U.S. patent application number 14/878720 was filed with the patent office on 2016-04-14 for peptide inhibitors of sodium channels.
The applicant listed for this patent is Spyryx Biosciences, Inc., The University of North Carolina at Chapel Hill. Invention is credited to Dale J. Christensen, Robert Tarran.
Application Number | 20160102121 14/878720 |
Document ID | / |
Family ID | 55653774 |
Filed Date | 2016-04-14 |
United States Patent
Application |
20160102121 |
Kind Code |
A1 |
Tarran; Robert ; et
al. |
April 14, 2016 |
Peptide Inhibitors of Sodium Channels
Abstract
The present invention relates to the ability of specialized
non-naturally occurring peptides to bind to sodium channels and
inhibit activation of the sodium channels. The invention further
relates to methods for regulating of sodium absorption and fluid
volume and treating disorders responsive to modulating sodium
absorption by modulating the binding of specialized non-naturally
occurring peptides to sodium channels.
Inventors: |
Tarran; Robert; (Chapel
Hill, NC) ; Christensen; Dale J.; (Cary, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill
Spyryx Biosciences, Inc. |
Chapel Hill
Durham |
NC
NC |
US
US |
|
|
Family ID: |
55653774 |
Appl. No.: |
14/878720 |
Filed: |
October 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62061461 |
Oct 8, 2014 |
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Current U.S.
Class: |
514/17.4 ;
435/375; 530/326; 530/327; 530/328 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
11/08 20180101; A61P 11/06 20180101; A61P 11/12 20180101; A61P
13/12 20180101; C07K 7/08 20130101; C07K 7/06 20130101; A61K 38/00
20130101; C07K 14/705 20130101; A61P 1/04 20180101; C07K 5/06026
20130101; C07K 2319/10 20130101; C07K 2319/00 20130101; A61P 11/00
20180101; A61P 1/00 20180101; A61K 9/0073 20130101; A61P 43/00
20180101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06 |
Claims
1. A peptide comprising the sequence:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8
(SEQ ID NO:116) wherein: X.sub.1 is leucine or a conservative
substitution with a natural or non-natural amino acid; X.sub.2 is
proline or a conservative substitution with a natural or
non-natural amino acid; X.sub.3 is valine or a conservative
substitution with a natural or non-natural amino acid; X.sub.4 is
proline or a conservative substitution with a natural or
non-natural amino acid; X.sub.5 is leucine or a conservative
substitution with a natural or non-natural amino acid; X.sub.6 is
aspartic acid or a conservative substitution with a natural or
non-natural amino acid; X.sub.7 is glutamine or a conservative
substitution with a natural or non-natural amino acid; and X.sub.8
is threonine or a conservative substitution with a natural or
non-natural amino acid; or a functional fragment thereof.
2. A peptide comprising the sequence:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12 (SEQ ID NO:117) wherein: X.sub.1 is
leucine, norleucine, or valine; X.sub.2 is proline,
4-hydroxyproline, (2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid,
or 3,4-dehydro-L-proline; X.sub.3 is valine, leucine, norleucine,
or N-methylvaline; X.sub.4 is proline, 4-hydroxyproline,
(2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid, or
3,4-dehydro-L-proline; X.sub.5 is leucine, norleucine, or valine;
X.sub.6 is aspartic acid or glutamic acid; X.sub.7 is glutamine or
asparagine; and X.sub.8 is threonine, serine, or
L-.alpha.-methylserine; or a functional fragment thereof.
3. The peptide of claim 1, comprising the sequence of any one of
SEQ ID NOS:4-115.
4. A peptide comprising the sequence:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15 (SEQ ID
NO:118) wherein: X.sub.1 is leucine or a conservative substitution
with a natural or non-natural amino acid; X.sub.2 is proline or a
conservative substitution with a natural or non-natural amino acid;
X.sub.3 is valine or a conservative substitution with a natural or
non-natural amino acid; X.sub.4 is proline or a conservative
substitution with a natural or non-natural amino acid; X.sub.5 is
leucine or a conservative substitution with a natural or
non-natural amino acid; X.sub.6 is aspartic acid or a conservative
substitution with a natural or non-natural amino acid; X.sub.7 is
glutamine or a conservative substitution with a natural or
non-natural amino acid; X.sub.8 is threonine or a conservative
substitution with a natural or non-natural amino acid; X.sub.9 is
threonine or a conservative substitution with a natural or
non-natural amino acid; X.sub.10 is leucine or a conservative
substitution with a natural or non-natural amino acid; X.sub.11 is
proline or a conservative substitution with a natural or
non-natural amino acid; X.sub.12 is asparagine or a conservative
substitution with a natural or non-natural amino acid; X.sub.13 is
valine or a conservative substitution with a natural or non-natural
amino acid; X.sub.14 is asparagine or a conservative substitution
with a natural or non-natural amino acid; and X.sub.15 is proline
or a conservative substitution with a natural or non-natural amino
acid; or a functional fragment thereof.
5. The peptide of claim 4, comprising the sequence of SEQ ID
NO:120.
6. A peptide comprising the sequence of SEQ ID NO:127 or SEQ ID
NO:128.
7. The peptide of claim 1, wherein the N-terminus is acetylated
and/or the C-terminus is amidated.
8. The peptide of claim 1, wherein the peptide comprises at least
one non-natural amino acid or at least one terminal
modification.
9. The peptide of claim 1, further comprising at least one
D-alanine at the N-terminus and/or the C-terminus.
10. A kit comprising the peptide of claim 1.
11. A method of inhibiting the activation of a sodium channel,
comprising contacting a sodium channel with the peptide of claim
1.
12. The method of claim 11, wherein the sodium channel is an
epithelial sodium channel (ENaC).
13. The method of claim 11, wherein the sodium channel is present
in an isolated cell.
14. The method of claim 13, wherein the isolated cell is part of an
epithelial cell culture.
15. The method of claim 11, wherein the sodium channel is present
in a cell in an animal.
16. The method of claim 15, wherein the animal is a disease
model.
17. The method of claim 11, wherein contacting the sodium channel
with the peptide comprises delivering the peptide to a cell
comprising the sodium channel.
18. The method of claim 11, wherein activation of the sodium
channel is inhibited by at least 20%.
19. The method of claim 11, wherein activation of the sodium
channel is inhibited by at least 50%.
20. The method of claim 11, wherein activation of the sodium
channel is inhibited by at least 90%.
21. A method of inhibiting sodium absorption through a sodium
channel, comprising contacting the sodium channel with the peptide
of claim 1.
22. The method of claim 21, wherein the sodium channel is ENaC.
23. A method of increasing the volume of fluid lining an epithelial
mucosal surface, comprising contacting a sodium channel present on
the epithelial mucosal surface with the peptide of claim 1.
24. The method of claim 23, wherein the sodium channel is ENaC.
25. A method of reducing the level of a sodium channel present on
the surface of a cell, comprising contacting the sodium channel
with the peptide of claim 1.
26. The method of claim 25, wherein the sodium channel is ENaC.
27. A method of treating a disorder responsive to inhibition of
sodium absorption across an epithelial mucosal surface in a subject
in need thereof, comprising delivering to the subject a
therapeutically effective amount of the peptide of claim 1.
28. The method of claim 27, wherein the sodium channel is ENaC.
29. The method of claim 27, wherein said disorder is a lung
disorder.
30. The method of claim 27, wherein said lung disorder is cystic
fibrosis.
31. The method of claim 27, wherein said lung disorder is
non-cystic fibrosis bronchiectasis.
32. The method of claim 27, wherein said lung disorder is chronic
obstructive pulmonary disease.
33. The method of claim 27, wherein said lung disorder is acute or
chronic bronchitis.
34. The method of claim 27, wherein said lung disorder is
asthma.
35. The method of claim 27, wherein said disorder is a
gastrointestinal disorder.
36. The method of claim 35, wherein said gastrointestinal disorder
is inflammatory bowel disease.
37. The method of claim 27, wherein said disorder is a kidney
disorder.
38. A method of regulating salt balance, blood volume, and/or blood
pressure in a subject in need thereof, comprising delivering to the
subject a therapeutically effective amount of the peptide of claim
1.
39. The method of claim 27, wherein delivering the peptide reduces
the level of a sodium channel present on the surface of a cell in
the subject.
40. A method for treating a symptom of a lung disorder, a
gastrointestinal disorder, a kidney disorder, or a cardiovascular
disorder in a subject in need thereof, comprising administering to
the subject a therapeutically effective amount of the peptide of
claim 1.
41. The method of claim 40, wherein the lung disorder is cystic
fibrosis, non-cystic fibrosis bronchiectasis, chronic obstructive
pulmonary disease, acute or chronic bronchitis, or asthma.
42. The method of claim 40, wherein the gastrointestinal disorder
is inflammatory bowel disease.
Description
STATEMENT OF PRIORITY
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/061,461, filed Oct. 8, 2014, the entire
contents of which are incorporated by reference herein.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn.1.821, entitled 5470-721_ST25.txt, 56,808 bytes in
size, generated on Oct. 8, 2015 and filed via EFS-Web, is provided
in lieu of a paper copy. The Sequence Listing is incorporated
herein by reference into the specification for its disclosures.
FIELD OF THE INVENTION
[0003] The present invention relates to optimized peptides that are
specialized non-naturally occurring peptides with improved ability
to bind to sodium channels and inhibit activation of the sodium
channels. The invention further relates to methods for regulating
sodium absorption and fluid volume and treating disorders
responsive to modulating sodium absorption by activity of sodium
channels.
BACKGROUND OF THE INVENTION
[0004] Epithelial mucosal surfaces are lined with fluids whose
volume and composition are precisely controlled. In the airways, a
thin film of airway surface liquid helps protect mammalian airways
from infection by acting as a lubricant for efficient mucus
clearance (Hobbs et al., J. Physiol. 591: 4377 (2013), Knowles et
al., J. Clin. Invest. 109:571 (2002)). This layer moves cephalad
during mucus clearance and excess liquid that accumulates as two
airways converge is eliminated by Na.sup.+-modulated airway surface
liquid absorption with Na.sup.+ passing through the epithelial
Na.sup.+ channel (ENaC) (Hobbs et al., J. Physiol. 591: 4377
(2013), Knowles et al., J. Clin. Invest. 109:571 (2002)).
Critically, the mechanism by which ENaC activity is regulated in
the airways is poorly understood. Recently, evidence has been
accumulating that molecular regulators in the airway surface liquid
can serve as volume sensing signals whose dilution or concentration
can alter specific cell surface receptors that control ion
transport rates to either absorb or secrete airway surface liquid
as needed (Chambers et al., Respir. Physiol. Neurobiol. 159:256
(2007)). As one of the regulated targets, ENaC must be cleaved by
intracellular furin-type proteases and/or extracellular channel
activating proteases (CAPs) such as prostasin to be active and to
conduct Na.sup.+ (Planes et al., Curr. Top. Dev. Biol. 78:23
(2007); Rossier, Proc. Am. Thorac. Soc. 1:4 (2004); Vallet et al.,
Nature 389:607 (1997); Chraibi et al., J. Gen. Physiol. 111:127
(1998)). ENaC can also be cleaved and activated by exogenous serine
proteases such as trypsin, an action that is attenuated by the
protease inhibitor aprotinin (Vallet et al., Nature 389:607
(1997)). When human bronchial epithelial cultures are mounted in
Ussing chambers where native airway surface liquid is washed away,
ENaC is predominantly active, suggesting that cell attached
proteases are predominant (Bridges et al., Am. J Physiol. Lung
Cell. Mol. Physiol. 281:L16 (2001); Donaldson et al., J. Biol.
Chem. 277:8338 (2002)). In contrast, under thin film conditions,
where native airway surface liquid is present, ENaC activity is
reduced, suggesting that airway surface liquid contains soluble
protease inhibitors (Myerburg et al., J. Biol. Chem. 281:27942
(2006); Tarran et al., J. Gen. Physiol. 127:591 (2006); Gaillard et
al., 2010 Pfleugers Arch, 460: 1-17).
[0005] Recently, it has been shown that the Short Palate Lung and
Nasal epithelial Clone (SPLUNC1) protein comprises up to 10% of the
total protein in the airway surface liquid and can readily be
detected in both nasal lavage and tracheal secretions (Bingle, C.
