1, wherein the agent further comprising an
organ-specific or cell-specific homing peptide or Fab antibody
fragment recognizing an organ- or cell-specific epitope.
13. (canceled)
14. A method for treating fibrotic disease in a subject, comprising
administering to the subject a therapeutically effective amount of
a composition comprising an agent that inhibits nuclear
translocation of Fused in Sarcoma (FUS), wherein the agent
comprises the peptide of claim 1.
15. The method of claim 14, wherein the agent inhibits FUS from
binding transportin or competes with FUS for binding to
transportin.
16. (canceled)
17. (canceled)
18. The method of claim 14, wherein the subject has a kidney
disease or damage and wherein the method inhibits
glomerulosclerosis in the subject; wherein the subject has a liver
disease or damage and wherein the method inhibits cirrhosis in the
subject; wherein the subject has a lung disease or damage and
wherein the method inhibits pulmonary fibrosis in the subject;
wherein the subject has a skin disease or damage and wherein the
method inhibits skin fibrosis (scleroderma) in the subject; or
wherein the subject has a fibrosarcoma or osteosarcoma tumor and
wherein the method inhibits collagen production by the tumor.
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. The method of claim 14, further comprising administering a
corticosteroid or a non-steroidal anti-inflammatory agent.
24. (canceled)
25. The method of claim 14, wherein the agent further comprises an
organ-specific or cell-specific homing peptide or Fab antibody
fragment recognizing the organ-specific or cell-specific
epitope.
26. (canceled)
27. A method for decreasing FUS-mediated collagen production by a
cell, comprising contacting the cell with an effective amount of a
composition comprising an agent that inhibits nuclear translocation
of Fused in Sarcoma (FUS), wherein the agent comprises the peptide
of claim 1.
28. The method of claim 27, wherein the agent inhibits FUS from
binding transportin or competes with FUS for binding to
transportin.
29. (canceled)
30. (canceled)
31. The method of claim 27, wherein the agent comprises the peptide
of claim 1 further comprising organ-specific homing peptide or Fab
antibody fragment recognizing the organ-specific or cell-specific
epitope.
32. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/451,636 filed Jan. 27, 2017, which
is fully incorporated herein by reference in its entirety.
BACKGROUND
[0003] End stage glomerular disease is the most common cause of
chronic kidney failure and represents a major cause of morbidity
and mortality for Veterans and civilian patients. Despite the fact
that glomerular disease has multiple etiologies, the final
pathology is characterized by overproduction and deposition of
extracellular matrix (ECM) and ensuing glomerulosclerosis (Borza,
C. M., et al. 2015. Curr Top Membr 76:231-253). In
glomerulosclerosis, the synthesis and remodeling of ECM components
(mainly collagens) are uncontrolled thus leading to scarred
glomeruli characterized by abnormal collagen deposition,
particularly collagens type I, IV, V and VI. Although many pathways
have been implicated in both initiation and progression to
glomerular fibrosis, to date there are very few therapeutic options
to treat glomerulosclerosis. Thus, there is the need of identifying
key factors contributing to the initiation and/or progression to
glomerulosclerosis, and fibrotic diseases in other organs (e.g.
liver, lungs, skin, retroperitoneal space), with the expectation
that targeting such factors will help in slowing and ideally
suppressing fibrotic responses, and ultimately reducing end stage
kidney disease as well as other organs' fibrotic diseases.
SUMMARY
[0004] Disclosed herein are compositions and methods for inhibiting
collagen production mediated by the Fused in Sarcoma (FUS)
ribonucleoprotein. These compositions and methods can therefore be
used to treat and prevent fibrotic disease in a subject, such as a
subject with liver, kidney, or lung disease or damage.
[0005] As disclosed herein, the C terminal domain of FUS contains
an uncommon nuclear localization sequence (NLS) motif called PY-NLS
that binds the nuclear import receptor transportin. Phosphorylation
of FUS leads to its association with transportin and nuclear
translocation with consequent increased in collagen production.
Therefore, disclosed herein is an isolated peptide that comprises a
transportin-binding moiety, which inhibits FUS from binding
transportin, linked to a membrane translocating motif.
[0006] In some embodiments, the transportin-binding moiety
comprises a C-terminal fragment of a FUS ribonucleoprotein. For
example, the transportin-binding moiety can comprise the amino acid
sequence SRGEHRQDRRERPY (SEQ ID NO:1), or a conservative variant
thereof.
[0007] In some embodiments, the membrane translocating motif
comprises a signal sequence hydrophobic region (SSHR). For example,
the SSHR can be derived from an integrin 133 protein, such as a
human integrin 133 protein, or from a fibroblast growth factor 4
(FGF4) protein, such as a human FGF4 protein. In some embodiments,
the membrane translocating motif comprises the amino acid sequence
XXXXLLPXXLLALLAP (SEQ ID NO:2) or XXXXLLPXXLLAVLAP (SEQ ID NO:3),
wherein X is any amino acid or absent. In some embodiments, the
membrane translocating motif comprises the amino acid sequence
AAVALLPAVLLALLAP (SEQ ID NO:4) or AAVALLPAVLLAVLAP (SEQ ID
NO:5).
[0008] In some embodiments, the polypeptide comprises the amino
acid sequence AAVALLPAVLLALLAP--SRGEHRQDRRERPY (SEQ ID NO:6) or
AAVALLPAVLLAVLAP--SRGEHRQDRRERPY (SEQ ID NO:7), wherein "--" is a
linker or peptide bond. Linkers can be short peptide sequences that
occur between protein domains. The linkers can be flexible or
rigid. Flexible linkers are often composed of flexible residues
like glycine and serine so that the adjacent protein domains are
free to move relative to one another. In particular, the linker can
be a polyglycine (e.g. 3, 4, or 5 glycine), a polyserine (e.g. 3,
4, or 5 serine), or a combination of glycine and serine including
repeating combinations. For example, the linker can be a glycine
and serine linker, such as, for example, a G4S, GSG4, G2SG3SG2,
G2SG, G3S linker, or any other linker known in the art where the
base linker sequence can optionally be repeated 2, 3, 4, or more
times. In some embodiments, the polypeptide comprises the amino
acid sequence
TABLE-US-00001 (SEQ ID NO: 8) AAVALLPAVLLALLAPSRGEHRQDRRERPY or
(SEQ ID NO: 9) AAVALLPAVLLAVLAPSRGEHRQDRRERPY.
[0009] The disclosed peptide can further include one or more
additional moieties. For example, the peptide can contain a homing
peptide or organ-specific or cell-specific Fab antibody fragment
for targeted delivery to an organ, such as the lung, kidney, skin,
heart, pancreas, uterus, retina, intestines, prostate, or liver.
The peptide can also contain a label, such as a fluorescent
dye.
[0010] Also disclosed is a method for decreasing FUS-mediated
collagen production by a cell, comprising contacting the cell with
an effective amount of a composition comprising an agent that
inhibits nuclear translocation of FUS. Also disclosed is a method
for treating fibrotic disease in a subject that involves
administering to the subject a therapeutically effective amount of
a composition comprising an agent that inhibits nuclear
translocation of FUS.
[0011] In some embodiments, the agent used in the disclosed methods
inhibits FUS from binding transportin. For example, the agent can
compete with FUS for binding to transportin, or can compete with
transportin for binding to FUS. In some embodiments, the agent
comprises a peptide disclosed herein having a transportin-binding
moiety linked to a membrane translocating motif.
[0012] The disclosed method can be used to treat any condition
involving abnormal FUS-mediated collagen formation. In particular,
the method can be used to treat a fibrosis involving abnormally
excessive collagen accumulation. For example, the subject can have
a kidney disease or damage, wherein the method inhibits
glomerulosclerosis in the subject. The subject can have a liver
disease or damage, wherein the method inhibits cirrhosis in the
subject. The subject can have a lung disease or damage, wherein the
method inhibits pulmonary fibrosis in the subject. The subject can
have a retroperitoneal fibrosis, wherein the method inhibits the
formation of fibrous tissue in the retroperitoneum. The subject can
have skin fibrosis (scleroderma) associated with systemic sclerosis
in which integrins and transforming growth factor beta as well as
connective tissue growth factor play significant role (Ray K. Nat
Rev Rheumatol 2013, 11:637
[0013] The subject can have a fibrosarcoma or osteosarcoma tumor,
wherein the method inhibits collagen production by the tumor.
