U.S. patent application number 09/682706 was filed with the patent office on 2002-06-27 for stresscopins and their uses.
Invention is credited to Hsu, Sheau Yu, Hsueh, Aaron J.W..
Application Number | 20020082409 09/682706 |
Document ID | / |
Family ID | 26936324 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082409 |
Kind Code |
A1 |
Hsu, Sheau Yu ; et
al. |
June 27, 2002 |
Stresscopins and their uses
Abstract
The invention provides novel nucleic acids and polypeptides,
referred to herein as stresscopin 1 and stresscopin 2, which
preferentially activate the CRH-R2 receptor over the R1 receptor.
Stresscopins, analogs and mimetics, and related CRH-R2 agonists
suppress food intake and heat-induced edema; but do not induce
substantial release of ACTH. Stresscopin also finds use in the
recovery phase of stress responses, as an anti-inflammatory agent,
as a hypotensive agent, as a cardioprotective agent, and in the
treatment of psychiatric and anxiolytic disorders. Stresscopin
nucleic acid compositions find use in identifying homologous or
related proteins and the DNA sequences encoding such proteins; in
producing compositions that modulate the expression or function of
the protein; and in studying associated physiological pathways.
Inventors: |
Hsu, Sheau Yu; (Menlo Park,
CA) ; Hsueh, Aaron J.W.; (Stanford, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
26936324 |
Appl. No.: |
09/682706 |
Filed: |
October 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60244128 |
Oct 26, 2000 |
|
|
|
60276615 |
Mar 15, 2001 |
|
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|
Current U.S.
Class: |
536/23.5 ;
435/320.1; 435/7.1; 530/326; 530/387.9 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
25/22 20180101; A61P 29/00 20180101; G01N 2500/02 20130101; G01N
2500/10 20130101; C07K 14/57509 20130101; A61P 3/04 20180101; A61P
3/00 20180101; A61P 9/12 20180101; C07K 14/47 20130101; A61K 38/00
20130101 |
Class at
Publication: |
536/23.5 ;
530/326; 514/2; 435/320.1; 530/387.9; 435/7.1 |
International
Class: |
A01N 037/18; A61K
038/00; G01N 033/53; C07H 021/04; C12N 015/00; C12N 015/09; C12N
015/63; C12N 015/70; C12N 015/74; C07K 005/00; C07K 007/00; C07K
016/00; C07K 017/00; A61K 038/04; C12P 021/08 |
Claims
1. A composition comprising a stresscopin peptide, wherein said
stresscopin peptide comprises at least 18 contiguous amino acids of
the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:5 or SEQ ID NO:6.
2. A composition according to claim 1, wherein said peptide
comprises at least 30 contiguous amino acids of the sequence set
forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID
NO:6.
3. The composition according to claim 1, wherein said composition
further comprises a pharmaceutically acceptable carrier.
4. A method of appetite suppression, the method comprising
administering to an individual the composition of claim 3.
5. A method for cardioprotection, the method comprising
administering to an individual the composition of claim 3.
6. A method for reduction of edema, the method comprising
comprising administering to an individual the composition of claim
3.
7. A method for reduction of inflammation, and organ graft
rejection the method comprising administering to an individual the
composition of claim 3.
8. A method for the reduction of hypertension, the method
comprising administering to an individual the composition of claim
3.
9. A method for the treatment of stress related to trauma, the
method comprising administering to an individual the composition of
claim 3.
10. A method of treatment for affective disorders, the method
comprising comprising administering to an individual the
composition of claim 3.
11. An isolated nucleic acid molecule comprising a cDNA sequence
encoding a mammalian stresscopin protein that will hybridize under
stringent conditions of 50.degree. C. or higher in the presence of
0.1.times.SSC to the sequence set forth in any one of SEQ ID NO:1
or SEQ ID NO:4, or encodes the peptide in any one of SEQ ID NO:3 or
SEQ ID NO:6.
12. An isolated nucleic acid according to claim 11, wherein said
cDNA sequence is of human origin.
13. An isolated nucleic acid molecule according to claim 12,
wherein said human stresscopin protein comprises the sequence set
forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID
NO:6.
14. An isolated nucleic acid molecule according to claim 13,
wherein said nucleic acid comprises the nucleotide sequence of SEQ
ID NO:1 or SEQ ID NO:4.
15. The nucleic acid of claim 11, further comprising a vector
sequence.
16. The nucleic acid of claim 15, wherein said vector comprises a
transcription cassette operably linked to said stresscopin cDNA
sequence.
17. The nucleic acid of claim 15, wherein said vector is a
plasmid.
18. The nucleic acid of claim 15, wherein said vector is a
retrovirus.
19. The nucleic acid of claim 1 5, wherein said vector is an
adenovirus.
20. An antibody that specifically recognizes a stresscopin
peptide.
21. A non-human transgenic animal model for stresscopin gene
function wherein said transgenic animal comprises an introduced
alteration in a stresscopin gene.
22. A method of screening for biologically active agents that
modulate stresscopin function, the method comprising: combining a
candidate biologically active agent with any one of:(a) a mammalian
stresscopin peptide;(b) a cell comprising a nucleic acid encoding a
mammalian stresscopin peptide; or(c) a non-human transgenic animal
model for stresscopin gene function comprising one of:(i) a
knockout of an stresscopin gene; (ii) an exogenous and stably
transmitted mammalian stresscopin gene sequence; and determining
the effect of said agent on stresscopin function.
Description
CROSS REFERENCE OF RELATED APPLICATIONS
[0001] This application claims benefit of prior U.S. provisional
application serial Nos. 60/244,1 28 filed Oct. 26, 2000 and
60/276,615 filed Mar. 15, 2001, both of which are incorporated
herein in their entirety by reference.
INTRODUCTION
[0002] Mammals respond to stress through interlinked endocrine,
neuroendocrine, autonomic and behavioral pathways. Activation of
the autonomic nervous system elicits the release of catecholamines,
whereas hypothalamic secretion of corticotropin releasing hormone
(CRH) leads to pituitary secretion of adrenocorticotrophic hormone
(ACTH), which, in turn, stimulates glucocorticoid secretion by the
adrenal cortex. These stress-responses can provide a vital
short-term metabolic lift, but when triggered inappropriately can
also cause severe diseases. For example, anxiety and depression
affect over 100 million patients worldwide every year, and
depression is the leading cause of suicide, which claims thousands
of lives each year in the U.S. Anxiety is among the most commonly
observed group of CNS disorders, which includes phobias or
irrational fears, panic attacks, obsessive-compulsive disorders and
other fear and tension syndromes. Another potentially deleterious
response to stress is hypertension, which can lead to fatal heart
disease and stroke.
[0003] The physiological response to stress is integrated through
corticotropin releasing hormone (CRH), and related factors
(Shibahara et al. (1983) EMBO J. 2(5):775-779). CRH is a 41-amino
acid peptide synthesized in the hypothalamus. It is a ligand for
two receptors, CRH-R1 and CRH-R2. Another known ligand for the CRH
receptors is urocortin, which is a 40 amino acid peptide having
substantial sequence similarity with the fish protein urotensin and
to CRH (Donaldson et al (1996) Endocrinology 737 (5):2167-2170).
These receptors have a seven-transmembrane structure, and belong to
the family of G-protein coupled receptors, whose actions are
mediated through activation of adenylate cyclase. The type-1
receptor is expressed in many areas of the brain, as well as in the
pituitary, gonads, and skin (Chen et al. (1993) P.N.A.S.
90:8967-8971). The type-2 receptor is expressed in the brain,
cardiac and skeletal muscle, epididymis, and the gastrointestinal
tract (Liaw et al. (1996) Endocrinology 137(1):72-77). In addition,
there is a CRH-R2 splice isoform found in human brain
(Grammatopoulos et al. (1999) Mol. Endocrinol.
73(12):2189-2202).
[0004] Although the two receptors share 70% sequence identity, they
differ in their ligand binding affinity. CRH itself has a much
higher affinity for CRH-R1, while urocortin is equally effective at
binding both the R1 and the R2 receptors. It is also believed that
the receptors differ in their physiological role. An inverse
relationship between the CRH-R1 and CRH-R2 receptor systems have
been reported in an anxiety model, suggesting that CRH neuronal
systems may be comprised of two separate, but interrelated,
subdivisions that can be coordinately and inversely regulated by
stress, anxiety, or anxiolytic drugs.
[0005] Mice lacking CRH-R1 display markedly reduced anxiety, and
fail to exhibit the normal hormonal response to stress (Smith et al
(1998) Neuron 20:1093-1102). Animals having a targeted disruption
in CRH-R2 have normal initiation of stress responses, but have
deficiencies in the maintenance and recovery phases. For example,
stress coping behaviors associated with de-arousal were reduced in
these knock-out mice. The mice were also hypersensitive to stress,
and displayed increased anxiety-like behavior (Bale et al. (2000)
Nat. Genet. 24:410-414). CRH-R2 may also mediate peripheral human
dynamic effects, including enhanced cardiac performance and reduced
blood pressure, as well as cardiovascular homeostasis (Coste et al.
(2000) Nat. Genet. 24 :403-409). CRH-R2 signaling is essential for
coping with the hypertension initiated during stress. Mutant mice
have normal basal feeding and weight gain, but decreased food
intake following food deprivation, suggesting a role of CRH-R2 in
feeding behavior and reduced gastric emptying. In addition, CRH-R2
signaling may be involved in the suppression of immune responses
associated with stress.
SUMMARY OF THE INVENTION
[0006] Stresscopin nucleic acid compositions and their encoded
polypeptides and variants thereof are provided. Stresscopins are
novel and selective ligands for the CRH-R2 receptor, and thus find
use where it is desirable to specifically induce the CRH-R2 and not
the CRH-R1 response pathway. In addition to use as a therapeutic
agent, stresscopins are utilized in screening and research methods
for the determination of specific analogs, agonists, antagonists
and mimetics.
[0007] Stresscopins, analogs and mimetics, and related CRH-R2
agonists suppress food intake and heat-induced edema, but unlike
CRH and urocortin, stresscopins do not induce substantial release
of pituitary ACTH and adrenal glucocorticoids. Stresscopins also
find use in the recovery phase of stress responses, as an
anti-inflammatory agent, as a hypotensive agent, as a
cardioprotective agent, and in the treatment of psychiatric and
anxiolytic disorders.
[0008] The invention also provides diagnostics and therapeutics
comprising stresscopin nucleic acids, their corresponding genes and
gene products, antisense nucleotides, and antibodies specific for
one or more epitopes of the stresscopin polypeptide. The nucleic
acid compositions find use in identifying homologous or related
genes; for production of the encoded protein; in producing
compositions that modulate the expression or function of its
encoded protein; for gene therapy; mapping functional regions of
the protein; and in studying associated physiological pathways. In
addition, modulation of the gene activity in vivo is used for
prophylactic and therapeutic purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0009] Fig. 1A. Nucleic acid sequence and amino acid sequence of
stresscopin 1 (SEQ ID NOS:1-2). The pre-pro region of stresscopin 1
polypeptide is 46 amino acids and is lightly shaded, while the
putative mature stresscopin 1 peptide is highlighted darkly on the
background (SEQ ID NO:3). The methionine start site and the
putative C-terminal amidation donor residue are in bold
letters.
[0010] FIG. 1B. Nucleic acid sequence and amino acid sequence of
stresscopin 2 (SEQ ID NOS:4-5). The pre-pro region of stresscopin 2
peptide is 96 amino acids and is lightly shaded, while the putative
mature stresscopin 2 peptide is shaded darkly (SEQ ID NO:6).
[0011] Fig. 1C. Comparison of the mature regions of CRH-related
peptides from mammals, fish, and frog. Amino acid numbering is
given on the left. The putative secondary structures of these
polypeptides are indicated above the upper row of the alignment.
CRH, urocortin, urotensin 1, and sauvagine shared a similar
structure with an N-terminal random coil followed by an extended
.alpha.-helix structure. In contrast, the N-terminal sequences of
human and pufferfish stresscopin peptides adopted an extended
strand structure followed by a short random coil. Lightly shaded
residues are conserved in the majority of aligned sequences.
Residues that are identical in peptides of each subgroup are
highlighted by a dark background. The legend is h is human; m is
mouse; g is goldfish (Carassius auratus); s is sucker (Catostomus
commersoni); f is leaf frog (Phyllomedusa sauvagei); and p is Fugu
pufferfish (Takifugu rubripes). CRH family peptides all have a
stretch of 30 residues at their C-termini and adopt an extended
.alpha.-helical structure. Alignment of the mature pepetides with
elevated CRH family hormones indicated that mature stresscopins
from human and pufferfish, but not the pre-pro regions show 35-38%
identity to other family proteins. SEQ ID NOS:4, 6-15.