D., and Craven, C. J. (2002) PLUNC: a novel family of candidate
host defense proteins expressed in the upper airways and
nasopharynx Hum Mol Genet 11, 937; Campos, M. A., et al. (2004)
Purification and characterization of PLUNC from human
tracheobronchial secretions Am J Respir Cell Mol Biol 30, 184;
Lindahl, M., Stahlbom, B., and Tagesson, C. (2001); Identification
of a new potential airway irritation marker, palate lung nasal
epithelial clone protein, in human nasal lavage fluid with
two-dimensional electrophoresis and matrix-assisted laser
desorption/ionization-time of flight Electrophoresis 22, 1795).
SPLUNC1 appears to be a volume sensing molecule since it can be
secreted onto the mucosal surface of the superficial epithelia
where ENaC is expressed (Bartlett et al., J. Leukoc. Biol. 83:1201
(2008); Bingle et al., J. Pathol. 205:491 (2005)). Furthermore,
SPLUNC1 has been demonstrated to contain a subdomain that functions
as an inhibitor of ENaC through its N-terminal domain.
[0006] The present invention discloses novel specialized
non-naturally occurring peptides that mimic the properties of
SPLUNC1 in regulation of sodium channels by binding to and
inhibiting ion transport to regulate sodium absorption and fluid
volume and treat disorders responsive to modulating sodium
absorption.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, on the design of
specialized non-naturally occurring peptides to regulate the
activity of sodium channels. Accordingly, in one aspect the
invention relates to a method of inhibiting the activation of a
sodium channel, comprising contacting a sodium channel with a
specialized non-naturally occurring peptide or a functional
fragment thereof. In one embodiment, the sodium channel is an
epithelial sodium channel (ENaC). In one embodiment, the
specialized non-naturally occurring peptide or a functional
fragment thereof binds to the sodium channel.
[0008] Another aspect of the invention relates to a method of
inhibiting sodium absorption through a sodium channel, comprising
contacting the sodium channel with a specialized non-naturally
occurring peptide or a functional fragment thereof. In one
embodiment, the specialized non-naturally occurring peptide or a
functional fragment thereof binds to the sodium channel.
[0009] A further aspect of the invention relates to a method of
increasing the volume of fluid lining an epithelial mucosal
surface, comprising contacting a sodium channel present on the
epithelial mucosal surface with a specialized non-naturally
occurring peptide or a functional fragment thereof. In one
embodiment, the specialized non-naturally occurring peptide or a
functional fragment thereof binds to the sodium channel.
[0010] Another aspect of the invention relates to a method of
reducing the level of a sodium channel present on the surface of a
cell, comprising contacting the sodium channel with a specialized
non-naturally occurring peptide or a functional fragment thereof.
In one embodiment, the specialized non-naturally occurring peptide
or a functional fragment thereof binds to the sodium channel.
[0011] A further aspect of the invention relates to a method of
treating a disorder responsive to inhibition of sodium absorption
across an epithelial mucosal surface in a subject in need thereof,
comprising delivering to the subject a therapeutically effective
amount of a specialized non-naturally occurring peptide or a
functional fragment thereof. In one embodiment, the specialized
non-naturally occurring peptide or a functional fragment thereof
binds to the sodium channel.
[0012] Another aspect of the invention relates to a method of
regulating salt balance, blood volume, and/or blood pressure in a
subject in need thereof, comprising delivering to the subject a
therapeutically effective amount of a specialized non-naturally
occurring peptide or a functional fragment thereof. In one
embodiment, the specialized non-naturally occurring peptide or a
functional fragment thereof binds to the sodium channel.
[0013] Another aspect of the invention relates to a specialized
non-naturally occurring peptide or a functional fragment thereof
that mimics the sodium channel binding domain of a PLUNC protein
and binds to a sodium channel, wherein cleavage of the sodium
channel by a protease is inhibited when bound to the peptide.
[0014] Another aspect of the invention relates to a kit comprising
the peptide of the invention.
[0015] Another aspect of the invention relates to the use of a
specialized non-naturally occurring peptide or a functional
fragment thereof for the preparation of a medicament to treat a
disorder responsive to inhibition of sodium absorption in a subject
in need thereof.
[0016] These and other aspects of the invention are set forth in
more detail in the description of the invention below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the sequence of S18 (SEQ ID NO:1). Highlighted
in red are residues essential for ENaC interaction, while
highlighted in blue are residues that were found not to
contribute.
[0018] FIGS. 2A-2B show the 1.sup.st half (LPVPLDQT (SEQ ID NO:113)
but not the remainder (DQTLPLNVNP (SEQ ID NO:114) of S18 is
required for inhibition of ENaC and preservation of ASL height.
[0019] FIG. 3A shows that S18 (SEQ ID NO:1) and aaLPVPLDQTLPLNVNPaa
(SEQ ID NO:2) have equal potency and efficacy, despite SEQ ID NO:2
being flanked by D-alanines. FIG. 3B shows that S18, and
aaLPVPLDQTaa (SEQ ID NO:3) have equal potency and efficacy, despite
(SEQ ID NO:3) being flanked by D-alanines. FIG. 3A also shows the
relative potency of a sample of peptides, including aaLPNlePLDQTaa
(SEQ ID NO:5), which displays increased potency, relative to S18 or
SEQ ID NO:4.
[0020] FIG. 4 shows an additional comparison of S18 and SEQ ID
NO:5, and increased potency of SEQ ID NO:5.
[0021] FIGS. 5A-5U show the results of experiments analyzing the
effects of various peptides on internalization of alpha-ENaC in
HEK293T cells and effects on cell viability when GFP-tagged
alpha-ENaC is co-expressed with beta and gamma ENaC. EC50 values
are shown at the bottom of several of the figures. In these
figures, ADG is a negative control peptide with the sequence:
NH3-ADGGLLLLNNPPPPQTVV-NH2 (SEQ ID NO:143). The peptides used in
these experiments were S18 (SEQ ID NO:1), SEQ ID NO:2, SEQ ID
NO:141, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:128, SEQ ID NO:131,
SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID
NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:131, SEQ ID NO:139;
SEQ ID NO:140, SEQ ID NO:144 and SEQ ID NO:127. Also used in these
experiments were peptides of SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:136, each with an
aa cap at both the N- and the C-terminus. Also shown is amiloride,
which inhibits ENaC by another mechanism and does not reduce the
amount of functional receptor on the surface of cells.
[0022] FIG. 6 shows the results of an experiment analyzing the
effects of various peptides on internalization of alpha-ENaC in
HEK293T cells when GFP-tagged alpha-ENaC is co-expressed only with
gamma ENaC and no beta-ENaC. In this figure, SEQ ID NO:143
(negative control peptide) and water (vehicle) controls are used
along with S18 (SEQ ID NO:1) and SEQ ID NO:128. While SEQ ID NO:1
AND SEQ ID NO:128 are effective in reducing alpha-ENaC when
beta-ENaC is co-expressed, no effect is observed in this experiment
when beta-ENaC is not present.
[0023] FIG. 7A shows the results of an experiment analyzing the
effect on percent survival of .beta.ENaC-Tg C57BL:FVB mice after
treatment with the S18 peptide (SEQ ID NO:1). S18 (100 mM solution)
was administered 3 times per day at 1 .mu.L/g of body weight.
[0024] FIG. 7B shows the results of an experiment analyzing the
effect on percent survival of .beta.ENaC-Tg C57BL:FVB mice after
treatment with inhaled SEQ ID NO:142 or SEQ ID NO:128.
[0025] FIGS. 7C and 7D show the results of an experiment analyzing
the effect on percent survival of .beta.ENaC-Tg C57BL:C3H mice
after treatment with SEQ ID NO:129 or SEQ ID NO:128.
[0026] FIG. 7E show the results of an experiment analyzing the
effect on percent survival (left panel) and weight gain (right
panel) of .beta.ENaC-Tg C57BL:C3H mice after treatment with a once
daily dose of SEQ ID NO:128.
[0027] FIG. 7F shows the results of an experiment analyzing the
effect on percent survival of .beta.ENaC-Tg C57BL:C3H mice after
treatment with SEQ ID NO:127, SEQ ID NO:134, or SEQ ID NO:136.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention will now be described in more detail
with reference to the accompanying drawings, in which preferred
embodiments of the invention are shown. This invention may,
however, be embodied in different forms and should not be construed
as limited to the embodiments set forth herein. Rather, these
embodiments are provided so that this disclosure will be thorough
and complete, and will fully convey the scope of the invention to
those skilled in the art.
[0029] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which this invention belongs. The terminology
used in the description of the invention herein is for the purpose
of describing particular embodiments only and is not intended to be
limiting of the invention. All publications, patent applications,
patents, patent publications and other references cited herein are
incorporated by reference in their entireties for the teachings
relevant to the sentence and/or paragraph in which the reference is
presented.
[0030] Amino acids are represented herein in the manner recommended
by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino
acids) by either the one-letter code, or the three letter code,
both in accordance with 37 C.F.R. .sctn.1.822 and established
usage.
[0031] As used in the description of the invention and the appended
claims, the singular forms "a," "an," and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
[0032] Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0033] The term "consists essentially of" (and grammatical
variants), as applied to a peptide sequence of this invention,
means a peptide that consists of both the recited sequence (e.g.,
SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10) additional amino acids on the N-terminal and/or
C-terminal ends of the recited sequence such that the function of
the peptide is not materially altered. The total of ten or less
additional amino acids includes the total number of additional
amino acids on both ends added together. The term "materially
altered," as applied to peptides of the invention, refers to an
increase or decrease in binding activity (e.g., to a sodium channel
or specialized non-naturally occurring peptide) of at least about
50% or more as compared to the activity of a peptide consisting of
the recited sequence.
[0034] The term "modulate," "modulates," or "modulation" refers to
enhancement (e.g., an increase) or inhibition (e.g., a decrease) in
the specified level or activity.
[0035] The term "enhance" or "increase" refers to an increase in
the specified parameter of at least about 1.25-fold, 1.5-fold,
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold,
twelve-fold, or even fifteen-fold.
[0036] The term "inhibit" or "reduce" or grammatical variations
thereof as used herein refers to a decrease or diminishment in the
specified level or activity of at least about 15%, 25%, 35%, 40%,
50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments,
the inhibition or reduction results in little or essentially no
detectible activity (at most, an insignificant amount, e.g., less
than about 10% or even 5%).
[0037] The term "contact" or grammatical variations thereof as used
with respect to a specialized non-naturally occurring peptide and a
sodium channel, refers to bringing the specialized non-naturally
occurring peptide and the sodium channel in sufficiently close
proximity to each other for one to exert a biological effect on the
other. In some embodiments, the term contact means binding of the
specialized non-naturally occurring peptide to the sodium
channel.
[0038] A "therapeutically effective" amount as used herein is an
amount that provides some improvement or benefit to the subject.
Alternatively stated, a "therapeutically effective" amount is an
amount that will provide some alleviation, mitigation, or decrease
in at least one clinical symptom in the subject. Those skilled in
the art will appreciate that the therapeutic effects need not be
complete or curative, as long as some benefit is provided to the
subject.
[0039] By the terms "treat," "treating," or "treatment of," it is
intended that the severity of the subject's condition is reduced or
at least partially improved or modified and that some alleviation,
mitigation or decrease in at least one clinical symptom is
achieved.
[0040] The term "fragment," as applied to a peptide, will be
understood to mean an amino acid sequence of reduced length
relative to a reference peptide or amino acid sequence and
comprising, consisting essentially of, and/or consisting of an
amino acid sequence of contiguous amino acids identical to the
reference peptide or amino acid sequence. Such a peptide fragment
according to the invention may be, where appropriate, included in a
larger polypeptide of which it is a constituent. In some
embodiments, such fragments can comprise, consist essentially of,
and/or consist of peptides having a length of at least about 4, 5,
6, 7, 8, 9, 10, or more consecutive amino acids of a peptide or
amino acid sequence according to the invention. In other
embodiments, such fragments can comprise, consist essentially of,
and/or consist of peptides having a length of less than about 10,
9, 8, 7, 6, 5, 4, or less consecutive amino acids of a peptide or
amino acid sequence according to the invention.
[0041] As used herein, the terms "protein" and "polypeptide" are
used interchangeably and encompass both peptides and proteins,
unless indicated otherwise.