[0014] The disclosed compositions can further contain or be
administered with other diagnostic or therapeutic agents for
fibrosis. For example, the disclosed composition can contain or be
administered with a corticosteroid or a non-steroidal
anti-inflammatory agent. In some embodiments, the disclosed
composition contains or is administered with a nuclear transport
modifier (NTM) that targets nuclear transport by an importin, such
as those described in U.S. Pat. Nos. 8,932,559, 9,044,433, and
9,492,544, which are incorporated by reference in their entirety
for the teaching of these NTM molecules and uses thereof.
[0015] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0016] FIGS. 1A to 1E. (A) Images of glomeruli from BALB/c WT and
Itg.alpha.1KO mice 8 weeks after ADR injection. Note that crossing
the Itg.alpha.1KO mice with the wave-2 mice or treating them with
erlotinib improved glomerular injury (A), albuminuria (mean.+-.SEM
of 5-7 mice/group) (B, C), and kidney collagen IV levels (erlotinib
group shown only, mean.+-.SEM of 3 mice/group) (D, E).
[0017] FIGS. 2A to 2D. (A, B) Kidney paraffin sections of the mice
indicated were co-stained with anti-FUS (green) and anti-phospho
EGFR (red) antibodies. Note that FUS is highly expressed and
co-localize with phospho EGFR in the glomeruli of Itg.alpha.1KO
mice (B mean.+-.SEM of 10 glom/mice with 3 mice evaluated). (C, D)
Nuclear fractionation of glomeruli isolated from 5 WT and 5
Itg.alpha.1KO mice showed significantly higher nuclear FUS levels
in the Itg.alpha.1KO mice.
[0018] FIGS. 3A to 3C. (A) Kidney paraffin sections of eNOSKO or
eNOSKO mice crossed with a mouse model of type 2 diabetes (db/db)
were stained with anti-FUS antibody. Note the presence of FUS in
the glomeruli of diabetic mice only (24 weeks old mice). (B,C)
BALB/c WT mice were injected with adriamycin and then sacrificed at
the time indicated. Nuclear fractions from isolated glomeruli
blotted with anti-FUS or anti-HDAC2 (as loading control) (C,
mean.+-.SEM of 6 mice/treatment).
[0019] FIGS. 4A to 4C. (A, B) Nuclear (N) and non-nuclear (NN)
fractions of WT and Itg.alpha.1KO mesangial cells showing
significantly higher levels of FUS in the nuclei of Itg.alpha.1KO
cells. (B, mean.+-.SEM of 6 samples). (C) Non-nuclear and nuclear
fractions were immuno-precipitated with the anti-pY antibody 4G10
or IgG control and then analyzed by Western Blot for levels of FUS.
Note that tyrosine phosphorylated FUS is detected primarily in the
nuclei of Itg.alpha.1KO cells.
[0020] FIGS. 5A to 5F. WT and Itg.alpha.1KO mesangial cells were
either kept untreated or treated with erlotinib (ERL) and the
levels of phospho EGFR (A, B), nuclear FUS (A, C), collagen IV (D,
E) and nuclear phosphorylated FUS (F,) were analyzed by Western
blot. In Itg.alpha.1KO cells, ERL significantly decreased EGFR
activation, nuclear FUS levels, collagen IV levels and tyrosine
phosphorylated FUS. (B, C mean.+-.SEM of 6 samples).
NN=non-nuclear; N=nuclear.
[0021] FIGS. 6A to 6F. WT and Itg.alpha.1KO mesangial cells were
treated with EGF for 0 or 30 minutes. The levels of phospho-EGFR
and EGFR were then analyzed by Western blot (A) and quantified by
densitometry analysis (B, mean SEM of 6 samples). (C) WT (W) and
Itg.alpha.1KO (K) cells were transiently transfected with RFP or
RFP-FUS cDNA and levels of endogenous FUS and RPF-FUS were analyzed
by Western blot with anti-RFP or anti-FUS antibody. (D) RFP-FUS
transfected cells were treated with EGF for 0 or 30 minutes and
then nuclear RFP-FUS (counterstaining with DAPI) was evaluated. (E)
The number of RFP-FUS and DAPI cells per microscopic field was
counted and expressed as RFP-FUS/DAPI (mean.+-.SEM of 150 cells).
WT and Itg.alpha.1KO mesangial cells were treated with EGF for 0 or
24 hours. The levels of Collagen IV and AKT (as loading control)
were analyzed by Western blot and quantified by densitometry
analysis (F, mean.+-.SEM of 3 samples).
[0022] FIGS. 7A to 7C. (A, B) Itg.alpha.1KO mesangial cells were
treated with scrambled-(Ser) or FUS-siRNA. 48 hours later the
levels of FUS and collagen IV were analyzed by WB and quantified by
densitometry analysis. (B, mean.+-.SEM of 3 samples). (C)
Itg.alpha.1KO cells were treated with Scr- or FUS-siRNA. 24 hours
later they were transiently transfected with the collagen IV
enhancer (E)/firefly luciferase or enhancer/promoter (E/P)/firefly
luciferase constructs together with renilla luciferase cDNAs. 24
hours later, the levels of firefly/renilla luciferase activity were
analyzed (mean.+-.SEM of 4 samples).
[0023] FIGS. 8A to 8D. (A) Itg.alpha.1KO mesangial cells were
treated with 0.1 .mu.M FUS PY-NLS derived peptide or its mutant
form for 24 hours and then left untreated or treated with EGF (20
ng/ml) for 3 hours. Cells where stained with anti-FUS antibody
(Red) or DAPI (Blue) to visualize FUS localization. (B) The
intensity of FUS nuclear staining was measured using Image-J and
expressed as mean of intensity/cell (mean.+-.SEM of 50 cells). WT
and Itg.alpha.1KO mesangial cells were treated with EGF for 0 or 24
hours in the presence of either FUS PY-NLS derived peptide or its
mutant form for 24 hours. The levels of Collagen IV and FAK (as
loading control) were then analyzed by Westem blot (C) and
quantified by densitometry analysis (D, mean.+-.SEM of 3
samples).
[0024] FIGS. 9A to 9C. (A) Lysates from WT (W) and Itg.alpha.1KO
(K) mesangial cells were immuno-precipitated with anti-EGFR
antibody or IgG and then analyzed by Western blot for levels of
EGFR, phospho EGFR and FUS. (B, C) The levels of phosphor EGFR,
EGFR and FUS were analyzed by densitometry and expressed as
pEGFR/EGFR and FUS/EGFR ratio (n=4 experiments).
[0025] FIGS. 10A and 10B. Schematic representation of a possible
Itg.alpha.1.beta.1/FUS interaction in healthy WT (A) or
Itg.alpha.1KO (B) mesangial cells. It was hypothesize that in
healthy cells (A), Itg.alpha.1.beta.1 prevents FUS tyrosine
phosphorylation, nuclear translocation, and activation of collagen
IV synthesis in a both EGFR-dependent and -independent manner. In
Itg.alpha.1KO cells (B), increased phosphorylation of FUS leads to
its association with transportin and nuclear translocation with
consequent increased collagen IV synthesis.
[0026] FIG. 11. In vivo delivery of FAM FUS-PY-NLS peptide injected
5 times every 2 hours. Mice were then sacrificed and kidney and
liver frozen sections were analyzed under an epifluorescence
microscope. Fluorescent peptide is displayed intracellularly in
kidney glomeruli and liver cells.
DETAILED DESCRIPTION
[0027] The term "subject" refers to any individual who is the
target of administration or treatment. The subject can be a
vertebrate, for example, a mammal. Thus, the subject can be a human
or veterinary patient. The term "patient" refers to a subject under
the treatment of a clinician, e.g., physician.
[0028] The term "therapeutically effective" refers to the amount of
the composition used is of sufficient quantity to ameliorate one or
more causes or symptoms of a disease or disorder. Such amelioration
only requires a reduction or alteration, not necessarily
elimination.
[0029] The term "pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other
problems or complications commensurate with a reasonable
benefit/risk ratio.
[0030] The terms "treatment" and "treating" refer to the medical
management of a patient with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0031] The term "prevent" refers to a treatment that forestalls or
slows the onset of a disease or condition or reduced the severity
of the disease or condition. Thus, if a treatment can treat a
disease in a subject having symptoms of the disease, it can also
prevent that disease in a subject who has yet to suffer some or all
of the symptoms.
