U.S. patent application number 09/818009 was filed with the patent office on 2003-02-13 for urocortin peptides.
This patent application is currently assigned to The Salk Institute for Biological Studies. Invention is credited to Donaldson, Cynthia J., Lewis, Kathy A., Perrin, Marilyn H., Rivier, Jean E.F., Sawchenko, Paul, Vale, Wylie W. JR., Vaughan, Joan.
Application Number | 20030032587 09/818009 |
Document ID | / |
Family ID | 27357112 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032587 |
Kind Code |
A1 |
Vale, Wylie W. JR. ; et
al. |
February 13, 2003 |
Urocortin peptides
Abstract
Urocortin (Ucn) is a native mammalian peptide generally related
to Urotensin I and Corticotropin Releasing Factor (CRF). Human Ucn
has the formula:
Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr--
Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Il-
e-Ile-Phe-Asp-Ser-Val-NH.sub.2 (SEQ ID NO: 15). Rat-derived Ucn is
identical but for 2 substitutions, Asp.sup.2 for Asn.sup.2 and
Pro.sup.4 for Ser.sup.4. Ucn or analogs thereof or pharmaceutically
acceptable salts can be administered to humans and other mammals to
achieve substantial elevation of ACTH, .beta.-endorphin,
.beta.-lipotropin, other products of the pro-opiomelanocortin gene
and corticosterone. They can also be used to lower blood pressure
over an extended period of time, as stimulants to elevate mood and
to improve memory and learning performance, as well as
diagnostically. Shortened fragments may be administered to release
endogenous CRF and/or Ucn in the brain and peripherally. Ucn
antagonists can be used to block the action of Ucn and/or CRF, as
can antibodies to Ucn. Labelled Ucn agonists and antagonists can be
used in drug screening assays along with CRF receptors; they may
also be used diagnostically along with Ucn antibodies.
Inventors: |
Vale, Wylie W. JR.; (La
Jolla, CA) ; Vaughan, Joan; (Oceanside, CA) ;
Donaldson, Cynthia J.; (San Diego, CA) ; Lewis, Kathy
A.; (San Diego, CA) ; Sawchenko, Paul;
(Encinitas, CA) ; Rivier, Jean E.F.; (La Jolla,
CA) ; Perrin, Marilyn H.; (La Jolla, CA) |
Correspondence
Address: |
FITCH EVEN TABIN AND FLANNERY
120 SOUTH LA SALLE STREET
SUITE 1600
CHICAGO
IL
60603-3406
US
|
Assignee: |
The Salk Institute for Biological
Studies
|
Family ID: |
27357112 |
Appl. No.: |
09/818009 |
Filed: |
March 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09818009 |
Mar 26, 2001 |
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08981189 |
Dec 10, 1997 |
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6214797 |
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08981189 |
Dec 10, 1997 |
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PCT/US96/10240 |
Jun 12, 1996 |
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60028144 |
Jun 13, 1995 |
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60002223 |
Aug 11, 1995 |
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Current U.S.
Class: |
514/4.9 ;
435/7.2; 514/10.8; 514/17.6; 514/17.8; 530/399 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/57509 20130101 |
Class at
Publication: |
514/12 ; 530/399;
435/7.2 |
International
Class: |
A61K 038/22; G01N
033/53; G01N 033/567; C07K 014/575 |
Goverment Interests
[0002] This invention was made with Government support under Grant
Number DK-26741 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
What is claimed is:
1. A peptide having SEQ ID NO: 15 of human urocortin (Ucn) or an
analogous sequence having only conservative substitutions to the
amino acid residues therein or an N-terminally shortened fragment
of either which is biologically active to increase ACTH
production.
2. Antibodies which bind specifically to a urocortin peptide or to
a fragment thereof according to claim 1.
3. Antibodies according to claim 2 which specifically bind to and
biologically inactivate said urocortin peptide so it no longer
increases production of ACTH.
4. A method for screening for ligands for CRF receptors, which
method comprises carrying out a competitive binding assay with a
CRF receptor, a peptide according to claim 1 which contains a
suitable label, and a candidate ligand and determining the ability
of said candidate ligand to displace said labelled peptide.
5. The screening method according to claim 4 wherein said CRF
receptor is CRF Receptor 2 and said labelled peptide is
.sup.125I-Tyr.sup..smallcircl- e.-Ucn.
6. A method for stimulating secretion of ACTH and
.beta.-endorphin-like-ac- tivities (.beta.-END-LI) in mammals
comprising peripherally administering to said mammal an effective
amount of the peptide according to claim 1 or a nontoxic salt
thereof and a pharmaceutically acceptable carrier therefor.
7. A method of modifying blood flow and/or blood pressure which
comprises administering an effective amount of the peptide
according to claim 1 or of an N-terminally shortened antagonist
peptide thereof.
8. A method according to claim 7 for modulating blood flow in a
desired vascular bed which comprises peripherally administering
said effective peptide amount.
9. A method of increasing coronary blood flow which comprises
peripherally administering an effective amount of the peptide
according to claim 1.
10. A method of decreasing swelling and/or inflammation and/or
vascular permeability which comprises parenterally administering an
effective amount of the peptide according to claim 1.
11. A pharmaceutical composition which comprises an effective
amount of the peptide according to claim 1 in combination with a
pharmaceutically acceptable carrier, which amount is effective to
modulate the transactivation of CRF receptors, or to increase
intestinal transit rate.
12. A method of treatment comprising peripherally administering to
a mammal an effective amount of the peptide according to claim 1 or
a nontoxic salt thereof and a pharmaceutically acceptable carrier
therefor, which amount is effective to stimulate secretion of ACTH
and .beta.-endorphin-like-activities (.beta.-END-LI) in such
mammal.
13. A Ucn antagonist peptide which comprises the following amino
acid sequence:
Asp-Leu-R.sub.10-R.sub.11-His-Leu-Leu-Arg-Thr-Leu-Leu-R.sub.19--
Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-R.sub.29-Ala-Glu-R.sub.32-Asn-Arg-Ile--
R.sub.36-Phe-R.sub.38-Ser-Val-NH.sub.2 (residues 90-122 SEQ ID NO:
15), wherein R.sub.10 is Pro, D-Pro, Thr or D-Tyr; R.sub.11 is
D-Phe or D-Tyr or another D-amino acid; R.sub.19 is Glu or Ala;
R.sub.29 is Arg or Glu; R.sub.32 is Gln, Lys or Orn; R.sub.36 is
Ile, C.sup..alpha.MeIle or C.sup..alpha.MeLeu; R.sub.38. is Asp or
Ala; provided that when R.sub.29 is Glu, R.sub.32 is either Lys or
Orn and the side chains thereof are linked by an amide bond and
provided further that Glu in the 31-position can be D-Glu or
another D-amino acid and that the N-terminus can be shortened by 1,
2 or 3 residues.
14. The Ucn antagonist peptide according to claim 13 which is
(cyclo 29-32)D-Phe.sup.11,Glu.sup.29,Lys.sup.32-Ucn(11-40).
15. A method for screening for antagonists for CRF receptors which
bind with high affinity to such receptors which method comprises
carrying out a competitive binding assay with a CRF receptor, with
the peptide according to claim 14 which contains a suitable label,
and with a candidate antagonist and determining the ability of said
candidate antagonist to displace said labelled peptide.
16. A compound useful for blocking CRF-binding protein (CRF-BP) to
thereby increase availability of endogenous CRF and/or Ucn, which
compound is a peptide having the amino acid sequence:
Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-P-
he-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-
-Ala-Glu-Gln (residues 85-114 of SEQ ID NO: 15), or a biologically
active fragment thereof which is formed by deleting 1 to 8 residues
consecutively from the N-terminus, or 1 to 5 residues consecutively
from the C-terminus, or both or which has at least 80% homology
with said peptide or with said fragment and binds to CRF-BP.
17. The compound according to claim 16 selected from the group
consisting of Ucn(5-32), Ucn(8-32) and hUcn(3-27).
18. A method for increasing the in vivo level of CRF and/or Ucn,
which method comprises administering an effective amount of the
compound according to claim 16.
19. The method according to claim 18 wherein said effective amount
is sufficient to promote parturition in a pregnant female.
20. The method according to claim 18 wherein said amount of said
compound administered is effective so as to result in an increase
in free endogenous CRF and/or Ucn in the brain which causes (a)
improvement in short to medium term memory in a subject afflicted
with Alzheimer's disease; (b) relief from chronic fatigue syndrome;
(c) suppression of appetite; (d) stimulation of the respiratory
system, (e) improvement in learning performance; (f) improvement in
memory; (g) improvement in alertness; (h) reduction of depression
and/or (i) lessening of anxiety.
21. The method according to claim 20 wherein said compound is
administered so that it reaches the brain.
Description
[0001] This application is a division of Ser. No. 08/981,189, filed
Dec. 10, 1997 as a national stage entry of PCT/US96/10240 which
claims priority from Provisional Applications Serial Nos.
60/028,144, filed Jun. 13, 1995 and 60/002,223, filed Aug. 11,
1995.
[0003] This invention is directed to peptide hormones, to methods
for treatment of mammals, including humans, using such peptides, to
antibodies which bind such peptides, to methods for diagnosis and
drug screening using such peptides and/or antibodies, and to
nucleic acid encoding such peptides. More specifically, the
invention relates to a native peptide having certain
pharmacological properties in common with urotensin and with CRF,
which is termed urocortin (Ucn), to analogs and fragments thereof
(broadly termed Ucn-like peptides), to pharmaceutical compositions
containing such Ucn peptides and to methods of treatment of
mammals, method of diagnosis and methods of screening using such
Ucn peptides and antibodies thereto.