[0042] A "fusion protein" is a polypeptide produced when two
heterologous nucleotide sequences or fragments thereof coding for
two (or more) different polypeptides not found fused together in
nature are fused together in the correct translational reading
frame. Illustrative fusion polypeptides include fusions of a
peptide of the invention (or a fragment thereof) to all or a
portion of glutathione-S-transferase, maltose-binding protein, or a
reporter protein (e.g., Green Fluorescent Protein,
.beta.-glucuronidase, .beta.-galactosidase, luciferase, etc.),
hemagglutinin, c-myc, FLAG epitope, etc.
[0043] As used herein, a "functional" peptide or "functional
fragment" is one that substantially retains at least one biological
activity normally associated with that peptide (e.g., binding to or
inhibiting a sodium channel). In particular embodiments, the
"functional" peptide or "functional fragment" substantially retains
all of the activities possessed by the unmodified peptide. By
"substantially retains" biological activity, it is meant that the
peptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%,
90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the
native polypeptide (and can even have a higher level of activity
than the native peptide). A "non-functional" peptide is one that
exhibits little or essentially no detectable biological activity
normally associated with the peptide (e.g., at most, only an
insignificant amount, e.g., less than about 10% or even 5%).
Biological activities such as protein binding and sodium channel
inhibitory activity can be measured using assays that are well
known in the art and as described herein.
[0044] The term "about," as used herein when referring to a
measurable value such as an amount of polypeptide, dose, time,
temperature, enzymatic activity or other biological activity and
the like, is meant to encompass variations of .+-.20%, .+-.10%,
.+-.5%, .+-.1%, .+-.0.5%, or even .+-.0.1% of the specified
amount.
[0045] A first aspect of the invention relates to the ability of
specialized non-naturally occurring peptides to bind to a sodium
channel and prevent activation of the sodium channel, thereby
inhibiting the flow of sodium ions. Thus, one aspect of the present
invention relates to a method of inhibiting the activation of a
sodium channel, comprising contacting (e.g., binding) a sodium
channel with a specialized non-naturally occurring peptide or a
functional fragment thereof. In one embodiment, the sodium channel
is an epithelial sodium channel (ENaC), e.g., human ENaC, or a
non-human mammalian ENaC. In another embodiment, the sodium channel
is one that is similar in sequence and/or structure to ENaC, such
as acid-sensing ion channels (ASIC). The inhibition of sodium
channel activation can be measured by any method known in the art
or disclosed herein, including, without limitation, measuring
sodium flow or change in potential across a membrane, across a
cell, or across a natural or artificial lining. The inhibition can
be at least about 20%, e.g., at least about 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 100%.
[0046] The method of inhibiting the activation of a sodium channel
can be carried out, e.g., on an isolated sodium channel, a sodium
channel in an artificial membrane, or a sodium channel in a cell.
In one embodiment, the sodium channel is present in an isolated
cell, e.g., a cultured primary cell or cell line. In another
embodiment, the isolated cell is part of an epithelial cell
culture, e.g., a natural or artificial epithelial lining, e.g., a
cell culture in a device (such as an Ussing chamber) in which
characteristics such as ion flow and/or potential can be measured
across lining. In another embodiment, the cell is part of an
isolated tissue or a tissue culture. In a further embodiment, the
cell can be present in an animal, e.g., an animal that is a disease
model or a subject in need of treatment.
[0047] In one embodiment, the step of contacting (e.g., binding)
the sodium channel with a specialized non-naturally occurring
peptide comprises delivering the specialized non-naturally
occurring peptide or a functional fragment or homolog thereof to a
cell comprising the sodium channel.
[0048] As used herein, the term "homolog" is used to refer to a
polypeptide which differs from a the disclosed specialized
non-naturally occurring peptide by modifications to the specialized
non-naturally occurring peptide, but which significantly retains a
biological activity of the disclosed non-naturally occurring
peptide. Minor modifications include, without limitation, changes
in one or a few amino acid side chains, changes to one or a few
amino acids (including deletions, insertions, and substitutions),
changes in stereochemistry of one or a few atoms, and minor
derivatizations, including, without limitation, methylation,
glycosylation, phosphorylation, acetylation, myristoylation,
prenylation, palmitoylation, amidation, and addition of
glycosylphosphatidyl inositol. The term "substantially retains," as
used herein, refers to a fragment, homolog, or other variant of a
peptide that retains at least about 20% of the activity of the
naturally occurring peptide (e.g., binding to a sodium channel),
e.g., about 30%, 40%, 50% or more. Other biological activities,
depending on the peptide, may include enzyme activity, receptor
binding, ligand binding, induction of a growth factor, a cell
signal transduction event, etc.
[0049] In one embodiment, the method comprises delivering to a cell
comprising a sodium channel an isolated specialized non-naturally
occurring peptide. In exemplary embodiments, the specialized
non-naturally occurring peptide comprises, consists essentially of,
or consists of the disclosed specialized non-naturally occurring
peptide or a functional fragment thereof. In another embodiment,
the isolated specialized non-naturally occurring peptide comprises,
consists essentially of, or consists of an amino acid sequence that
is at least 70% identical, e.g., at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to the publicly known amino acid
sequence or a functional fragment thereof. In some embodiments, the
peptides comprise a portion of the natural amino acid sequence of a
PLUNC protein with one or more conservative substitutions with
natural or non-natural amino acids and/or one or more additions of
non-natural amino acids. Conservative substitutions are described
below. In some embodiments, the peptides comprise one or more
terminal modifications as described below.
[0050] Non-limiting examples of peptides of the invention are
disclosed in Table 1 below. In some embodiments, the peptides of
the invention may comprise one or more additional residues at the
amino- and/or carboxyl-terminal ends. In some embodiments, the one
or more additional residues are D-alanines. For example, a peptide
may comprise one or two D-alanines at the amino- and/or
carboxyl-terminal ends.
[0051] The specialized non-naturally occurring peptides of the
invention also include functional portions or fragments. The length
of the fragment is not critical as long as it substantially retains
the biological activity of the peptide (e.g., sodium channel
binding activity). Illustrative fragments comprise at least about
4, 5, 6, 7, 8, 9, 10, or more contiguous amino acids of a
specialized non-naturally occurring peptide. In other embodiments,
the fragment comprises no more than about 10, 9, 8, 7, 6, 5, or 4
contiguous amino acids of a specialized non-naturally occurring
peptide.
[0052] Likewise, those skilled in the art will appreciate that the
present invention also encompasses fusion polypeptides comprising a
specialized non-naturally occurring peptides peptide or a
functional fragment thereof. As another alternative, the fusion
protein can comprise a reporter molecule. In other embodiments, the
fusion protein can comprise a polypeptide that provides a function
or activity that is the same as or different from the activity of
the peptide, e.g., a targeting, binding, or enzymatic activity or
function.
[0053] Likewise, it will be understood that the peptides
specifically disclosed herein will typically tolerate substitutions
in the amino acid sequence and substantially retain biological
activity. To identify peptides of the invention other than those
specifically disclosed herein, amino acid substitutions may be
based on any characteristic known in the art, including the
relative similarity or differences of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like.
TABLE-US-00001 TABLE 1 SEQ ID No N-term 1 NH3 G G L P V P L D Q T L
P L N V N P A NH2 138 NH3 G G L P I P L D Q T L P L N V N P A NH2
119 NH3 L P V P L D Q T L P L N V N P NH2 120 NH3 L P V P L D Q T
NH2 121 NH3 Nle P V P L D Q T NH2 122 NH3 L P Nle P L D Q T NH2 142
NH3 a a L P Nle P L D Q T a a NH2 135 NH3 a L P Nle P L D Q T a NH2
123 NH3 L P V P Nle D Q T NH2 124 NH3 L P V P L D N T NH2 125 NH3 L
P V P L E Q T NH2 126 NH3 L P V P L D Q S NH2 127 NH3 L P I P L D Q
T NH2 128 NH3 a a L P I P L D Q T a a NH2 134 Ac L P I P L D Q T
NH2 10 Ac L P V P L D Q T NH2 11 Ac L DHP V P L D Q T NH2 136 NH3 L
DHP V P L D Q T NH2 12 NH3 L P V DHP L D Q T NH2 13 NH3 L DHP V DHP
L D Q T NH2 14 NH3 L 2PP V P L D Q T NH2 15 NH3 L P V 2PP L D Q T
NH2 16 NH3 L HYP V P L D Q T NH2 17 NH3 L P V HYP L D Q T NH2 18
NH3 L HYP V HYP L D Q T NH2 19 NH3 L P V P L E Q S NH2 20 NH3 L P V
P L E Q MS NH2 21 NH3 L P V P V D Q T NH2 22 NH3 L P V P V E Q S
NH2 23 NH3 L P V P V D Q S NH2 24 NH3 V P V P L D Q T NH2 25 NH3 L
P L P L D Q S NH2 26 NH3 V P L P L D Q S NH2 27 NH3 L P L P L D Q T
NH2 141 NH3 a a L P L P L D Q T a a NH2 28 NH3 V HYP L HYP V E Q S
NH2 29 NH3 V P L P V E Q S NH2 30 NH3 L P L P L E Q S NH2 31 NH3 L
P mV P L D Q S NH2 32 NH3 L P Nle P L D Q T NH2 33 NH3 L P Nle P V
E Q S NH2 34 NH3 L P Nle P V E Q S NH2 35 NH3 L HYP Nle HYP L E Q S
NH2 36 NH3 L HYP Nle HYP V E Q S NH2 37 Ac L P V DHP L D Q T NH2 38
Ac L DHP V DHP L D Q T NH2 39 Ac L 2PP V P L D Q T NH2 40 Ac L P V
2PP L D Q T NH2 41 Ac L HYP V P L D Q T NH2 42 Ac L P V HYP L D Q T
NH2 43 Ac L HYP V HYP L D Q T NH2 44 Ac L P V P L E Q S NH2 45 Ac L
P V P L E Q MS NH2 46 Ac L P V P V D Q T NH2 47 Ac L P V P V E Q S
NH2 48 Ac L P V P V D Q S NH2 49 Ac V P V P L D Q T NH2 50 Ac L P L
P L D Q S NH2 51 Ac V P L P L D Q S NH2 52 Ac L P L P L D Q T NH2
53 Ac V HYP L HYP V E Q S NH2 54 Ac V P L P V E Q S NH2 55 Ac L P L
P L E Q S NH2 56 Ac L P mV P L D Q S NH2 57 Ac L P Nle P L D Q T
NH2 58 Ac L P Nle P V E Q S NH2 59 Ac L P Nle P V E Q S NH2 60 Ac L
HYP Nle HYP L E Q S NH2 61 Ac L HYP Nle HYP V E Q S NH2 62 Ac L DHP
V P L D Q T NH2 63 NH3 L P V DHP L D Q T NH2 64 NH3 L DHP V DHP L D
Q T NH2 65 NH3 L 2PP V P L D Q T NH2 66 NH3 L P V 2PP L D Q T NH2
67 NH3 L HYP V P L D Q T NH2 68 NH3 L P V HYP L D Q T NH2 69 NH3 L
HYP V HYP L D Q T NH2 70 NH3 L P V P L E Q S NH2 71 NH3 L P V P L E
Q MS NH2 72 NH3 L P V P V D Q T NH2 73 NH3 L P V P V E Q S NH2 74
NH3 L P V P V D Q S NH2 75 NH3 V P V P L D Q T NH2 137 NH3 V P V P
L D Q S NH2 76 NH3 L P L P L D Q S NH2 77 NH3 V P L P L D Q S NH2
78 NH3 L P L P L D Q T NH2 79 NH3 V HYP L HYP V E Q S NH2 80 NH3 V
P L P V E Q S NH2 81 NH3 L P L P L R Q S NH2 82 NH3 L P mV P L D Q
S NH2 83 NH3 L P Nle P L D Q T NH2 84 NH3 L P Nle P V E Q S NH2 85
NH3 L P Nle P V E Q S NH2 86 NH3 L HYP Nle HYP L E Q S NH2 87 NH3 L
HYP Nle HYP V E Q S NH2 88 Ac L P V DHP L D Q T NH2 89 Ac L DHP V
DHP L D Q T NH2 90 Ac L 2PP V P L D Q T NH2 91 Ac L P V 2PP L D Q T
NH2 92 Ac L HYP V P L D Q T NH2 93 Ac L P V HYP L D Q T NH2 94 Ac L
HYP V HYP L D Q T NH2 95 Ac L P V P L E Q S NH2 96 Ac L P V P L E Q
MS NH2 97 Ac L P V P V D Q T NH2 98 Ac L P V P V E Q S NH2 99 Ac L
P V P V D Q S NH2 100 Ac V P V P L D Q T NH2 101 Ac L P L P L D Q S
NH2 102 Ac V P L P L D Q S NH2 103 Ac L P L P L D Q T NH2 104 Ac V
HYP L HYP V E Q S NH2 105 Ac V P L P V E Q S NH2 106 Ac L P L P L E
Q S NH2 107 Ac L P mV P L D Q S NH2 108 Ac L P Me P L D Q T NH2 109
Ac L P Nle P V E Q S NH2 110 Ac L P Nle P V E Q S NH2 111 Ac L HYP
Nle HYP L E Q S NH2 112 Ac L HYP Nle HYP V E Q S NH2 113 NH3 L P V
P L D Q T NH2 114 NH3 D Q T L P L N V N P NH2
115 NH3 L P V P L D Q T L P L NH2 129 NH3 L P Ahp P L D Q T NH2 130
NH3 L P F P L D Q T NH2 131 NH3 a a L HYP Nle P L D Q T a a NH2 132
NH3 a a L P Nle HYP L D Q T a a NH2 133 NH3 G G L P Nle P L D Q T L
P L N V N P A NH2 139 NH3 L HYP I P L D Q T NH2 140 NH3 G G L P I P
L D Q T L P L N V N P A NH2 144 NH3 a L P I P L E Q S a NH2 Where a
= D-alanine, Nle = Norleucine, HYP = 4-hydroxyproline, DHP =
3,4-dehydro-L-proline, Ahp = aminoheptanoic acid, 2PP = (2R,
5S)-5-phenyl-pyrrolidine-2-carboxylic acid, MS =
L-.alpha.-methylserine, and mV = N-methylvaline
[0054] In identifying amino acid sequences encoding peptides other
than those specifically disclosed herein, the hydropathic index of
amino acids may be considered. The importance of the hydropathic
amino acid index in conferring interactive biologic function on a
protein is generally understood in the art (see, Kyte and
Doolittle, J. Mol. Biol. 157:105 (1982); incorporated herein by
reference in its entirety). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like.