[0032] The term "inhibit," "reduce," or "suppress" refers to a
decrease in an activity, response, condition, disease, or other
biological parameter. This can include but is not limited to the
complete ablation of the activity, response, condition, or disease.
This may also include, for example, a 10% reduction in the
activity, response, condition, or disease as compared to the native
or control level. Thus, the reduction can be a 10, 20, 30, 40, 50,
60, 70, 80, 90, 100%, or any amount of reduction in between as
compared to native or control levels.
[0033] The terms "peptide," "polypeptide," and "protein" are used
interchangeably to refer to a natural or synthetic molecule
comprising two or more amino acids linked by the carboxyl group of
one amino acid to the alpha amino group of another.
[0034] In addition, as used herein, the term "polypeptide" refers
to amino acids joined to each other by peptide bonds or modified
peptide bonds, e.g., peptide isoesters, etc. and may contain
modified amino acids other than the 20 gene-encoded amino acids.
The polypeptides can be modified by either natural processes, such
as post-translational processing, or by chemical modification
techniques which are well known in the art. Modifications can occur
anywhere in the polypeptide, including the peptide backbone, the
amino acid side-chains and the amino or carboxyl termini. The same
type of modification can be present in the same or varying degrees
at several sites in a given polypeptide. Also, a given polypeptide
can have many types of modifications. Modifications include,
without limitation, acetylation, acylation, ADP-ribosylation,
amidation, covalent cross-linking or cyclization, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of a phosphytidylinositol, disulfide bond formation,
demethylation, formation of cysteine or pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristolyation, oxidation,
pergylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, and transfer-RNA mediated
addition of amino acids to protein such as arginylation. (See
Proteins--Structure and Molecular Properties 2nd Ed., T. E.
Creighton, W.H. Freeman and Company, New York (1993);
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York, pp. 1-12 (1983)).
[0035] As used herein, "peptidomimetic" means a mimetic of a
peptide which includes some alteration of the normal peptide
chemistry. Peptidomimetics typically enhance some property of the
original peptide, such as increase stability, increased efficacy,
enhanced delivery, increased half-life, etc. Methods of making
peptidomimetics based upon a known polypeptide sequence is
described, for example, in U.S. Pat. Nos. 5,631,280; 5,612,895; and
5,579,250. Use of peptidomimetics can involve the incorporation of
a non-amino acid residue with non-amide linkages at a given
position. One embodiment of the present invention is a
peptidomimetic wherein the compound has a bond, a peptide backbone
or an amino acid component replaced with a suitable mimic. Some
non-limiting examples of unnatural amino acids which may be
suitable amino acid mimics include .beta.-alanine, L-.alpha.-amino
butyric acid, L-.gamma.-amino butyric acid, L-.alpha.-amino
isobutyric acid, L-.epsilon.-amino caproic acid, 7-amino heptanoic
acid, L-aspartic acid, L-glutamic acid,
N-.epsilon.-Boc-N-.alpha.-CBZ-L-lysine,
N-.epsilon.-Boc-N-.alpha.-Fmoc-L-lysine, L-methionine sulfone,
L-norleucine, L-norvaline, N-.alpha.-Boc-N-.delta.CBZ-L-ornithine,
N-.delta.-Boc-N-.alpha.-CBZ-L-ornithine,
Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and
Boc-L-thioproline.
[0036] The term "protein domain" refers to a portion of a protein,
portions of a protein, or an entire protein showing structural
integrity; this determination may be based on amino acid
composition of a portion of a protein, portions of a protein, or
the entire protein.
[0037] The term "residue" as used herein refers to an amino acid
that is incorporated into a polypeptide. The amino acid may be a
naturally occurring amino acid and, unless otherwise limited, may
encompass known analogs of natural amino acids that can function in
a similar manner as naturally occurring amino, acids.
[0038] A "fusion protein" refers to a polypeptide formed by the
joining of two or more polypeptides through a peptide bond formed
between the amino terminus of one polypeptide and the carboxyl
terminus of another polypeptide. The fusion protein can be formed
by the chemical coupling of the constituent polypeptides or it can
be expressed as a single polypeptide from nucleic acid sequence
encoding the single contiguous fusion protein. A single chain
fusion protein is a fusion protein having a single contiguous
polypeptide backbone. Fusion proteins can be prepared using
conventional techniques in molecular biology to join the two genes
in frame into a single nucleic acid, and then expressing the
nucleic acid in an appropriate host cell under conditions in which
the fusion protein is produced.
[0039] The C terminal domain of FUS contains an uncommon nuclear
localization sequence (NLS) motif called PY-NLS that binds the
nuclear import receptor transportin. Phosphorylation of FUS leads
to its association with transportin and nuclear translocation with
consequent increased in collagen production. Therefore, disclosed
herein is an isolated peptide (or peptidomimetic thereof)
comprising a transportin-binding moiety, which inhibits FUS from
binding transportin, linked to a membrane translocating motif. In
some embodiments, the disclosed peptide has a binding affinity
greater than about 10.sup.5 (e.g., 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11, and 10.sup.12 or more) moles/liter
for transportin.
[0040] In some embodiments, the transportin-binding moiety
comprises a C-terminal fragment of a FUS ribonucleoprotein. For
example, the transportin-binding moiety can comprise the amino acid
sequence SRGEHRQDRRERPY (SEQ ID NO:1), or a conservative variant
thereof.
[0041] Non-limiting examples of membrane translocating motifs
include Polyarginine (e.g., R9), Antennapedia sequences, TAT,
HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II,
Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion,
pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC
(Bis-Guanidinium-Spermidine-Cholesterol, and BGTC
(Bis-Guanidinium-Tren-Cholesterol).
[0042] In some embodiments, the membrane translocating motif
comprises a signal sequence hydrophobic region (SSHR). For example,
the SSHR can be derived from an integrin .beta..sub.3 protein, such
as a human integrin .beta..sub.3 protein, or from a fibroblast
growth factor 4 (FGF4) protein, such as a human FGF4 protein. In
some embodiments, the membrane translocating motif comprises the
amino acid sequence XXXXLLPXXLLALLAP (SEQ ID NO:2) or
XXXXLLPXXLLAVLAP (SEQ ID NO:3), wherein X is any amino acid or
absent. In some embodiments, the membrane translocating motif
comprises the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4) or
AAVALLPAVLLAVLAP (SEQ ID NO:5).
[0043] In some embodiments, the polypeptide comprises the amino
acid sequence AAVALLPAVLLALLAP--SRGEHRQDRRERPY (SEQ ID NO:6) or
AAVALLPAVLLAVLAP--SRGEHRQDRRERPY (SEQ ID NO:7), wherein "--" is a
linker or peptide bond. Linkers can be short peptide sequences that
occur between protein domains. The linkers can be flexible or
rigid. Flexible linkers are often composed of flexible residues
like glycine and serine so that the adjacent protein domains are
free to move relative to one another. In particular, the linker can
be a polyglycine (e.g. 3, 4, or 5 glycine), a polyserine (e.g. 3,
4, or 5 serine), or a combination of glycine and serine including
repeating combinations. For example, the linker can be a glycine
and serine linker, such as, for example, a G4S, GSG4, G2SG3SG2,
G2SG, G3S linker, or any other linker known in the art where the
base linker sequence can optionally be repeated 2, 3, 4, or more
times. In some embodiments, the polypeptide comprises the amino
acid sequence
TABLE-US-00002 (SEQ ID NO: 8) AAVALLPAVLLALLAPSRGEHRQDRRERPY or
(SEQ ID NO: 9) AAVALLPAVLLAVLAPSRGEHRQDRRERPY.
[0044] The disclosed peptide can further include one or more
additional moieties. For example, the peptide can contain a homing
peptide or organ-specific or cell-specific Fab antibody fragment
for targeted delivery to an organ, such as the lung, kidney, skin,
heart, pancreas, uterus, retina, intestines, prostate, or liver.
The peptide can also contain a label, such as a fluorescent dye.