BACKGROUND OF THE INVENTION
[0004] Experimental and clinical observations have supported the
concept that the hypothalamus plays a key role in the regulation of
adenohypophysial corticotropic cells secretory functions. Although
over 40 years ago, Guillemin, Rosenberg and Saffran and Schally
independently demonstrated the presence of factors in hypothalamus
which would increase the rate of ACTH secretion by the pituitary
gland incubated in vitro or maintained in an organ culture, a
physiologic corticotropin releasing factor (CRF) was not
characterized until ovine CRF (oCRF) was characterized in 1981. It
was disclosed in U.S. Pat. No. 4,415,558, as having the amino acid
sequence (SEQ ID NO: 1):
[0005]
H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-G-
lu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-
-Lys-Leu-Leu-Asp-Ile-Ala-NH2.
[0006] Although originally isolated and characterized on the basis
of its role in this hypothalamopituitary-adrenal (HPA) axis, CRF
has been found to be distributed broadly throughout the central
nervous system as well as in extraneural tissues, such as the
adrenal glands, placenta and testes, where it may also act as a
paracrine regulator or a neurotransmitter. Moreover, the likely
involvement of CRF in affective disorders, such as anxiety,
depression, alcoholism and anorexia nervosa, and in modulating
reproduction and immune responses suggests that changes in CRF
expression may have important physiological and pathophysiological
consequences. For example, perturbations in the regulatory loops
comprising the HPA axis often produce chronically elevated levels
of circulating glucocorticoids; such patients display the physical
hallmarks of Cushing's syndrome, including truncal obesity,
muscle-wasting, and reduced fertility.
[0007] In addition to its role in mediating activation of the
hypothalamic-pituitary-adrenal, CRF has also been shown to modulate
autonomic and behavioral changes, some of which occur during the
stress response. Many of these behavioral changes have been shown
to occur independently of HPA activation in that they are not
duplicated by dexamethasone treatment and are insensitive to
hypophysectomy. In addition, direct infusion of CRF into the CNS
mimics autonomic and behavioral responses to a variety of
stressors. Because peripheral administration of CRF or a CRF
antagonist fails to affect certain of these changes, it appears
that CRF exhibits a direct brain action with respect to such
functions, which include appetite suppression, increased arousal
and learning ability. However, CRF antagonists given peripherally
attenuate stress-mediated increases in ACTH secretion, and when
delivered into the cerebral ventricles can mitigate stress-induced
changes in autonomic activity and behavior.
[0008] As a result of the extensive anatomical distribution and
multiple biological actions of CRF, this regulatory peptide is
believed to be involved in the regulation of numerous biological
processes. CRF has also been implicated in the regulation of
inflammatory responses. Although it has been observed that CRF
plays a pro-inflammatory role in certain animal models, CRF appears
to suppress inflammation in others by reducing injury-induced
increases in vascular permeability.
[0009] In about 1981, a 40-residue amidated peptide generally
similar to CRF was isolated from the skin of the South American
frog Phyllomedusa sauvagei; it is referred to as sauvagine. It was
characterized by Erspamer et al. and was described in Regulatory
Peptides, Vol. 2 (1981), pp. 1-13. Sauvagine has the amino acid
sequence (SEQ ID NO: 2):
[0010]
pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Me-
t-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu--
Leu-Leu-Asp-Thr-Ile-NH.sub.2. When given intravenously(iv),
sauvagine and oCRF have been reported to cause vasodilation of the
mesenteric arteries so as to lower blood pressure in mammals and
also in stimulating the secretion of ACTH and .beta.-endorphin.
However, when administered intracerebroventricularly(icv), there is
an elevation of heart rate and mean arterial blood pressure, which
are secondary to activation of the sympathetic nervous system.
[0011] Rat CRF(rCRF) was isolated, purified and characterized in
about 1982-1983 as a hentetracontapeptide having the amino acid
sequence (SEQ ID NO: 3):
[0012]
H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-G-
lu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-
-Lys-Leu-Met-Glu-Ile-Ile-NH.sub.2. The formula of human CRF was
subsequently determined to be the same as that of rCRF. The
compound is often referred to as r/hCRF and is covered in U.S. Pat.
No. 4,489,163.
[0013] At about the same time, two homologous polypeptides were
isolated from the urophyses of different species of fish. These
isolated peptides were generally homologous to CRF, i.e. about 54%
homology, and were termed Urotensin I (UI). Catostomus
commersoni(white sucker or suckerfish) UI is a polypeptide having
the amino acid sequence (SEQ ID NO: 4):
[0014]
H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-A-
sn-Met-Ile-Glu-Met-Ala-Arg-Ile-Glu-Asn-Glu-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-
-Lys-Tyr-Leu-Asp-Glu-Val-NH.sub.2; it is sometimes referred to as
suckerfish(sf) urotensin or sfUI. Its purification and
characterization are described in an article by Lederis et al.,
Science Vol. 218, No. 4568, 162-164 (Oct. 8, 1982). The homolog,
carp urotensin, was obtained from Cyprinus carpio and has the amino
acid sequence (SEQ ID NO: 5):
[0015]
H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-A-
sn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-
-Lys-Tyr-Leu-Asp-Glu-Val-NH.sub.2. Another urotensin homolog having
the following amino acid sequence (SEQ ID NO: 6):
[0016]
H-Ser-Glu-Glu-Pro-Pro-Met-Ser-Ile-Asp-Leu-Thr-Phe-His-Met-Leu-Arg-A-
sn-Met-Ile-His-Arg-Ala-Lys-Met-Glu-Gly-Glu-Arg-Glu-Gln-Ala-Leu-Ile-Asn-Arg-
-Asn-Leu-Leu-Asp-Glu-Val-NH.sub.2 was later isolated from the
urophyses of Hippoglossoides elassodon or Flathead (Maggy) Sole; it
is sometimes referred to as Maggy urotensin. Synthetic UIs have
been found to also stimulate ACTH and .beta.-endorphin activities
in vitro and in vivo and to have many of the same general
biological activities of CRFs and sauvagine.
[0017] Since the discovery of the original discoveries of CRFs in
mammals and urotensins in fish, CRFs have now been shown to exist
in other animal species. For example, fish CRF was found to be a
41-residue peptide having high homology to r/hCRF; it has the amino
acid sequence (SEQ ID NO: 7):
[0018]
H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-G-
lu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-
-Lys-Met-Met-Glu-Ile-Phe-NH.sub.2. Synthetic fish CRF (fCRF)
stimulates ACTH and .beta.-endorphin activities in vitro and in
vivo and has similar biological activities to mammalian CRFs.
Because of the high homology between fCRF and r/hCRF, it is thought
that other mammalian hormones may exist which would be the
counterparts of urotensin and/or sauvagine.
SUMMARY OF THE INVENTION
[0019] Another peptide 40 residues in length has now been
discovered, which is related to urotensin and CRF; it is
arbitrarily referred to as urocortin (Ucn). It has less than 50%
homology with rat/human CRF. Although it has the same length as
sauvagine, it shares less than 40% homology with sauvagine. It has
62.5% homology with the closest urotensin sequence, i.e. carp
urotensin. Thus Ucn has less than about 80% homology with any other
previously known native peptide. Rat Ucn has the following amino
acid sequence (SEQ ID NO: 8):
Asp-Asp-Pro-Pro-Leu-Ser-Ile-Asp-Leu-T-
hr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-
-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2. Human
Ucn has the following amino acid sequence (residues 83-122 of SEQ
ID NO: 15):
Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-G-
lu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-
-Asp-Ser-Val-NH.sub.2. Thus, hUcn is the same as rUcn except for
Asn.sup.2 and Ser.sup.4. Ucn has biological properties which are
considered to generally resemble those of known CRFs, urotensins
and sauvagine but is more biopotent in a number of respects.
[0020] The present invention provides Ucn-like peptides, including
human and rat Ucn and Analogs thereof, which have substantially all
the properties of known CRFs. These Ucn-like peptides not only are
potent hypotensive agents, but they have additional pharmacological
and physiological properties over and beyond those of heretofore
known CRFs. More specifically, agonists are provided for the
stimulation of the known CRF receptors (referred to as CRF-Rs),
i.e., CRF-R1 and CRF-R2 and their splice varients, as well as the
putative novel receptor for Ucn.
[0021] Ucn competitive antagonists are also provided which bind the
CRF-Rs and the putative Ucn receptor with high affinity but do not
significantly stimulate or activate such receptors. Such
antagonists are broadly created by deleting a sequence of from 7 to
10 residues beginning at the N-terminus from the amino acid
sequence of Ucn or from an analog sequence that is substantially
the same. Preferably 9 or 10 residues are deleted, and most
preferably 9 are deleted. It may be preferred that the shortened
N-terminus be acylated with a group having 7 or less carbon atoms,
e.g. [Ac-Thr.sup.10]-Ucn(10-40), and the inclusion of one, two or
three other residues at the N-terminus, e.g., Pro.sup.10, does not
markedly affect biopotency. Other substitutions may be effectively
made as described hereinafter. Particularly, D-Phe.sup.11 or
D-Tyr.sup.11 may be present at the N-terminus and/or a lactam bond
created between residues 29 and 32. These antagonists can be
administered to achieve at least the same physiological effects as
the known CRF antagonists, and such more effective methods of
treatment are thus provided.
[0022] Fragments of Ucn and Ucn Analogs that are useful to block
CRF-binding protein (CRF-BP) are further provided which are
effective to elevate levels of endogenous peptides normally cleared
by the binding protein. More specifically, these Ucn-like peptides
and blocking fragments bind to human CRF-binding protein with high
affinity and effectively compete with human CRF and human Ucn
(hUcn) in the formation of complexes with hCRF-BP; in this manner,
they increase the effective in vivo concentration of endogenous
hCRF and/or hUcn, as well as the effective concentration of any CRF
agonist or CRF antagonist that may be optionally administered along
therewith for the purpose of achieving a particular therapeutic
purpose. As a result of blocking the effect of CRF-BP, these
fragments effectively increase the concentration of endogenous CRF
in those regions of the body where CRF-BP is normally present.
[0023] The invention also provides pharmaceutical compositions
which include such Ucn-like peptides, or nontoxic salts thereof,
dispersed in a pharmaceutically acceptable liquid or solid carrier.