[0055] Each amino acid has been assigned a hydropathic index on the
basis of its hydrophobicity and charge characteristics (Kyte and
Doolittle, id), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0056] Accordingly, the hydropathic index of the amino acid (or
amino acid sequence) may be considered when modifying the peptides
specifically disclosed herein.
[0057] It is also understood in the art that the substitution of
amino acids can be made on the basis of hydrophilicity. U.S. Pat.
No. 4,554,101 (incorporated herein by reference in its entirety)
states that the greatest local average hydrophilicity of a protein,
as governed by the hydrophilicity of its adjacent amino acids,
correlates with a biological property of the protein.
[0058] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (.+-.3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.I); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0059] Thus, the hydrophilicity of the amino acid (or amino acid
sequence) may be considered when identifying additional peptides
beyond those specifically disclosed herein.
[0060] Peptides (and fragments thereof) of the invention include
peptides that have at least about 70%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or higher amino acid sequence identity with the
peptide sequences disclosed herein.
[0061] As is known in the art, a number of different programs can
be used to identify whether a polypeptide has sequence identity or
similarity to a known sequence. Sequence identity or similarity may
be determined using standard techniques known in the art,
including, but not limited to, the local sequence identity
algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),
by the sequence identity alignment algorithm of Needleman &
Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity
method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444
(1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Drive, Madison,
Wis.), the Best Fit sequence program described by Devereux et al.,
Nucl. Acid Res. 12:387 (1984), preferably using the default
settings, or by inspection.
[0062] An example of a useful algorithm is PILEUP. PILEUP creates a
multiple sequence alignment from a group of related sequences using
progressive, pairwise alignments. It can also plot a tree showing
the clustering relationships used to create the alignment. PILEUP
uses a simplification of the progressive alignment method of Feng
& Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar
to that described by Higgins & Sharp, CABIOS 5:151 (1989).
[0063] Another example of a useful algorithm is the BLAST
algorithm, described in Altschul et al., J. Mol. Biol. 215:403
(1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873
(1993). A particularly useful BLAST program is the WU-BLAST-2
program which was obtained from Altschul et al., Meth. Enzymol.,
266:460 (1996); blast.wustl/edu/blast/README.html. WU-BLAST-2 uses
several search parameters, which are preferably set to the default
values. The parameters are dynamic values and are established by
the program itself depending upon the composition of the particular
sequence and composition of the particular database against which
the sequence of interest is being searched; however, the values may
be adjusted to increase sensitivity.
[0064] An additional useful algorithm is gapped BLAST as reported
by Altschul et al., Nucleic Acids Res. 25:3389 (1997).
[0065] A percentage amino acid sequence identity value is
determined by the number of matching identical residues divided by
the total number of residues of the "longer" sequence in the
aligned region. The "longer" sequence is the one having the most
actual residues in the aligned region (gaps introduced by
WU-Blast-2 to maximize the alignment score are ignored).
[0066] The alignment may include the introduction of gaps in the
sequences to be aligned. In addition, for sequences which contain
either more or fewer amino acids than the peptides specifically
disclosed herein, it is understood that in one embodiment, the
percentage of sequence identity will be determined based on the
number of identical amino acids in relation to the total number of
amino acids. Thus, for example, sequence identity of sequences
shorter than a sequence specifically disclosed herein, will be
determined using the number of amino acids in the shorter sequence,
in one embodiment. In percent identity calculations relative weight
is not assigned to various manifestations of sequence variation,
such as insertions, deletions, substitutions, etc.
[0067] In one embodiment, only identities are scored positively
(+1) and all forms of sequence variation including gaps are
assigned a value of "0," which obviates the need for a weighted
scale or parameters as described below for sequence similarity
calculations. Percent sequence identity can be calculated, for
example, by dividing the number of matching identical residues by
the total number of residues of the "shorter" sequence in the
aligned region and multiplying by 100. The "longer" sequence is the
one having the most actual residues in the aligned region.
[0068] Peptides and fragments of the invention can be modified for
in vivo use by the addition, at the amino- and/or carboxyl-terminal
ends, of a blocking agent to facilitate survival of the relevant
polypeptide in vivo. This can be useful in those situations in
which the peptide termini tend to be degraded by proteases prior to
cellular uptake. Such blocking agents can include, without
limitation, additional related or unrelated peptide sequences that
can be attached to the amino and/or carboxyl terminal residues of
the peptide to be administered. This can be done either chemically
during the synthesis of the peptide or by recombinant DNA
technology by any suitable methods. For example, one or more
non-naturally occurring amino acids, such as D-alanine, can be
added to the termini. Alternatively, blocking agents such as
pyroglutamic acid or other molecules known in the art can be
attached to the amino and/or carboxyl terminal residues, or the
amino group at the amino terminus or carboxyl group at the carboxyl
terminus can be replaced with a different moiety. Additionally, the
peptide terminus can be modified, e.g., by acetylation of the
N-terminus and/or amidation of the C-terminus. Likewise, the
peptides can be covalently or noncovalently coupled to
pharmaceutically acceptable "carrier" proteins prior to
administration.
[0069] In one embodiment, the peptides or fragments thereof of the
invention are administered directly to a subject. Generally, the
compounds of the invention will be suspended in a
pharmaceutically-acceptable carrier (e.g., physiological saline)
and administered orally or by intravenous infusion, or administered
subcutaneously, intramuscularly, intrathecally, intraperitoneally,
intrarectally, intravaginally, intranasally, intragastrically,
intratracheally, or intrapulmonarily. In another embodiment, the
intratracheal or intrapulmonary delivery can be accomplished using
a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry
powder inhaler, or metered dose inhaler. They can be delivered
directly to the site of the disease or disorder, such as lungs,
kidney, or intestines. The dosage required depends on the choice of
the route of administration; the nature of the formulation; the
nature of the patient's illness; the subject's size, weight,
surface area, age, and sex; other drugs being administered; and the
judgment of the attending physician. Suitable dosages are in the
range of 0.01-100.0 .mu.g/kg. Wide variations in the needed dosage
are to be expected in view of the variety of peptides, fragments,
and homologs available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by i.v.
injection. Variations in these dosage levels can be adjusted using
standard empirical routines for optimization as is well understood
in the art. Administrations can be single or multiple (e.g., 2-,
3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold).
Encapsulation of the peptides, fragments, and homologs in a
suitable delivery vehicle (e.g., polymeric microparticles or
implantable devices) may increase the efficiency of delivery,
particularly for oral delivery.
[0070] According to certain embodiments, the peptides, or fragments
thereof can be targeted to specific cells or tissues in vivo.
Targeting delivery vehicles, including liposomes and targeted
systems are known in the art. For example, a liposome can be
directed to a particular target cell or tissue by using a targeting
agent, such as an antibody, soluble receptor or ligand,
incorporated with the liposome, to target a particular cell or
tissue to which the targeting molecule can bind. Targeting
liposomes are described, for example, in Ho et al., Biochemistry
25:5500 (1986); Ho et al., J. Biol. Chem. 262:13979 (1987); Ho et
al., J. Biol. Chem. 262:13973 (1987); and U.S. Pat. No. 4,957,735
to Huang et al., each of which is incorporated herein by reference
in its entirety).
[0071] Another aspect of the invention relates to a method of
inhibiting sodium absorption through a sodium channel, comprising
contacting (e.g., binding) the sodium channel with a specialized
non-naturally occurring peptide or fragment thereof. Inhibition of
sodium absorption can be measured by any technique known in the art
or disclosed herein.
[0072] Another aspect of the invention relates to a method of
increasing the volume of fluid lining an epithelial mucosal
surface, comprising contacting (e.g., binding) a sodium channel
present on the epithelial mucosal surface with a specialized
non-naturally occurring peptide or a functional fragment or homolog
thereof. The volume of fluid lining an epithelial mucosal surface
can be measured by any technique known in the art or disclosed
herein.
[0073] A further aspect of the invention relates to a method of
reducing the level of a sodium channel present on the surface of a
cell, comprising contacting (e.g., binding) the sodium channel with
a specialized non-naturally occurring peptide or a functional
fragment or homolog thereof.
[0074] An additional aspect of the invention relates to a method of
treating a disorder responsive to inhibition of sodium absorption
across an epithelial mucosal surface in a subject in need thereof,
comprising delivering to the subject a therapeutically effective
amount of a specialized non-naturally occurring peptide or a
functional fragment or homolog thereof. In one embodiment, the
invention encompasses a method for treating a symptom of a disorder
responsive to inhibition of sodium absorption across an epithelial
mucosal surface in a subject in need thereof, comprising
administering a peptide comprising a sequence selected from SEQ ID
NOS:2-128 to the subject. The disorder in the methods of the
invention can be, in non-limiting examples, a lung disorder (e.g.,
cystic fibrosis, non-cystic fibrosis bronchiectasis, chronic
obstructive pulmonary disease, acute or chronic bronchitis, or
asthma), a gastrointestinal disorder (e.g., inflammatory bowel
disease), a kidney disorder, or a cardiovascular disorder.
[0075] Another aspect of the invention relates to a method of
regulating salt balance, blood volume, and/or blood pressure in a
subject in need thereof, comprising delivering to the subject a
therapeutically effective amount of a specialized non-naturally
occurring peptide or a functional fragment or homolog thereof.
[0076] A third aspect of the invention relates to products that can
be used to carry out the methods disclosed herein. Thus, one aspect
of the invention relates to a peptide comprising the sequence:
[0077]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8
(SEQ ID NO:116) wherein: [0078] X.sub.1 is leucine or a
conservative substitution with a natural or non-natural amino acid;
[0079] X.sub.2 is proline or a conservative substitution with a
natural or non-natural amino acid; [0080] X.sub.3 is valine or a
conservative substitution with a natural or non-natural amino acid;
[0081] X.sub.4 is proline or a conservative substitution with a
natural or non-natural amino acid; [0082] X.sub.5 is leucine or a
conservative substitution with a natural or non-natural amino acid;
[0083] X.sub.6 is aspartic acid or a conservative substitution with
a natural or non-natural amino acid; [0084] X.sub.7 is glutamine or
a conservative substitution with a natural or non-natural amino
acid; and [0085] X.sub.8 is threonine or a conservative
substitution with a natural or non-natural amino acid; [0086] or a
functional fragment thereof.