[0012] FIG. 1D. Phylogenetic tree of CRH family proteins from
vertebrates. Phylogenetic inference based on mature regions of CRH
family proteins. Phylogenetic analysis of nine CRH family proteins
from fish, frog and mammals suggests the ancient evolution of three
subgroups of CRH family proteins, with the human and pufferfish
stresscopins clustered in a separate branch.
[0013] FIG. 2. Expression of stresscopin transcripts and proteins.
FIG. 2a, Expression of the stresscopin 1 (panels 1 and 2) and
stresscopin 2 (panels 3 and 4) transcripts in 23 different human
tissues as determined by PCR amplification. The specific cDNA bands
are indicated by an arrowhead on the left. PCR products from
experiments using two different concentrations of cDNA templates (1
ng template/reaction, panels 1 and 3; 10 pg template/reaction,
panels 2 and 4) are shown. PBL, peripheral blood cells. The
expression of -actin transcripts in cDNA templates from different
tissues are shown in panels 5 (1 ng template/reaction) and 6 (1 0
pg template/reaction). FIG. 2b. Expression of the stresscopin 1
transcript in 11 different human cardiac compartments as determined
by PCR amplification using a primer pair flanking part of the
C-terminal ORF and the 3'-untranslated region of stresscopin 1 cDNA
and 1 ng of template cDNA. Lane 1, atrioventricular node; lane 2,
atrioventricular septum; lane 3, aorta; lane 4, apex of the heart;
lane 5, left atrium; lane 6, right atrium; lane 7, dextra auricle;
lane 8, sinistra auricle; lane 9, left ventricle; lane 10, right
ventricle; lane 11, adult heart. The specific 177-bp stresscopin 1
cDNA bands are indicated by an arrowhead. FIG. 2c. Expression of
the stresscopin 2 transcript in 12 different human tissues of the
digestive system as determined by PCR amplification using a primer
pair flanking part of the ORF of stresscopin 2 cDNA and 1 ng of
template cDNAs. Lane 1, ascending colon; lane 2, descending colon;
lane 3, transverse colon; lane 4, duodenum; lane 5, lleocecum; lane
6, Ileum; lane 7, jejunum; lane 8, stomach; lane 9, cecum; lane 10,
rectum; lane 11, liver; lane 12, esophagus. The specific 237-bp
stresscopin 2 cDNA bands are indicated by an arrowhead. FIGS. 2d
and 2e. Stresscopin 1 expression in mouse cardiac (d) and rat
pituitary (e) sections using anti-stresscopin 1 antibody C2208.
Specific signals (black particles) are indicated by arrows (left
panel). Adjacent sections hybridized with anti-stresscopin 1
antibodies presaturated with the antigen peptide showed minimal
staining (right panels). A, atrium tissues; BV, blood vessel; AP,
anterior pituitary; PP, posterior pituitary; IL, intermediate lobe.
f, Stresscopin 2 expression in mouse intestinal sections using
anti-stresscopin 2 antibody C2221 (left panel). A negative control
with presaturated antibodies is shown on the right panel. MM,
muscularis mucosae; IG, intestinal glands; ME, muscularis
externa.
[0014] FIG. 3. Stresscopin 1 and stresscopin 2 preferentially
activate CRH R2. Hormonal stimulation of cAMP production by FIG.
3a. CRHR2-containing rat cardiac A7r5 cells, FIG. 3b.
CRHR1-containing human retinoblastoma Y79 cells, FIG. 3c.
recombinant CRHR2, FIG. 3d. recombinant CRHR2 and, FIG. 3e.
recombinant CRHR1. Cells incubated with the test peptide or vehicle
were harvested at 16 h after treatment and heated to 95 .degree. C.
for 5 min to inactivate phosphodiesterase activity before cAMP
measurement. FIG. 3f. Full-length 43-amino-acid stresscopin 1
(SCP1) and truncated derivatives with deletion of 1-5 amino acids
at the N-terminus (1 nM) stimulated cAMP production by recombinant
CRH R2 (hatched bars), but not CRH R1 (blank bars). Nonamidated
stresscopin 1 (SCP1-NA) showed a minimal stimulation of cAMP
production as compared to the amidated counterpart. Data are the
mean SEM (N=4). SCPL-0.1, stresscopin 1 (0.1 nM); SCP1, stresscopin
1 (1 nM); SCPI(2-43), truncated stresscopin 1 with the first amino
acid deleted; SCP1 (3-43), truncated stresscopin 1 with
2-amino-acid deletion; SCP1 (4-43), truncated stresscopin 1 with
3-amino-acid deletion; SCP1 (5-43), truncated stresscopin 1 with
4-amino-acid deletion; SCP1 (6-43), truncated stresscopin 1 with
5-amino-acid deletion; SCP1-NA-0.1, full-length stresscopin 1
devoid of amidation at the C-terminus (0.1 nM); SCP1-NA,
full-length stresscopin 1 devoid of amidation at the C-terminus (1
nM); UCN, urocortin. FIGS. 3g and 3h. Competitive displacement by
unlabeled CRH, urocortin, stresscopin 1, and stresscopin 2 of
.sup.125 l-labeled urocortin bound to membranes of 293T cells
transfected with CRHR2 (FIG. 3g.) or CRHR2 (FIG. 3h.) cDNA. Data
are mean SEM (N=3).
[0015] FIG. 4. CRH and urocortin, but not stresscopin 1 and
stresscopin 2, stimulate ACTH release by cultured anterior
pituitary cells in vitro (FIG. 4a) and induce ACTH secretion in
vivo (FIG. 4b). ACTH contents in culture media and serum were
determined using a radioimmunoassay. For in vivo studies, male rats
were injected i.p. with test peptides (2 nmoles/kg B.W.) and
sacrificed 30 min later. Data are mean SEM (N=4). FIG. 4c.
Anti-edema response regulated by stresscopins. Stresscopin 1 (20
nmoles/kg), stresscopin 2 (100 nmoles/kg), and related hormones (20
nmoles/kg) suppress heat-induced paw edema formation in
anaesthetized rats (N=6). FIG. 4d. Cumulative food intake in mice
treated with stresscopin 1 (left panel, N=6) and stresscopin 2
(right panel, N=4) peptides and other hormones at 2, 4, and 8 h
after treatment. FIG. 4e. Reduction of gastric emptying by
stresscopin 1 (blank bars, 8 nmoles/kg; hatched bars, 80
nmoles/kg), stresscopin 2 (blank bars, 80 nmoles/kg; hatched bars,
200 nmoles/kg), and related hormones (blank bars, 8 nmoles/kg;
hatched bars, 80 nmoles/kg) at 2 h after hormone treatment (N=6).
The rates of gastric emptying were calculated by the formula (wet
weight of stomach at 2 h after treatment/wet weight of stomach in
fed animals sacrificed at 0 h). SCP1 is stresscopin 1; SCP2 is
stresscopin 2; UCN is urocortin; SCP1 (11-43) is truncated
stresscopin 1 with the first 10 amino acids deleted.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0016] The invention provides novel nucleic acids and polypeptides,
referred to herein as stresscopin 1 and stresscopin 2, which are
members of the corticotropin releasing hormone family. Because
stresscopins preferentially activate the CRH-R2 receptor over the
R1 receptor, stresscopins, analogs and mimetics, and related CRH-R2
agonists suppress heat-induced edema as the result of their
hypotensive actions as well as food intake; but do not induce
substantial release of ACTH. Stresscopin also finds use in the
recovery phase of stress responses, as an anti-inflammatory agent,
as a cardioprotective agent, and in the treatment of psychiatric
and anxiolytic disorders.
[0017] The nucleic acid compositions of the subject invention find
use in identifying homologous or related genes; for production of
the encoded protein; in producing compositions that modulate the
expression or function of its encoded protein; for gene therapy;
mapping functional regions of the protein; and in studying
associated physiological pathways. In addition, modulation of the
gene activity in vivo is used for prophylactic and therapeutic
purposes. The proteins are useful as a therapeutic, as an immunogen
for producing specific antibodies, in screening for biologically
active agents that act in the CRH signaling pathways and for
therapeutic and prophylactic purposes.
[0018] Stresscopins are natural agonists of the CRH-R2 receptor,
where the term "agonist" refers to a compound that binds to, and
activates a receptor. Compounds that inhibit this effect are
referred to as "antagonists." Ligands, e.g. variants, derivatives
and mimetics of stresscopins, may evoke a spectrum of responses
ranging from full CRH-R2 activation by agonists to partial
activation and inhibition by partial or complete antagonists.
Stresscopin Polypeptides
[0019] The mature stresscopin 1 polypeptide is a 43 amino acid
peptide, derived from a 112 amino acid precursor protein. The amino
acid sequence of the precursor protein and mature protein are
provided as SEQ ID NO:2 and SEQ ID NO:3, respectively. The
nucleotide sequence of the human stresscopin 1 cDNA is provided as
SEQ ID NO:1. The mature stresscopin 2 is a peptide of 40 amino
acids, derived from a 161 precursor protein. The amino acid
sequence of the precursor protein and mature protein are provided
as SEQ ID NO:5 and SEQ ID NO:6. The nucleotide sequence of
stresscopin 2 is provided as SEQ ID NO:4.
[0020] Both human stresscopin ORFs contain a signal peptide for
secretion and the predicted mature regions are flanked by potential
proteolytic cleavage sites and an .alpha.-amidation donor residue.
The identity of the stresscopin 1 and 2 transcripts was confirmed
following PCR of cDNA from human testis and colon,
respectively.
[0021] For use in the subject methods, either of the native
stresscopin forms, modifications thereof, or a combination of forms
may be used. Peptides of interest include fragments of at least
about 12 contiguous amino acids, more usually at least about 20
contiguous amino acids, and may comprise 30 or more amino acids, up
to the provided peptide, and may extend further to comprise other
sequences present in the precursor protein.
[0022] A fragment of a stresscopin peptide may be selected to
achieve a specific purpose. For example, deletions at the amino
terminus of peptides having binding affinity for CRH-receptors have
the effect of turning an agonist peptide into an antagonist, by
retaining the receptor binding activity, but deleting the
activation activity (for example, see Ruhmann et al. (1998)
P.N.A.S. USA 95:15264-15269). Such deletions generally extend from
residue 1 through 10 of the peptide, and may further delete
additionally amino acids at residues 11, 12 or more. Smaller
deletions, of from 1 to to 5 amino acids, may be deleted in the
N-terminus and still retain the agonist properties.
[0023] The sequence of the stresscopin polypeptide may be altered
in various ways known in the art to generate targeted changes in
sequence. The polypeptide will usually be substantially similar to
the sequences provided herein, i.e. will differ by at least one
amino acid, and may differ by at least two but not more than about
ten amino acids. The sequence changes may be substitutions,
insertions or deletions. Scanning mutations that systematically
introduce alanine, or other residues, may be used to determine key
amino acids. Conservative amino acid substitutions typically
include substitutions within the following groups: (glycine,
alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic
acid); (asparagine, glutamine); (serine, threonine); (lysine,
arginine); or (phenylalanine, tyrosine).
[0024] Modifications of interest that do not alter primary sequence
include chemical derivatization of polypeptides, e.g., acetylation,
or carboxylation. Also included are modifications of glycosylation,
e.g. those made by modifying the glycosylation patterns of a
polypeptide during its synthesis and processing or in further
processing steps; e.g. by exposing the polypeptide to enzymes which
affect glycosylation, such as mammalian glycosylating or
deglycosylating enzymes. Also embraced are sequences that have
phosphorylated amino acid residues, e.g. phosphotyrosine,
phosphoserine, or phosphothreonine.
[0025] Also included in the subject invention are polypeptides that
have been modified using ordinary molecular biological techniques
and synthetic chemistry so as to improve their resistance to
proteolytic degradation or to optimize solubility properties or to
render them more suitable as a therapeutic agent. For examples, the
backbone of the peptide may be cyclized to enhance stability (see
Friedler et al. (2000)J. Biol. Chem. 275:23783-23789). Analogs of
such polypeptides include those containing residues other than
naturally occurring L-amino acids, e.g. D-amino acids or
non-naturally occurring synthetic amino acids.
[0026] The subject peptides may be prepared by in vitro synthesis,
using conventional methods as known in the art. Various commercial
synthetic apparatuses are available, for example, automated
synthesizers by Applied Biosystems, Inc., Foster City, Calif.,
Beckman, etc. By using synthesizers, naturally occurring amino
acids may be substituted with unnatural amino acids. The particular
sequence and the manner of preparation will be determined by
convenience, economics, purity required, and the like.
[0027] If desired, various groups may be introduced into the
peptide during synthesis or during expression, which allow for
linking to other molecules or to a surface. Thus cysteines can be
used to make thioethers, histidines for linking to a metal ion
complex, carboxyl groups for forming amides or esters, amino groups
for forming amides, and the like.