The administration of such peptides or pharmaceutically acceptable
salts thereof to mammals, particularly humans, in accordance with
the invention may be carried out for regulation of the secretion of
ACTH, .beta.-endorphin, .beta.-lipotropin, other products of the
pro-opiomelanocortin (POMC) gene and corticosterone and/or for
lowering blood pressure or increasing coronary flow and/or
decreasing swelling and inflammation and/or for affecting learning,
mood, behavior, appetite, gastrointestinal and intestinal functions
and autonomic nervous system activities.
[0024] The invention also provides antibodies which recognize Ucn,
and assays for practically employing Ucn and analogs and/or such
antibodies for the evaluation of the status of pituitary,
cardiovascular, reproductive, hepatic, immune, gastrointestinal or
central nervous system functions. For example, such antibodies can
be used diagnostically to monitor the level of therapeutically
administered Ucn, to facilitate the maintenance of therapeutically
effective amounts thereof, as well as for the diagnosis of
potential physiological disorders that result from abnormal levels
of Ucn. Antibodies of the invention may be therapeutically
administered to neutralize endogenous Ucn; alternatively DNA
encoding such antibodies might be employed in gene therapy.
Anti-Ucn antibodies can also be used to purify CRF-R protein as
well as to therapeutically counteract the biological action of Ucn
in vivo.
[0025] The invention also provides competitive binding assays which
are particularly useful for screening candidates for new drugs,
e.g. to identify new Ucn-like peptides or other compounds having
even greater or more selective binding affinity for CRF receptors
and/or for CRF-BP than Ucn, which candidates would therefore be
potentially useful as drugs. Such screening assays may be used to
screen for potential agonists of Ucn, and other assays employing a
labelled Ucn antagonist with high affinity may be used to screen
for more potent antagonists of Ucn. In addition, there is provided
a method for screening for particularly effective peptides or other
compounds which will block the ability of CRF-BP to bind to CRF and
Ucn and therefore increase the concentration of CRF and/or Ucn in
locations where hCRF-BP is present.
[0026] The present invention further provides nucleic acid
hybridization probes in the form of isolated nucleic acid encoding
native rat Ucn and isolated nucleic acid encoding native human Ucn,
which are useful for detecting other Ucn-encoding nucleic acids in
biological samples or in libraries of other species in order to
identify additional native Ucn or Ucn-like peptides. Such
nucleotide sequences also can be used as coding sequences for the
recombinant expression of complete Ucn-like peptides or desired
biologically active fragments thereof. Fragments of Ucn-encoding
nucleic acid can also be employed as primers for PCR amplification
of Ucn-encoding DNA. In addition, sequences derived from sequences
encoding Ucn or analogs thereof can also be used in gene therapy
applications to target the expression of vectors carrying useful
genes to specific cell types, and antisense polynucleotides that
hybridize with Ucn mRNA may also be used to reduce Ucn levels to
counteract certain conditions, e.g. Ucn-secreting tumors. More
specifically, the present invention further provides isolated
nucleic acids encoding Ucn as well as Ucn analogs containing
L-isomers of the 20 natural amino acids. Such nucleic acids
comprise:
[0027] (a) nucleic acids that encode the amino acid sequence of rat
Ucn set forth in SEQ ID NO: 8 and that encode the amino acid
sequence of human Ucn set forth in SEQ ID NO: 15;
[0028] (b) nucleic acids which hybridize to the nucleic acids of
(a) wherein said hybridizing nucleic acids encode biologically
active Ucn-like peptides; or
[0029] (c) nucleic acids which encode fragments of Ucn or analogs
thereof which are CRF-antagonists or CRF-BP blockers.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. The
nomenclature used to define the peptides is that specified by
Schroder & Lubke, "The Peptides", Academic Press (1965)
wherein, in accordance with conventional representation, the amino
group appears to the left and the carboxyl group to the right. The
standard 3-letter abbreviations to identify the alpha-amino acid
residues, and where the amino acid residue has isomeric forms, it
is the L-form of the amino acid that is represented unless
otherwise expressly indicated, e.g. Ser=L-serine. The nucleotides,
which occur in the various nucleic acids, are designated with the
standard single-letter designations used routinely in the art.
[0031] The term "homology" is used in its usual and well known
sense of indicating correspondence between members in a sequence,
e.g. either on an amino acid (AA) level or at the nucleotide level.
For purposes of this application, the term homologous refers to at
least about 70% correspondence, the term substantially homologous
refers to a correspondence of at least about 80%, and the term
highly homologous refers to a correspondence of at least about 90%
or preferably about 95% or higher. The term "homolog" is generally
considered to include analogous proteins, peptides and DNA
sequences from other mammalian species wherein insignificant
changes have evolved but the homolog still performs the same
biological function in substantially the same way.
[0032] Protein, polypeptide and peptide are used to designate
linear sequences of amino acid residues connected one to the other
by peptide bonds between the alpha-amino and alpha-carboxy groups
of adjacent residues. The term polypeptide may be used
interchangeably with peptide and with the term protein; unless
otherwise limited, protein is generally used to describe a sequence
of about 75 or more residues.
[0033] The term "analog" includes any polypeptide having an amino
acid residue sequence generally identical to a sequence
specifically shown herein, e.g. rUcn or hUcn, wherein one or more
residues has been replaced (with at least about 80% and preferably
at least about 90% of the residues being the same) and wherein the
analog displays the ability to biologically mimic the parent
molecule as described herein in some particular function.
Preferably, most if not all of such substitutions are replacements
of a residue with a functionally similar residue, i.e. conservative
substitutions. Examples of such conservative substitutions include:
the substitution of one non-polar (hydrophobic) residue, such as
isoleucine, valine, alanine, glycine, leucine or methionine for
another non-polar residue; the substitution of one polar
(hydrophilic) residue for another polar residue, such as arginine
for lysine, glutamine for asparagine, threonine for serine; the
substitution of one basic residue such as lysine, arginine or
histidine for another basic residue; and the substitution of one
acidic residue, i.e. aspartic acid or glutamic acid, for the other.
The phrase "conservative substitution" is also intended to include
the use of a chemically derivatized residue in place of a
non-derivatized residue provided that the resultant polypeptide
displays the requisite biological activity, e.g. binding activity.
For purposes of this application, two peptides are considered to be
substantially the same when they only differ from each other by
conservative substitutions. Examples of preferred conservative
substitutions are set forth in Table 1.
1TABLE 1 Preferred Original Conservative Most Preferred Residue
Substitutions Substitution Ala (A) Val; Leu; ILe Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C)
Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H)
Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Leu Phe; Nle Leu
(L) Nle; Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met
(M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala Leu Pro (P) Gly
Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp;
Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Nle
[0034] "Chemical derivative" refers to a subject polypeptide having
one or more residues chemically derivatized by reaction of a
functional side group. Such derivatized polypeptides include, for
example, those in which free amino groups have been derivatized to
form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups may be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups may be derivatized to from O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine may be derivatized
to form N-im-benzylhistidine. Chemical derivatives also include
those peptides which contain one or more naturally occurring amino
acid derivatives of the twenty standard amino acids. For example:
4-hydroxyproline may be substituted for proline; 5-hydroxylysine
may be substituted for lysine; 3-methylhistidine may be substituted
for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine. Peptides embraced by the
present invention also include peptides having one or more residue
additions and/or deletions relative to the specific peptide whose
sequence is shown herein, so long as the modified peptide maintains
the requisite biological activity.
[0035] As used herein, the terms "pharmaceutically acceptable",
"physiologically tolerable" and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to a mammal without the production of undesirable
physiological effects such as nausea, dizziness, gastric upset and
the like.
[0036] The term "biologically active fragment" as used herein
refers to (a) a fragment of a peptide of the invention which has
been truncated with respect to either the N- or C-termini, or both;
or (b) a fragment of nucleic acid corresponding to a coding region
for rUcn or a highly homologous native peptide of another mammalian
species which has been truncated at the 5' or 3' end, or both, and
is useful in antisense applications. The peptide fragment shown
performs substantially the same function or a directly related
biological function as does the parent.
[0037] The phrase "modulating the transactivation of CRF receptors"
as used herein refers to administering a therapeutically effective
amount of a physiologically tolerable composition containing a
Ucn-like peptide to thereby modulate CRF actions in mammals by
means of direct or induced antagonistic(competitive) association
with CRF receptors (CRF-Rs).
[0038] CRF-R is used to refer to a family of receptor protein
subtypes which participate in the G-protein-coupled response of
cells to CRF and Ucn-like ligands. CRF-Rs are coupled by
heterotrimeric G-proteins to various intracellular enzymes, ion
channels, and transporters. The G-proteins associate with the
receptor proteins at the intracellular face of the plasma membrane.
An agonist binding to a CRF-R catalyzes the exchanges of GTP for
GDP on the .alpha.-subunit (G-protein "activation"), resulting in
its dissociation and stimulation of one (or more) of the various
signal-transducing enzymes and channels. G-protein preferentially
stimulates particular effectors, and the specificity of signal
transduction may be determined, therefore, by the specificity of
G-protein/receptor interaction. CRF-R proteins mediate signal
transduction through the modulation of adenylate cyclase and
perhaps through PI turnover. For example, when CRF or Ucn binds to
and activates the CRF-R, adenylate cyclase causes an elevation in
the level of intracellular cAMP. An effective bioassay for
evaluating whether a test compound is capable of elevating
intracellular cAMP is carried out by culturing cells containing
cDNA which expresses CRF receptor proteins in the presence of a
potential agonist or antagonist whose ability to modulate signal
transduction activity of CRF receptor protein is sought to be
determined. Such transformed cells are monitored for either an
increase or decrease in the level of intracellular CAMP which
provides a determination of the effectiveness of the potential
agonist or antagonist. Methods for measuring intracellular levels
of cAMP, or measuring cyclase activity, are well known in the
art.