[0087] Another aspect of the invention relates to a peptide
comprising the sequence: [0088]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8
(SEQ ID NO:117) wherein: [0089] X.sub.1 is leucine, norleucine, or
valine; [0090] X.sub.2 is proline, 4-hydroxyproline,
(2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid, or
3,4-dehydro-L-proline; [0091] X.sub.3 is valine, leucine,
norleucine, or N-methylvaline; [0092] X.sub.4 is proline,
4-hydroxyproline, (2R,5S)-5-phenyl-pyrrolidine-2-carboxylic acid,
or 3,4-dehydro-L-proline; [0093] X.sub.5 is leucine, norleucine, or
valine; [0094] X.sub.6 is aspartic acid or glutamic acid; [0095]
X.sub.7 is glutamine or asparagine; and [0096] X.sub.8 is
threonine, serine, or L-.alpha.-methylserine;or a functional
fragment thereof.
[0097] A further aspect of the invention relates to a peptide
comprising the sequence: [0098]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15(SEQ ID NO:118)
wherein: X.sub.1 is leucine or a conservative substitution with a
natural or non-natural amino acid; [0099] X.sub.2 is proline or a
conservative substitution with a natural or non-natural amino acid;
[0100] X.sub.3 is valine or a conservative substitution with a
natural or non-natural amino acid; [0101] X.sub.4 is proline or a
conservative substitution with a natural or non-natural amino acid;
[0102] X.sub.5 is leucine or a conservative substitution with a
natural or non-natural amino acid; [0103] X.sub.6 is aspartic acid
or a conservative substitution with a natural or non-natural amino
acid; [0104] X.sub.7 is glutamine or a conservative substitution
with a natural or non-natural amino acid; [0105] X.sub.8 is
threonine or a conservative substitution with a natural or
non-natural amino acid; [0106] X.sub.9 is threonine or a
conservative substitution with a natural or non-natural amino acid;
[0107] X.sub.10 is leucine or a conservative substitution with a
natural or non-natural amino acid; [0108] X.sub.11 is proline or a
conservative substitution with a natural or non-natural amino acid;
[0109] X.sub.12 is asparagine or a conservative substitution with a
natural or non-natural amino acid; [0110] X.sub.13 is valine or a
conservative substitution with a natural or non-natural amino acid;
[0111] X.sub.14 is asparagine or a conservative substitution with a
natural or non-natural amino acid; [0112] X.sub.15 is proline or a
conservative substitution with a natural or non-natural amino acid;
or a functional fragment thereof.
[0113] In some embodiments, the peptide comprises a sequence
selected from the group consisting of SEQ ID NOS:4-142 and SEQ ID
NO:144. In one embodiment, the peptide comprises the sequence of
SEQ ID NO:5 or SEQ ID NO:122. In one embodiment, the peptide
comprises the sequence of SEQ ID NO:2 (aaLPVPLDQTLPLNVNPaa) or SEQ
ID NO:119. In one embodiment, the peptide comprises the sequence of
SEQ ID NO:127 or SEQ ID NO:128 (aaLPIPLDQTaa).
[0114] In certain embodiments, the peptides of the invention
comprise at least one modified terminus, e.g., to protect the
peptide against degradation. In some embodiments, the N-terminus is
acetylated and/or the C-terminus is amidated. In some embodiments,
the peptide comprises the sequence of any one of SEQ ID NOS:10-127,
129, 130, 133, 134, or 136-140, further comprising one or two
D-alanines at the amino- and/or carboxyl-terminal ends.
[0115] In certain embodiments, the peptides of the invention
comprise at least one non-natural amino acid (e.g., 1, 2, 3, or
more) or at least one terminal modification (e.g., 1 or 2). In some
embodiments, the peptide comprises at least one non-natural amino
acid and at least one terminal modification.
[0116] In certain embodiments, the peptide mimics the sodium
channel binding domain of a PLUNC protein. The sodium channel
binding domain is the minimal fragment of the PLUNC protein
required to have substantially the same binding activity to the
sodium channel as the full length PLUNC protein. The term
"substantially the same binding activity" refers to an activity
that is at least about 50% of the binding activity of the full
length protein, e.g., at least about 60%, 70%, 80%, or 90% of the
binding activity. In some embodiments, the peptide has at least the
same binding activity as the full length PLUNC protein. In one
embodiment, the sodium channel is ENaC, e.g., human ENaC. In
another embodiment, the sodium channel is one that is similar in
sequence and/or structure to ENaC, such as acid-sensing ion
channels (ASIC).
[0117] The peptides of the present invention can optionally be
delivered in conjunction with other therapeutic agents. The
additional therapeutic agents can be delivered concurrently with
the peptides of the invention. As used herein, the word
"concurrently" means sufficiently close in time to produce a
combined effect (that is, concurrently can be simultaneously, or it
can be two or more events occurring within a short time period
before or after each other). In one embodiment of the invention,
the specialized non-naturally occurring peptide is delivered to a
patient concurrently with a compound that modulates the function of
the Cystic fibrosis transmembrane conductance regulator (CFTR)
where the combined activity of the specialized non-naturally
occurring peptide and the CFTR-targeted agent have superior
activity to the CFTR-targeted agent alone. In another embodiment of
the invention, the specialized non-naturally occurring peptide is
delivered to a patient concurrently with a mucolytic compound where
the combined activity of the specialized non-naturally occurring
peptide and the mucolytic agent have superior activity to the
mucolytic agent alone. In yet another embodiment of the invention,
the specialized non-naturally occurring peptide is delivered to a
patient concurrently with a long acting B-agonist compound (LABA)
where the combined activity of the specialized non-naturally
occurring peptide and the LABA agent have superior activity to the
LABA alone. In yet another embodiment of the invention, the
specialized non-naturally occurring peptide is delivered to a
patient concurrently with a glucocorticoid agonist where the
combined activity of the specialized non-naturally occurring
peptide and the glucocorticoid agent have superior activity to the
glucocorticoid alone.
[0118] Another aspect of the invention relates to a kit comprising
the peptide of the invention and useful for carrying out the
methods of the invention. The kit may further comprise additional
reagents for carrying out the methods (e.g., buffers, containers,
additional therapeutic agents) as well as instructions.
[0119] As a further aspect, the invention provides pharmaceutical
formulations and methods of administering the same to achieve any
of the therapeutic effects (e.g., modulation of sodium absorption)
discussed above. The pharmaceutical formulation may comprise any of
the reagents discussed above in a pharmaceutically acceptable
carrier, e.g., a specialized non-naturally occurring peptide or
functional fragment thereof.
[0120] By "pharmaceutically acceptable" it is meant a material that
is not biologically or otherwise undesirable, i.e., the material
can be administered to a subject without causing any undesirable
biological effects such as toxicity.
[0121] The formulations of the invention can optionally comprise
medicinal agents, pharmaceutical agents, carriers, adjuvants,
dispersing agents, diluents, and the like.
[0122] The peptides of the invention can be formulated for
administration in a pharmaceutical carrier in accordance with known
techniques. See, e.g., Remington, The Science And Practice of
Pharmacy (9.sup.th Ed. 1995). In the manufacture of a
pharmaceutical formulation according to the invention, the peptide
(including the physiologically acceptable salts thereof) is
typically admixed with, inter alia, an acceptable carrier. The
carrier can be a solid or a liquid, or both, and is preferably
formulated with the peptide as a unit-dose formulation, for
example, a tablet, which can contain from 0.01 or 0.5% to 95% or
99% by weight of the peptide. One or more peptides can be
incorporated in the formulations of the invention, which can be
prepared by any of the well-known techniques of pharmacy.
[0123] A further aspect of the invention is a method of treating
subjects in vivo, comprising administering to a subject a
pharmaceutical composition comprising a peptide of the invention in
a pharmaceutically acceptable carrier, wherein the pharmaceutical
composition is administered in a therapeutically effective amount.
Administration of the peptides of the present invention to a human
subject or an animal in need thereof can be by any means known in
the art for administering compounds.
[0124] The formulations of the invention include those suitable for
oral, rectal, topical, buccal (e.g., sub-lingual), vaginal,
parenteral (e.g., subcutaneous, intramuscular including skeletal
muscle, cardiac muscle, diaphragm muscle and smooth muscle,
intradermal, intravenous, intraperitoneal), topical (i.e., both
skin and mucosal surfaces, including airway surfaces), intranasal,
transdermal, intraarticular, intrathecal, and inhalation
administration, administration to the liver by intraportal
delivery, as well as direct organ injection (e.g., into the liver,
into the brain for delivery to the central nervous system, into the
pancreas, or into a tumor or the tissue surrounding a tumor). The
most suitable route in any given case will depend on the nature and
severity of the condition being treated and on the nature of the
particular peptide which is being used.
[0125] For injection, the carrier will typically be a liquid, such
as sterile pyrogen-free water, sterile normal saline, hypertonic
saline, pyrogen-free phosphate-buffered saline solution,
bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.).
For other methods of administration, the carrier can be either
solid or liquid.
[0126] For oral administration, the peptide can be administered in
solid dosage forms, such as capsules, tablets, and powders, or in
liquid dosage forms, such as elixirs, syrups, and suspensions.
Peptides can be encapsulated in gelatin capsules together with
inactive ingredients and powdered carriers, such as glucose,
lactose, sucrose, mannitol, starch, cellulose or cellulose
derivatives, magnesium stearate, stearic acid, sodium saccharin,
talcum, magnesium carbonate and the like. Examples of additional
inactive ingredients that can be added to provide desirable color,
taste, stability, buffering capacity, dispersion or other known
desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium dioxide, edible white ink and the like. Similar
diluents can be used to make compressed tablets. Both tablets and
capsules can be manufactured as sustained release products to
provide for continuous release of medication over a period of
hours. Compressed tablets can be sugar coated or film coated to
mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric-coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient
acceptance.
[0127] Formulations suitable for buccal (sub-lingual)
administration include lozenges comprising the compound in a
flavored base, usually sucrose and acacia or tragacanth; and
pastilles comprising the compound in an inert base such as gelatin
and glycerin or sucrose and acacia.
[0128] Formulations of the present invention suitable for
parenteral administration comprise sterile aqueous and non-aqueous
injection solutions of the peptide, which preparations are
preferably isotonic with the blood of the intended recipient. These
preparations can contain anti-oxidants, buffers, bacteriostats and
solutes which render the formulation isotonic with the blood of the
intended recipient. Aqueous and non-aqueous sterile suspensions can
include suspending agents and thickening agents. The formulations
can be presented in unit/dose or multi-dose containers, for example
sealed ampoules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or water-for-injection
immediately prior to use.
[0129] Extemporaneous injection solutions and suspensions can be
prepared from sterile powders, granules and tablets of the kind
previously described. For example, in one aspect of the present
invention, there is provided an injectable, stable, sterile
composition comprising a peptide of the invention, in a unit dosage
form in a sealed container. The peptide or salt is provided in the
form of a lyophilizate which is capable of being reconstituted with
a suitable pharmaceutically acceptable carrier to form a liquid
composition suitable for injection thereof into a subject. The unit
dosage form typically comprises from about 1 mg to about 10 grams
of the peptide or salt. When the peptide or salt is substantially
water-insoluble, a sufficient amount of emulsifying agent which is
pharmaceutically acceptable can be employed in sufficient quantity
to emulsify the peptide or salt in an aqueous carrier. One such
useful emulsifying agent is phosphatidyl choline.
[0130] Formulations suitable for rectal administration are
preferably presented as unit dose suppositories. These can be
prepared by admixing the peptide with one or more conventional
solid carriers, for example, cocoa butter, and then shaping the
resulting mixture.
[0131] Formulations suitable for topical application to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which can be used include
petroleum jelly, lanoline, polyethylene glycols, alcohols,
transdermal enhancers, and combinations of two or more thereof.
[0132] Formulations suitable for transdermal administration can be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Formulations suitable for transdermal administration can also be
delivered by iontophoresis (see, for example, Tyle, Pharm. Res.
3:318 (1986)) and typically take the form of an optionally buffered
aqueous solution of the peptides. Suitable formulations comprise
citrate or bis/tris buffer (pH 6) or ethanol/water and contain from
0.1 to 0.2M of the compound.