[0028] The polypeptides may also be isolated and purified in
accordance with conventional methods of recombinant synthesis. A
lysate may be prepared of the expression host and the lysate
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, or other purification technique. For the
most part, the compositions which are used will comprise at least
20% by weight of the desired product, more usually at least about
75% by weight, preferably at least about 95% by weight, and for
therapeutic purposes, usually at least about 99.5% by weight, in
relation to contaminants related to the method of preparation of
the product and its purification. Usually, the percentages will be
based upon total protein.
Compound Screening
[0029] The availability of purified stresscopin and other
components in the signaling pathways, e.g. CRH-R1, CRH-R2, etc.,
allows in vitro reconstruction of the pathway. Two or more of the
components may be combined in vitro, and the behavior assessed in
terms of activation of transcription of specific target sequences;
modification of protein components, e.g. proteolytic processing,
phosphorylation, methylation, etc.; ability of different protein
components to bind to each other, etc. The components may be
modified by sequence deletion, substitution, etc. to determine the
functional role of specific residues.
[0030] Drug screening may be performed using an in vitro model, a
genetically altered cell or animal, or purified stresscopin
protein. One can identify ligands or substrates that compete with,
modulate or mimic the action of stresscopin. Areas of investigation
include the development of treatments for suppression of food
intake; suppression of edema; enhancing the recovery phase of
stress responses; as an anti-inflammatory agent; as a
cardioprotective agent; in the treatment of psychiatric and
anxiolytic disorders, etc.
[0031] Drug screening identifies agents that mimic stresscopin
activity, either as an antagonist or as an agonist. A wide variety
of assays may be used for this purpose, including labeled in vitro
protein-protein binding assays, electrophoretic mobility shift
assays, immunoassays for protein binding, and the like. Knowledge
of the 3-dimensional structure of stresscopin, derived from
crystallization of purified synthetic stresscopin protein, leads to
the rational design of small drugs that specifically inhibit
stresscopin activity.
[0032] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of altering or
mimicking the physiological function of stresscopin. Generally, a
plurality of assay mixtures are run in parallel with different
agent concentrations to obtain a differential response to the
various concentrations. Typically one of these concentrations
serves as a negative control, i.e., at zero concentration or below
the level of detection.
[0033] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0034] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0035] Where the screening assay is a binding assay, one or more of
the molecules may be joined to a label, where the label can
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles, and
the like. Specific binding molecules include pairs, such as biotin
and streptavidin, digoxin and antidigoxin, etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule that provides for detection, in accordance with
known procedures.
[0036] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc. that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc. may be used. The mixture of components are added in
any order that provides for the requisite binding.
[0037] Incubations are performed at any suitable temperature,
typically between 4 and 40.degree. C. Incubation periods are
selected for optimum activity, but may also be optimized to
facilitate rapid high-throughput screening. Typically between 0.1
and 1 hours will be sufficient.
[0038] For example, a number of molecules have been described as
antagonists or as agonists of CRH receptors. Screening assays that
utilize stresscopin permit the improved selection for compounds
having a desired specificity, of acting specifically on CRH-R2.
Examples of such CRH agonists and antagonists include, among
others, arylamino fused pyrimidines (U.S. Pat. No. 6,107,300);
thiazolo[4,5-d] pyrimidines and pyridines (U.S. Pat. No.
6,107,294); pyrazoles and pyrazolopyrimidines (U.S. Pat. No.
6,103,900); aryl- and arylamino- substituted heterocycles (U.S.
Pat. No. 6,103,737); tetrahydropteridines (U.S. Pat. No.
6,083,948); benzimidazole derivatives (U.S. Pat. No. 6,022,978);
substituted 4-phenylaminothiazoles (U.S. Pat. No. 5,880,135);
benzo(e)perimidine-4-carboxamide derivatives (U.S. Pat. No.
5,861,398); etc.
[0039] Also of interest are cyclic stresscopin analogs (see U.S.
Pat. No. 5,663,292). Certain cyclic analogs, e.g. of CRH, have been
found to act as antagonists, and have substantially no residual
agonist activity. These peptides may have a cyclizing bond
initiating, e.g. at the residues in the 32-position and may
optionally have a second such bond initiating, e.g. at the residues
in the 19- or the 20-positions. Either or both of these bonds may
be an amide bond (or lactam bridge) between side chain carboxyl and
amino groups. Alternative antagonists include fragments of the
stresscopin sequence, as previously described.
[0040] The compounds having the desired pharmacological activity
may be administered in a physiologically acceptable carrier to a
host for treatment of stress related disorders, etc The compounds
may also be used to enhance stresscopin function in weight
reduction, treatment of heart disease, reduction of edema,
suppression of anxiety, stress reduction following major surgery,
etc. The inhibitory agents may be administered in a variety of
ways, orally, topically, parenterally e.g. subcutaneously,
intraperitoneally, by viral infection, intravascularly, etc.
Depending upon the manner of introduction, the compounds may be
formulated in a variety of ways. The concentration of
therapeutically active compound in the formulation may vary from
about 0.1-10 wt %.
Antibodies Specific for Stresscopin Polypeptides
[0041] The present invention provides antibodies specific for
stresscopin polypeptides, e.g. any one of the variants,
polypeptides, or domains described above. Such antibodies are
useful, for example, in methods of detecting the presence of
stresscopin in a biological sample, and in methods of isolating
stresscopin from a biological sample.
[0042] The stresscopin polypeptides of the invention are useful for
the production of antibodies, where short fragments provide for
antibodies specific for the particular polypeptide, and larger
fragments or the entire protein allow for the production of
antibodies over the surface of the polypeptide. As used herein, the
term "antibodies" includes antibodies of any isotype, fragments of
antibodies which retain specific binding to antigen, including, but
not limited to, Fab, Fv, scFv, and Fd fragments, chimeric
antibodies, humanized antibodies, single-chain antibodies, and
fusion proteins comprising an antigen-binding portion of an
antibody and a non-antibody protein. The antibodies may be
detectably labeled, e.g., with a radioisotope, an enzyme that
generates a detectable product, a green fluorescent protein, and
the like. The antibodies may be further conjugated to other
moieties, such as members of specific binding pairs, e.g., biotin
(member of biotin-avidin specific binding pair), and the like. The
antibodies may also be bound to a solid support, including, but not
limited to, polystyrene plates or beads, and the like.
[0043] "Antibody specificity", in the context of antibody-antigen
interactions, is a term well understood in the art, and indicates
that a given antibody binds to a given antigen, wherein the binding
can be inhibited by that antigen or an epitope thereof which is
recognized by the antibody, and does not substantially bind to
unrelated antigens. Methods of determining specific antibody
binding are well known to those skilled in the art, and can be used
to determine the specificity of antibodies of the invention for a
stresscopin polypeptide, particularly a human stresscopin
polypeptide.
[0044] Antibodies are prepared in accordance with conventional
ways, where the expressed polypeptide or protein is used as an
immunogen, by itself or conjugated to known immunogenic carriers,
e.g. KLH, preHBsAg, other viral or eukaryotic proteins, or the
like. Various adjuvants may be employed, with a series of
injections, as appropriate. For monoclonal antibodies, after one or
more booster injections, the spleen is isolated, the lymphocytes
immortalized by cell fusion, and then screened for high affinity
antibody binding. The immortalized cells, i.e. hybridomas,
producing the desired antibodies may then be expanded. For further
description, see Monoclonal Antibodies: A Laboratory Manual, Harlow
and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor,
N.Y., 1988. If desired, the mRNA encoding the heavy and light
chains may be isolated and mutagenized by cloning in E. coli, and
the heavy and light chains mixed to further enhance the affinity of
the antibody. Alternatives to in vivo immunization as a method of
raising antibodies include binding to phage display libraries,
usually in conjunction with in vitro affinity maturation.
Uses of Stresscopin
[0045] In light of the pharmacologic activities of stresscopin,
numerous clinical indications are evident. For example, clinical
indications for which a stresscopin peptide or variants thereof may
find use include treatment of obesity, reduction of edema; as an
anti-inflammatory agent, as a cardioprotective agent, as a
hypotensive agent, as a stress-reducing agent, and in the treatment
of psychiatric and anxiolytic disorders.
[0046] Human obesity is a widespread and serious disorder,
affecting a high percentage of the adult population in developed
countries. In spite of an association with heart disease, type II
diabetes, cancer, and other conditions, few persons are able to
permanently achieve significant weight loss. The subject peptides
are administered to obese patients for purposes of appetite
suppression. Patients may use various criteria for determining
obesity. Conveniently, a body mass index (BMI) is calculated, where
a person having a BMI greater than 25 is overweight and may
considered for treatment with the subject peptides. Stresscopins
find use in promoting gastric stasis and anorexic behavior without
concomitant activation of the ACTH-glucocorticoid axis.
[0047] In a related embodiment, the treatment of
non-insulin-dependent diabetes mellitus (NIDDM) is closely related
to the treatment of obesity. NIDDM is a metabolic disease that
affects about 5% to 7% of the population in western countries (and
10% of individuals over age 70). It is characterized by
hyperglycemia and often accompanied by a number of other
conditions, including hypertension, obesity and lipid disturbances.
Patients are generally categorized as diabetic or hyperglycemic by
measuring the level of glucose in the blood, either directly or by
monitoring the level of glycosylated hemoglobin. Treatment is
recommended where fasting glucose levels are greater 140 mg/dl,
where bedtime glucose is greater than 160 mg/dl, or where HbA
.sub.1c is greater than 8%. The level of reduction that is
desirable depends on the condition of the patient, and the blood
glucose levels at the start of treatment, but generally about a 10
to 40% reduction is blood glucose is desirable, usually about a 25
to 35% reduction.
[0048] The effects of stresscopins on stress related disorders
provides a means of treating affective and mood disorders, which
are a group of mental disorders characterized by neuroendocrine
dysregulation and are characterized by a disturbance in the
regulation of mood, behavior, and affect. Affective and mood
disorders can have serious impact on an individual's functional
ability, interpersonal relationships and behavior. Neuroendocrine
dysregulation, specifically changes in the
hypothalamic-pituitary-adrenal (HPA) axis, has been investigated as
a biological correlate of depression. Overall, the HPA axis
regulates physiologic responses to stress. The hypothalamus
controls endocrine functions and the autonomic nervous system. It
is involved in behaviors related to fight, flight, feeding and
mating, many of which are altered during episodes of
depression.
[0049] The hypothalamus releases CRH and related peptides in
response to stress, which then stimulates the anterior pituitary to
secrete adrenocorticotrophichormone (ACTH). ACTH prompts the
adrenal cortex to release cortisol which, through elaborate
feedback mechanisms signals the hypothalamus to increase or
decrease CRH production. Under ordinary circumstances, activation
of hypothalamic CRH is terminated rapidly by the negative feedback
of rising glucocorticoid levels. However, in melancholic
depression, hypercortisolism does not adequately restrain the
production of CRH in the hypothalamus. Thus, in melancholic
depression, CRH levels are chronically elevated causing
hyperactivity of the HPA axis. Through administration of
stresscopins, the excessive release of ACTH is avoided.
[0050] Major depression is a syndromal, episodic and recurrent
illness with both psychological and biological components. A
diagnosis of bipolar disorder is given to those patients with
recurring depression and mania. Those patients with recurrent
depression alone have a unipolar pattern. Within the spectrum of
depressive illness, there are two distinct subtypes: melancholic
depression and atypical depression. Melancholic depression is
equally common among those with a pattern of unipolar and bipolar
depression. Melancholic depression is characterized by hyposomnia
(early morning awakening), anorexia and diurnal variation in mood,
and is associated with a state of hyperarousal. Atypical depression
is more common in bipolar patients than in unipolar depressed
patients. Atypical depression is characterized by a state which
seems to be opposite to that of melancholic depression. Patients
with atypical depression have a syndrome of hypoarousal with
hypersomnia, hyperphagia, weight gain and mood liability.
[0051] Dysthymia is a chronic disorder characterized by symptoms
that include poor appetite or overeating, low energy (decreased
arousal), insomnia or hypersomnia, and poor concentration. These
functions are modulated by neuropeptides in the brain, such as CRH
and stresscopins. Generally, dysthymia is characterized by
hypothalamic CRH levels that are higher than normal, thereby
causing hyperactivity of the HPA axis. However, in dysthymia,
hypothalamic CRH levels can be lower than normal, causing
hypoactivity of the HPA axis, in individuals with a higher than
normal body mass index (BMI). Thus, in dysthymia, hypothalamic CRH
levels are inversely related to the BMI of the individual.
[0052] Affective disorders are extremely common in general medical
practice, as well as in psychiatry. The severity of these
conditions covers an extraordinarily broad range, from normal grief
reactions to severe, incapacitating, and sometimes fatal psychosis.