[0039] A human CRF receptor was the first to be reported, and it
was cloned from a human Cushing pituitary tumor as described in
Chen R., et al, P.N.A.S., 90, 8967-8971 (October 1993). It is
referred to as hCRF-R1 or hCRF-RA and has 415 amino acids; a splice
variant thereof includes an insert of 29-amino acids. A rat CRF
receptor was isolated, approximately contemporaneously, by
hybridization from a rat brain cDNA library. It is referred to as
rCRF-R1; it has the 415 amino acid sequence which is set forth
hereinafter as SEQ ID NO: 10. It was disclosed in Perrin, M., et
al., Endocrinology, 133, 3058-3061 (1993). It was found to be 97%
identical at the amino acid level to the human CRF-R1, differing by
only 12 amino acids. The receptor has since been reported to be
widely distributed throughout the brain and the pituitary and to be
likely present in the adrenals and spleen.
[0040] A second subclass of CRF receptors has more recently been
found, and such receptors are arbitrarily referred to herein as
CRF-R2 but are sometimes referred to as CRF-RB. One such receptor,
having the amino acid sequence set forth hereinafter as SEQ ID NO:
11, was obtained by the cloning and characterization of a cDNA from
a mouse heart cDNA library. It is 431 amino acid residues in
length, and the details of the receptor are set forth in Perrin,
M., et al., P.N.A.S, 92, 2969-2973 (March 1995). It is hereinafter
referred to as CRF-R2.beta., but has been referred to as
CRF-RB.sub.L.
[0041] Another, slightly shorter receptor of this second subclass
was independently obtained from a rat hypothalamus cDNA library. It
is referred to herein as CRF-R2.alpha. and has the 411 amino acid
residue sequence set forth hereinafter as SEQ ID NO: 12. The
details of its cloning are set forth in Lovenberg, T., et al.,
P.N.A.S., 92, 836-840 (January 1995), wherein a second spliced
variant was also identified via PCR as being a putative protein of
431 amino acids that would be the rat homolog of mCRF-R2.beta.
identified above. The 431 amino acid sequence is set forth
hereinafter as SEQ ID NO: 13 and can be seen to be homologous with
mCRF-R2.beta.. The distribution of Ucn throughout the rat brain is
consistent with its being the endogenous ligand for CRF-R2, as is
the fact that it exhibits a much higher binding affinity, than does
CRF, for the receptor, particularly the R2s which is believed to be
the main CRF-R in the brain.
[0042] Ucn-like peptides, including rUcn, hUcn and analogs thereof,
can be easily synthesized as described in Example I hereinafter and
then individually tested for binding affinity. Binding affinity
refers to the strength of interaction between ligand and receptor.
To demonstrate binding affinity for a CRF receptor, the peptides of
the invention are easily evaluated using a tracer ligand of known
affinity, such as .sup.125I-radiolabeled oCRF, in binding assay
experiments which are well known in this art. The results of such
assays indicate the affinity at which each Ucn-like ligand binds to
a CRF receptor, expressed in terms of K.sub.i, an inhibitory
binding affinity constant relative to such a known standard.
K.sub.i (inhibitory binding affinity constant) is determined using
a "standard" or "tracer" radioactive ligand and thus measures the
displacement of the tracer from the receptor or binding protein; it
is most properly expressed with reference to such tracer. So long
as these assays are carefully performed under specific conditions
with relatively low concentrations of receptor or the like, the
calculated K.sub.i will be substantially the same as its
dissociation constant K.sub.D. It is particularly efficient to test
for K.sub.i because only a single tracer need be labelled, e.g.
radioiodinated. Dissociation constant K.sub.D is representative of
the concentration of ligand necessary to occupy one-half (50%) of
the binding sites of a receptor or the like. A given ligand having
a high binding affinity for a CRF receptor will require the
presence of very little ligand to bind at least 50% of the
available binding sites so that the K.sub.D value for that ligand
and receptor will be a small number. On the other hand, a given
ligand having a low binding affinity for a particular CRF receptor
will require the presence of a relatively high level of the ligand
to bind 50% of the sites, so that the K.sub.D value for that ligand
and receptor will be a large number.
[0043] With respect to a particular receptor protein, a Ucn-like
peptide having a K.sub.D of about 10 nM or less means that a
concentration of the ligand (i.e., the Ucn-like peptide) of no
greater than about 10 nM will be required to occupy at least 50% of
the active binding sites of the receptor protein. Such values may
be fairly determined from the results obtained using a
radioiodinated standard and no more than approximately 0.8 nM of
the receptor (approximately 10-20 pmol receptor/mg membrane
protein). Preferred Ucn-like peptides have a binding affinity
(K.sub.D) such that a ligand concentration of about 10 nanomolar or
less is required in order to occupy (or bind to) at least 50% of
the receptor binding sites, and particularly preferred Ucn-like
peptides have a binding affinity of 1 nM or less. Generally, a
dissociation constant of about 5 nanomolar or lower is considered
to be an indication of fairly strong affinity, and a K.sub.D of
about 1 nanomolar or less is an indication of very strong affinity.
For example, rUcn binds CRF-R1 with very strong affinity, having a
K.sub.D=about 0.18 nanomolar and binds CRF-R2.beta. with similar
strong affinity. It is also considered to be particularly
advantageous to provide Ucn-like peptides which have a
substantially higher affinity for CRF-R2, compared to CRF-R1, and
which will thus be selective in their biological effect. Because
CRF-R2 receptors are distributed widely throughout the body, Ucn
will have a substantially greater effect than CRF in modulating
many peripheral actions, and because the native peptide or
fragments thereof should not be immunogenic, it should be a very
good drug physiologically.
[0044] These binding assays employing CRF receptors are
straightforward to perform and can be readily carried out with
initially identified or synthesized peptides to determine whether
such peptides are effective agonists of CRF, or alternatively to
determine whether other shortened candidates are effective
antagonists of CRF. Such binding assays can be carried out in a
variety of ways as well known to one of skill in the art. A
detailed example of such an assay is set forth in Perrin, M., et
al., Endocrinology, 118, 1171-1179 (1986). Competitive binding
assays employing Ucn are particularly contemplated to evaluate
whether candidate peptides are effective agonists with respect to
each of the receptors previously described, i.e. CRF-R1,
CRF-R2.beta. and CRF-R2.alpha. as well as assays with Ucn
antagonists to determine whether candidates are effective
antagonists. In such assays, Ucn can be appropriately labeled with
a substance that is readily detected, such as a radioactive
isotope, e.g. .sup.125I, or an enzyme or some other suitable tag.
For example, suitably labelled agonists, such as
.sup.125I-Tyr.sup..smallcircle.-Ucn, or suitably labelled
antagonists, such as .sup.125I-(cyclo 29-32) [D-Tyr.sup.11,
Glu.sup.29, Lys.sup.32]-Ucn(11-40), are particularly useful tracers
for use in such receptor assays. Such receptor assays can be used
as screens for potential drugs which interact with CRF and/or CRF
receptors.
[0045] Very generally, the invention provides Ucn-like peptides,
including Ucn and analogs of Ucn, having an amino acid sequence
which is substantially the same as the following amino acid
sequence based upon SEQ ID NO: 8 and upon SEQ ID NO: 15 (Formula
I): Y-R.sub.1-Pro-R.sub.4-Le-
u-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr--
Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub-
.2, wherein Y is an acyl group having 7 or less carbon atoms,
preferably acetyl, or hydrogen; R.sub.1 is Asp-Asp or Asp-Asn or
Asp or Asn or desR.sub.1; and R.sub.4 is Pro or Ser; as well as
nontoxic salts thereof. When the N-terminus is shortened by the
deletion of 2 residues, it is preferably acylated, e.g. acetylated.
These peptides have pharmacological properties somewhat similar to
those of oCRF or r/hCRF and additional properties as described
hereinafter. As indicated hereinbefore, analogs of the above having
at least about 80% homology with the amino acid sequence of either
hUcn or rUcn are preferred for the Ucn-like peptides of the
invention, although peptides having at least 66% homology with
either hUcn or rUcn, wherein all or all but one of the
substitutions are conservative substitutions, are considered to be
biologically active and to have advantages over known CRF peptides.
Particularly preferred are analogs which are substantially the same
as either hUcn or rUcn (as defined hereinbefore) and which have
D-isomer amino acid substitutions and/or cyclizing bonds between
the side chains of specific residues in the sequence which are
known to increase ligand binding affinity for CRF receptors.
[0046] In addition to the foregoing general group of Ucn-like
peptides, two additional groups are disclosed hereinafter based
upon related syntheses and testing carried out in this general area
with regard to ligands which bind to CRF-R.
[0047] The following group of analogs of Ucn does not merely
include one or more conservative substitutions. Instead, bioactive
Ucn analogs are found to be defined according to the following
amino acid sequence:
Y-Asp-R.sub.2-Pro-R.sub.4-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-T-
hr-Leu-Leu-R.sub.19-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-R.sub.29-Ala-Glu-R-
.sub.32-Asn-Arg-Ile-R.sub.36-Phe-R.sub.38-Ser-Val-NH.sub.2, wherein
Y is an acyl group having 7 or less carbon atoms or hydrogen;
R.sub.2 is Asp or Asn; R.sub.4 is Pro or Ser; R.sub.19 is Glu or
Ala; R.sub.29 is Arg, Glu, Lys or Orn; R.sub.32 is Gln, Lys, Orn or
Glu; R.sub.36 is Ile, C.sup..alpha.MeIle or C.sup..alpha.MeLeu;
R.sub.38 is Asp or Ala; provided that when R.sub.29 is Glu,
R.sub.32 is either Lys or Orn and the side chains thereof are
linked by an amide bond and that when R.sub.32 is Glu, R.sub.29 is
either Lys or Orn and the side chains thereof are linked by an
amide bond; and provided further that D-Phe.sup.11 can be
substituted by another D-isomer amino acid, preferably a D-isomer
of a natural amino acid, such as D-Leu and more preferably one
other than D-Cys; that Glu in the 31-position can be substituted by
any D-amino acid, e.g. D-Glu, D-Asp, D-Arg, (imBzl)D-His,
.beta.-(2naphthyl)-D-Ala, etc., again preferably a D-isomer of a
natural amino acid other than Cys; and that the N-terminus can be
shortened by 1 or 2 residues. One particularly preferred Ucn analog
is (cyclo 29-32) [Lys.sup.29, D-Glu.sup.31, Glu.sup.32]-Ucn, with
Ucn being either hUcn or rUcn; others are described in the Examples
hereinafter. When N-terminally shortened by 7 to 10 residues, these
Ucn analogs are effective antagonists.