The methods for selecting homing peptides or Fab antibody fragments
are available as described in several publications. For example,
those skilled in the art can use published protocols in Korbelin J
t al 2016 Mol. Therapy, 24(6):1050-1061), Pulmonary Targeting of
Adeno-associated Viral Vectors by Next-generation Sequencing-guided
Screening of Random Capsid Displayed peptide Libraries, Rosowski S
et al Microb Cell Fact. 2018 Jan. 9; 17(1):3. doi:
10.1186/s12934-017-0853-z A novel one-step approach for the
construction of yeast surface display Fab antibody libraries, and
Kelly R L et al 2018 J. Mol. Biol. 430(1):119-130,doi:
10.1016/j.jmb.2017.11.008. Epub 2017 Nov. 26. Examples of homing
peptides include but are not limited to the lysine glutamine
(K2E3).sub.3K peptide which has renal specificity; CARSKNKDC (SEQ
ID NO: 12) which has vascular specificity; and the lung homing
peptide X.sub.1-G-F-E-X.sub.2(SEQ ID NO: 13), where X.sub.1 and
X.sub.2 each is 1 to 10 independently selected amino acids
including, for example, the sequence CGFECVRQCPERC (SEQ ID NO: 14)
or CGFELETC (SEQ ID NO: 15). In some aspects, the disclosed peptide
comprises the amino acid sequence
TABLE-US-00003 (SEQ ID NO: 10) XXXXLLPXXLLA$LAP-SRGEHRQDRRERPY,
[0045] wherein "X" is any amino acid or a peptide bond,
[0046] wherein "$" is a valine or a leucine, and
[0047] wherein "--" is a linker or a peptide bond.
[0048] In some aspects, the disclosed peptide comprises the amino
acid sequence
TABLE-US-00004 (SEQ ID NO: 11) AAVALLPAVLLA$LAP-SRGEHRQDRRERPY,
[0049] wherein "X" is any amino acid or a peptide bond,
[0050] wherein "$" is a valine or a leucine, and
[0051] wherein "--" is a linker or a peptide bond.
[0052] In some aspects, the disclosed polypeptide comprises a
conservative variant of a disclosed amino acid sequence. For
example, in some aspects, the disclosed polypeptide comprises a
disclosed amino acid sequence having 1, 2, 3, or 4 conservative
amino acid substitutions.
[0053] The disclosed peptide can have a variety of lengths and
structures as described herein. In some aspects, the disclosed
peptide can consist essentially of from about 25 to about 100 amino
acids, including about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, or more amino acids. The disclosed peptide can
comprise less than about 100 amino acid residues, including less
than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35,
or 30 amino acid residues. The disclosed peptide can comprise more
than about 25 amino acid residues, including more than about 25,
30, 35, 40, 45, or 50 amino acid residues.
[0054] The disclosed polypeptides can be artificial sequences and
can be synthesized in vitro and/or recombinantly. The disclosed
polypeptides can be peptides that are not naturally occurring
proteins and can be peptides that have at least two contiguous
sequences that are not contiguous in a naturally occurring
protein.
[0055] Fusion proteins, also known as chimeric proteins, are
proteins created through the joining of two or more genes which
originally coded for separate proteins. Translation of this fusion
gene results in a single polypeptide with function properties
derived from each of the original proteins. Recombinant fusion
proteins can be created artificially by recombinant DNA technology
for use in biological research or therapeutics. Chimeric mutant
proteins occur naturally when a large-scale mutation, typically a
chromosomal translocation, creates a novel coding sequence
containing parts of the coding sequences from two different
genes.
[0056] The functionality of fusion proteins is made possible by the
fact that many protein functional domains are modular. In other
words, the linear portion of a polypeptide which corresponds to a
given domain, such as a tyrosine kinase domain, may be removed from
the rest of the protein without destroying its intrinsic enzymatic
capability. Thus, any of the herein disclosed functional domains
can be used to design a fusion protein.
[0057] A recombinant fusion protein is a protein created through
genetic engineering of a fusion gene. This typically involves
removing the stop codon from a cDNA sequence coding for the first
protein, then appending the cDNA sequence of the second protein in
frame through ligation or overlap extension PCR. That DNA sequence
will then be expressed by a cell as a single protein. The protein
can be engineered to include the full sequence of both original
proteins, or only a portion of either.
[0058] If the two entities are proteins, often linker (or "spacer")
peptides are also added which make it more likely that the proteins
fold independently and behave as expected. Especially in the case
where the linkers enable protein purification, linkers in protein
or peptide fusions are sometimes engineered with cleavage sites for
proteases or chemical agents which enable the liberation of the two
separate proteins. This technique is often used for identification
and purification of proteins, by fusing a GST protein, FLAG
peptide, or a hexa-his peptide (aka: a 6.times.his-tag) which can
be isolated using nickel or cobalt resins (affinity
chromatography). Chimeric proteins can also be manufactured with
toxins or anti-bodies attached to them in order to study disease
development.
[0059] Alternatively, internal ribosome entry sites (IRES) elements
can be used to create multigene, or polycistronic, messages. IRES
elements are able to bypass the ribosome scanning model of 5'
methylated Cap dependent translation and begin translation at
internal sites (Pelletier and Sonenberg, 1988). IRES elements from
two members of the picornavirus family (polio and
encephalomyocarditis) have been described (Pelletier and Sonenberg,
1988), as well an IRES from a mammalian message (Macejak and
Sarnow, 1991). IRES elements can be linked to heterologous open
reading frames. Multiple open reading frames can be transcribed
together, each separated by an IRES, creating polycistronic
messages. By virtue of the IRES element, each open reading frame is
accessible to ribosomes for efficient translation. Multiple genes
can be efficiently expressed using a single promoter/enhancer to
transcribe a single message (U.S. Pat. Nos. 5,925,565 and
5,935,819; PCT/US99/05781). IRES sequences are known in the art and
include those from encephalomycarditis virus (EMCV) (Ghattas, I. R.
et al., Mol. Cell. Biol., 11:5848-5849 (1991); BiP protein (Macejak
and Sarnow, Nature, 353:91 (1991)); the Antennapedia gene of
drosophilia (exons d and e) [Oh et al., Genes & Development,
6:1643-1653 (1992)); those in polio virus [Pelletier and Sonenberg,
Nature, 334:320325 (1988); see also Mountford and Smith, TIG,
11:179-184 (1985)).
[0060] The disclosed peptide can further include one or more
additional moieties. For example, the peptide can contain a homing
peptide for targeted delivery to an organ, such as the lung,
kidney, skin, heart, pancreas, uterus, retina, intestines,
prostate, or liver. The peptide can also contain a label, such as a
fluorescent dye. In one aspect, the homing peptide can be an Fab
antibody fragment specific for an organ-specific or cell-specific
epitope (such as, for example, a cell-specific or organ-specific
peptide, polypeptide, or protein). It is understood and herein
contemplated that by "organ-specific" and "cell-specific" epitope
is meant an epitope (such as, for example, a peptide, polypeptide,
or protein) whose expression is limited to that cell-type or
organ.
[0061] Therapeutic molecules, such as the polypeptides disclosed
herein, can be used therapeutically in combination with a
pharmaceutically acceptable carrier. The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problems or complications
commensurate with a reasonable benefit/risk ratio.
[0062] Pharmaceutical carriers suitable for administration of the
molecules provided herein include any such carriers known to those
skilled in the art to be suitable for the particular mode of
administration. Pharmaceutical compositions may include thickeners,
diluents, buffers, preservatives, surface active agents and the
like in addition to the molecule of choice. Pharmaceutical
compositions may also include one or more active ingredients such
as antimicrobial agents, anti-inflammatory agents, anesthetics, and
the like.
[0063] Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, or otherwise mixing
a molecules as defined above and optional pharmaceutical adjuvants
in a carrier, such as, for example, water, saline, aqueous
dextrose, glycerol, glycols, ethanol, and the like, to thereby form
a solution or suspension. If desired, the pharmaceutical
composition to be administered may also contain minor amounts of
nontoxic auxiliary substances such as wetting agents, emulsifying
agents, solubilizing agents, pH buffering agents and the like, for
example, acetate, sodium citrate, cyclodextrin derivatives,
sorbitan monolaurate, triethanolamine sodium acetate,
triethanolamine oleate, and other such agents.
[0064] The compounds described herein can be formulated for
parenteral administration. Parenteral formulations can be prepared
as aqueous compositions using techniques is known in the art.
Typically, such compositions can be prepared as injectable
formulations, for example, solutions or suspensions; solid forms
suitable for using to prepare solutions or suspensions upon the
addition of a reconstitution medium prior to injection; emulsions,
such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions,
and microemulsions thereof, liposomes, or emulsomes.