Typically these disorders are treated with antidepressant agents or
lithium salts. Nevertheless, many shortcomings and problems
continue to be associated with all drugs used to treat affective
disorders. In addition to less than-dramatic efficacy in some
cases, virtually all the drugs used to treat disorders of mood are
potentially lethal when acute over dosage occurs and can cause
appreciable morbidity even with careful clinical use. Stresscopins
find use as an anxiolytic agent.
[0053] Hypertension is a disease which, if untreated, strongly
predisposes to atherosclerotic cardiovascular disease. It is
estimated that as many as 1 in 4 adult Americans have hypertension.
Hypertension is approximately twice as common in persons with
diabetes as in those without. The prevalence of hypertension
increases with age.
[0054] Hypertension should not be diagnosed on the basis of a
single measurement. Initial elevated readings should be confirmed
on at least two subsequent visits over one week or more with
average diastolic blood pressure of 90 mmHg or greater or systolic
blood pressure of 140 mmHg or greater required for diagnosis of
hypertension. Special care is warranted in diagnosing hypertension
in persons with diabetes because of greater variability of blood
pressure and a much greater likelihood of isolated systolic
hypertension. A goal blood pressure of less than 1 30/85 mmHg is
recommended for these patients.
[0055] In addition to dietary changes, pharmacological treatment
may be required to control high blood pressure. The subject
peptides may be administered to reduce arterial blood pressure. In
addition, a secondary effect of reducing hypertension is reduction
of edema and inflammatory exudate volume.
[0056] After substantial stress, e.g. major surgery, severe burn,
emotional trauma, organ transplantation, and other life threatening
situations, the subject peptides may be administered to enhance the
stress coping responses. The regulation of the
hypothalamo-pituitary-adre- nal (HPA) axis in the operative and
perioperative period of major surgical procedures is necessary for
successful adaption to surgical stress. For example, plasma ACTH
has been found to be highly elevated during a surgical procedure;
which were temporally related to CRH levels. The immediate
postoperative period may be associated with profound elevations of
plasma ACTH, cortisol, and epinephrine. Results have indicated an
altered regulation of the HPA axis in the postoperative period of
patients after surgery, which are compatible with similar results
in patients after major abdominal surgery, burned patients, and
critically ill patients.
[0057] Pharmaceutical compositions containing stresscopin peptides
and derivatives therefrom are useful as cardioprotective agents,
e.g. to ameliorate ischemic injury or myocardial infarct size
consequent to myocardial ischemia. The development of new
therapeutic agents capable of limiting the extent of myocardial
injury, i.e. , the extent of myocardial infarction, following acute
myocardial ischemia is a major concern of modern cardiology. There
has also been interest in the development of therapies capable of
providing additional myocardial protection which could be
administered in conjunction with thrombolytic therapy, or alone,
since retrospective epidemiological studies have shown that
mortality during the first few years following infarction appears
to be related to original infarct size.
[0058] Myocardial ischemia is the result of an imbalance of
myocardial oxygen supply and demand and includes exertional and
vasospastic myocardial dysfunction. Exertional ischemia is
generally ascribed to the presence of critical atherosclerotic
stenosis involving large coronary arteries resulting in a reduction
in subendocardial flow. Vasospastic ischemia is associated with a
spasm of focal variety, whose onset is not associated with exertion
or stress. The spasm is better defined as an abrupt increase in
vascular tone.
[0059] The compounds of this invention can be normally administered
orally or parenterally, in the treatment of patients in need of
cardioprotective therapy. The dosage regimen is that which insures
maximum therapeutic response until improvement is obtained and
thereafter the minimum effective level which gives relief. Thus, in
general, the dosages are those that are therapeutically effective
in producing a cardioprotective effect, i.e., amelioration of
ischemic injury or myocardial infarct size consequent to myocardial
ischemia. It is also anticipated that the peptides would be useful
as an injectable dosage form which may be administered in an
emergency to a patient suffering from myocardial ischemia, etc.
[0060] The prevention or inhibition of illness leading to
inflammation is of significant concern, particularly for those
afflicted with autoimmune diseases such as arthritis and different
injuries, including sports-related injuries and musculoskeletal
ailments. Pain usually accompanies inflammation and vice versa.
Inflammation involves capillary dilation, with accumulation of
fluid and migration of phagocytic leukocytes, such as granulocytes
and monocytes, to the site of injury or lesion. Inflammation is
important in defending a host against a variety of infections, but
can also have undesirable consequences in inflammatory disorders.
Inflammatory conditions include autoimmune diseases; inflammation
caused by bacterial and viral infection, including response to
vaccination; local inflammation in response to trauma; graft
rejection; graft v. host disease, and the like. Stresscopin also
finds use in the treatment of different skin diseases.
[0061] Conditions of interest for treatment with the subject
peptides include musculoskeletal conditions, both inflammatory and
non-inflammatory in nature, and acute, subacute or chronic
presentation. For example, the composition may be used in the
treatment of both the early and late stages of inflammatory
arthritis, as well as non-infectious inflammatory arthropathy such
as rheumatoid arthritis, bursitis, tendinitis, soft tissue
injuries, Sjogren's syndrome, systemic lupus erythematous,
psoriatic arthritis, gout and other crystalline arthropathies,
capsulitis, carpal tunnel syndrome, myositis, polymyalgia,
rheumatica, synovitis and Reiter's syndrome. The compositions of
this invention may also be used in the prevention or treatment of
erosive osteoarthritis. Acute and chronic pain and inflammation are
often treated with anti-inflammatory/analgesic compounds such as
aspirin, ibuprofen and naproxen. The subject peptides may find use
in combinations with these compounds.
[0062] The stresscopin peptides and derivatives therefrom also find
use in the reduction of edema, for example in rheumatoid arthritis,
edema secondary to brain tumors or irradiation for cancer, edema
resulting from stroke, head trauma or spinal cord injury,
post-surgical edema, asthma and other respiratory diseases and
cystoid macular edema of the eye.
Formulations
[0063] The compounds of this invention can be incorporated into a
variety of formulations for therapeutic administration.
Particularly, agents that modulate stresscopin activity, or
stresscopin polypeptides and analogs thereof are formulated for
administration to patients for the treatment of stresscopin
dysfunction, where the stresscopin activity is undesirably high or
low. More particularly, the compounds of the present invention can
be formulated into pharmaceutical compositions by combination with
appropriate, pharmaceutically acceptable carriers or diluents, and
may be formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants, gels,
microspheres, and aerosols. As such, administration of the
compounds can be achieved in various ways, including oral, buccal,
rectal, parenteral, intraperitoneal, intradermal, transdermal,
intracheal, etc., administration. The stresscopin may be systemic
after administration or may be localized by the use of an implant
that acts to retain the active dose at the site of
implantation.
[0064] In pharmaceutical dosage forms, the compounds may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0065] For oral preparations, the compounds can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch;
[0066] with binders, such as crystalline cellulose, cellulose
derivatives, acacia, corn starch or gelatins; with disintegrators,
such as corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants, such as talc or magnesium
stearate; and if desired, with diluents, buffering agents,
moistening agents, preservatives and flavoring agents.
[0067] The compounds can be formulated into preparations for
injections by dissolving, suspending or emulsifying them in an
aqueous or nonaqueous solvent, such as vegetable or other similar
oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0068] The compounds can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0069] Furthermore, the compounds can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0070] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more compounds of the present invention.
Similarly, unit dosage forms for injection or intravenous
administration may comprise the compound of the present invention
in a composition as a solution in sterile water, normal saline or
another pharmaceutically acceptable carrier.
[0071] Implants for sustained release formulations are well-known
in the art. Implants are formulated as microspheres, slabs, etc.
with biodegradable or non-biodegradable polymers. For example,
polymers of lactic acid and/or glycolic acid form an erodible
polymer that is well-tolerated by the host. The implant is placed
in proximity to the site of infection, so that the local
concentration of active agent is increased relative to the rest of
the body.
[0072] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0073] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0074] Typical dosages for systemic administration range from 0.1
.mu.g to 100 milligrams per kg weight of subject per
administration. A typical dosage may be one tablet taken from two
to six times daily, or one time-release capsule or tablet taken
once a day and containing a proportionally higher content of active
ingredient. The time-release effect may be obtained by capsule
materials that dissolve at different pH values, by capsules that
release slowly by osmotic pressure, or by any other known means of
controlled release.
[0075] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Some of the specific compounds are more potent than others.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means. A preferred means
is to measure the physiological potency of a given compound.
[0076] The use of liposomes as a delivery vehicle is one method of
interest. The liposomes fuse with the cells of the target site and
deliver the contents of the lumen intracellularly. The liposomes
are maintained in contact with the cells for sufficient time for
fusion, using various means to maintain contact, such as isolation,
binding agents, and the like. In one aspect of the invention,
liposomes are designed to be aerosolized for pulmonary
administration. Liposomes may be prepared with purified proteins or
peptides that mediate fusion of membranes, such as Sendai virus or
influenza virus, etc. The lipids may be any useful combination of
known liposome forming lipids, including cationic lipids, such as
phosphatidylcholine. The remaining lipid will normally be neutral
lipids, such as cholesterol, phosphatidyl serine, phosphatidyl
glycerol, and the like.
[0077] For preparing the liposomes, the procedure described by Kato
et al. (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the
lipids and lumen composition containing the nucleic acids are
combined in an appropriate aqueous medium, conveniently a saline
medium where the total solids will be in the range of about 1-10
weight percent. After intense agitation for short periods of time,
from about 5-60 sec., the tube is placed in a warm water bath, from
about 25-40.degree. C. and this cycle is repeated about 5-10 times.
The composition is then sonicated for a convenient period of time,
generally from about 1-10 sec. and may be further agitated by
vortexing. The volume is then expanded by adding aqueous medium,
generally increasing the volume by about from 1-2 fold, followed by
shaking and cooling. This method allows for the incorporation into
the lumen of high molecular weight molecules.
[0078] For use in the above described formulations, stresscopin or
derivatives therefrom may be synthesized and stored as a solid
lyophilized powder which is reconstituted into a pharmaceutically
acceptable liquid immediately prior to use. Such formulations are
usually preferred because it is recognized by those skilled in the
art that lyophilized preparations generally maintain pharmaceutical
activity better over time than their liquid counterparts.
[0079] In addition, stresscopins and their analogs could be applied
topically on the skin as well as administered as aerosal
sprays.
[0080] Alternatively, the peptides may be formulated as a liquid,
erg. comprising a buffer at a concentration of from about 1 mM to
about 50 mM that functions to maintain the pH, wherein the anion of
said buffer may be selected from the group consisting of acetate,
phosphate, carbonate, succinate, citrate, borate, tartrate,
fumarate and lactate; and an alcohol which may be selected from the
group consisting of mannitol, sorbitol, ribotol, arabitol, xylitol,
inositol, galactitol, methanol, ethanol and glycerol. Other
additives may include amino acids such as methionine, arginine,
lysine, glutamic acid, cysteine, glutathione, and the like, where
amino acids are generally present in concentrations ranging from
about 1 mM to about 100 mM. Various sugars are optionally included
in the formulations, including, for example, glucose, sucrose,
lactose, fructose, trehalose, mannose, and the like. Additive
sugars are generally present in concentrations ranging from about 1
% to about 10%.
Stresscopin Nucleic Acids
[0081] The invention includes nucleic acids having a sequence set
forth in SEQ ID NO:1 and SEQ ID NO:4; nucleic acids that hybridize
under stringent conditions, particularly conditions of high
stringency, to the sequences set forth in SEQ ID NO:1 and SEQ ID
NO:2; genes corresponding to the provided nucleic acids; sequences
encoding stresscopins; and fragments and derivatives thereof. Other
nucleic acid compositions contemplated by and within the scope of
the present invention will be readily apparent to one of ordinary
skill in the art when provided with the disclosure here.
[0082] The nucleic acids of the invention include nucleic acids
having sequence similarity or sequence identity to SEQ ID NO:1 and
SEQ ID NO:4. Nucleic acids having sequence similarity are detected
by hybridization under low stringency conditions, for example, at
50.degree. C. and 10.times.SSC (0.9 M saline/0.09 M sodium citrate)
and remain bound when subjected to washing at 55.degree. C. in
1.times.SSC. Sequence identity can be determined by hybridization
under stringent conditions, for example, at 50.degree. C. or higher
and 0.1.times.SSC (9 mM saline/0.9 mM sodium citrate).
Hybridization methods and conditions are well known in the art,
see, e.g., U.S. Pat. No. 5,707,829. Nucleic acids that are
substantially identical to the provided nucleic acid sequence, e.g.
allelic variants, genetically altered versions of the gene, etc.,
bind to SEQ ID NO:1 or SEQ ID NO:4 under stringent hybridization
conditions. By using probes, particularly labeled probes of DNA
sequences, one can isolate homologous or related genes. The source
of homologous genes can be any species, e.g. primate species,
particularly human; rodents, such as rats and mice; canines,
felines, bovines, ovines, equines, fish, yeast, nematodes, etc.