[0048] Extensive synthesis and testing over the past 10+ years have
shown that ligands for the CRF receptors can tolerate a number of
changes in amino acid sequence of native r/hCRF which do not result
in significant changes in bioactivity, such as would be indicative
of the resulting analog being no longer able to bind and/or
activate CRF receptors, particularly CRF-R1. As a result of this
extensive earlier work, it has been found that one, two, or three
substitutions can be made in the Ucn amino acid sequence, within
certain limits as set forth hereinafter, that will result in Ucn
analogs which retain the bioactivity of Ucn and, in some instances,
may have even more desirable pharmacological characteristics.
[0049] Using the amino acid sequence of Ucn as a reference, these
analogs should differ only by one, two or three substitutions from
SEQ ID NO: 8 or from SEQ ID NO: 15. The invention thus provides Ucn
analogs according to the following amino acid sequence (SEQ ID NO:
14):
Y-Xaa.sub.1-Xaa.sub.2-Pro-Xaa.sub.4-Xaa.sub.5-Ser-Xaa.sub.7-Asp-Leu-Xaa.s-
ub.10-Xaa.sub.11-Xaa.sub.12-Xaa.sub.13-Leu-Arg-Xaa.sub.16-Xaa.sub.17-Xaa.s-
ub.18-Xaa.sub.19-Xaa.sub.20-Xaa.sub.21-Xaa.sub.22-Xaa.sub.23-Xaa.sub.24-Xa-
a.sub.25-Xaa.sub.26-Xaa.sub.27-Xaa.sub.28-Xaa.sub.29-Ala-Xaa.sub.31-Xaa.su-
b.32-Asn-Arg-Xaa.sub.35-Xaa.sub.36-Xaa.sub.37-Xaa.sub.38-Xaa.sub.39-Xaa.su-
b.40-NH.sub.2, wherein Y is an acyl group having 7 or less carbon
atoms or hydrogen; Xaa.sub.1 is Asp, Glu or Gln; Xaa.sub.2 is Asn,
Asp, Glu or Gly; Xaa.sub.4 is Ser or Pro; Xaa.sub.5 is Leu, Ile or
Met; Xaa.sub.7 is Ile or Leu; Xaa.sub.10 is Thr or Ser; Xaa.sub.11
is Phe or Leu; Xaa.sub.12 is His or Glu; Xaa.sub.13 is Leu or Met;
Xaa.sub.16 is Thr, Asn, Glu, or Lys; Xaa.sub.17 is Leu, Met or Val;
Xaa.sub.18 is Leu or Ile; Xaa.sub.19 is Glu or His; Xaa.sub.20 is
Leu, Met, Ile or Arg; Xaa.sub.21 is Ala, Glu or Thr; Xaa.sub.22 is
Arg or Lys; Xaa.sub.23 is any natural amino acid and preferably
Thr, Ser, Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or
Asp; Xaa.sub.24 is Gln, Glu or Asp; Xaa.sub.25 is any natural amino
acid and preferably Ser, Thr, Ala, Ile, Met, Val, Asn, Gln, Gly,
Lys, His, Leu, Glu or Asp; Xaa.sub.26 is Gln, Leu or Glu;
Xaa.sub.27 is Arg, Ala or Lys; Xaa.sub.28 is Glu or Gln; Xaa.sub.29
is Arg or Gln; Xaa.sub.31 is any natural amino acid and preferably
Ala, Ile, Met, Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp;
Xaa.sub.32 is any natural amino acid and preferably Ala, Ile, Met,
Val, Asn, Gln, Gly, Lys, His, Leu, Glu or Asp; Xaa.sub.35 is Ile,
Lys, Leu or Asn; Xaa.sub.36 is Ile, Tyr, Met or Leu; Xaa.sub.37 is
Phe, Leu or Met; Xaa.sub.38 is Asp or Glu; Xaa.sub.39 is Ser, Ile,
Glu or Thr; and Xaa.sub.40 is Val, Ile, Phe or Ala; provided that
there are no more than 3 residues different from the hUcn or the
rUcn amino acid sequence and that the N-terminus may be shortened
by 1 or 2 residues. Moreover, these Ucn analogs when N-terminally
shortened by 7 to 10 residues constitute effective antagonists.
[0050] The Ucn-like peptides of the invention may be chemically
synthesized by any suitable method, such as by exclusively
solid-phase techniques, by partial solid-phase techniques, by
fragment condensation or by classical solution addition.
[0051] Ucn may also be synthesized by recombinant DNA techniques as
may its analogs which include only natural amino acids. The amino
acid sequence for rUcn (SEQ ID NO: 8) was deduced from a partial
cDNA clone isolated from a rat brain cDNA library. Set forth in
Table 2 hereinafter is the native rat nucleic acid sequence
encoding Ucn (SEQ ID NO: 9). The additional codon encoding glycine
that is present at the end of the native sequence is expected to
account for the C-terminal amidation of rUcn.
2TABLE 2 GAC GAC CCG CCG TTG TCC ATC GAC CTC ACC TTC CAC CTG CTG
CGG ACC Asp Asp Pro Pro Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg
Thr 1 5 10 15 CTG CTA GAG CTA GCT CGG ACA CAG AGC CAG CGC GAG CGC
GCA GAG CAG Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala
Glu Gln 20 25 30 AAC CGC ATC ATA TTC GAT TCG GTG GGCAAGTGA Asn Arg
Ile Ile Phe Asp Ser Val 35 40
[0052] Using the nucleic acid encoding rUcn as a probe, the nucleic
acid encoding the mature hUcn was isolated from a human genomic
placental library. Set forth in TABLE 2A hereinafter is the portion
of native human nucleic acid sequence encoding the mature Ucn
peptide (see SEQ ID NO: 16), with the additional codon for glycine
at the end being expected to account for C-terminal amidation.
3TABLE 2A GAC AAC CCT TCT CTG TCC ATT GAC CTC ACC TTT CAC CTG CTG
CGG ACC Asp Asn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg
Thr 1 5 10 15 CTG CTG GAG CTG GCG CGG ACG CAG AGC CAG CGG GAG CGC
GCC GAG CAG Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala
Glu Gln 20 25 30 AAC CGC ATC ATA TTC GAC TCG GTG GGCAAGTGA Asn Arg
Ile Ile Phe Asp Ser Val 35 40
[0053] Synthesis by the use of recombinant DNA techniques, for
purposes of this application, should be understood to include the
suitable employment isolated nucleic acid encoding for Ucn or an
appropriate analog, as is well known in the art at the present
time. As explained in detail hereinafter, synthetic Ucn peptides
may be obtained by transforming a microorganism using an expression
vector including appropriate regulatory sequences associated with
nucleic acid encoding a Ucn-like peptide and causing such
transformed microorganism to express the Ucn peptide.
[0054] Because of the relative shortness of the Ucn-like peptides,
about 40 residues or less, chemical or chain elongation synthesis
is presently felt to be the method of choice. Analogs of hUcn or
rUcn having one or more substitutions can be readily synthesized in
this manner and then tested for biological activity in a
straightforward manner to determine the specific biological effect
of such substitution(s). Common to such chemical syntheses of
peptides is the protection of the labile side chain groups of the
various amino acid moieties with suitable protecting groups which
will prevent a chemical reaction from occurring at that site until
the group is ultimately removed. Also common is the protection of
an alpha-amino group on an amino acid or a short peptide fragment
while that entity reacts at the free carboxyl group to effect chain
elongation, followed by the selective removal of the alpha-amino
protecting group to allow subsequent reaction to take place at that
location. Accordingly, it is common that, as a step in the
synthesis, an intermediate compound is produced which includes each
of the amino acid residues located in its desired sequence in the
peptide chain with various of these residues having side-chain
protecting groups.
[0055] Thus, in such chemical syntheses, intermediates are formed
having a protected amino acid sequence such as the following which
is based on hUcn SEQ ID NO: 15 (Formula II):
X.sup.1-Asp(X.sup.5)-Asn(X.sup.4)-Pro-Se-
r(X.sup.2)-Leu-Ser(X.sup.2)-Ile-Asp(X.sup.5)-Leu-Thr(X.sup.2)-Phe-His(X.su-
p.6)-Leu-Leu-Arg(X.sup.3)-Thr(X.sup.2)-Leu-Leu-Glu(X.sup.5)-Leu-Ala-Arg(X.-
sup.3)-Thr(X.sup.2)-Gln
(X.sup.4)-Ser(X.sup.2)-Gln(X.sup.4)-Arg(X.sup.3)-G-
lu(X.sup.5)-Arg(X.sup.3)-Ala-Glu(X.sup.5)-Gln(X.sup.4)-Asn(X.sup.4)-Arg(X.-
sup.3)-Ile-Ile-Phe-Asp(X.sup.5)-Ser(X.sup.2)-Val-X.sup.7 (or
suitably N-terminally shortened versions thereof) wherein:
[0056] X.sup.1 is either hydrogen or an alpha-amino protecting
group. The alpha-amino protecting groups contemplated by X.sup.1
are those known to be useful in the art in the step-wise synthesis
of polypeptides. Among the classes of alpha-amino protecting groups
covered by X.sup.1 are (1) acyl-type protecting groups, such as
formyl, acrylyl(Acr), benzoyl(Bz) and acetyl(Ac) which are
preferably used only at the N-terminal; (2) aromatic urethan-type
protecting groups, such as benzyloxycarbonyl(Z) and substituted Z,
such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbony- l,
p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic
urethan protecting groups, such as t-butyloxycarbonyl (BOC),
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups,
such as fluorenylmethyloxycarbonyl (FMOC), cyclopentyloxycarbonyl,
adamantyloxycarbonyl,and cyclohexyloxycarbonyl; and (5)
thiourethan-type protecting groups, such as phenylthiocarbonyl. The
preferred alpha-amino protecting group is BOC if the synthesis
employs acid-catalyzed removal of the alpha-amino protecting
groups; however, for syntheses employing a base-catalyzed removal
strategy, FMOC is preferred, in which case, more acid-labile side
chain protecting groups can be used, including t-Butyl esters or
ethers as well as BOC.