[0065] Solutions and dispersions of the active compounds as the
free acid or base or pharmacologically acceptable salts thereof can
be prepared in water or another solvent or dispersing medium
suitably mixed with one or more pharmaceutically acceptable
excipients including, but not limited to, surfactants, dispersants,
emulsifiers, pH modifying agents, and combination thereof.
[0066] Suitable surfactants may be anionic, cationic, amphoteric or
nonionic surface active agents. Suitable anionic surfactants
include, but are not limited to, those containing carboxylate,
sulfonate and sulfate ions. Examples of anionic surfactants include
sodium, potassium, ammonium of long chain alkyl sulfonates and
alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate;
dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene
sulfonate; dialkyl sodium sulfosuccinates, such as sodium
bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as
sodium lauryl sulfate. Cationic surfactants include, but are not
limited to, quaternary ammonium compounds such as benzalkonium
chloride, benzethonium chloride, cetrimonium bromide, stearyl
dimethylbenzyl ammonium chloride, polyoxyethylene and coconut
amine. Examples of nonionic surfactants include ethylene glycol
monostearate, propylene glycol myristate, glyceryl monostearate,
glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose
acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene
monolaurate, polysorbates, polyoxyethylene octylphenylether,
PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene
glycol butyl ether, Poloxamer.RTM. 401, stearoyl
monoisopropanolamide, and polyoxyethylene hydrogenated tallow
amide. Examples of amphoteric surfactants include sodium
N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,
myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0067] The formulation can contain a preservative to prevent the
growth of microorganisms. Suitable preservatives include, but are
not limited to, parabens, chlorobutanol, phenol, sorbic acid, and
thimerosal. The formulation may also contain an antioxidant to
prevent degradation of the active agent(s).
[0068] The formulation is typically buffered to a pH of 3-8 for
parenteral administration upon reconstitution. Suitable buffers
include, but are not limited to, phosphate buffers, acetate
buffers, and citrate buffers.
[0069] Water soluble polymers are often used in formulations for
parenteral administration. Suitable water-soluble polymers include,
but are not limited to, polyvinylpyrrolidone, dextran,
carboxymethylcellulose, and polyethylene glycol.
[0070] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent or dispersion medium with one or more of the
excipients listed above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those listed above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
solution thereof. The powders can be prepared in such a manner that
the particles are porous in nature, which can increase dissolution
of the particles. Methods for making porous particles are well
known in the art.
[0071] Pharmaceutical formulations can be designed for immediate
release, sustained release, delayed release and/or burst release of
one or more polypeptides in a therapeutically effective amount. In
a preferred embodiment, the formulation provides an initial burst
release of a "loading dosage", followed by a sustained release to
maintain the therapeutically effective dosage. This can be
accomplished using a delayed and/or extended release
formulation.
[0072] Disclosed herein are methods for reducing, inhibiting,
preventing, or treating a fibrotic disease in a subject comprising
administering to the subject a therapeutically effective amount of
a composition comprising an agent that inhibits nuclear
translocation of Fused in Sarcoma (FUS). Similarly, disclosed
herein are methods for reducing, inhibiting, preventing, or
treating FUS-mediated collagen production by a cell comprising
administering to the subject a therapeutically effective amount of
a composition comprising an agent that inhibits nuclear
translocation of Fused in Sarcoma (FUS). It is understood and
herein contemplated that the agent for reducing, inhibiting,
preventing, or treating a fibrotic disease or FUS collagen
production can be any isolated peptides disclosed herein comprising
a transportin-binding moiety linked to a membrane translocating
motif.
[0073] As disclosed herein, fibrotic diseases can include, but are
not limited to pulmonary fibrosis (including, cystic fibrosis and
radiation induced lung injury), atrial fibrosis,
glomerulosclerosis, kidney damage, skin fibrosis (scleroderma),
scleroderma from a systemic fibrosis, cirrhosis, Crohn's Disease,
Keloid, Myelofibrosis, arthrofibrosis, fibrosarcoma, osteosarcoma
tumor, or collagen production by a tumor.
[0074] In particular embodiments, the method involves administering
a polypeptide disclosed herein. For example, the disclosed
polypeptides can in some cases be administered in a dose equivalent
to parenteral administration of about 0.1 ng to about 100 g per kg
of body weight, about 10 ng to about 50 g per kg of body weight,
about 100 ng to about 1 g per kg of body weight, from about 1 .mu.g
to about 100 mg per kg of body weight, from about 1 .mu.g to about
50 mg per kg of body weight, from about 1 mg to about 500 mg per kg
of body weight; and from about 1 mg to about 50 mg per kg of body
weight. Alternatively, the amount of polypeptide administered to
achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng,
100 ng, 1 .mu.g, 10 .mu.g, 100 .mu.g, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg,
6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg,
16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70
mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or
greater.
[0075] In some embodiments, the dose of polypeptide to be
administered provides a final plasma level of polypeptide of about
100 ng/ml to about 1000 ng/ml, about 1100 ng/ml to about 1450
ng/ml, 100 ng/ml to about 250 ng/ml, about 200 ng/ml to about 350
ng/ml, about 300 ng/ml to about 450 ng/ml, about 350 ng/ml to about
450 ng/ml, about 400 ng/ml to about 550 ng/ml, about 500 ng/ml to
about 650 ng/ml, about 600 ng/ml to about 750 ng/ml, about 700
ng/ml to about 850 ng/ml, about 800 ng/ml to about 950 ng/ml, about
900 ng/ml to about 1050 ng/ml, about 1000 ng/ml to about 1150
ng/ml, about 100 ng/ml to about 1250 ng/ml, about 1200 ng/ml to
about 1350 ng/ml, about 1300 ng/ml to about 1500 ng/ml.
[0076] The herein disclosed compositions, including pharmaceutical
composition, may be administered in a number of ways depending on
whether local or systemic treatment is desired, and on the area to
be treated. For example, the disclosed compositions can be
administered intravenously, intraperitoneally, intramuscularly,
subcutaneously, intracavity, transdermally, or topically.
[0077] The disclosed composition can be administered
therapeutically, to treat, prevent, or reduce fibrotic disease or
FUS-mediated collagen production in a subject or prophylactically,
to patients or subjects at risk for fibrosis. Accordingly, the
compositions may be administered prior to the onset of fibrosis
(including, for example, prior to exposure to radiation which could
result in fibrotic injury). In one aspect, the disclosed
compositions can be administered to the patient or subject as a
single one time injection or as multiple administrations. For
example, the disclosed compositions can be administered at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day. The
compositions can be administered to the patient or subject at least
once about every 4, 6, 8, 12, 24 hours, or every day, every other
day, every third day, every fourth day, every fifth day, every
sixth day, once a week, once every two weeks, once every three
weeks, once a month, once every two months, once every three
months, once every four months, once every five months, once every
six months, once every seven months, once every eight months, once
every nine months, once every ten months, once every eleven months,
once every year, once every eighteen months, once every two year,
once every three years, once every four years, or once every five
years. Treatment can be continued as long as needed to reduce,
inhibit, prevent, or eliminate the fibrotic disease or symptoms
associated with the disease.
[0078] The disclosed polypeptides can be administered adjunctively
with other active compounds such as analgesics, anti-inflammatory
drugs, antipyretics, antiepileptics, antihistamines, antimigraine
drugs, antimuscarinics, anxioltyics, sedatives, hypnotics,
antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular
drugs, corticosteroids, doparninergics, electrolytes,
parasympathomimetics, stimulants, anorectics and
anti-narcoleptics.
[0079] As noted above, the compositions disclosed herein may be
administered prophylactically to patients or subjects who are at
risk for fibrosis. Thus, the method can further comprise
identifying a subject at risk for fibrosis prior to administration
of the herein disclosed compositions.
[0080] A number of embodiments of the invention have been
described.
[0081] Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
EXAMPLES
Example 1
[0082] Integrins are transmembrane receptors for ECM components
composed of non-covalently bound .alpha. and .beta. subunits that
heterodimerize to produce 24 different transmembrane receptors
(Hynes, R. 2002. Cell 110:673-687; Pan, L., et al. 2016.