[0083] In one embodiment, hybridization is performed using at least
18 contiguous nucleotides (nt) of SEQ ID NO:1 and SEQ ID NO:4, or a
DNA encoding the polypeptide of SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:5 or SEQ ID NO:6. Such a probe will preferentially hybridize
with a nucleic acid comprising the complementary sequence, allowing
the identification and retrieval of the nucleic acids that uniquely
hybridize to the selected probe. Probes of more than 18 nt can be
used, e.g., probes of from about 18 nt to about 25, 50,100, 250, or
500 nt, but 18 nt usually represents sufficient sequence for unique
identification.
[0084] Nucleic acids of the invention also include naturally
occurring variants of the nucleotide sequences (e.g., degenerate
variants, allelic variants, etc. ). Variants of the nucleic acids
of the invention are identified by hybridization of putative
variants with nucleotide sequences disclosed herein, preferably by
hybridization under stringent conditions. For example, by using
appropriate wash conditions, variants of the nucleic acids of the
invention can be identified where the allelic variant exhibits at
most about 25-30% base pair (bp) mismatches relative to the
selected nucleic acid probe. In general, allelic variants contain
15-25% bp mismatches, and can contain as little as even 5-15%, or
2-5%, or 1-2% bp mismatches, as well as a single bp mismatch.
[0085] The invention also encompasses homologs corresponding to the
nucleic acids of SEQ ID NO:1 and SEQ ID NO:4, or a DNA encoding the
polypeptide of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID
NO:6, where the source of homologous genes can be any mammalian
species, e.g., primate species, particularly human; rodents, such
as rats; canines, felines, bovines, ovines, equines, fish, yeast,
nematodes, etc. Between mammalian species, e.g., human and mouse,
homologs generally have substantial sequence similarity, e.g., at
least 75% sequence identity, usually at least 90%, more usually at
least 95% between nucleotide sequences. Sequence similarity is
calculated based on a reference sequence, which may be a subset of
a larger sequence, such as a conserved motif, coding region,
flanking region, etc. A reference sequence will usually be at least
about 18 contiguous nt long, more usually at least about 30 nt
long, and may extend to the complete sequence that is being
compared. Algorithms for sequence analysis are known in the art,
such as gapped BLAST, described in Altschul et al. Nucl. Acids Res.
(1997) 25:3389-3402.
[0086] The subject nucleic acids can be cDNAs or genomic DNAs, as
well as fragments thereof, particularly fragments that encode a
biologically active polypeptide and/or are useful in the methods
disclosed herein (e.g., in diagnosis, as a unique identifier of a
differentially expressed gene of interest, etc. ). The term "cDNA"
as used herein is intended to include all nucleic acids that share
the arrangement of sequence elements found in native mature mRNA
species, where sequence elements are exons and 3" and 5" non-coding
regions. Normally mRNA species have contiguous exons, with the
intervening introns, when present, being removed by nuclear RNA
splicing, to create a continuous open reading frame encoding a
polypeptide of the invention.
[0087] A genomic sequence of interest comprises the nucleic acid
present between the initiation codon and the stop codon, as defined
in the listed sequences, including all of the introns that are
normally present in a native chromosome. It can further include the
3" and 5" untranslated regions found in the mature mRNA. It can
further include specific transcriptional and translational
regulatory sequences, such as promoters, enhancers, etc., including
about 1 kb, but possibly more, of flanking genomic DNA at either
the 5" and 3" end of the transcribed region. The genomic DNA can be
isolated as a fragment of 100 kbp or smaller; and substantially
free of flanking chromosomal sequence. The genomic DNA flanking the
coding region, either 3" and 5", or internal regulatory sequences
as sometimes found in introns, contains sequences required for
proper tissue, stage-specific, or disease-state specific
expression.
[0088] The nucleic acid compositions of the subject invention can
encode all or a part of the subject polypeptides. Double or single
stranded fragments can be obtained from the DNA sequence by
chemically synthesizing oligonucleotides in accordance with
conventional methods, by restriction enzyme digestion, by PCR
amplification, etc. Isolated nucleic acids and nucleic acid
fragments of the invention comprise at least about 18, about 50,
about 100, to about 500 contiguous nt selected from the nucleic
acid sequence as shown in SEQ ID NO:1 and SEQ ID NO:4. For the most
part, fragments will be of at least 18usually at least 25 and up to
at least about 50 contiguous nt in length or more.
[0089] Probes specific to the nucleic acid of the invention can be
generated using the nucleic acid sequence disclosed in SEQ ID NO:1
and SEQ ID NO:4, or a DNA encoding the polypeptide of SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6. The probes are preferably
at least about 18 nt, 25 nt or more of the corresponding contiguous
sequence. The probes can be synthesized chemically or can be
generated from longer nucleic acids using restriction enzymes. The
probes can be labeled, for example, with a radioactive,
biotinylated, or fluorescent tag. Preferably, probes are designed
based upon an identifying sequence of one of the provided
sequences. More preferably, probes are designed based on a
contiguous sequence of one of the subject nucleic acids that remain
unmasked following application of a masking program for masking low
complexity (e.g., BLASTX) to the sequence, i.e., one would select
an unmasked region, as indicated by the nucleic acids outside the
poly-n stretches of the masked sequence produced by the masking
program.
[0090] The nucleic acids of the subject invention are isolated and
obtained in substantial purity, generally as other than an intact
chromosome. Usually, the nucleic acids, either as DNA or RNA, will
be obtained substantially free of other naturally-occurring nucleic
acid sequences, generally being at least about 50%, usually at
least about 90% pure and are typically "recombinant," e.g., flanked
by one or more nucleotides with which it is not normally associated
on a naturally occurring chromosome.
[0091] The nucleic acids of the invention can be provided as a
linear molecule or within a circular molecule, and can be provided
within autonomously replicating molecules (vectors) or within
molecules without replication sequences. Expression of the nucleic
acids can be regulated by their own or by other regulatory
sequences known in the art. The nucleic acids of the invention can
be introduced into suitable host cells using a variety of
techniques available in the art, such as transferrin
polycation-mediated DNA transfer, transfection with naked or
encapsulated nucleic acids, liposome-mediated DNA transfer,
intracellular transportation of DNA-coated latex beads, protoplast
fusion, viral infection, electroporation, gene gun, calcium
phosphate-mediated transfection, and the like.
Modulation of Stresscopin Expression
[0092] The stresscopin genes, gene fragments, or the encoded
protein or protein fragments are useful in gene therapy to treat
disorders associated with stresscopin defects. From a therapeutic
point of view, inhibiting stresscopin activity has a therapeutic
effect on a number of disorders relating to stress. Inhibition is
achieved in a number of ways. Antisense stresscopin sequences may
be administered to inhibit expression. Competitive binding
antagonists, for example, a peptide that mimics stresscopin binding
may be used to inhibit activity. Other inhibitors are identified by
screening for biological activity in a stresscopin-based binding
assay.
[0093] Expression vectors may be used to introduce the stresscopin
gene into a cell. Such vectors generally have convenient
restriction sites located near the promoter sequence to provide for
the insertion of nucleic acid sequences. Transcription cassettes
may be prepared comprising a transcription initiation region, the
target gene or fragment thereof, and a transcriptional termination
region. The transcription cassettes may be introduced into a
variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus;
adenovirus; and the like, where the vectors are able to transiently
or stably be maintained in the cells, usually for a period of at
least about one day, more usually for a period of at least about
several days to several weeks.
[0094] The gene or stresscopin peptide may be introduced into
tissues or host cells by any number of routes, including viral
infection, microinjection, or fusion of vesicles. Jet injection may
also be used for intramuscular administration, as described by
Furth et al. (1992) Anal Biochem 205:365-368. The DNA may be coated
onto gold microparticles, and delivered intradermally by a particle
bombardment device, or "gene gun" as described in the literature
(see, for example, Tang et al. (1992) Nature 356:152-154), where
gold microprojectiles are coated with the stresscopin or DNA, then
bombarded into skin cells.
[0095] Antisense molecules can be used to down-regulate expression
of stresscopin in cells. The anti-sense reagent may be antisense
oligonucleotides (ODN), particularly synthetic ODN having chemical
modifications from native nucleic acids, or nucleic acid constructs
that express such anti-sense molecules as RNA. The antisense
sequence is complementary to the mRNA of the targeted gene, and
inhibits expression of the targeted gene products. Antisense
molecules inhibit gene expression through various mechanisms, e.g.
by reducing the amount of mRNA available for translation, through
activation of RNAse H, or steric hindrance. One or a combination of
antisense molecules may be administered, where a combination may
comprise multiple different sequences.
[0096] Antisense molecules may be produced by expression of all or
a part of the target gene sequence in an appropriate vector, where
the transcriptional initiation is oriented such that an antisense
strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a synthetic oligonucleotide. Antisense oligonucleotides
will generally be at least about 7, usually at least about 12, more
usually at least about 20 nucleotides in length, and not more than
about 500, usually not more than about 50, more usually not more
than about 35 nucleotides in length, where the length is governed
by efficiency of inhibition, specificity, including absence of
cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and
selective inhibitors of gene expression (see Wagner et al. (1996)
Nature Biotechnol. 14 :840-844).
[0097] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in an in
vitro or animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0098] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in vitro or
in an animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0099] Antisense oligonucleotides may be chemically synthesized by
methods known in the art (see, Wagner et al. (1993), supra and
Milligan et al., supra). Preferred oligonucleotides are chemically
modified from the native phosphodiester structure, in order to
increase their intracellular stability and binding affinity. A
number of such modifications have been described in the literature
which alter the chemistry of the backbone, sugars or heterocyclic
bases.
[0100] Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.alpha.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .beta.-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
[0101] As an alternative to anti-sense inhibitors, catalytic
nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc.
may be used to inhibit gene expression. Ribozymes may be
synthesized in vitro and administered to the patient, or may be
encoded on an expression vector, from which the ribozyme is
synthesized in the targeted cell (for example, see International
patent application WO 95/23225, and Beigelman et al. (1995) Nucl.
Acids Res. 23 :4434-4442). Examples of oligonucleotides with
catalytic activity are described in WO 9506764. Conjugates of
anti-sense ODN with a metal complex, e.g. terpyridylCu(II), capable
of mediating mRNA hydrolysis are described in Bashkin et al. (1995)
Appl. Biochem. Biotechnol. 54 :43-56.
[0102] Agents that block stresscopin activity provide a point of
intervention in an important signaling pathway. Numerous agents are
useful in reducing stresscopin activity, including agents that
directly modulate stresscopin expression as described above, e.g.
expression vectors, anti-sense specific for stresscopin; and agents
that act on the stresscopin protein, e.g. stresscopin specific
antibodies and analogs thereof, small organic molecules that block
stresscopin binding activity, etc.
Diagnostic Uses
[0103] DNA-based reagents derived from the sequence of
stresscopins, e.g. PCR primers, oligonucleotide or cDNA probes, as
well as antibodies against stresscopins, are used to screen patient
samples, e.g. biopsy-derived tissues, blood samples, etc. , for
amplified stresscopin DNA, or increased expression of stresscopin
mRNA or proteins. DNA-based reagents are also designed for
evaluation of chromosomal loci implicated in certain diseases e.g.
for use in loss-of-heterozygosity (LOH) studies, or design of
primers based on stresscopin coding sequence.
[0104] The polynucleotides of the invention can be used to detect
differences in expression levels between two samples. A difference
between the protein levels, or the mRNA in the two tissues that are
compared, for example, in molecular weight, amino acid or
nucleotide sequence, or relative abundance, indicates a change in
the gene, or a gene which regulates it, in the tissue of the human
that was suspected of being diseased.
[0105] The subject nucleic acid and/or polypeptide compositions may
be used to analyze a patient sample for the presence of
polymorphisms associated with a disease state or genetic
predisposition to a disease state. Biochemical studies may be
performed to determine whether a sequence polymorphism in a
stresscopin coding region or control regions is associated with
disease, particularly stress related disorders, e.g. anxiety
disorders. Disease associated polymorphisms may include deletion or
truncation of the gene, mutations that alter expression level, that
affect the binding activity of the protein, the kinase activity
domain, etc.
[0106] Changes in the promoter or enhancer sequence that may affect
expression levels of stresscopin can be compared to expression
levels of the normal allele by various methods known in the art.
Methods for determining promoter or enhancer strength include
quantitation of the expressed natural protein; insertion of the
variant control element into a vector with a reporter gene such as
.beta.-galactosidase, luciferase, chloramphenicol
acetyltransferase, etc. that provides for convenient quantitation;
and the like.