[0133] The peptide can alternatively be formulated for nasal
administration or otherwise administered to the lungs of a subject
by any suitable means, e.g., administered by an aerosol suspension
of respirable particles comprising the peptide, which the subject
inhales. The respirable particles can be liquid or solid. The term
"aerosol" includes any gas-borne suspended phase, which is capable
of being inhaled into the bronchioles or nasal passages.
Specifically, aerosol includes a gas-borne suspension of droplets,
as can be produced in a metered dose inhaler or nebulizer, or in a
mist sprayer. Aerosol also includes a dry powder composition
suspended in air or other carrier gas, which can be delivered by
insufflation from an inhaler device, for example. See Ganderton
& Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood
(1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier
Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth.
27:143 (1992). Aerosols of liquid particles comprising the peptide
can be produced by any suitable means, such as with a
pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is
known to those of skill in the art. See, e.g., U.S. Pat. No.
4,501,729. Aerosols of solid particles comprising the peptide can
likewise be produced with any solid particulate medicament aerosol
generator, by techniques known in the pharmaceutical art.
[0134] Alternatively, one can administer the peptide in a local
rather than systemic manner, for example, in a depot or
sustained-release formulation.
[0135] Further, the present invention provides liposomal
formulations of the peptides disclosed herein and salts thereof.
The technology for forming liposomal suspensions is well known in
the art. When the peptide or salt thereof is an aqueous-soluble
salt, using conventional liposome technology, the same can be
incorporated into lipid vesicles. In such an instance, due to the
water solubility of the peptide or salt, the peptide or salt will
be substantially entrained within the hydrophilic center or core of
the liposomes. The lipid layer employed can be of any conventional
composition and can either contain cholesterol or can be
cholesterol-free. When the peptide or salt of interest is
water-insoluble, again employing conventional liposome formation
technology, the salt can be substantially entrained within the
hydrophobic lipid bilayer which forms the structure of the
liposome. In either instance, the liposomes which are produced can
be reduced in size, as through the use of standard sonication and
homogenization techniques.
[0136] The liposomal formulations containing the peptides disclosed
herein or salts thereof, can be lyophilized to produce a
lyophilizate which can be reconstituted with a pharmaceutically
acceptable carrier, such as water, to regenerate a liposomal
suspension.
[0137] In the case of water-insoluble peptides, a pharmaceutical
composition can be prepared containing the water-insoluble peptide,
such as for example, in an aqueous base emulsion. In such an
instance, the composition will contain a sufficient amount of
pharmaceutically acceptable emulsifying agent to emulsify the
desired amount of the peptide. Particularly useful emulsifying
agents include phosphatidyl cholines and lecithin.
[0138] In particular embodiments, the peptide is administered to
the subject in a therapeutically effective amount, as that term is
defined above. Dosages of pharmaceutically active peptides can be
determined by methods known in the art, see, e.g., Remington's
Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). The
therapeutically effective dosage of any specific peptide will vary
somewhat from peptide to peptide, and patient to patient, and will
depend upon the condition of the patient and the route of delivery.
As a general proposition, a dosage from about 0.1 to about 50 mg/kg
will have therapeutic efficacy, with all weights being calculated
based upon the weight of the peptide, including the cases where a
salt is employed. Toxicity concerns at the higher level can
restrict intravenous dosages to a lower level such as up to about
10 mg/kg, with all weights being calculated based upon the weight
of the peptide, including the cases where a salt is employed. A
dosage from about 10 mg/kg to about 50 mg/kg can be employed for
oral administration. Typically, a dosage from about 0.5 mg/kg to 5
mg/kg can be employed for intramuscular injection. Particular
dosages are about 1 .mu.mol/kg to 50 .mu.mol/kg, and more
particularly to about 22 .mu.mol/kg and to 33 .mu.mol/kg of the
peptide for intravenous or oral administration, respectively.
[0139] In particular embodiments of the invention, more than one
administration (e.g., two, three, four, or more administrations)
can be employed over a variety of time intervals (e.g., hourly,
daily, weekly, monthly, etc.) to achieve therapeutic effects.
[0140] The present invention finds use in veterinary and medical
applications. Suitable subjects include both avians and mammals,
with mammals being preferred. The term "avian" as used herein
includes, but is not limited to, chickens, ducks, geese, quail,
turkeys, and pheasants. The term "mammal" as used herein includes,
but is not limited to, humans, bovines, ovines, caprines, equines,
felines, canines, lagomorphs, etc. Human subjects include neonates,
infants, juveniles, and adults.
[0141] The present invention is more particularly described in the
following examples that are intended as illustrative only since
numerous modifications and variations therein will be apparent to
those skilled in the art.
EXAMPLE 1
Experimental Methods
[0142] Tissue procurement and cell culture: Cells were harvested by
enzymatic digestion from human bronchial tissue as previously
described under a protocol approved by the UNC School of Medicine
IRB (Tarran et al., J. Gen. Physiol. 127:591 (2006)). Human excess
donor lungs and excised recipient lungs were obtained at the time
of lung transplantation from portions of main stem or lumbar
bronchi and cells were harvested by enzymatic digestion. All
preparations were maintained at an air-liquid interface in a
modified bronchial epithelial medium and used 2-5 weeks after
seeding on 12 mm T-Clear inserts (Corning Costar) coated with human
placental type VI collagen (Sigma). Phosphate buffered saline (PBS)
was used for washing human bronchial epithelial culture mucosal
surfaces.
[0143] Confocal microscopy: To label airway surface liquid, Ringer
containing Texas Red-dextran (2 mg/ml; Invitrogen) was added to
human bronchial epithelial culture mucosal surfaces.
Perfluorocarbon was added mucosally to prevent evaporation of the
airway surface liquid and the culture placed in a chamber
containing 100 .mu.l Ringer on the stage of a Leica SP8 confocal
microscope with a 63.times. glycerol immersion objective. 10 points
per culture were scanned and an average airway surface liquid
height determined. For confocal microscopy human bronchial
epithelial cultures were bathed serosally in a modified Ringer
solution containing (mM): 116 NaCl, 10 NaHCO.sub.3, 5.1 KCl, 1.2
CaCl.sub.2, 1.2 MgCl.sub.2, 20 TES, 10 glucose, pH 7.4). At all
other times, human bronchial epithelial cultures were maintained in
a modified BEGM growth medium which contained 24 mM NaHCO.sub.3
gassed with 5% CO.sub.2. Perfluorocarbon (FC-77) was obtained from
3M and had no effect on ASL height as previously reported.
[0144] Internalization of alpha-ENaC: HEK293T cells were
transfected with gfp-.alpha.ENaC where gfp was fused at the
N-terminus of ENaC and unlabeled .beta.ENaC and .gamma.ENaC using
lipofectamine in 384 well plates as per the manufacturer's
instructions and as published previously (see, e.g., Hobbs et al.
Am J Physiol Lung Cell Mol Physiol. 2013 December;
305(12):L990-L1001. doi: 10.1152/ajplung.00103.2013. Epub 2013
October; Garland et al. Proc Natl Acad Sci USA. 2013 Oct. 1;
110(40):15973-8. doi: 10.1073/pnas.1311999110. Epub 2013 Sep. 16;
Tan et al. J Physiol. 2014 Dec. 1; 592(Pt 23):5251-68). Twenty-four
hours later, peptide or vehicle control were added at t=0 and
fluorescence was read using a Tecan M1000 plate reader 3 h later.
Fluorescence was read at 488 and 510 nm.
[0145] FIG. 6 shows the results of an experiment analyzing the
effects of various peptides on internalization of alpha-ENaC in
HEK293T cells when CFP-tagged alpha-ENaC is co-expressed only with
gamma ENaC and no beta-ENaC. In this figure, SEQ ID NO:143
(negative control peptide) and water (vehicle) controls are used
along with S18 (SEQ ID NO:1, and SEQ ID NO:128. While SEQ ID NO:1
AND SEQ ID NO:128 are effective in reducing alpha-ENaC when
beta-ENaC is co-expressed, no effect is observed in this experiment
when beta-ENaC is not present.
[0146] Efficacy testing in .beta.ENaC mice: Like CF patients,
.beta.ENaC-Tg mice are disease free at birth, but soon develop
obstructive lung disease (Zhou, Z., et al. Am J Respir Crit Care
Med 178, 1245-1256 (2008)). C57BL:FVB and C57BL:C3H mixed strains
have 90% and 50% mortality, respectively, and the reproducibility
of the disease on these backgrounds is sufficiently high that
reliable data are produced with n of about 8-10 animals/group.
Furthermore, the .beta.ENaC-Tg mouse responds to therapeutic
interventions in a fashion similar to CF/COPD in humans, and the
development of lung disease can be prevented following inhibition
of lung disease at birth (Livraghi, A., et al. J Immunol 182,
4357-4367 (2009)). S18-derived peptides or vehicle were dosed one
to three times a day by intranasal instillation (1 .mu.l/g body
weight) for 14 days in parallel cohorts of mice. Mice were weighed
daily and, if a diuretic effect was found, the volume excreted was
replaced by sub-cutaneous injections of sterile saline. At the end
of the treatment, mice were sacrificed for phenotypic analysis.
[0147] Statistical analyses: All data are presented as the
mean.+-.SE for n experiments. Airway cultures derived from three or
more separate donors were used for each study and each oocyte study
was repeated on three separate occasions. Differences between means
were tested for statistical significance using paired or unpaired t
tests or their non parametric equivalent as appropriate to the
experiment. From such comparisons, differences yielding
P.ltoreq.0.05 were judged to be significant. All binding assays
were fitted to the Hill equation.
EXAMPLE 2
Identification of Specialized Non-Naturally Occurring Peptides
[0148] Based on the ability of normal human bronchial epithelial
cultures to regulate airway surface liquid height to 7 .mu.m, which
was paralleled by a decrease in trypsin-sensitive ENaC activity, we
previously demonstrated that SPLUNC1 derived peptides (namely, S18;
FIG. 1) can inhibit ENaC with EC.sub.50 in the sub micromolar range
for up to 24 h following a single dose (Hobbs et al., 2013). While
S18 is resistant to proteolysis and heat-stable, we tested whether
we could reduce the size of S18, increase its stability and/or
increase its potency. Any of these actions would increase its
utility as a drug.
[0149] To confirm our alanine-scan data, we made a peptide of
subsequence A (LPVPLDQT (SEQ ID NO:113); FIG. 1). As a control, we
made an overlapping 2nd peptide, which contained the charged
mid-region (DQT), as well as subsequence B (DQTLPLNVNP (SEQ ID
NO:114)). As shown in FIG. 2, LPVPLDQT (SEQ ID NO:113) retained
similar activity to S18, whilst DQTLPLNVNP (SEQ ID NO:114) was
inactive. However, additional experiments, where we examined
susceptibility to proteases, demonstrated that whilst LPVPLDQT (SEQ
ID NO:113) could inhibit ENaC-led fluid absorption, this peptide
was more susceptible to proteolytic degradation. Thus, we generated
new peptides where we flanked the C- and N-termini with a pair of
D-alanines (shown as lower case `a`). Peptides were added mucosally
to HBECs at 30 .mu.M and ASL height measured 3 h later by
XZ-confocal microscopy. FIG. 2A shows the comparison of the
N-terminal region of S18 (LPVPLDQT) (SEQ ID NO:113) vs. an
overlapping, but C-terminal segment (DQTLPLNVNP) (SEQ ID NO:114).
In FIG. 2B, D-alanines were added to Sub-S18 peptides LPVPLDQT (SEQ
ID NO:113) and LPVPLDQTLPL (SEQ ID NO:115) to increase the
stability of LPVPLDQT (SEQ ID NO:113). D-alanines are noted as
lowercase a. All n=6-9. Data are shown as mean.+-.SEM.
[0150] The 15-mer and 8-mer showed identical activity when compared
against S18 (FIGS. 2A-2B). We then generated full dose-responses
for S18 vs. the peptide aaLPVPLDQTaa (SEQ ID NO:3). S18 and
aaLPVPLDQTaa (SEQ ID NO:3) produced identical dose responses (FIG.
3A), indicating that shortening the peptide and flanking it with
unnatural amino acids did not affect its potency or efficacy. Next,
we made several new peptides based on the sequence of SEQ ID NO:2.