Springerplus 5:1094). Integrin .alpha.1.beta.1 (Itg.alpha.1.beta.1)
is a major collagen IV receptor that is highly expressed by
podocytes, endothelial cells and mesangial cells of the glomerulus
(Patey, N., et al. 1994. Cell Adhes Commun 2:159-167). Absence of
Itg.alpha.1.beta.1 does not affect the normal glomerular function;
however, this integrin plays an important role in regulating the
glomerulus response to injury. Itg.alpha.1.beta.1 has been
identified as a negative, inhibitory, modulator of glomerular
injury. To this end, Itg.alpha.1.beta.1 prevents excessive
injury-mediated glomerulosclerosis by negatively regulating EGF
receptor (EGFR) tyrosine phosphorylation, by preventing the
assembly of the NADPH oxidase and generation of profibrotic ROS,
and by negatively regulating collagen levels at both translational
and transcriptional levels (Chen, X., et al. 2007. Mol Cell Biol
27:3313-3326; Chen, X., et al. 2004. Am J Pathol 165:617-630; Chen,
X., et al. 2010. Mol Cell Biol 30:3048-3058; Wang, H., et al. 2015.
Kidney Int 87:948-962; Gardner, H., et al. 1999. J Cell Sci
112:263-272). Itg.alpha.1.beta.1 exerts its anti-fibrotic role by
regulating both the level and tyrosine phosphorylation of
caveolin-1 a scaffolding protein that controls EGFR activation
(Chen, X., et al. 2010. Mol Cell Biol 30:3048-3058; Borza, C. M.,
et al. 2010. J Biol Chem 285:40114-40124). TGF-.beta. receptor II
has also been identified as another target of Itg.alpha.1.beta.1.
Itg.alpha.1.beta.1 also negatively regulates TGF-.beta. receptor
II-mediated SMAD3 activation and pro-fibrotic signaling by
downregulating the tyrosine phosphorylation levels of TGF-.beta.
receptor II (Chen, X., et al. 2014. J Clin Invest
124:3295-3310).
[0083] A mechanism whereby Itg.alpha.1.beta.1 negatively regulates
the tyrosine phosphorylation levels of several growth factor
receptors as well as scaffolding proteins is by recruiting and
activating the tyrosine phosphatase TCPTP (Mattila, E., et al.
2005. Nat Cell Biol 7:78-85). Consistent with this finding, cells
lacking Itg.alpha.1.beta.1 do not recruit and activate TCPTP thus
showing increased basal levels of tyrosine phosphorylated proteins
(Chen, X., et al. 2007. Mol Cell Biol 27:3313-3326). Mice lacking
Itg.alpha.1.beta.1 manifest excessive and accelerated
glomerulosclerosis following various models of glomerular injury,
including partial renal ablation, adriamycin injection, oxidative
stress, and type 1 diabetes (Chen, X., et al. 2004. Am J Pathol
165:617-630; Wang, H., et al. 2015. Kidney Int 87:948-962; Borza,
C. M., et al. 2012. J Am Soc Nephrol 23:1027-1038; Zent, R., et al.
2006. Kidney Int 70:460-470; Yu, L., et al. 2012. Kidney Int
81:1086-1097).
[0084] A key question is how Itg.alpha.1.beta.1, in addition to the
targets indicated above, controls collagen levels at the
transcriptional level. The activation of many transcription factors
and their nuclear translocation are regulated by tyrosine
phosphorylation (Rebelo, S., et al. 2015. Cell Signal 27:2589-2598;
Thapar, R. 2015. ACS Chem Biol 10:652-666). Thus,
immunoprecipitation of nuclear proteins from wild type (WT) and
Itg.alpha.1KO mesangial cells was performed using
anti-phosphotyrosine antibody. The complexes were analyzed by mass
spectrometry in order to identify highly tyrosine phosphorylated
nuclear proteins only in Itg.alpha.1KO cells. As disclosed herein,
increased levels of total and tyrosine phosphorylated nuclear
ribonucleoprotein Fused in Sarcoma (FUS) in Itg.alpha.1KO cells are
associated with increased collagen production, and reducing FUS
levels diminishes collagen production. Thus, Itg.alpha.1.beta.1
plays an anti-fibrotic action by decreasing the tyrosine
phosphorylation and nuclear levels of FUS.
[0085] EGFR is a receptor tyrosine kinase activated by several
ligands including EGF, TGF-.alpha., and HB-EGF. This receptor is
expressed by mesangial cells and podocytes and plays an important
role in the development of the kidney (Zhuang, S., et al. 2014.
Kidney Int Suppl (2011) 4:70-74). In addition, EGFR is a key
determinant in the initiation, development and progression of
kidney glomerular injury. In the nephrectomy model, for example,
inhibition of EGFR reduces glomerular fibrosis suggesting that
activation of EGFR occurs in the course of glomeruli injury and
contributes to fibrosis (Liu, N., et al. 2012. PLoS ONE 7:e36194).
In both mice and humans with rapidly progressive glomerulonephritis
expression of HB-EGF by podocytes promotes EGFR phosphorylation and
activation thus contributing to glomerular injury (Bollee, G., et
al. 2011. Nat Med 17:1242-1250). In addition, mice lacking HB-EGF
expression specifically in endothelial cells, show decrease
glomerular EGFR activation and decreased angiotensin-II mediated
glomerular injury (Zeng, F., et al. 2016. Am J Physiol Renal
Physiol:ajprenal 311(4):F695-F707).
[0086] A key negative regulator of EGFR activation and pro-fibrotic
function is Itg.alpha.1.beta.1. At least two mechanisms account for
this negative regulation: Itg.alpha.1.beta.1 binds and activates
TCPTP and interacts with the membrane scaffolding protein caveolin
1, two negative regulators of EGFR activation (Chen, X., et al.
2010. Mol Cell Biol 30:3048-3058; Borza, C. M., et al. 2010. J Biol
Chem 285:40114-40124; Borza, C. M., et al. 2010. J Biol Chem
285:40114-40124; Abulrob, A., et al. 2004. Oncogene 23:6967-6979).
To further determine the contribution of EGFR to glomerular injury
in Itg.alpha.1KO mice, a genetic and a pharmacological approach was
used.
[0087] In the first model, Itg.alpha.1KO mice were crossed with
mice expressing a functionally hypomorphic EGFR (waved-2 mice)
(Luetteke, N.C., et al. 1994. Genes Dev 8:399-413) and then
subjected to adriamycin (ADR)-mediated injury. In the second model,
wild type (WT) and Itg.alpha.1KO mice were injected with ADR and
then left untreated or treated with the EGFR inhibitor erlotinib
(20 mg/Kg/day i.p.). Compared to WT mice, Itg.alpha.1KO mice
developed significantly more glomerular injury, proteinuria and
glomerular collagen synthesis 8 weeks after ADR treatment (FIG.
1A-E). Crossing the Itg.alpha.1KO mice with the wave-2 mice or
treating them with erlotinib significantly improved glomerular
injury, proteinuria and collagen synthesis (FIG. 1A-E).
[0088] Although this data suggests that blocking EGFR with
available receptor tyrosine kinase inhibitors might be a promising
strategy for the treatment and management of glomerular injury, it
is important to notice that prolonged treatment with receptor
kinase inhibitors, including erlotinib, can cause some severe side
effects. The most common side effects include skin rash,
cardiovascular and pulmonary toxicities, electrolyte depletion,
diarrhea and renal complications (reviewed in (Liu, F., et al.
2016. Int J Mol Sci 17). Thus, the identification of key downstream
signaling molecules activated by the integrins/EGFR axis or
integrins alones, might represent a valid tool to better target
kidney disease and avoid severe side effects. In this regard, FUS
is shown herein to contain Tyr6 and Tyr296 as two EGFR
phosphorylatable and TCPTP dephosphorylatable tyrosines. In
addition, levels of nuclear FUS seem to be associated with levels
of activated EGFR.
[0089] FUS, also known as translocated in liposarcoma (TLS), is a
heterogeneous ribonucleoprotein able to bind RNA and proteins
(Sama, R. R., et al. 2014. ASN Neuro 6). FUS consists of an
N-terminal end involved in transcriptional activation and a
C-terminal end involved in protein-RNA and protein-protein
interactions (Sama, R. R., et al. 2014. ASN Neuro 6). The C
terminal domain also contains an uncommon nuclear localization
sequence (NLS) motif called PY-NLS because the PY is localized at
the C-terminus of the protein. The PY-NLS binds the nuclear import
receptor transportin (or karyopherin .beta.2) (Dormann, D., et al.