[0107] A number of methods are available for analyzing nucleic
acids for the presence of a specific sequence, e.g. a disease
associated polymorphism. Where large amounts of DNA are available,
genomic DNA is used directly. Alternatively, the region of interest
is cloned into a suitable vector and grown in sufficient quantity
for analysis. Cells that express stresscopin may be used as a
source of mRNA, which may be assayed directly or reverse
transcribed into cDNA for analysis. The nucleic acid may be
amplified by conventional techniques, such as the polymerase chain
reaction (PCR), to provide sufficient amounts for analysis. The use
of the polymerase chain reaction is described in Saiki et al.
(1985) Science 239 :487, and a review of techniques may be found in
Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press
1989, pp. 14.2-14.33.
[0108] A detectable label may be included in an amplification
reaction. Suitable labels include fluorochromes, e.g. fluorescein
isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,
allophycocyanin,6-carboxyflu-
orescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein
(JOE), 6-carboxy-X-rhodamine (ROX),
6-carboxy-2,4,7,4,7-hexachlorofluorescein (HEX),
5-carboxyfluorescein (5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhoda-
mine (TAMRA), radioactive labels, e.g. .sup.32P, .sup.35S, .sup.3H;
etc. The label may be a two stage system, where the amplified DNA
is conjugated to biotin, haptens, etc. having a high affinity
binding partner, e.g. avidin, specific antibodies, etc. , where the
binding partner is conjugated to a detectable label. The label may
be conjugated to one or both of the primers. Alternatively, the
pool of nucleotides used in the amplification is labeled, so as to
incorporate the label into the amplification product.
[0109] The sample nucleic acid, e.g., amplified or cloned fragment,
is analyzed by one of a number of methods known in the art. The
nucleic acid may be sequenced by dideoxy or other methods, and the
sequence of bases compared to a wild-type stresscopin sequence.
Hybridization with the variant sequence may also be used to
determine its presence, by Southern blots, dot blots, etc. The
hybridization pattern of a control and variant sequence to an array
of oligonucleotide probes immobilized on an array, may also be used
as a means of detecting the presence of variant sequences. Single
strand conformational polymorphism (SSCP) analysis, denaturing
gradient gel electrophoresis (DGGE), and heteroduplex analysis in
gel matrices are used to detect conformational changes created by
DNA sequence variation as alterations in electrophoretic mobility.
Alternatively, where a polymorphism creates or destroys a
recognition site for a restriction endonuclease, the sample is
digested with that endonuclease, and the products size fractionated
to determine whether the fragment was digested. Fractionation is
performed by gel or capillary electrophoresis, particularly
acrylamide or agarose gels.
[0110] Screening for mutations in stresscopins may be based on the
functional or antigenic characteristics of the protein. Protein
truncation assays are useful in detecting deletions that may affect
the biological activity of the protein. Various immunoassays
designed to detect polymorphisms in stresscopin proteins may be
used in screening. Where many diverse genetic mutations lead to a
particular disease phenotype, functional protein assays have proven
to be effective screening tools. The activity of the encoded
stresscopin protein in binding assays, etc., may be determined by
comparison with the wild-type protein. Proteins may also be
screened for the presence of post-translational modification of the
stresscopin proteins, e.g. under pathological conditions, including
proteolytic fragments, amidation, acetylation etc.
[0111] Antibodies specific for stresscopin may be used in staining
or in immunoassays. Samples, as used herein, include biological
fluids such as blood, cerebrospinal fluid, dialysis fluid and the
like; organ or tissue culture derived fluids; and fluids extracted
from physiological tissues. Also included in the term are
derivatives and fractions of such fluids. The cells may be
dissociated, in the case of solid tissues, or tissue sections may
be analyzed. Alternatively a lysate of the cells may be
prepared.
[0112] Diagnosis may be performed by a number of methods to
determine the absence or presence or altered amounts of normal or
abnormal stresscopin in patient cells. For example, detection may
utilize staining of cells or histological sections, performed in
accordance with conventional methods. Cells are permeabilized to
stain cytoplasmic molecules. The antibodies of interest are added
to the cell sample, and incubated for a period of time sufficient
to allow binding to the epitope, usually at least about 10 minutes.
The antibody may be labeled with radioisotopes, enzymes,
fluorescers, chemiluminescers, or other labels for direct
detection. Alternatively, a second stage antibody or reagent is
used to amplify the signal. Such reagents are well known in the
art. For example, the primary antibody may be conjugated to biotin,
with horseradish peroxidase-conjugated avidin added as a second
stage reagent. Alternatively, the secondary antibody conjugated to
a fluorescent compound, e.g. fluorescein rhodamine, Texas red, etc.
Final detection uses a substrate that undergoes a color change in
the presence of the peroxidase. The absence or presence of antibody
binding may be determined by various methods, including flow
cytometry of dissociated cells, microscopy, radiography,
scintillation counting, etc.
[0113] In some embodiments, the methods are adapted for use in
vivo. In these embodiments, a detectably-labeled moiety, e.g., an
antibody, which is specific for stresscopin is administered to an
individual (e.g., by injection), and labeled cells are located
using standard imaging techniques, including, but not limited to,
magnetic resonance imaging, computed tomography scanning, and the
like.
[0114] Diagnostic screening may also be performed for polymorphisms
that are genetically linked to a disease predisposition,
particularly through the use of microsatellite markers or single
nucleotide polymorphisms. Frequently the microsatellite
polymorphism itself is not phenotypically expressed, but is linked
to sequences that result in a disease predisposition. However, in
some cases the microsatellite sequence itself may affect gene
expression. Microsatellite linkage analysis may be performed alone,
or in combination with direct detection of polymorphisms, as
described above. The use of microsatellite markers for genotyping
is well documented. For examples, see Mansfield et al. (1994)
Genomics 24 :225-233; Ziegle et al. (1992) Genomics 74:1026-1031;
Dib et al., supra.
[0115] Diagnostic screening may also be performed for polymorphisms
that are genetically linked to a predisposing mutation,
particularly through the use of microsatellite markers or single
nucleotide polymorphisms. Frequently the microsatellite
polymorphism itself is not phenotypically expressed, but is linked
to sequences that result in a disease predisposition. However, in
some cases the microsatellite sequence itself may affect gene
expression. Microsatellite linkage analysis may be performed alone,
or in combination with direct detection of polymorphisms, as
described above. The use of microsatellite markers for genotyping
is well documented. For examples, see Mansfield et al. (1994)
Genomics 24:225-233;
[0116] Ziegle et al. (1992) Genomics 74 :1026-1031; Dib et al.,
supra.
[0117] The detection methods can be provided as part of a kit.
Thus, the invention further provides kits for detecting the
presence of an mRNA encoding stresscopin, and/or a polypeptide
encoded thereby, in a biological sample. Procedures using these
kits may be performed by clinical laboratories, experimental
laboratories, medical practitioners, or private individuals. The
kits of the invention for detecting a polypeptide comprise a moiety
that specifically binds the polypeptide, which may be a specific
antibody. The kits of the invention for detecting a nucleic acid
comprise a moiety that specifically hybridizes to such a nucleic
acid. The kit may optionally provide additional components that are
useful in the procedure, including, but not limited to, buffers,
developing reagents, labels, reacting surfaces, means for
detection, control samples, standards, instructions, and
interpretive information.
Genetically Altered Cell or Animal Models for Stresscopin
Function
[0118] The subject nucleic acids can be used to generate transgenic
animals or site specific gene modifications in cell lines.
Transgenic animals may be made through homologous recombination,
where the normal stresscopin locus is altered. Alternatively, a
nucleic acid construct is randomly integrated into the genome.
Vectors for stable integration include plasmids, retroviruses and
other animal viruses, YACs, and the like.
[0119] The modified cells or animals are useful in the study of
stresscopin function and regulation. For example, a series of small
deletions and/or substitutions may be made in the stresscopin gene
to determine the role of different residues in receptor binding,
signal transduction, etc. Of interest is the use of stresscopin to
construct transgenic animal models for stress related disorders,
where expression of stresscopin is specifically reduced or absent.
Specific constructs of interest include anti-sense stresscopin,
which will block stresscopin expression and expression of dominant
negative stresscopin mutations. A detectable marker, such as lac Z
may be introduced into the stresscopin locus, where up-regulation
of stresscopin expression will result in an easily detected change
in phenotype.
[0120] One may also provide for expression of the stresscopin gene
or variants thereof in cells or tissues where it is not normally
expressed or at abnormal times of development. By providing
expression of stresscopin protein in cells in which it is not
normally produced, one can induce changes in cell behavior, e.g. in
the control of cell growth and tumorigenesis.
[0121] DNA constructs for homologous recombination will comprise at
least a portion of the stresscopin gene with the desired genetic
modification, and will include regions of homology to the target
locus. The regions of homology may include coding regions, or may
utilize intron and/or genomic sequence. DNA constructs for random
integration need not include regions of homology to mediate
recombination. Conveniently, markers for positive and negative
selection are included. Methods for generating cells having
targeted gene modifications through homologous recombination are
known in the art. For various techniques for transfecting mammalian
cells, see Keown et al. (1990) Methods in Enzymology
185:527-537.
[0122] For embryonic stem (ES) cells, an ES cell line may be
employed, or embryonic cells may be obtained freshly from a host,
e.g. mouse, rat, guinea pig, etc. Such cells are grown on an
appropriate fibroblast-feeder layer or grown in the presence of
leukemia inhibiting factor (LIF). When ES or embryonic cells have
been transformed, they may be used to produce transgenic animals.
After transformation, the cells are plated onto a feeder layer in
an appropriate medium. Cells containing the construct may be
detected by employing a selective medium. After sufficient time for
colonies to grow, they are picked and analyzed for the occurrence
of homologous recombination or integration of the construct. Those
colonies that are positive may then be used for embryo manipulation
and blastocyst injection. Blastocysts are obtained from 4 to 6 week
old superovulated females. The ES cells are trypsinized, and the
modified cells are injected into the blastocoel of the blastocyst.
After injection, the blastocysts are returned to each uterine horn
of pseudopregnant females. Females are then allowed to go to term
and the resulting offspring screened for the construct. By
providing for a different phenotype of the blastocyst and the
genetically modified cells, chimeric progeny can be readily
detected.
[0123] The chimeric animals are screened for the presence of the
modified gene and males and females having the modification are
mated to produce homozygous progeny. If the gene alterations cause
lethality at some point in development, tissues or organs can be
maintained as allogeneic or congenic grafts or transplants, or in
culture. The transgenic animals may be any non-human mammal, such
as laboratory animals, domestic animals, etc. The transgenic
animals may be used in functional studies, drug screening, etc., to
determine the effect of a candidate drug on stress responses.
Experimental
[0124] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc. ) but some experimental
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, molecular weight is weight
average molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
[0125] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0126] The present invention has been described in terms of
particular embodiments found or proposed by the present inventor to
comprise preferred modes for the practice of the invention. It will
be appreciated by those of skill in the art that, in light of the
present disclosure, numerous modifications and changes can be made
in the particular embodiments exemplified without departing from
the intended scope of the invention. For example, due to codon
redundancy, changes can be made in the underlying DNA sequence
without affecting the protein sequence. Moreover, due to biological
functional equivalency considerations, changes can be made in
protein structure without affecting the biological action in kind
or amount. All such modifications are intended to be included
within the scope of the appended claims.
Materials and Methods
[0127] Cloning, Sequencing and expression analysis of human
stresscopin cDNA. The identity of stresscopin 1 mRNA was deduced by
comparisons of multiple human genomic DNA sequences (AC024179 and
AC005903) and a partial EST sequence (BE390203). The deduced ORF of
stresscopin 1 was verified by PCR using nested gene-specific
primers and Marathon-ready cDNA templates (Clontech, Inc., Palo
Alto, Calif.) from human testis and prostate. Stresscopin 2 was
initially identified as a partial cDNA (AW293249) from a human
subtracted library NCl_CGAP_Sub4. The identity of this gene was
verified by rapid amplification of 3" cDNA ends using a human
Marathon-ready colon cDNA library.
[0128] Amplified cDNA fragments were gel-purified and subcloned
following blunt-end ligation. To determine the expression profile
of the stresscopin 1 gene, stresscopin transcript in 23 human
tissues was amplified by high stringency PCR using a panel of
genomic DNA-free first strand cDNAs primed with oligo-d(T) primer
(Origene Technologies, Inc., Rockville, Md.) as template.
[0129] Immunohistochemical analysis. Specific rabbit
anti-stresscopin 1 and anti-stresscopin 2 antibodies were generated
using synthetic peptides corresponding to the mature region of
stresscopin 1 and stresscopin 2, respectively, as the antigen
(Strategic Biosolutions, Ramona, Calif.). The stresscopin peptides
were conjugated to the carrier protein keyhole limpet hemocyanin
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
before immunization. Antibodies were purified using
antigen-conjugated affinity columns and their titers determined by
ELISA.