[0057] X.sup.2 is hydrogen or a protecting group for the hydroxyl
group of Thr and Ser and is preferably acetyl (Ac), benzoyl (Bz),
tert-butyl, triphenylmethyl (trityl), tetrahydropyranyl, benzyl
ether (Bzl) or 2,6-dichlorobenzyl (DCB). The most preferred
protecting group is Bzl.
[0058] X.sup.3 is hydrogen or a protecting group for the guanidino
group of Arg, preferably selected from nitro, p-toluenesulfonyl
(Tos), Z, adamantyloxycarbonyl and BOC. Tos is preferred for a BOC
strategy, and 4-methoxy-2,3,6-trimethyl benzenesulfonyl (MTR) or
pentamethyl chroman-6-sulfonyl (PMC) is preferred for FMOC
strategies.
[0059] X.sup.4 is hydrogen or a protecting group for the side chain
amido group of Asn or Gln, preferably xanthyl (Xan). Asn or Gln is
preferably coupled without side chain protection in the presence of
hydroxybenzotriazole (HOBt).
[0060] X.sup.5 is hydrogen or an ester-forming protecting group for
the .beta.- or .gamma.-carboxyl group of Asp or Glu, preferably
cyclohexyl (OChx), benzyl (OBzl), 2,6-dichlorobenzyl, methyl, ethyl
and t-butyl ester (Ot-Bu). Chx is preferred for a BOC strategy and
Ot-Bu for FMOC strategy.
[0061] X.sup.6 is hydrogen or a protecting group for the side chain
imidazole nitrogen of His, such as Tos.
[0062] The selection of a side chain amino protecting group is not
critical except that the protecting group should be one which is
not removed during deprotection of the alpha-amino groups during
the synthesis. Hence, the alpha-amino protecting group and the
side-chain-amino protecting group cannot be the same.
[0063] X.sup.7 is NH.sub.2, a protecting group, such as an ester,
or is an anchoring bond of the type used in solid phase synthesis
for linking the peptide being synthesized to a solid resin support,
preferably one represented by the formulae:
[0064] --NH-benzhydrylamine (BHA) resin support and
[0065] --NH-paramethylbenzhydrylamine (MBHA) resin support.
[0066] Cleavage from a BHA or MBHA resin directly gives the
Ucn-like peptide in amidated form. By employing a methyl-derivative
of such a resin, if desired, the corresponding, equivalent,
methyl-substituted amide can be created. Alternatively, using an
appropriate resin support, the ethylamide, which is also considered
to be an equivalent, can be created as well known in this art.
[0067] In the formula for the intermediate, at least one of
X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5 and X.sup.6 is a
protecting group. In selecting a particular side chain protecting
group to be used in the synthesis of the peptides, the following
rules are followed: (a) the protecting group should be stable to
the reagent, and under the reaction conditions, selected for
removing the alpha-amino protecting group at each step of the
synthesis, (b) the protecting group should retain its protecting
properties and not be split off under coupling conditions and (c)
the protecting group must be removable upon completion of the
synthesis under reaction conditions that will not alter the peptide
chain.
[0068] For the acyl group at the N-terminus of a Ucn-like agonist
peptide, which is represented by Y, acetyl, formyl, acrylyl and
benzoyl are preferred. Moreover, as indicated earlier, the
N-terminus can be slightly shortened by removal of the N-terminal
residue or the first two N-terminal residues without significantly
affecting biological potency of the peptide to function as a Ucn
agonist, and when such shortening occurs, acylation of the residue
at the shortened N-terminus may be preferred. More extensive
shortening of the N-terminus by a sequence of 7 to 11 residues
results in the creation of Ucn antagonists which strongly bind
CRF-R without activating the receptor as discussed hereinafter.
[0069] Overall, there is broadly provided a process for the
manufacture of peptides defined by Formula I or analogs thereof
comprising (a) forming an intermediate peptide according to Formula
II or an analog thereof wherein there is at least one protective
group, with X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5 and X.sup.6
being each either hydrogen or a protective group, and X.sup.7 being
either a protective group or an anchoring bond to resin support or
NH.sub.2 and (b) splitting off the protective group or groups or
anchoring bond from the intermediate peptide of the Formula II and
(c) if desired, converting the resulting peptide into a nontoxic
salt thereof.
[0070] When the peptides of the invention are prepared by chemical
synthesis, they are preferably prepared using solid phase
synthesis, such as that described by Merrifield, J. Am. Chem. Soc.,
85, p 2149 (1964), although other equivalent chemical syntheses
known in the art can also be used as previously mentioned. Thus,
Ucn-like peptides can be prepared in a straightforward manner and
then simply tested for biological activity. This facilitates the
ready preparation and evaluation of Ucn-like peptides which are
analogs of hUcn or rUcn. Solid-phase synthesis is preferably
commenced from the C-terminus of the peptide by coupling a
protected alpha-amino acid to a suitable resin as generally set
forth in U.S. Pat. No. 4,244,946 issued Jan. 21, 1981 to Rivier et
al. by coupling with the free carboxyl group. The synthesis of Ucn
can be initiated by coupling alpha-amino-protected Val to a BHA
resin using methylene chloride and dimethylformamide (DMF).
Following the coupling of BOC-Val to the resin support, the
alpha-amino protecting group may be removed using trifluoroacetic
acid(TFA) in methylene chloride, TFA alone or with HCl in dioxane.
Preferably 50 volume % TFA in methylene chloride is used with 0-5
weight % 1,2 ethanedithiol. The deprotection is carried out at a
temperature between about 0.degree. C. and room temperature. Other
standard cleaving reagents and conditions for removal of specific
alpha-amino protecting groups may be used as described in Schroder
& Lubke, "The Peptides", 1, pp 72-75 (Academic Press 1965).
[0071] After removal of the alpha-amino protecting group of Val,
the remaining alpha-amino- and side chain-protected amino acids are
coupled stepwise in the desired order to obtain the intermediate
compound of Formula II. As an alternative to adding each amino acid
separately in the synthesis, some of them may be coupled to one
another prior to addition to the solid phase reactor. The selection
of an appropriate coupling reagent is within the skill of the art.
Particularly suitable as coupling reagents are N,N'-dicyclohexyl
carbodiimide (DCCI) and N,N'-diisopropyl carbodiimide (DICI).
[0072] Activating reagents used in the solid phase synthesis of the
peptides of the invention are well known in the peptide art.
Examples of suitable activating reagents are carbodiimides, such as
N,N'-diisopropyl carbodiimide and
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide. Other activating
reagents and their use in peptide coupling are described by
Schroder & Lubke, supra, in Chapter III and by Kapoor, J. Phar.
Sci., 59, pp 1-27 (1970). P-nitrophenyl ester (ONp) may also be
used to activate the carboxyl end of Asn or Gln for coupling. For
example, BOC-Asn (ONp) can be coupled overnight using one
equivalent of HOBt in a 50% mixture of DMF and methylene chloride,
in which case no DCCI is added.
[0073] Each protected amino acid or amino acid sequence is
introduced into the solid phase reactor in about a fourfold excess,
and the coupling is carried out in a medium of dimethylformamide
(DMF): CH.sub.2Cl.sub.2 (1:1) or in DMF or CH.sub.2Cl.sub.2 alone.
In instances where the coupling is carried out manually, the
success of the coupling reaction at each stage of the synthesis is
monitored by the ninhydrin reaction, as described by E. Kaiser et
al., Anal. Biochem. 34, 595 (1970). In cases where incomplete
coupling occurs, the coupling procedure is repeated before removal
of the alpha-amino protecting group prior to the coupling of the
next amino acid. The coupling reactions can be performed
automatically, as on a Beckman 990 automatic synthesizer, using a
program such as that reported in Rivier et al., Biopolymers, 1978,
17, pp.1927-1938.
[0074] After the desired amino acid sequence has been completed,
the intermediate peptide is removed from the resin support by
treatment with a suitable clearing agent, such as liquid hydrogen
fluoride, which not only cleaves the peptide from the resin but
also cleaves all remaining side chain protecting groups X.sup.2,
X.sup.3, X.sup.4, X.sup.5 and X.sup.6 and the alpha-amino
protecting group X.sup.1 (unless it is an acyl group which is
intended to be present in the final peptide). When using hydrogen
fluoride for cleaving, anisole or cresole and methylethyl sulfide
are preferably included in the reaction vessel as scavengers. The
BOC protecting group at the N-terminus is preferably cleaved with
trifluoroacetic acid(TFA)/ethanedithiol prior to the cleaving of
the peptide from the resin.
[0075] The determination of whether any Ucn-like peptide of about
40 residues in length, or a fragment thereof, or an antagonist
version thereof, will have desirable pharmacological properties can
be made in a straightforward manner. First, assays are run to
determine the effect of a candidate agonist or antagonist peptide
on the different CRF receptors; then, the ability of the peptide to
promote or inhibit production of ACTH is determined. Fragments
which function as CRF-BP blockers can be likewise similarly assayed
using an inhibitory binding assay with hCRF-BP and a known labelled
ligand.