Here, since prolines typically provide kinks in a peptide, they
were not altered and instead, we systematically made conservative
substitutions of the other amino acids. The dose responses are
shown in FIG. 3B. Note, since there are several lines on this
graph, the error bars and data symbols were omitted. However, these
data were obtained simultaneously using paired airway cultures, so
their comparison is valid. Clearly, SEQ ID NO:5 (aaLPNlePLDQTaa)
shows a log-fold increase in potency as compared to S18, whilst SEQ
ID NO:9 (aaLPVPLDQSaa) and SEQ ID NO:6 (aaLPVPNleDQTaa) show
diminished potency and efficacy respectively (FIG. 3B). Other
sequences included in FIG. 3B are SEQ ID NO:4 (aaNlePVPLDQTaa), SEQ
ID NO:7 (naLPVPLDNTaa) and SEQ ID NO:8 (aaLPVPLEQTaa).
[0151] Using a fresh batch of airway cells, we performed an
additional dose response to SEQ ID NO:5 (FIG. 4). As can be seen,
this peptide gave a significant increase in potency, as compared to
S18, and the IC.sub.50 was 30 nM.
[0152] The ability of the peptides to modulate internalization of
.alpha.ENaC was tested. Results from these experiments are shown in
FIGS. 5A-5U. ADG is a negative control peptide with the sequence:
NH3-ADGGLLLLNNPPPPQTVV-NH2 (SEQ ID NO:143). The peptides used in
these experiments were S18 (SEQ ID NO:1), SEQ ID NO:2, SEQ ID
NO:141, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:128, SEQ ID NO:131,
SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID
NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:131, SEQ ID NO:139;
SEQ ID NO:140, SEQ ID NO:144 and SEQ ID NO:127. Also used in these
experiments were peptides of SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:136, each with an
aa cap at both the N- and the C-terminus. Also shown is amiloride,
which inhibits ENaC by another mechanism and does not reduce the
amount of functional receptor on the surface of cells. These
results demonstrate that the SPLUNC peptides trigger the
destruction of the ENaC function to inhibit its activity.
[0153] The efficacy of the peptides was tested in .beta.ENaC mice.
Results from these experiments are shown in FIGS. 7A-7F.
Fourteen-day treatment with inhaled SEQ ID NO:128 was more
effective than inhaled SEQ ID NO:142 at increasing percent survival
of .beta.ENaC-Tg C57BL:FVB mice (FIG. 7B). Fourteen-day treatment
with SEQ ID NO:128 also increased percent survival of .beta.ENaC-Tg
C57BL:C3H mice (FIGS. 7C and 7D). Once daily dosing with SEQ ID
NO:128 increased percent survival of .beta.ENaC-Tg C57BL:C3H mice
(FIG. 7E, left panel). The right panel of FIG. 7E shows that SEQ ID
NO:128 did not have a diuretic effect. FIG. 7F shows that treatment
with SEQ ID NO:127 increased percent survival of ENaC-Tg C57BL:C3H
mice. Sequences of other peptides used in the experiment were SEQ
ID NO:129, SEQ ID NO:134, and SEQ ID NO:136.
[0154] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
Sequence CWU 1
1
144118PRTArtificialSodium channel activity inhibiting peptide
sequence 1Gly Gly Leu Pro Val Pro Leu Asp Gln Thr Leu Pro Leu Asn
Val Asn 1 5 10 15 Pro Ala 219PRTArtificialSodium channel activity
inhibiting peptide sequence 2Xaa Xaa Leu Pro Val Pro Leu Asp Gln
Thr Leu Pro Leu Asn Val Asn 1 5 10 15 Pro Xaa Xaa
312PRTArtificialSodium channel activity inhibiting peptide sequence
3Xaa Xaa Leu Pro Val Pro Leu Asp Gln Thr Xaa Xaa 1 5 10
412PRTArtificialSodium channel activity inhibiting peptide sequence
4Xaa Xaa Xaa Pro Val Pro Leu Asp Gln Thr Xaa Xaa 1 5 10
512PRTArtificialSodium channel activity inhibiting peptide sequence
5Xaa Xaa Leu Pro Xaa Pro Leu Asp Gln Thr Xaa Xaa 1 5 10
612PRTArtificialSodium channel activity inhibiting peptide sequence
6Xaa Xaa Leu Pro Val Pro Xaa Asp Gln Thr Xaa Xaa 1 5 10
712PRTArtificialSodium channel activity inhibiting peptide sequence
7Xaa Xaa Leu Pro Val Pro Leu Asp Asn Thr Xaa Xaa 1 5 10
812PRTArtificialSodium channel activity inhibiting peptide sequence
8Xaa Xaa Leu Pro Val Pro Leu Glu Gln Thr Xaa Xaa 1 5 10
912PRTArtificialSodium channel activity inhibiting peptide sequence
9Xaa Xaa Leu Pro Val Pro Leu Asp Gln Ser Xaa Xaa 1 5 10
108PRTArtificialSodium channel activity inhibiting peptide sequence
10Leu Pro Val Pro Leu Asp Gln Thr 1 5 118PRTArtificialSodium
channel activity inhibiting peptide sequence 11Leu Xaa Val Pro Leu
Asp Gln Thr 1 5 128PRTArtificialSodium channel activity inhibiting
peptide sequence 12Leu Pro Val Xaa Leu Asp Gln Thr 1 5
138PRTArtificialSodium channel activity inhibiting peptide sequence
13Leu Xaa Val Xaa Leu Asp Gln Thr 1 5 148PRTArtificialSodium
channel activity inhibiting peptide sequence 14Leu Xaa Val Pro Leu
Asp Gln Thr 1 5 158PRTArtificialSodium channel activity inhibiting
peptide sequence 15Leu Pro Val Xaa Leu Asp Gln Thr 1 5
168PRTArtificialSodium channel activity inhibiting peptide sequence
16Leu Xaa Val Pro Leu Asp Gln Thr 1 5 178PRTArtificialSodium
channel activity inhibiting peptide sequence 17Leu Pro Val Xaa Leu
Asp Gln Thr 1 5 188PRTArtificialSodium channel activity inhibiting
peptide sequence 18Leu Xaa Val Xaa Leu Asp Gln Thr 1 5
198PRTArtificialSodium channel activity inhibiting peptide sequence
19Leu Pro Val Pro Leu Glu Gln Ser 1 5 208PRTArtificialSodium
channel activity inhibiting peptide sequence 20Leu Pro Val Pro Leu
Glu Gln Xaa 1 5 218PRTArtificialSodium channel activity inhibiting
peptide sequence 21Leu Pro Val Pro Val Asp Gln Thr 1 5
228PRTArtificialSodium channel activity inhibiting peptide sequence
22Leu Pro Val Pro Val Glu Gln Ser 1 5 238PRTArtificialSodium
channel activity inhibiting peptide sequence 23Leu Pro Val Pro Val
Asp Gln Ser 1 5 248PRTArtificialSodium channel activity inhibiting
peptide sequence 24Val Pro Val Pro Leu Asp Gln Thr 1 5
258PRTArtificialSodium channel activity inhibiting peptide sequence
25Leu Pro Leu Pro Leu Asp Gln Ser 1 5 268PRTArtificialSodium
channel activity inhibiting peptide sequence 26Val Pro Leu Pro Leu
Asp Gln Ser 1 5 278PRTArtificialSodium channel activity inhibiting
peptide sequence 27Leu Pro Leu Pro Leu Asp Gln Thr 1 5
288PRTArtificialSodium channel activity inhibiting peptide sequence
28Val Xaa Leu Xaa Val Glu Gln Ser 1 5 298PRTArtificialSodium
channel activity inhibiting peptide sequence 29Val Pro Leu Pro Val
Glu Gln Ser 1 5 308PRTArtificialSodium channel activity inhibiting
peptide sequence 30Leu Pro Leu Pro Leu Glu Gln Ser 1 5
318PRTArtificialSodium channel activity inhibiting peptide sequence
31Leu Pro Xaa Pro Leu Asp Gln Ser 1 5 328PRTArtificialSodium
channel activity inhibiting peptide sequence 32Leu Pro Xaa Pro Leu
Asp Gln Thr 1 5 338PRTArtificialSodium channel activity inhibiting
peptide sequence 33Leu Pro Xaa Pro Val Glu Gln Ser 1 5
348PRTArtificialSodium channel activity inhibiting peptide sequence
34Leu Pro Xaa Pro Val Glu Gln Ser 1 5 358PRTArtificialSodium
channel activity inhibiting peptide sequence 35Leu Xaa Xaa Xaa Leu
Glu Gln Ser 1 5 368PRTArtificialSodium channel activity inhibiting
peptide sequence 36Leu Xaa Xaa Xaa Val Glu Gln Ser 1 5
378PRTArtificialSodium channel activity inhibiting peptide sequence
37Leu Pro Val Xaa Leu Asp Gln Thr 1 5 388PRTArtificialSodium
channel activity inhibiting peptide sequence 38Leu Xaa Val Xaa Leu
Asp Gln Thr 1 5 398PRTArtificialSodium channel activity inhibiting
peptide sequence 39Leu Xaa Val Pro Leu Asp Gln Thr 1 5
408PRTArtificialSodium channel activity inhibiting peptide sequence
40Leu Pro Val Xaa Leu Asp Gln Thr 1 5 418PRTArtificialSodium
channel activity inhibiting peptide sequence 41Leu Xaa Val Pro Leu
Asp Gln Thr 1 5 428PRTArtificialSodium channel activity inhibiting
peptide sequence 42Leu Pro Val Xaa Leu Asp Gln Thr 1 5
438PRTArtificialSodium channel activity inhibiting peptide sequence
43Leu Xaa Val Xaa Leu Asp Gln Thr 1 5 448PRTArtificialSodium
channel activity inhibiting peptide sequence 44Leu Pro Val Pro Leu
Glu Gln Ser 1 5 458PRTArtificialSodium channel activity inhibiting
peptide sequence 45Leu Pro Val Pro Leu Glu Gln Xaa 1 5
468PRTArtificialSodium channel activity inhibiting peptide sequence
46Leu Pro Val Pro Val Asp Gln Thr 1 5 478PRTArtificialSodium
channel activity inhibiting peptide sequence 47Leu Pro Val Pro Val
Glu Gln Ser 1 5 488PRTArtificialSodium channel activity inhibiting
peptide sequence 48Leu Pro Val Pro Val Asp Gln Ser 1 5
498PRTArtificialSodium channel activity inhibiting peptide sequence
49Val Pro Val Pro Leu Asp Gln Thr 1 5 508PRTArtificialSodium
channel activity inhibiting peptide sequence 50Leu Pro Leu Pro Leu
Asp Gln Ser 1 5 518PRTArtificialSodium channel activity inhibiting
peptide sequence 51Val Pro Leu Pro Leu Asp Gln Ser 1 5
528PRTArtificialSodium channel activity inhibiting peptide sequence
52Leu Pro Leu Pro Leu Asp Gln Thr 1 5 538PRTArtificialSodium
channel activity inhibiting peptide sequence 53Val Xaa Leu Xaa Val
Glu Gln Ser 1 5 548PRTArtificialSodium channel activity inhibiting
peptide sequence 54Val Pro Leu Pro Val Glu Gln Ser 1 5
558PRTArtificialSodium channel activity inhibiting peptide sequence
55Leu Pro Leu Pro Leu Glu Gln Ser 1 5 568PRTArtificialSodium
channel activity inhibiting peptide sequence 56Leu Pro Xaa Pro Leu
Asp Gln Ser 1 5 578PRTArtificialSodium channel activity inhibiting
peptide sequence 57Leu Pro Xaa Pro Leu Asp Gln Thr 1 5
588PRTArtificialSodium channel activity inhibiting peptide sequence
58Leu Pro Xaa Pro Val Glu Gln Ser 1 5 598PRTArtificialSodium
channel activity inhibiting peptide sequence 59Leu Pro Xaa Pro Val
Glu Gln Ser 1 5 608PRTArtificialSodium channel activity inhibiting
