Title: Urocortin-III and uses thereof
United States Patent: 6,953,838
Issued: October 11, 2005
Inventors: Vale, Jr.; Wylie W. (La Jolla, CA); Rivier; Jean
E. (La Jolla, CA); Kunitake; Koichi S. (San Diego, CA); Lewis; Kathy A. (San
Diego, CA); Perrin; Marilyn H. (La Jolla, CA); Gulyas; Jozsef (Julian, CA)
Assignee: Research Development Foundation (Carson City, NV)
Appl. No.: 771224
Filed: February 3, 2004
A search of the public human genome database identified a human EST,
GenBank accession number AW293249, which has high homology to known
pufferfish urocortin sequences. The full length sequence was amplified from
human genomic DNA and sequenced. Sequence homology comparisons of the novel
sequence with human urocortin I and urocortin II revealed that the sequence
encoded a novel human urocortin, which was designated urocortin III (UcnIII).
While urocortin III does not have high affinity for either CRF-R1 or CRF-R2,
the affinity for CRF-R2 is greater than the affinity for CRF-R1. Urocortin
III is capable stimulating cyclic AMP production in cells expressing CRF-R2α
or β. Thus, the affinity is high enough that urocortin III could act as a
native agonist of CRF-R2. However, it is also likely that urocortin III is a
stronger agonist of a yet to be identified receptor.
Description of the Invention
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of neuroendocrinology
and neuropeptide chemistry. More specifically, the instant invention relates
to protein factors involved in the regulation of neuroendocrine and
paracrine responses to stress. Most specifically, the present invention
discloses a corticotropin releasing factor related peptide designated
2. Description of the Related Art
Corticotropin releasing factor (CRF) and its related family of peptides were
recognized initially for their regulation of the
hypothalamic-pituitary-adrenal axis (HPA) under basal and stress conditions
(1, 2). Corticotropin releasing factor (CRF) is a 41 amino acid peptide that
was first isolated from ovine hypothalamus (3) and shown to play an
important role in the regulation of the pituitary-adrenal axis, and in
endocrine, autonomic and behavioral responses to stress (4). The CRF family
of neuropeptides also includes structurally related mammalian and
non-mammalian peptides such as urocortin (Ucn), a 40 amino acid peptide
originally identified in rat brain (5), fish urotensin I (Uro) (6), and
amphibian sauvagine (Svg) (7).
It has been hypothesized that members of the CRF family are involved in
neuroendocrine and paracrine responses in many tissues. In addition to their
effects on the pituitary and central nervous system, members of the CRF
family have been shown to modulate cardiovascular and gastrointestinal
functions and inflammatory processes in mammals to integrate endocrine,
autonomic and behavioral responses to stressors. These peptides may also be
implicated in the control of appetite, arousal, and cognitive functions.
Severe psychological and physiological consequences can occur as a result of
the long term effects of stress, such as anxiety disorders, anorexia
nervosa, gastrointestinal dysfunction and melancholic depression.
CRF family members mediate their biological actions by specifically binding
to CRF receptors with high affinities (8, 9). CRF receptors are G-protein
coupled receptors that act through adenylate cyclase and are structurally
related to the secretin receptor family. This family also includes GRF, VIP,
PTH, and the calcitonin receptors.
The CRF receptors are derived from two distinct genes, CRF receptor type 1
(CRF-R1) (10-12) and CRF receptor type 2 (CRF-R2) (13-15). CRF-R1 and CRF-R2
have distinct pharmacologies and differ in their anatomical distribution
(16). The type 1 CRF receptor (CRF-R1) gene has 13 exons; several splice
variants of this receptor have been found. The CRF-R1 is distributed
throughout the brain and is found in sensory and motor relay sites (17). The
rodent type 2α receptor (CRF-R2α) is distributed in lateral septum, ventral
medial hypothalamus, nucleus of the solitary tract and the dorsal raphe
nucleus, which are areas where CRF-R1 is expressed very little or not at all
(18). The rodent type 2β receptor (CRF-R2β) is found mostly in peripheral
sites including the heart, blood vessels, gastrointestinal tract, epididymis,
lung and skin (9, 19).
The pharmacology of the two types of receptors differs in that CRF has a
modest affinity for CRF-R2 [Ki=5-100 nM] but high affinity for CRF-R1 [Ki=1-2
nM]. Other related peptides such as carp urotensin, frog sauvagine, and
urocortin have a high affinity for both CRF-R1 and CRF-R2. CRF-R2 knockout
mice demonstrate an increased anxiety-like behavior caused by
hypersensitivity to stressors (5, 20).
Recently, searches of the public human genome database identified a region
with significant sequence homology to the CRF neuropeptide family. The
entire human sequence was amplified and sequenced. The human sequence,
however, lacks a consensus proteolytic cleavage site that would allow for
C-terminal processing of the peptide, and is therefore referred to as an
urocortin-related peptide (URP) sequence. Using homologous primers deduced
from the human sequence, a mouse cDNA was isolated from whole brain poly
(A+) RNA that encodes a predicted 38 amino acid peptide, designated
urocortin II, which is structurally related to the other known mammalian
family members, CRF and urocortin (Ucn). The question of whether human
urocortin-related peptide represents the mouse Ucn II ortholog remains open
until additional mouse genes are identified. Ucn II binds selectively to the
type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1.