[0076] The candidate peptide is easily evaluated in binding assays
with the various CRF receptors earlier discussed using assays as
described in Perrin, M., et al., Endocrinology, 118, 1171-1179
(1986). A binding assay with human CRF-R1 is preferably carried out
using a radioligand oCRF analog; such a binding assay utilizing
CRF-R1 receptor is described in Chen, et al., P.N.A.S., 90,
supra.
[0077] A straightforward assay using rat anterior pituitary cells
in monolayer culture can be carried out to determine whether a
candidate peptide thereof will function as a CRF agonist and
stimulate ACTH secretion by activating CRF receptors on such cells.
The procedure which is used is that generally set forth in
Endocrinology, 91, supra. A very similar assay is used to test for
antagonistic properties, using a challenge dose of oCRF or the
like.
[0078] By the in vivo administration to mammals of peptides which
have a high affinity to human CRF binding protein and which thus
compete with endogenous CRF and Ucn for binding to hCRF-BP, CRF-BP
is effectively blocked. This leaves endogenous CRF and Ucn
available in higher concentrations to carry out their usual
biological functions throughout the body, particularly in localized
areas where the peptide is administered and/or where CRF-BP is
present.
[0079] More specifically, fragments of Ucn, or analogs of Ucn,
between about 19 and 28 residues in length have a very high
affinity to hCRF-BP, but generally exhibit relatively low
propensity for binding CRF receptors. As a result, these blocking
fragments can be administered to prevent the clearance of
endogenous CRF and/or Ucn from particular regions in the body and
thereby stimulate the biological effect of CRF and/or Ucn in vivo.
In certain instances, it may be advantageous to administer such
peptides along with CRF or Ucn or an agonist thereof. The very
nature of these fragments is such that potentially undesirable
immunogenic side effects are minimized or totally obviated. They
might also be administered along with antagonists to prevent the
clearance of antagonists having a fairly high binding affinity to
hCRF-BP from a target region. These blocking fragments are useful
for therapeutic treatment to promote parturition in pregnancy, to
stimulate the respiratory system, to combat obesity, and to
counteract the effects of Alzheimer's disease, and of chronic
fatigue syndrome; for the latter four indications, the blocking
fragments are preferably administered in a manner so as to be
delivered to the brain.
[0080] Ucn peptides can be used in diagnostic methods to detect the
level of Ucn present in a body sample as well as in an inoculum for
the preparation of antibodies that immunoreact with epitopes on
Ucn. Antibodies generated against Ucn can be employed for
diagnostic applications, therapeutic applications, and the like.
Such antibodies can be prepared employing standard techniques, as
are well known to those of skill in the art, using Ucn or a
fragment thereof, as an antigen. Antibodies of the present
invention are typically produced by immunizing a mammal, e.g.
rabbit, sheep, goat, etc., with an inoculum containing Ucn or
fragment thereof thereby inducing the production of antibody
molecules having immunospecificity for the immunizing agent.
[0081] Antibodies which recognize Ucn are raised against either the
entire 40-residue amino acid sequence or against a synthetic
fragment of a sequence of at least about 5 or preferably 6
residues. For example, such antibodies can be raised against the 6
N-terminal residues, or against the 6 C-terminal residues, or
against an interior sequence, such as the sequence embracing
residues 18-23. Such antibodies will bind to and thus can be
employed to indicate the presence of Ucn; they are therefore useful
in assays. Moreover, certain of these antibodies will bind to and
biologically inactivate Ucn, and such antibodies can be
administered to animals for the purpose of neutralizing endogenous
Ucn. In an instance where endogenous antibodies are being created
that would bind Ucn, short amino acid sequences from Ucn might be
administered as antibody blockers. To generate such blocking
antibodies, either the entire 40-residue sequence can be used, or a
short peptide sequence can be synthesized constituting one region
of particular interest. Such a synthetic short chain peptide is
generally conjugated to a large carrier molecule, and the conjugate
is then used as inoculum to induce a mammalian immune system in
rabbits or sheep or the like. Details of the production of such
polyclonal antibodies are set forth in U.S. Pat. No. 4,864,019
(Sep. 5, 1989). If instead of polyclonal antibodies, it is desired
to produce monoclonal antibodies, such can be made in a
straightforward manner using similar inoculum by employing
hybridoma techniques now well established in this art. Details of
exemplary monoclonal antibody production are set forth in U.S. Pat.
No. 5,032,521 (Jul. 16, 1991), and U.S. Pat. No. 5,051,364 (Sep.
24, 1991).
[0082] Antibodies so produced can be used in diagnostic methods and
systems to detect the level of Ucn present in a human or other
mammalian body sample, such as tissue or fluid. The anti-Ucn
antibodies may also be used for immunoaffinity or affinity
chromatography purification of Ucn, the details of which are well
known in this art. In addition, an anti-Ucn antibody can be used in
human therapeutic methods. Moreover, it is contemplated that DNA
encoding such antibodies may be injected via gene therapy methods
to raise desired antibodies within a patient or alternatively to
provide antibody blockers in an appropriate situation.
[0083] The lack of evolutionary digression with respect to the
amino acid sequences of the 41-residue CRF biological messenger in
rats and humans (which sequences are identical) is fairly
indicative of the probability of conserved regions in the
corresponding amino acid sequences of Ucn in mammalian species,
such as human, bovine, porcine, ovine, caprine, murine, canine,
feline, baboon, monkey, rabbit, etc. The corollary is that, once
one has a significant portion of the Ucn nucleic acid sequence of
one mammalian species, i.e. the rat sequence as disclosed herein,
it is a straightforward exercise to obtain naturally occurring
variant homolog nucleic acid sequences of other animal species
which encode homologs, such as CRF-binding proteins (see e.g.,
Potter et al., Nature, 349, 423-426 (1991), where it was shown that
the cDNA coding region for human serum-derived CRF binding protein
was of sufficient homology in the rat cDNA coding region to permit
identification of the latter. The first disclosed nucleic acid
sequence (SEQ ID NO: 9) is of the native rat species; in addition
to its being useful in an expression vector to express a Ucn
peptide, it is also useful to obtain the DNA of other mammalian
species encoding the respective counterpart Ucn peptides. In this
respect, either the entire nucleic acid sequence or nucleic acid
sequences at least about 14 nucleotides in length can be used as
hybridization probes to obtain and clone counterpart mammalian
sequences, as is presently well known in this art. Similarly,
primers based upon the foregoing nucleic acid sequence can be used
along with PCR (Polymerase Chain Reaction) techniques to amplify
nucleic acid sequences of other mammalian species using suitable
sources of DNA.
[0084] As used herein, a nucleic acid "probe" may be a
single-stranded DNA or RNA that has a nucleotide sequence of at
least 14, and preferably at least 20 or more, contiguous bases that
are the same as (or the complement of) any 14 or more contiguous
bases set forth in SEQ ID NO: 9.
[0085] Labeled nucleic acid encoding Ucn, or fragments thereof, can
be employed to probe cDNA libraries, genomic libraries and the like
for additional nucleotide sequences encoding other novel mammalian
members of the Ucn family. Such screening may be initially carried
out under stringency conditions employing a temperature of about
42.5.degree. C., a formamide concentration of about 20%, and a salt
concentration of about 5.times. standard saline citrate (SSC;
20.times. SSC contains 3M sodium chloride, 0.3M sodium citrate, pH
7.5). Such conditions will allow the identification of sequences
having substantial similarity with the probe sequence, without
requiring perfect homology. By "substantial similarity" is meant
nucleotide sequences which share at least about 50% homology. It
may be desirable to select hybridization conditions which will
identify only sequences having at least 70% homology with the
probe, while discriminating against sequences which have a lower
degree of homology with the probe; such is effected by increasing
the stringency used to exceed the above-stated conditions as is
well known in this art.
[0086] For example, using established methods well known to those
skilled in the art (see e.g., Molecular Cloning, A Laboratory
Manual 2Ed, Chapter 8, Construction and Analysis of cDNA Libraries,
J. Sambrook et al. (1989)), either the entire SEQ ID NO: 9 or
portions thereof of at least about 14 or 17 or 20 nucleotides in
length may be used to screen mammalian genomic or cDNA libraries to
identify and isolate homologous nucleic acids encoding Ucn from
human and other mammalian species. Oligonucleotide sequences of
about 14 or 17 or 20 nucleotides or longer can be prepared by
conventional in vitro synthesis techniques. Screening with such
oligonucleotides as probes is preferably carried out under high
stringency conditions as defined in Sambrook et al., supra, Chapter
11, pp. 11.45-11.57.
[0087] As indicated above, possession of a native DNA sequence
encoding a specific peptide hormone of one mammalian species allows
one to obtain the homologous DNA sequence of other mammalian
species. Isolation of nucleotide sequences encoding Ucn peptides of
other species often involves utilization of either a genomic
library or a cDNA library made from RNA isolated from tissue
containing Ucn. If such a source is available, it will generally be
preferable to create a cDNA library for isolation of nucleotide
sequences encoding Ucn so as to avoid any possible problems arising
from attempts to determine intron/exon borders. Libraries can be
made in either eukaryotic or prokaryotic host cells. Widely
available cloning vectors, such as plasmids, cosmids, phage and the
like, can be used to generate genetic libraries suitable for the
isolation of nucleotide sequences encoding Ucn peptides.
[0088] Methods for screening genetic libraries for the presence of
target nucleotide sequences include using such probes based on the
sequence of a known nucleotide sequence are described in detail in
Chapter 11 of Sambrook et al., supra. In the present situation, it
may be preferable to use the entire length of the rat nucleotide
sequence SEQ ID NO: 9 labeled with radionuclides, enzymes, biotin,
fluoresces, or the like, as a probe for screening such genetic
libraries.
[0089] Hybridization refers to the binding of complementary strands
of nucleic acid (i.e., sense:antisense strands or probe:target-DNA)
to each other through hydrogen bonds, similar to the bonds that
naturally occur in chromosomal DNA. Stringency levels used to
hybridize a given probe with target-nucleic acid can be readily
varied by those of skill in the art. As used herein, the phrase
"high stringency" hybridization refers to conditions that permit
target-nucleic acid to bind a complementary nucleic acid that has
at least about 80% homology to the target-nucleic acid. An example
of such high stringency conditions would be conditions being
minimally equivalent to hybridization in 50% formamide, 5.times.