peptide sequence 60Leu Xaa Xaa Xaa Leu Glu Gln Ser 1 5
618PRTArtificialSodium channel activity inhibiting peptide sequence
61Leu Xaa Xaa Xaa Val Glu Gln Ser 1 5 628PRTArtificialSodium
channel activity inhibiting peptide sequence 62Leu Xaa Val Pro Leu
Asp Gln Thr 1 5 638PRTArtificialSodium channel activity inhibiting
peptide sequence 63Leu Pro Val Xaa Leu Asp Gln Thr 1 5
648PRTArtificialSodium channel activity inhibiting peptide sequence
64Leu Xaa Val Xaa Leu Asp Gln Thr 1 5 658PRTArtificialSodium
channel activity inhibiting peptide sequence 65Leu Xaa Val Pro Leu
Asp Gln Thr 1 5 668PRTArtificialSodium channel activity inhibiting
peptide sequence 66Leu Pro Val Xaa Leu Asp Gln Thr 1 5
678PRTArtificialSodium channel activity inhibiting peptide sequence
67Leu Xaa Val Pro Leu Asp Gln Thr 1 5 688PRTArtificialSodium
channel activity inhibiting peptide sequence 68Leu Pro Val Xaa Leu
Asp Gln Thr 1 5 698PRTArtificialSodium channel activity inhibiting
peptide sequence 69Leu Xaa Val Xaa Leu Asp Gln Thr 1 5
708PRTArtificialSodium channel activity inhibiting peptide sequence
70Leu Pro Val Pro Leu Glu Gln Ser 1 5 718PRTArtificialSodium
channel activity inhibiting peptide sequence 71Leu Pro Val Pro Leu
Glu Gln Xaa 1 5 728PRTArtificialSodium channel activity inhibiting
peptide sequence 72Leu Pro Val Pro Val Asp Gln Thr 1 5
738PRTArtificialSodium channel activity inhibiting peptide sequence
73Leu Pro Val Pro Val Glu Gln Ser 1 5 748PRTArtificialSodium
channel activity inhibiting peptide sequence 74Leu Pro Val Pro Val
Asp Gln Ser 1 5 758PRTArtificialSodium channel activity inhibiting
peptide sequence 75Val Pro Val Pro Leu Asp Gln Thr 1 5
768PRTArtificialSodium channel activity inhibiting peptide sequence
76Leu Pro Leu Pro Leu Asp Gln Ser 1 5 778PRTArtificialSodium
channel activity inhibiting peptide sequence 77Val Pro Leu Pro Leu
Asp Gln Ser 1 5 788PRTArtificialSodium channel activity inhibiting
peptide sequence 78Leu Pro Leu Pro Leu Asp Gln Thr 1 5
798PRTArtificialSodium channel activity inhibiting peptide sequence
79Val Xaa Leu Xaa Val Glu Gln Ser 1 5 808PRTArtificialSodium
channel activity inhibiting peptide sequence 80Val Pro Leu Pro Val
Glu Gln Ser 1 5 818PRTArtificialSodium channel activity inhibiting
peptide sequence 81Leu Pro Leu Pro Leu Glu Gln Ser 1 5
828PRTArtificialSodium channel activity inhibiting peptide sequence
82Leu Pro Xaa Pro Leu Asp Gln Ser 1 5 838PRTArtificialSodium
channel activity inhibiting peptide sequence 83Leu Pro Xaa Pro Leu
Asp Gln Thr 1 5 848PRTArtificialSodium channel activity inhibiting
peptide sequence 84Leu Pro Xaa Pro Val Glu Gln Ser 1 5
858PRTArtificialSodium channel activity inhibiting peptide sequence
85Leu Pro Xaa Pro Val Glu Gln Ser 1 5 868PRTArtificialSodium
channel activity inhibiting peptide sequence 86Leu Xaa Xaa Xaa Leu
Glu Gln Ser 1 5 878PRTArtificialSodium channel activity inhibiting
peptide sequence 87Leu Xaa Xaa Xaa Val Glu Gln Ser 1 5
888PRTArtificialSodium channel activity inhibiting peptide sequence
88Leu Pro Val Xaa Leu Asp Gln Thr 1 5 898PRTArtificialSodium
channel activity inhibiting peptide sequence 89Leu Xaa Val Xaa Leu
Asp Gln Thr 1 5 908PRTArtificialSodium channel activity inhibiting
peptide sequence 90Leu Xaa Val Pro Leu Asp Gln Thr 1 5
918PRTArtificialSodium channel activity inhibiting peptide sequence
91Leu Pro Val Xaa Leu Asp Gln Thr 1 5 928PRTArtificialSodium
channel activity inhibiting peptide sequence 92Leu Xaa Val Pro Leu
Asp Gln Thr 1 5 938PRTArtificialSodium channel activity inhibiting
peptide sequence 93Leu Pro Val Xaa Leu Asp Gln Thr 1 5
948PRTArtificialSodium channel activity inhibiting peptide sequence
94Leu Xaa Val Xaa Leu Asp Gln Thr 1 5 958PRTArtificialSodium
channel activity inhibiting peptide sequence 95Leu Pro Val Pro Leu
Glu Gln Ser 1 5 968PRTArtificialSodium channel activity inhibiting
peptide sequence 96Leu Pro Val Pro Leu Glu Gln Xaa 1 5
978PRTArtificialSodium channel activity inhibiting peptide sequence
97Leu Pro Val Pro Val Asp Gln Thr 1 5 988PRTArtificialSodium
channel activity inhibiting peptide sequence 98Leu Pro Val Pro Val
Glu Gln Ser 1 5 998PRTArtificialSodium channel activity inhibiting
peptide sequence 99Leu Pro Val Pro Val Asp Gln Ser 1 5
1008PRTArtificialSodium channel activity inhibiting peptide
sequence 100Val Pro Val Pro Leu Asp Gln Thr 1 5
1018PRTArtificialSodium channel activity inhibiting peptide
sequence 101Leu Pro Leu Pro Leu Asp Gln Ser 1 5
1028PRTArtificialSodium channel activity inhibiting peptide
sequence 102Val Pro Leu Pro Leu Asp Gln Ser 1 5
1038PRTArtificialSodium channel activity inhibiting peptide
sequence 103Leu Pro Leu Pro Leu Asp Gln Thr 1 5
1048PRTArtificialSodium channel activity inhibiting peptide
sequence 104Val Xaa Leu Xaa Val Glu Gln Ser 1 5
1058PRTArtificialSodium channel activity inhibiting peptide
sequence 105Val Pro Leu Pro Val Glu Gln Ser 1 5
1068PRTArtificialSodium channel activity inhibiting peptide
sequence 106Leu Pro Leu Pro Leu Glu Gln Ser 1 5
1078PRTArtificialSodium channel activity inhibiting peptide
sequence 107Leu Pro Xaa Pro Leu Asp Gln Ser 1 5
1088PRTArtificialSodium channel activity inhibiting peptide
sequence 108Leu Pro Xaa Pro Leu Asp Gln Thr 1 5
1098PRTArtificialSodium channel activity inhibiting peptide
sequence 109Leu Pro Xaa Pro Val Glu Gln Ser 1 5
1108PRTArtificialSodium channel activity inhibiting peptide
sequence 110Leu Pro Xaa Pro Val Glu Gln Ser 1 5
1118PRTArtificialSodium channel activity inhibiting peptide
sequence 111Leu Xaa Xaa Xaa Leu Glu Gln Ser 1 5
1128PRTArtificialSodium channel activity inhibiting peptide
sequence 112Leu Xaa Xaa Xaa Val Glu Gln Ser 1 5
1138PRTArtificialSodium channel activity inhibiting peptide
sequence 113Leu Pro Val Pro Leu Asp Gln Thr 1 5
11410PRTArtificialSodium channel activity inhibiting peptide
sequence 114Asp Gln Thr Leu Pro Leu Asn Val Asn Pro 1 5 10
11511PRTArtificialSodium channel activity inhibiting peptide
sequence 115Leu Pro Val Pro Leu Asp Gln Thr Leu Pro Leu 1 5 10
1168PRTArtificialSodium channel activity inhibiting peptide
sequence 116Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
1178PRTArtificialSodium channel activity inhibiting peptide
sequence 117Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
11815PRTArtificialSodium channel activity inhibiting peptide
sequence 118Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 11915PRTArtificialSodium channel activity inhibiting
peptide sequence 119Leu Pro Val Pro Leu Asp Gln Thr Leu Pro Leu Asn
Val Asn Pro 1 5 10 15 1208PRTArtificialSodium channel activity
inhibiting peptide sequence 120Leu Pro Val Pro Leu Asp Gln Thr 1 5
1218PRTArtificialSodium channel activity inhibiting peptide
sequence 121Xaa Pro Val Pro Leu Asp Gln Thr 1 5
1228PRTArtificialSodium channel activity inhibiting peptide
sequence 122Leu Pro Xaa Pro Leu Asp Gln Thr 1 5
1238PRTArtificialSodium channel activity inhibiting peptide
sequence 123Leu Pro Val Pro Xaa Asp Gln Thr 1 5
1248PRTArtificialSodium channel activity inhibiting peptide
sequence 124Leu Pro Val Pro Leu Asp Asn Thr 1 5
1258PRTArtificialSodium channel activity inhibiting peptide
sequence 125Leu Pro Val Pro Leu Glu Gln Thr 1 5
1268PRTArtificialSodium channel activity inhibiting peptide
sequence 126Leu Pro Val Pro Leu Asp Gln Ser 1 5
1278PRTArtificialSodium channel activity inhibiting peptide
sequence 127Leu Pro Ile Pro Leu Asp Gln Thr 1 5
12812PRTArtificialSodium channel activity inhibiting peptide
sequence 128Xaa Xaa Leu Pro Ile Pro Leu Asp Gln Thr Xaa Xaa 1 5 10
1298PRTArtificialSodium channel activity inhibiting peptide
sequence 129Leu Pro Xaa Pro Leu Asp Gln Thr 1 5
1308PRTArtificialSodium channel activity inhibiting peptide
sequence 130Leu Pro Phe Pro Leu Asp Gln Thr 1 5
13112PRTArtificialSodium channel activity inhibiting peptide
sequence 131Xaa Xaa Leu Xaa Xaa Pro Leu Asp Gln Thr Xaa Xaa 1 5 10
13212PRTArtificialSodium channel activity inhibiting peptide
sequence 132Xaa Xaa Leu Pro Xaa Xaa Leu Asp Gln Thr Xaa Xaa 1 5 10
13318PRTArtificialSodium channel activity inhibiting peptide
sequence 133Gly Gly Leu Pro Xaa Pro Leu Asp Gln Thr Leu Pro Leu Asn
Val Asn 1 5 10 15 Pro Ala 1348PRTArtificialSodium channel activity
inhibiting peptide sequence 134Leu Pro Ile Pro Leu Asp Gln Thr 1 5
13510PRTArtificialSodium channel activity inhibiting peptide
sequence 135Xaa Leu Pro Xaa Pro Leu Asp Gln Thr Xaa 1 5 10
1368PRTArtificialSodium channel activity inhibiting peptide
sequence 136Leu Xaa Val Pro Leu Asp Gln Thr 1 5
1378PRTArtificialSodium channel activity inhibiting peptide
sequence 137Val Pro Val Pro Leu Asp Gln Ser 1 5
13818PRTArtificialSodium channel activity inhibiting peptide
sequence 138Gly Gly Leu Pro Ile Pro Leu Asp Gln Thr Leu Pro Leu Asn
Val Asn 1 5 10 15 Pro Ala 1398PRTArtificialSodium channel activity
inhibiting peptide sequence 139Leu Xaa Ile Pro Leu Asp Gln Thr 1 5
14018PRTArtificialSodium channel activity inhibiting peptide
sequence 140Gly Gly Leu Pro Ile Pro Leu Asp Gln Thr Leu Pro Leu Asn
Val Asn 1 5 10 15 Pro Ala 14112PRTArtificialSodium channel activity
inhibiting peptide sequence 141Xaa Xaa Leu Pro Leu Pro Leu Asp Gln
Thr Xaa Xaa 1 5 10 14212PRTArtificialSodium channel activity
inhibiting peptide sequence 142Xaa Xaa Leu Pro Xaa Pro Leu Asp Gln
Thr Xaa Xaa 1 5 10 14318PRTArtificialADG negative control peptide
sequence 143Ala Asp Gly Gly Leu Leu Leu Leu Asn Asn Pro Pro Pro Pro
Gln Thr 1 5 10 15 Val Val 14410PRTArtificialSodium channel activity
inhibiting peptide sequence 144Xaa Leu Pro Ile Pro Leu Glu Gln Ser
Xaa 1 5 10
* * * * *