Transcripts encoding Ucn II are expressed in discrete regions of the rodent
CNS, including stress-related cell groups in the hypothalamus (paraventricular
and arcuate nuclei) and brainstem (locus coeruleus). These findings identify
Ucn II as a new member of the CRF family of neuropeptides, which is
expressed centrally and binds selectively to CRF-R2. Initial functional
studies are consistent with Ucn II involvement in central autonomic and
appetitive control, but not in generalized behavioral activation (21).
The prior art is deficient in the recognition of the human Urocortin-III
gene and protein and uses thereof. The present invention fulfills this
longstanding need and desire in the art.
SUMMARY OF THE INVENTION
A human urocortin, Urocortin-III (Ucn-III) with homology to known
pufferfish urocortins was identified from the public human genome database.
From the sequence of the human gene, a mouse ortholog was isolated. The
present invention relates to these novel genes and uses thereof.
In one aspect, the instant invention is directed to an isolated and purified
urocortin III protein, which may be either mouse or human urocortin III. The
mouse protein preferably has a n amino acid sequence of SEQ ID No. 5, which
is derived from a precursor peptide of SEQ ID No. 4. The human protein
preferably has an amino acid sequence of SEQ ID No. 3 derived from a
precursor peptide of SEQ ID No. 2.
The instant invention is also directed to human urocortin III containing one
or more amino acid substitutions derived from the mouse amino acid sequence.
The sequence of mouse urocortin III (SEQ ID No. 5) differs from human
urocortin III (SEQ ID No. 3) by four amino acids, specifically Ile14,
Asp19, Lys27, and Gln33. Substitution of
the Leu14 residue in the human protein with Ile is contemplated
to be especially useful.
The instant invention is also directed to a pharmaceutical composition
comprising a urocortin III protein and to a method of treating a
pathophysiological state using this pharmaceutical composition. This
pharmaceutical composition could be administered to activate the CRF-R2
receptor to remedy a pathophysiological state such as high body temperature,
appetite dysfunction, congestive heart failure, vascular disease, stress and
The instant invention is also directed to modification of a urocortin III
protein. The N-terminus of urocortin III may be extended with additional
amino acids or peptides such as Threonine-Lysine (the preceding two residues
in the precursor protein), D-tyrosine, L-tyrosine, D-tyrosine-glycine, or
L-tyrosine-glycine. In addition, one or more methionine residues in
urocortin III, such as those at position 12 and 35 of SEQ ID No. 3, may be
replaced with Nle residues. Alternatively, the N-terminus may be extended
with D-iodotyrosine, L-iodotyrosine, D-iodotyrosine-glycine, and L-iodotyrosine-glycine
and the methionine residues at positions 12 and 35 replaced with Nle. The
iodotyrosine residues may be labeled with 125I.
Additional substitutions are suggested by amino acid residues conserved in
other urocortin and urocortin-related proteins which differ in urocortin
III. Such urocortin analogs may be comprised of urocortin III with one or
more amino acid substitutions selected from the group consisting of Ile3,
Nle3, CαMe-Leu3, Ile5, Nle5,
CαMe-Leu5, Leu7, Nle7, Thr8,
Ile9, Phe9, Gly10, His10, Leu11,
Nle11, Leu12, Nle12, Arg13, Gln13,
Nle14, CαMe-Leu14, Nle15, CαMe-Leu15,
Leu16, Nle16, Glu17, Asp17, Arg20,
Nle24, CαMe-Leu24, Arg32, Ile34,
Nle34, CαMe-Leu34, Leu35, Nle35,
Asp36, Glu36, and Val38.
The instant invention is also directed to a CRF-R2 receptor antagonist
comprising urocortin III protein or a urocortin III analog wherein the first
five to eight N-terminal amino acids of the protein have been deleted. This
antagonist may be incorporated into a pharmaceutical composition and used to
treat congestive heart failure, vascular disease, gastrointestinal
dysfunction and migraine headaches or as an angiogenesis inhibitor.
In yet another embodiment of the instant invention, Urocortin III may also
be modified to contain a fluorescent label or a complexing agent for
radionuclides. The resulting labeled urocortin III can be used to identify
cells expressing urocortin III receptors. Alternatively, urocortin III may
be linked to a toxin molecule.
In yet another embodiment of the instant invention, a n antibody directed
against urocortin III is provided. In a preferred embodiment, the antibody
is a monoclonal antibody. The antibody may be conjugated to a molecular
label such as a fluorescent label, photoaffinity label or radioactive
markers. Alternatively, the antibody could be conjugated to a cytotoxic
compound to form a n immunotoxin.
Claim 1 of 28 Claims
1. A method of treating a pathophysiological state, comprising the step of
administering a pharmaceutical composition comprising a urocortin III
protein and a pharmaceutically acceptable carrier to an individual in need
of such treatment, the urocortin III protein having the amino acid
sequence of SEQ ID NO:5 with an N-terminal deletion selected from the
group consisting of the first five amino acids, the first six amino acids,
the first seven amino acids and the first eight amino acids.
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