[0130] Tissues were obtained from adult male mice and embedded in
paraffin following fixation in Bouin"s solution. Following
incubation in xylene, tissue sections were blocked with 5% goat
serum in PBS to saturate nonspecific binding sites. Sections were
then incubated with the specific anti-stresscopin antibody for 2 h
at room temperature in a moist chamber before washing for three
times (20 min each) in PBS with 0.1 %Tween 20. Following incubation
with the primary antibody, sections were incubated with
gold-conjugated goat anti-rabbit IgG. Sections were then washed
before being stained with the SilvEnhance solution (Zymed
Laboratory, Inc., South San Francisco, Calif.) and counterstained
with hematoxylin. Negative controls were performed by substituting
the primary antibodies with antibodies presaturated with the
peptide antigen.
[0131] Peptide systhesis and analysis. The stresscopin peptides
with greater than 95% purity were synthesized using a
Symphony/Multiplex.TM. automated peptide synthesizer based on the
solid phase fluorenylmethoxycarbonyl protocol. All peptides
synthesized were routinely analyzed by reverse phase HPLC with a
Vydac C18 analytical column and Mass Spectrometry using a MALDI-TOF
(matrix-assisted laser desorption ionization-time of flight)
Voyager-DE RP Biospectrometry Workstation. Full-length stresscopin
prepared with this protocol agrees with a calculated 4691 MW of the
amidated form of stresscopin. CRH and urocortin were purchased from
Sigma Biochemicals, Inc. (St Louis, Mo.). Astressin was purchased
from Bachem Feinchemicalien, Bubendorf, Switzerland. Radiolabeled
[.sup.125I] human urocortin (2000 Cu/mmole) tracer was purchased
from Amersham Pharmacia (Arlington Heights, ILL.).
[0132] Construction of expression vectors for CRH receptors and the
cAMP assay. To obtain full-length type I and type II CRH receptor
cDNAs for functional assays, nested gene-specific primers flanking
the receptor ORF were used to PCR full-length cDNA fragments of CRH
R1, CRH R2-.alpha., and CRH R2 .beta. receptors. The receptor cDNA
fragments were subcloned into the pcDNA3.1/Zeo vector with a
prolactin signal peptide for secretion and triple FLAG/M1 epitope
cassette upstream of the multiple cloning site. CRH receptor
expression vectors were transfected into human 293T cells under
serum-free conditions for 2 h followed by incubation in DMEM/F12
medium with 10% fetal bovine serum (FBS) for another 48 h. For the
estimation of adenylate cyclase activation by stresscopin and
related hormones, transfected cells were seeded at a concentration
of 100,000 cell/well in 48 well culture plates in DMEM/F12 medium
containing 0.1% BSA and 2.5 mM IBMX for 16 h. Contents of cAMP in
whole cell lysate were determined using a cAMP radioimmunoassay. To
analyze the effects of stresscopin and related hormones on cAMP
accumulation in cells constitutively expressing CRH receptors, a
human retinoblastoma cell line (Y79) and a rat cardiac cell line
(A7r5) were purchased from ATCC (Manassas, Va.) and maintained in
DMEM/F12 medium with 20% FBS before treatment with different
hormones.
[0133] Phylogenetic inference. A multiple alignment of selected
vertebrate CRH family protein sequences was constructed with
ClustaIX and corrected using a published alignment of mature
protein data. Phylogenetic analysis was carried out by the
neighboring-joining method as well as the BlockMaker using Gibbs
method.
[0134] Recptor binding assay. Human 293T cells expressing different
isoforms of CRH R2 were incubated with 50,000 cpm (0.1 g) of
.sup.125I-urocortin and various concentrations of nonradioactive
peptides diluted in a binding buffer consisting of PBS, a protease
inhibitor cocktail (Sigma Biochemicals, Inc.), and 0.1% BSA. After
60 min incubation at 37.degree. C., the cell-associated ligand was
estimated following centrifugation and repeated washing.
Radioactivity of samples was determined in a GENESYS-counter
(Laboratory Technologies, Inc., Maple Park, ILL.).
[0135] Food intake and body weight in food-restricted mice. Six
week-old male mice (20-25 g b.w.) of the inbreed Balb/C strain were
housed individually in a regulated environment with 12L/12D light
cycle. Before feeding tests, mice were deprived of food for 16 h
with free access to water and injected with testing reagents i.p.
at 10 AM the following day. Food intake was measured by placing
preweighed pellets in the cage and weighing the uneaten pellets at
2, 4 and 8 h after injection. Body weight was also monitored at 0,
2, 4, and 8 h.
[0136] Effects on gastric emtying in food-restricted mice. To study
the effects on gastric emptying, mice were deprived of food for 16
h with free access to water. The fasted mice were then given free
access to preweighed pellets for 90 min. and were injected i.p.
with different hormones or saline. The mice were deprived of food
again after i.p. injection and sacrificed by cervical dislocation
at 2 h. after injection. The stomach was excised at point of the
pylorus and cardia before determination of its wet weight. Gastric
emptying was calculated by comparison with stomach weight of
control mice sacrificed at 0 h. following injection. See, Asakawa,
A. et al. (1999) Gastroenterology 116: 1287-1292.
[0137] Effects on heat-induced paw edema formation in anaesthetized
rats. The anti-inflammatory effect of stresscopin and related
peptides were assayed using an established model (Turnbull et al.
(1996) Euro. J. Pharmacol. 303 :213-216). Briefly, 5-week-old male
Sprague-Dawley rats were injected with 20 nM of the testing peptide
and anaesthetized with ketamine (100 mg/Kg). Thirty min. later, paw
edema was induced following a one minute exposure to hot water at
58.degree. C. The animals were sacrificed 30 min later. Both paws
were removed at the ankle joint and weighed. The degree of edema
was estimated as the differences in weight gain between the heated
and unheated paw divided by the weight of the unheated paw.
[0138] In Vivo and in Vitro assay of pituitary ACTH releasing
activity. Anterior pituitaries were obtained from 6-week-old male
Sprague-Dawley rats. Following dispersion using collagenase and
mechanical pipetting, cells were resuspended and cultured for 3
days in DMEM with 10% FBS (2.times.10.sup.5 cells/well). Before
hormonal treatment, cells were washed twice with serum-free medium
followed by incubation with DMEM/F12 media containing 0.1% BSA, 2.5
mM IBMX, and different hormones (1 nM). After 2 h at 37.degree. C.,
media were collected and assayed for ACTH contents using a
radioimmunoassay from Diagnostic Systems Laboratories, Inc.
(Webster, Tex.). To detect the ACTH-releasing activity of
stresscopins and related peptides in vivo, 6-week-old male
Sprague-Dawley rats were injected with different hormones i.p. (2
nmoles/kg) and sacrificed 30 min following treatment. Whole blood
was collected with 60 lU/ml heparin and serum obtained for ACTH
measurement.
Results
[0139] As shown in FIG. 1, there is significant homology between
the provided stresscopin sequences, and other genes in the
corticitropin releasing hormone gene family. Two human paralogs of
CRH/urocortin have been identified, stresscopin 1 and stresscopin
2. A putative stresscopin ortholog from Japanese pufferfish
(Takifugu rubripes) is also identified. Stresscopin 1 encodes a
prepro-protein of 112 amino acids and a putative mature protein of
43 amino acids whereas the 161-amino-acid open reading frame (ORF)
of stresscopin 2 contains a predicted 41-amino-acid mature peptide
(FIGS. 1a and 1b).
[0140] Although the overall amino acid sequences of stresscopins
from human and fish showed no similarity to known proteins, a
stretch of 30 residues at their C-termini adopted an extended
.alpha.-helical structure shared by all CRH family peptides (FIG.
1c). Both human stresscopin ORFs contain a signal peptide for
secretion. The predicted mature regions are flanked by potential
proteolytic cleavage sites and an alpha-amidation donor residue.
The identity of stresscopin 1 and stresscopin 2 transcripts was
confirmed following PCR of cDNAs from human testis and colon,
respectively.
[0141] Alignment of the mature peptides with related CRH family
hormones indicated that mature stresscopins from human and
pufferfish, but not the prepro-regions, show 35-38% identity to
other family proteins (FIG. 1c). These novel peptides share
identical secondary structures, although the predicted structures
of mature stresscopin 1 and stresscopin 2 are distinct from that of
other family peptides at their N-terminus (FIG. 1c). Phylogenetic
analysis of nine CRH family proteins from fish, frog, and mammals
suggested the ancient evolution of three subgroups of CRH family
proteins, with the human and pufferfish stresscopins clustered in a
separate branch (Fig. 1d).
[0142] Based on PCR analysis using a panel of human cDNAs (1
ng/reaction) from 23 different tissues, stresscopin 1 transcript
was found in brain and multiple peripheral tissues (heart, kidney,
spleen, lung, muscle, stomach, testis, placenta, thyroid, adrenal,
pancreas, ovary, peripheral blood cells, bone marrow, and fetal
liver) (FIG. 2a, panel 1). The same analysis using diluted template
cDNA (10 pg/reaction) shows the stresscopin 1 transcript only in
brain, heart, adrenal, and peripheral blood cells, suggesting a
relatively greater abundance of stresscopin 1 expression in these
tissues (FIG. 2a, panel 2).
[0143] Likewise, PCR analysis showed that the stresscopin 2
transcript could be detected in most tissues analyzed with colon,
small intestine, muscle, stomach, thyroid, adrenal, and pancreas
showing greater levels of expression (FIG. 2a, panels 3 and 4).
Because heart and digestive tissues showed relatively higher
expression of the stresscopin 1 and stresscopin 2 transcripts,
respectively, a comparative analysis was performed for stresscopin
1 mRNA in different cardiac compartments and stresscopin 2
transcript in the digestive system.
[0144] As shown in FIG. 2b and 2c, the stresscopin 1 transcript
could be amplified in various regions of human heart whereas the
stresscopin 2 transcript was detected in the ascending colon,
descending colon, transverse colon, duodenum, Ileum, jejunum,
stomach, but not in Ileocecum, cecum, rectum, liver, and esophagus.
In addition, immunohistochemical analysis using an anti-stresscopin
1 antibody detected specific stresscopin 1 signals in different
regions of mouse heart with the atrium tissues showing a higher
level of expression (FIG. 2d, left panel). In addition,
immunoreactive stresscopin 1 was detected in the posterior
pituitary (FIG. 2e, left panel). In contrast, specific staining for
stresscopin 2 was detected in the muscularis mucosae of the small
intestine using an anti-stresscopin 2 polyclonal antibody (FIG. 2f,
left panel). Negative control staining using antibodies
presaturated with free antigens showed no specific signals (FIGS.
2d, 2e, and 2f; right panels).
[0145] While stresscopin peptides could be the ligand for orphan
GPCRs, the observation that ligand-receptor pairs usually
co-evolved in diverse vertebrates suggests that the putative
receptors for stresscopin peptides are likely related to the known
CRH receptors and other group B GPCRs. Global sequence analysis
based on all GPCR sequences in the GenBank using both pairwise
sequence comparison and phylogenetic tree building indicated that
the type-1 and type-2 CRH receptors are the most likely candidates
to mediate the action of stresscopins because other closely related
GPCRs are known to bind ligands with highly diverged structures
(e.g. secretin and glucagon-related peptides) whereas no known
orphan GPCR has an intermediate similarity. Two cell lines
expressing CRHR1 (human retinoblastoma cell Y79) or CRHR2 (rat
cardiac cell A7r5) were treated with the synthetic stresscopin 1
peptide. As shown in FIG. 3a, treatment with stresscopin 1
stimulated cAMP production by the cardiac cell line.
[0146] Surprisingly, this peptide was ineffective in Y79 cells
(FIG. 3b), suggesting that stresscopin 1 activates only the type-2
CRH receptor. In contrast, both CRH and urocortin stimulated cAMP
production in both cell lines. To expand this observation, 293T
cells transiently transfected with different CRH receptor cDNAs
were treated with synthetic stresscopin 1, stresscopin 2, or the
pufferfish stresscopin peptide. Analysis of cAMP production showed
that stresscopins are potent agonists for two isoforms of
recombinant CRHR2, but not CRHR1 (FIGS. 3c-3e). Again, CRH and
urocortin stimulated cAMP production mediated by all three
receptors. While both human stresscopins and the pufferfish
ortholog selectively activated type-2 CRH receptors, stresscopin 1
appeared to have a higher potency (>10-fold) as compared to
stresscopin 2 and the pufferfish peptide.