2010. Embo J 29:2841-2857). In 2009, two groups analyzed several
unrelated families who presented with amyotrophic lateral sclerosis
(ALS) phenotype and found 14 mutations in the FUS gene, thus
providing the first evidence that FUS is linked to familiar ALS
(Kwiatkowski, T. J., Jr., et al. 2009. Science 323:1205-1208;
Vance, C., et al. 2009. Science 323:1208-1211). Indeed, mutations
in the C-terminal domain of FUS that prevent nuclear translocation
thus causing increased cytoplasmic localization and formation of
stress granule-like structures account for .about.5% of familiar
ALS cases (reviewed in (Sama, R. R., et al. 2014. ASN Neuro 6). In
addition to mutations, overexpression of FUS can also be pathogenic
in human patients (Sabatelli, M., et al. 2013. Hum Mol Genet
22:4748-4755). After these findings, mouse models of ALS
overexpressing FUS or carrying the same FUS mutations identified in
humans have been generated (Picher-Martel, V., et al. 2016. Acta
Neuropathol Commun 4:70). Mice have been generated that express
human FUSWT or the pathological mutation FUSR521G (no longer able
to translocate to the nucleus) under the control of the
cytomegalovirus immediate early enhancer-chicken .beta.-actin
hybrid promoter. These mice express wild type or mutated FUS only
when crossed with a Cre mouse line. When crossed with a global Cre
mouse line, thus forcing expression of these two proteins in all
cells, these mice are born alive but develop severe motor deficits
phenocopying the human diseases (Sephton, C. F., et al. 2014. Proc
Natl Acad Sci U.S.A. 111:E4769-4778). These mice have been crossed
with PDGFR-Cre mice in order to drive expression of WT and mutated
form of FUS preferentially in mesangial cells. FUShet mice were
also obtained. While FUSKO mice die immediately after birth on a
C57/B6 background (Hicks, G. G., et al. 2000. Nat Genet
24:175-179), their survival rate increases on the BALB/c
background. These mice are used to analyze the contribution of FUS
in the regulation of collagen production in both physiological and
pathological conditions.
[0090] Increased Nuclear Phosphorylated FUS in Itg.alpha.1KO
Mesangial Cells.
[0091] A key question is to understand the molecular mechanisms
whereby Itg.alpha.1.beta.1 controls collagen levels at the
transcriptional level. The nuclear translocation and activation of
many transcription factors are processes regulated by tyrosine
phosphorylation (Rebelo, S., et al. 2015. Cell Signal 27:2589-2598;
Thapar, R. 2015. ACS Chem Biol 10:652-666). Cells lacking
Itg.alpha.1.beta.1 have increased basal levels of tyrosine
phosphorylated proteins (e.g., EGFR, TGF.beta. receptor II and
caveolin-1) (Chen, X., et al. 2007. Mol Cell Biol 27:3313-3326;
Borza, C. M., et al. 2010. J Biol Chem 285:40114-40124; Chen, X.,
et al. 2014. J Clin Invest 124:3295-3310) due to inability to
recruit and activate the tyrosine phosphatase TCPTP (Mattila, E.,
et al. 2005. Nat Cell Biol 7:78-85). In order to identify highly
tyrosine phosphorylated nuclear proteins only in Itg.alpha.1KO, but
not wild type (WT) cells, immuno-precipitation of nuclear proteins
from WT and Itg.alpha.1KO mesangial cells was performed using
anti-phosphotyrosine antibody and the complexes analyzed by mass
spectrometry. Five potential hits were identified with 1 of them
being the ribonucleoprotein Fused in Sarcoma (FUS).
[0092] FUS is a Ribonucleoprotein Regulated by TCPTP and EGFR.
[0093] FUS is a RNA-protein binding molecule that consists of an
N-terminal end involved in transcriptional activation and a
C-terminal end involved in protein and RNA binding. The rationale
for selecting this candidate for study is as following: 1) FUS
binds Sp1 (Dhar, S. K., et al. 2014. Antioxid Redox Signal
20:1550-1566) a transcriptional activator involved in collagen
synthesis and fibrosis (Ghosh, A. K., et al. 2013. Exp Biol Med
(Maywood) 238:461-481). 2) Patients with ALS show decreased levels
of collagen in skin and blood (34, 35). 3) Collagen IV is a
multimeric protein composed of 3 .alpha. subunits. These subunits
are encoded by 6 different genes (.alpha.1-.alpha.6), each of which
can form a triple helix with 2 other subunits to form type IV
collagen. The .alpha.1 and .alpha.2 chains form the
.alpha.1.alpha.2.alpha.1 type IV collagen and their transcription
is regulated by a bidirectional promoter (846 bp) and a enhancer
(329 bp) located in the first intron of the .alpha.1(IV) chain gene
(Burbelo, P. D., et al. 1988. Proc Natl Acad Sci USA 85:9679-9682).
Analysis of the murine enhancer and promoter sequence with
ALGGEN-PROMO-v3 revealed the presence of 4 and 9 FUS responsive
element in the enhancer and promoter, respectively. 4) FUS has 36
tyrosines and analysis of FUS with PhosphoMotif Finder revealed
Tyr6 and Tyr296 as two EGFR phosphorylatable and TCPTP
dephosphorylatable tyrosines. 5) Studies in Drosophila suggest a
genetic link between Cabeza (orthologue of human FUS) and
rhomboid-1, a key component of the EGFR signaling pathway
(Shimamura, M., et al. 2014. Exp Cell Res 326:36-45). 6) Data shown
below clearly suggest a link between nuclear localization of FUS
and collagen synthesis.
[0094] Increased Levels of FUS in Itg.alpha.1KO Glomeruli.
[0095] To validate the mass spectrometry analysis, the nuclear
levels of FUS in glomeruli from WT and Itg.alpha.1KO mice was
analyzed. Nuclear FUS was detected in the glomeruli of both WT and
Itg.alpha.1KO mice, although it was significantly more in the
latter group (FIG. 2A-D). Interestingly nuclear FUS was found to
localize with activated EGFR, which was evident only in glomeruli
of Itg.alpha.1KO, but not WT mice (FIG. 2A) supporting the finding
of increased basal level activation of EGFR in the absence of
Itg.alpha.1.beta.1 (Chen, X., et al. 2010. Mol Cell Biol
30:3048-3058).
[0096] Increased Glomerular FUS Expression in Human and Mouse
Diseased Kidneys.
[0097] To determine whether levels of glomerular FUS are increased
in kidney disease, FUS levels were analyzed in the glomeruli of
control and type 2 diabetic mice. While no expression of this
ribonucleoprotein was detected in the glomeruli of non-diabetic
mice, FUS expression became evident in the glomeruli of type 2
diabetic mice (FIG. 3A). To further confirm that the levels of FUS
increase following injury, WT mice were treated with Adriamycin
(ADR) and a significant increase in nuclear FUS levels was observed
in glomeruli isolated 3 days after ADR treatment (FIG. 3B,C).
Interestingly, analysis of kidneys from healthy human subjects or
individuals with early and late diabetic nephropathy, revealed
expression of nuclear FUS only in the glomeruli of diabetic
subjects, clearly suggesting that FUS is upregulated in kidney
disease.
[0098] Increased FUS Nuclear Levels Directly Correlate to Collagen
Synthesis.
[0099] To further confirm the in vivo data, mesangial cells were
isolated from WT and Itg.alpha.1KO mice and the basal level of
nuclear FUS was analyzed. FUS was detected in the nuclei of both WT
and Itg.alpha.1KO mesangial cells, although its levels were higher
and more tyrosine phosphorylated in the latter group (FIG. 4A-C).
To determine whether nuclear translocation of FUS is dependent on
EGFR activation, mesangial cells were treated with erlotinib. This
EGFR inhibitor decreased EGFR activation (5A,B) and significantly
decreased nuclear FUS (FIG. 5A,C) and collagen IV levels (FIG.
5D,E), and these events were more pronounced in Itg.alpha.1KO
mesangial cells. Treatment with erlotinib also significantly
decreased the levels of nuclear tyrosine phosphorylated FUS (FIG.
5F), suggesting a potential link between EGFR activation, FUS
phosphorylation and nuclear FUS localization.