[0147] Because the proteolytic processing sites flanking the mature
stresscopin 1 peptide do not correspond to those found in other CRH
family peptides, 293T cells expressing CRHR1 or CRHR2 were treated
with a panel of truncated stresscopin 1 peptides with deletions of
1 to 5 amino acids at the N-terminus and a nonamidated stresscopin
1 peptide to test the structural requirement for the selective
activation of type-2 CRHR by stresscopin 1. As shown in FIG. 3f,
full-length stresscopin 1 and truncated peptides all stimulated
cAMP production by recombinant CRHR2, but not CRHR1. However, a
stresscopin 1 peptide without amidation at the C-terminus showed a
>50-fold reduction in its ability to stimulate cAMP production,
suggesting that .alpha.-amidation is important for generating
bioactive stresscopin 1. Furthermore, radioligand receptor binding
assays using labeled urocortin confirmed the ability of stresscopin
1 and 2 peptides to bind CRHR2 and CRHR2 (FIGS. 3g and 3h).
[0148] To confirm the specific activation of CRHR2 by stresscopin
peptides, tests were conducted to determine the ability of human
stresscopins and related hormones to stimulate ACTH secretion by
cultured rat anterior pituitary cells, and to elicit ACTH release
in intact male rats. As shown in FIGS. 4a and 4b, both in vitro and
in vivo treatments with CRH and urocortin, but not stresscopin 1 or
stresscopin 2, stimulated the release of ACTH, which is presumably
mediated by CRH R1. Earlier studies indicated that CRH R2 mutant
mice failed to show the enhanced cardiac performance or reduced
blood pressure associated with systemic urocortin, but exhibited
increased edema formation in response to thermal exposure. Based on
the known association between urocortin-induced hypotension and
anti-edema responses, the effects of stresscopins on heat-induced
edema were tested to determine if edema is mediated by CRH R2. As
shown in FIG. 4c, i.p. administration with stresscopin 1 or
stresscopin 2 suppressed heat-induced edema formation in
anesthetized rats, similar to that induced by urocortin and CRH.
Because CRHR2 is essential for sustained feeding suppression
induced by urocortin, the ability of stresscopins to regulate
anorexic responses was also studied based on cumulative food intake
in fasting mice.
[0149] As shown in FIG. 4d, i.p. treatment with stresscopin 1 (left
panel) or stresscopin 2 (right panel), like CRH and urocortin,
dose-dependently decreased food intake in fasting mice. In
contrast, a truncated stresscopin 1 peptide with a deletion of the
first 10 amino acids at the N-terminus (SCP1 (11-43)) has no effect
on food intake in mice (left panel). Furthermore, stresscopin 1 and
stresscopin 2 also suppressed gastric emptying activity as found
for urocortin and CRH (FIG. 4e). This suggests that the anorexic
effects of stresscopins are partly mediated at the level of the
stomach.
[0150] As a result, these tests show stresscopin 1 and stresscopin
2 are novel selective and cogent ligands for CRHR2, and are likely
important in the mediation of anorexic and vascular responses
following stress. Unlike CRH and urocortin, stresscopins do not
elicit ACTH release and the resultant elevations in
glucocorticoids.
[0151] Initial stress-induced responses, such as gluconeogenesis
and increases in arterial pressure and heart rate, provide a vital
short-term metabolic lift, but prolonged or inappropriate exposure
to stress can compromise homeostasis thereby leading to disease.
Because CRHR2 is believed to be important in the regulation of the
recovery phase of the stress response, the present findings suggest
that stresscopin peptides represent important hormones in the
protection of the organism to avoid damage incurred by prolonged
and excessive exposure to the initial "flight or fight" response.
This response is characterized by the activation of the
CRH/ACTH/glucocorticoid axis and the release of catecholamines by
the sympathetic adrenomedullary network. The stress-coping or
"countershock" responses mediated by stresscopins in both central
and peripheral tissues likely include the hypotensive,
cardioprotective, anxiolytic, and anorexic responses mediated by
CRH R2 expressed in brain, posterior pituitary, cardiac and
skeletal muscle, spleen, and the gastrointestinal tract.
[0152] It is clear that adaptive responses induced by stressors are
mediated by the autonomic nervous system and two interrelated and
somewhat antagonistic CRH receptor pathways. Although the four
mammalian CRH-related peptide hormones, CRH, urocortin, stresscopin
1, and stresscopin 2 show overlapping specificity to CRH R1 and CRH
R2, optimal responses to stress depend on an integrated release of
these endocrine/paracrine ligands in a tissue-specific and
time-coordinated manner.
[0153] Table 1, shown below, describes the effects on body weight
change and accumulative food intake in mice treated with
stresscopin 1 or stresscopin 2. At 2 h after treatment, body weight
and food intake were reduced in stresscopin-treated animals as
compared to control mice receiving saline vehicle.
1TABLE 1 Effects of Stresscopin 1 and Stresscopin 2 on food intake
and body weight change in fasted animals. Stresscopin Stresscopin
Stresscopin Control 1 (20 nM) 2 (2 nM) 2 (20 nM) Average Body 0.98
.+-. 0.2 0.31 .+-. 0.14* -0.19 .+-. 0.24* 0.11 .+-. 0.1* weight
change/mouse (grams, N = 5) Average Food 0.675 0.185 0.176 0.152
intake/mouse (grams, N = 5) *Significantly different from control
animals.
[0154] The experiments described herein should not be considered
limiting on the invention, but merely illustrative to one skilled
in the art, of the wide spectrum of possibilities for using
stresscopin 1 and 2.
Sequence CWU 1
1
15 1 339 DNA Homo Sapiens 1 atgaccaggt gtgctctgct gttgctgatg
gtcctgatgt tgggcagagt cctggttgtc 60 ccagtgaccc ctatcccaac
cttccagctc cgccctcaga attctcccca gaccactccc 120 cgacctgcgg
cctcagagag cccctcagct gctcccacat ggccgtgggc tgcccagagc 180
cactgcagcc ccacccgcca ccctggctcg cgcattgtcc tatcgctgga tgtccccatc
240 ggcctcttgc agatcttact ggagcaagcc cgggccaggg ctgccaggga
gcaggccacc 300 accaacgccc gcatcctggc ccgtgtcggc cactgctga 339 2 112
PRT Homo Sapiens 2 Met Thr Arg Cys Ala Leu Leu Leu Leu Met Val Leu
Met Leu Gly Arg 1 5 10 15 Val Leu Val Val Pro Val Thr Pro Ile Pro
Thr Phe Gln Leu Arg Pro 20 25 30 Gln Asn Ser Pro Gln Thr Thr Pro
Arg Pro Ala Ala Ser Glu Ser Pro 35 40 45 Ser Ala Ala Pro Thr Trp
Pro Trp Ala Ala Gln Ser His Cys Ser Pro 50 55 60 Thr Arg His Pro
Gly Ser Arg Ile Val Leu Ser Leu Asp Val Pro Ile 65 70 75 80 Gly Leu
Leu Gln Ile Leu Leu Glu Gln Ala Arg Ala Arg Ala Ala Arg 85 90 95
Glu Gln Ala Thr Thr Asn Ala Arg Ile Leu Ala Arg Val Gly His Cys 100
105 110 3 43 PRT Homo sapiens 3 His Pro Gly Ser Arg Ile Val Leu Ser
Leu Asp Val Ile Leu Gly Leu 1 5 10 15 Leu Gln Ile Leu Leu Glu Gln
Ala Arg Ala Arg Ala Ala Arg Glu Gln 20 25 30 Ala Thr Thr Asn Ala
Arg Ile Leu Ala Arg Val 35 40 4 486 DNA Homo sapiens 4 atg ctg atg
ccg gtc cac ttc ctg ctg ctc ctg ctg ctg ctc ctg ggg 48 ggc ccc agg
aca ggc ctc ccc cac aag ttc tac aaa gcc aag ccc atc 96 ttc agc tgc
ctc aac acc gcc ctg tct gag gct gag aag ggc cag tgg 144 gag gat gca
tcc ctg ctg agc aag agg agc ttc cac tac ctg cgc agc 192 aga gac gcc
tct tcg gga gag gag gag gag ggc aaa gag aaa aag act 240 ttc ccc atc
tct ggg gcc agg ggt gga gcc gga ggc acc cgt tac aga 288 tac gtg tcc
caa gca cag ccc agg gga aag cca cgc cag gac aca gcc 336 aag agt ccc
cac cgc acc aag ttc acc ctg tcc ctc gac gtc ccc acc 384 aac atc atg
aac ctc ctc ttc aac atc gcc aag gcc aag aac ctg cgt 432 gcc cag gcg
gcc gcc aat gcc cac ctg atg gcg caa att ggg agg aag 480 aag tag 486
5 161 PRT Homo sapiens 5 Met Leu Met Pro Val His Phe Leu Leu Leu
Leu Leu Leu Leu Leu Gly 1 5 10 15 Gly Pro Arg Thr Gly Leu Pro His
Lys Phe Tyr Lys Ala Lys Pro Ile 20 25 30 Phe Ser Cys Leu Asn Thr
Ala Leu Ser Glu Ala Glu Lys Gly Gln Trp 35 40 45 Glu Asp Ala Ser
Leu Leu Ser Lys Arg Ser Phe His Tyr Leu Arg Ser 50 55 60 Arg Asp
Ala Ser Ser Gly Glu Glu Glu Glu Gly Lys Glu Lys Lys Thr 65 70 75 80
Phe Pro Ile Ser Gly Ala Arg Gly Gly Ala Gly Gly Thr Arg Tyr Arg 85
90 95 Tyr Val Ser Gln Ala Gln Pro Arg Gly Lys Pro Arg Gln Asp Thr
Ala 100 105 110 Lys Ser Pro His Arg Thr Lys Phe Thr Leu Ser Leu Asp
Val Pro Thr 115 120 125 Asn Ile Met Asn Leu Leu Phe Asn Ile Ala Lys
Ala Lys Asn Leu Arg 130 135 140 Ala Gln Ala Ala Ala Asn Ala His Leu
Met Ala Gln Ile Gly Arg Lys 145 150 155 160 Lys 6 40 PRT Homo
sapiens 6 Thr Lys Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met
Asn Leu 1 5 10 15 Leu Phe Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala
Gln Ala Ala Ala 20 25 30 Asn Ala His Leu Met Ala Gln Ile 35 40 7 42
PRT Homo sapiens 7 Arg Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr
Phe His Leu Leu 1 5 10 15 Arg Glu Val Leu Glu Met Ala Arg Ala Glu
Gln Leu Ala Gln Gln Ala 20 25 30 His Ser Asn Arg Lys Leu Met Glu
Ile Ile 35 40 8 42 PRT Mus musculus 8 Arg Ser Glu Glu Pro Pro Ile
Ser Leu Asp Leu Thr Phe His Leu Leu 1 5 10 15 Arg Glu Val Leu Glu
Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala 20 25 30 His Ser Asn
Arg Ile Ile Phe Asp Ser Val 35 40 9 42 PRT Homo sapiens 9 Arg Arg
Asp Asn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu 1 5 10 15
Arg Thr Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala 20
25 30 Glu Gln Asn Arg Ile Ile Phe Asp Ser Val 35 40 10 42 PRT Mus
musculus 10 Arg Arg Asp Asp Pro Pro Leu Ser Ile Asp Leu Thr Phe His
Leu Leu 1 5 10 15 Arg Thr Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln
Arg Glu Arg Ala 20 25 30 Glu Gln Asn Arg Ile Ile Phe Asp Ser Val 35
40 11 42 PRT Carassius auratus 11 Arg Asn Asp Asp Pro Pro Ile Ser
Ile Asp Leu Thr Phe His Leu Leu 1 5 10 15 Arg Asn Met Ile Glu Met
Ala Arg Asn Glu Asn Gln Arg Glu Gln Ala 20 25 30 Gly Leu Asn Arg
Lys Tyr Leu Asp Glu Val 35 40 12 42 PRT Catostomus commersoni 12
Arg Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu 1 5
10 15 Arg Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln
Ala 20 25 30 His Ser Asn Arg Lys Met Met Glu Ile Phe 35 40 13 42
PRT Catostomus commersoni 13 Arg Ser Glu Glu Pro Pro Ile Ser Leu
Asp Leu Thr Phe His Leu Leu 1 5 10 15 Arg Glu Val Leu Glu Met Ala
Arg Ala Glu Gln Leu Val Gln Gln Ala 20 25 30 His Ser Asn Arg Lys
Met Met Glu Ile Phe 35 40 14 40 PRT Phyllomedusa sauvagei 14 Gln
Gly Pro Pro Ile Ser Ile Asp Leu Ser Leu Glu Leu Leu Arg Lys 1 5 10
15 Met Ile Glu Ile Glu Lys Gln Glu Lys Glu Lys Gln Gln Ala Ala Asn
20 25 30 Asn Arg Leu Leu Leu Asp Thr Ile 35 40 15 40 PRT Takifugu
rubripes 15 Ser Arg Leu Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met
Asn Val 1 5 10 15 Leu Phe Asp Val Ala Lys Ala Lys Asn Leu Arg Ala
Lys Ala Ala Glu 20 25 30 Asn Ala Arg Leu Leu Ala His Ile 35 40
* * * * *