[0100] FUS Nuclear Translocation is Dependent Upon EGFR
Activation.
[0101] Mesangial cells were transiently transfected with murine FUS
cDNA inserted downstream the Red Fluorescent Protein gene (RFP-FUS)
(FIG. 6C) and its basal nuclear localization was determined.
RFP-FUS was detected in the nuclei of both WT and Itg.alpha.1KO
cells, although it was significantly more in the latter group (FIG.
6D,E). When cells were treated for 30 minutes with EGF, increased
activation of EGFR was observed in both WT and Itg.alpha.1KO cells,
although it was more evident in the Itg.alpha.1KO cells (FIG.
6A,B). Treatment with EGF, also significantly promoted more RFP-FUS
nuclear translocation in Itg.alpha.1KO cells compared to WT cells
(FIG. 6D,E).
[0102] Downregulation of FUS Decreased Basal Collagen Production in
Itg.alpha.1KO Cells.
[0103] To determine whether the increased total and phosphorylated
levels of nuclear FUS observed in Itg.alpha.1KO cells (FIGS. 4,5)
are responsible for increased levels of collagen production in
these cells (FIG. 5D,E), Itg.alpha.1KO cells were treated with
either scrambled (Scr) or FUS siRNA and then the levels of FUS and
collagen IV were analyzed. The focus was on collagen IV, as it is
the major Itg.alpha.1.beta.1 binding collagen (Gardner, H., et al.
1996. Dev Biol 175:301-313); and the collagen IV promoter and
enhancer region contain several FUS responsive elements. FUS-siRNA,
but not Scr-siRNA, significantly downregulated FUS levels and this
event was accompanied by a significant decrease in collagen IV
production (FIG. 7A,B). Thus, FUS either directly and/or indirectly
controls collagen levels.
[0104] FUS Knockdown Decreases Collagen Transcription Levels.
[0105] As the collagen IV enhancer/promoter contains FUS responsive
elements, whether FUS can control collagen at the transcriptional
levels was analyzed. Itg.alpha.1KO cells were treated with Scr- or
FUS-siRNA and then the cells were transfected with a firefly
luciferase reporter gene under the control of the collagen IV
enhancer or enhancer/promoter. Analysis of luciferase activity
(normalized to renilla) in cells treated with Scr-siRNA revealed
the collagen IV enhancer by itself failed to promote luciferase
transcription, while the collagen IV enhancer/promoter promoted
robust luciferase transcription (FIG. 7C). Downregulation of FUS
resulted in .about.50% reduction in the collagen IV
enhancer/activity, suggesting that FUS can control collagen IV
production the transcriptional level (FIG. 7C).
[0106] Design and Testing of Cell-Penetrating Peptides that Inhibit
FUS Nuclear Translocation.
[0107] At present there are no inhibitors available to prevent FUS
function and/or nuclear translocation. FUS has an uncommon nuclear
localization sequence (NLS) motif called PY-NLS because the PY is
localized at the C-terminus of the protein
(RGGRGGGDRGGFGPGKMDSRGEHRQDRRERPY, SEQ ID NO:12). This
non-classical NLS motif is recognized by transportin and
methylation of the arginine in the RGG motif or phosphorylation of
the tyrosine in the PY motif alters FUS/transportin interaction and
interferes with FUS nuclear translocation (Zhang, Z. C., et al.
2012. Proc Natl Acad Sci USA 109:12017-12021).
[0108] Based on this finding, a peptide
AAVALLPAVLLALLAPSRGEHRQDRRERPY (SEQ ID NO:8) was designed carrying
a FUS PY-NLS derived peptide (bold) fused with the signal sequence
hydrophobic region of FGF4 (Italicized). Signal sequence
hydrophobic region was designed as a membrane translocating
fragment that enables NLS to cross cell membrane bypassing
endosomal pathway (Veach, R. A., et al. 2004. J Biol Chem
279:11425-11431). The mutated version of the fragment-designed
peptide AAVALLPAVLLALLAPSEGEHRADEEERGA (SEQ ID NO:13) contained
amino acid replacements in PY-NLS of FUS.
[0109] Both peptides were purified and tested for cytotoxicity at
the concentrations used in these experiments. Itg.alpha.1KO
mesangial cells were pre-treated with these peptides (0.1 .mu.M)
for 24 hours and then left untreated or treated with EGF for 3
hours. FUS localization was then analyzed by immunofluorescence
using anti-FUS antibody. FUS PY-NSL derived peptide, but not its
mutated version, significantly inhibited both basal and
EGF-mediated FUS nuclear translocation (FIG. 8A, B). Cells treated
with the FUS PY-NSL derived peptide also showed cytoplasmic FUS
indicating that the peptide efficiently prevents FUS nuclear
translocation (FIG. 8A).
[0110] Based on the finding that cells lacking Itg.alpha.1.beta.1
show increased tyrosine phosphorylated and nuclear levels of FUS
and that FUS nuclear levels are positively associated to collagen
production, it is proposed that, in the course of glomerular
injury, Itg.alpha.1.beta.1 attenuates excessive and unwanted
collagen synthesis by negatively regulating FUS tyrosine
phosphorylation, nuclear translocation, and activation of collagen
transcription (FIGS. 10A and 10B).
[0111] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0112] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
15114PRTArtificial Sequencesynthetic construct 1Ser Arg Gly Glu His
Arg Gln Asp Arg Arg Glu Arg Pro Tyr1 5 10216PRTArtificial
Sequencesynthetic constructmisc_feature(1)..(4)Xaa can be any
naturally occurring amino acidmisc_feature(8)..(9)Xaa can be any
naturally occurring amino acid 2Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa
Leu Leu Ala Leu Leu Ala Pro1 5 10 15316PRTArtificial
Sequencesynthetic constructmisc_feature(1)..(4)Xaa can be any
naturally occurring amino acidmisc_feature(8)..(9)Xaa can be any
naturally occurring amino acid 3Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa
Leu Leu Ala Val Leu Ala Pro1 5 10 15416PRTArtificial
Sequencesynthetic construct 4Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Leu Leu Ala Pro1 5 10 15516PRTArtificial
Sequencesynthetic construct 5Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Val Leu Ala Pro1 5 10 15630PRTArtificial
Sequencesynthetic construct 6Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Leu Leu Ala Pro1 5 10 15Ser Arg Gly Glu His Arg Gln Asp
Arg Arg Glu Arg Pro Tyr 20 25 30730PRTArtificial Sequencesynthetic
construct 7Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Val Leu
Ala Pro1 5 10 15Ser Arg Gly Glu His Arg Gln Asp Arg Arg Glu Arg Pro
Tyr 20 25 30830PRTArtificial Sequencesynthetic construct 8Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Ser
Arg Gly Glu His Arg Gln Asp Arg Arg Glu Arg Pro Tyr 20 25
30930PRTArtificial Sequencesynthetic construct 9Ala Ala Val Ala Leu
Leu Pro Ala Val Leu Leu Ala Val Leu Ala Pro1 5 10 15Ser Arg Gly Glu
His Arg Gln Asp Arg Arg Glu Arg Pro Tyr 20 25 301029PRTArtificial
Sequencesynthetic constructmisc_feature(1)..(4)Xaa can be any
naturally occurring amino acidmisc_feature(8)..(9)Xaa can be any
naturally occurring amino acid 10Xaa Xaa Xaa Xaa Leu Leu Pro Xaa
Xaa Leu Leu Ala Leu Ala Pro Ser1 5 10 15Arg Gly Glu His Arg Gln Asp
Arg Arg Glu Arg Pro Tyr 20 251129PRTArtificial Sequencesynthetic
construct 11Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Ala
Pro Ser1 5 10 15Arg Gly Glu His Arg Gln Asp Arg Arg Glu Arg Pro Tyr
20 25129PRTArtificial Sequencesynthetic construct 12Cys Ala Arg Ser
Lys Asn Lys Asp Cys1 5135PRTArtificial Sequencesynthetic
constructmisc_feature(1)..(1)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 13Xaa Gly Phe Glu Xaa1 51413PRTArtificial
Sequencesynthetic construct 14Cys Gly Phe Glu Cys Val Arg Gln Cys
Pro Glu Arg Cys1 5 10158PRTArtificial Sequencesynthetic construct
15Cys Gly Phe Glu Leu Glu Thr Cys1 5
* * * * *