Denhart's solution, 5.times. SSPE, 0.2% SDS at 42.degree. C.,
followed by washing in 0.2.times. SSPE, 0.2% SDS, at 65.degree. C.
Denhart's solution and SSPE are desirable in Sambrook et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989) and are well known to those of skill in
this art; there are other suitable hybridization buffers that may
be used. It may be preferred to use stringency conditions requiring
greater than about 90% homology to target-DNA.
[0090] As an example, the human form of Ucn was successfully cloned
from a human placental genomic library. Approximately
0.6.times.10.sup.6 phage plaques of a human placental genomic
library in the EMBL3 SP6/T7 vector (Clontech) were screened by
hybridization using a probe corresponding to the mature peptide
region of rat Ucn. The 160 bp probe encoding the rat Ucn mature
peptide was synthesized by PCR using the following oligos (sense:
5'-TGCAGGCGAGCGGCAACGACGAGACGA-3') (SEQ ID NO: 18) and (antisense:
5'-ATACGGGGCCGATCACTTGCCCACCGAG-3') (SEQ ID NO: 19) and
[.alpha..sup.32P-dCTP]. Hybridization was carried out at 42.degree.
C. in standard buffers with 20% formamide. Final washes were at
42.degree. C. in 2.times. SSC/0.1% SDS. The phage DNA from an
individual positive plaque for the clone was purified and then
subcloned into pBluescript (Strategene). Dideoxy sequencing was
done using the Sequenase kit (US Biochemical).
[0091] The genomic clone isolated from the library which contains
the gene for human Ucn has an insert size of approximately 15 kb. A
large number of base pairs of the insert have been sequenced (see
SEQ ID NO: 16) in the region corresponding to the precursor and the
mature peptide region of human Ucn. The portion of nucleotide
sequence initially sequenced encoding the mature human Ucn peptide,
as compared to the mature rat Ucn peptide, was set forth in TABLE
2A hereinbefore. The nucleotide sequence for the mature human Ucn
peptide is 88% similar to the nucleotide sequence for the rat Ucn
peptide. The amino acid sequence encoded by this region shows 95%
similarity between human Ucn and rat Ucn. The mature human peptide
(see residues 83-122 of SEQ ID NO: 15) is 40 residues of the total
of 124.
[0092] Another suitable technique which may be used in the present
situation involves the use of primers based on sequences derived
from rat Ucn nucleic acid and the polymerase chain reaction (PCR)
to amplify target nucleic acid. The target can then be isolated
using a specific hybridization probe based on the amplified
segment, which is then analyzed for its overall sequence and the
polypeptide which it encodes.
[0093] To synthesize a peptide of the invention by recombinant DNA,
a double-stranded DNA which encodes the desired peptide can be
synthetically constructed. Although PCR techniques might nowadays
be the method of choice to produce a DNA sequence, for example, SEQ
ID NO: 9, DNA encoding Ucn can be designed using certain codons
that are particularly efficient for polypeptide expression in a
certain organism, i.e. selection might employ those codons which
are most efficient for expression in the type of organism which is
to serve as the host for the recombinant vector. However, any
correct set of codons will encode a desired product, although
perhaps slightly less efficiently. Codon selection may also depend
upon vector construction considerations; for example, it may be
necessary to avoid placing a particular restriction site in the DNA
coding sequence if, subsequent to inserting the coding sequence,
the vector is to be manipulated using a restriction enzyme that
cleaves at such a site. Of course one would avoid placing
restriction sites in the DNA coding sequence if the host organism,
which is to be transformed with the recombinant vector, is known to
produce a restriction enzyme that would cleave at such a site
within the DNA chain.
[0094] Isolated nucleotide sequences encoding Ucn and analogs
thereof can be used to produce purified Ucn by either recombinant
DNA methodology or by in vitro polypeptide synthesis techniques.
The term "isolated" refers to a nucleotide sequence or a
polypeptide sequence that has been manually produced and is
separated from its native, in vivo, cellular environment and is
present in the substantial absence of other biological molecules of
the same type. As a result of this human intervention, the
recombinant, isolated and/or substantially pure DNAs, RNAs,
polypeptides and proteins of the invention can be produced in large
quantities and are useful in ways that the DNAs, RNAs, polypeptides
or proteins as they naturally occur are not, such as identification
of selective drugs or compounds. The term "purified" as used herein
for nucleotide sequences preferably means at least 95% by weight,
and most preferably at least 99% by weight, of biological
macromolecules of the same type present (but water, buffers, and
other small molecules, can be present).
[0095] To assemble a synthetic, nonchromosomal nucleic acid
sequence, oligonucleotides are constructed by conventional
procedures such as those described in Sambrook et al., supra. Sense
and antisense oligonucleotide chains, up to about 70 nucleotide
residues long, are synthesized, preferably on automated
synthesizers well known in this art. The oligonucleotide chains are
constructed so that portions of the sense and antisense
oligonucleotides overlap, associating with each other through
hydrogen bonding between complementary base pairs and thereby
forming double-stranded chains, in most cases with gaps in the
strands. Subsequently, the gaps in the strands are filled in, and
oligonucleotides of each strand are joined end to end with
nucleotide triphosphates in the presence of appropriate DNA
polymerases and/or with ligases. As an alternative to such stepwise
construction of a synthetic nucleic acid sequence, DNA or cDNA
encoding the complete structure of a native polypeptide as obtained
by screening a library is used. As indicated hereinbefore,
amplification is preferably carried out by using PCR, and the
isolated and purified DNA is then incorporated into recombinant
molecules.
[0096] The desired nucleic acid coding sequence to be inserted into
a vector preferably has linkers at its ends to facilitate insertion
into restriction sites within the cloning vector. Optionally, the
nucleic acid coding sequence may be constructed so as to encode the
desired peptide as a portion of a fusion polypeptide; and if so,
the coding sequence will generally contain terminal sequences that
encode amino acid residue sequences that serve as proteolytic
processing sites, whereby the encoded polypeptide may be
proteolytically cleaved from the remainder of the fusion
polypeptide. The terminal portions of the nucleic acid coding
sequence may also contain appropriate start and stop signals.
[0097] The desired peptide is then expressed by recombinant
techniques after the nucleic acid coding sequence is functionally
inserted into a vector. By "functionally inserted" is meant in
proper reading frame and orientation, as is well understood by
those skilled in this art. For example, when producing a genetic
construction containing a complete Ucn reading frame, the preferred
starting material is a cDNA library isolate encoding Ucn rather
than a genomic library isolate. Typically, the Ucn-encoding
sequence will be inserted downstream from a promoter and will be
followed by a stop codon, although production as a hybrid protein
followed by cleavage may be used, if desired. In general,
host-cell-specific sequences which improve the production yield of
Ucn will be used, and appropriate control sequences will be added
to the expression vector, such as enhancer sequences,
polyadenylation sequences, and ribosome binding sites.
[0098] The production of Ucn can be carried out in both prokaryotic
and eukaryotic cell lines to provide protein for biological and
therapeutic use. While Ucn synthesis is easily demonstrated using
either bacteria or yeast cell lines, the synthetic genes should
also be insertable for expression in cells of higher animals, such
as Chinese hamster ovary (CHO) cells or mammalian tumor cells as
described in detail in Sambrook et al, supra. Some mammalian cells
may be grown, for example, as peritoneal tumors in host animals,
and Ucn harvested from the peritoneal fluid. Descriptions of
mammalian expression systems, baculovirus expression systems,
bacterial expression systems and yeast expression systems are set
forth in U.S. Pat. No. 5,212,074 (May 18, 1993).
[0099] The following Examples set forth preferred chemical methods
for synthesizing Ucn, Ucn analogs, Ucn fragments, and Ucn
antagonists, by the solid-phase chain elongation technique.
EXAMPLE I
[0100] The synthesis of rat Ucn having the formula (SEQ ID NO:
8):
[0101]
Asp-Asp-Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-
-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-I-
le-Phe-Asp-Ser-Val-NH.sub.2 is conducted in a stepwise manner on a
MBHA hydrochloride resin, such as available from Bachem, Inc.,
having a substitution range of about 0.1 to 0.5 mmoles/gm. resin.
The synthesis is performed on an automatic Beckman 990B peptide
synthesizer using a suitable program, such as the following:
4 STEP REAGENTS AND OPERATIONS MIX TIMES MIN. 1 CH.sub.2Cl.sub.2
wash-80 ml. (2 times) 3 2 Methanol (MeOH) wash-30 ml. 3 (2 times) 3
CH.sub.2Cl.sub.2 wash-80 ml. (3 times) 3 4 50 percent TFA plus 5
percent 12 1,2-ethanedithiol in CH.sub.2Cl.sub.2- 70 ml. (2 times)
5 Isopropanol wash-80 ml. 3 (2 times) 6 TEA 12.5 percent in
CH.sub.2Cl.sub.2-70 ml. 5 (2 times) 7 MeOH wash-40 ml. (2 times) 2
8 CH.sub.2Cl.sub.2 wash-80 ml. (3 times) 3 9 Boc-amino acid (10
mmoles in 30-300 30 ml. of either DMF or CH.sub.2Cl.sub.2,
depending upon the solubility of the particular protected amino
acid, (1 time), plus DCCI (10 mmoles) in CH.sub.2Cl.sub.2
[0102] Coupling of BOC-Val results in the substitution of about
0.35 mmol Val per gram of resin. All solvents that are used are
carefully degassed, preferably by sparging with an inert gas, e.g.
helium or nitrogen, to insure the absence of oxygen.
[0103] After deprotection and neutralization, the peptide chain is
built step-by-step on the resin. Generally, one to two mmol. of
BOC-protected amino acid in methylene chloride is used per gram of
resin, plus one equivalent of 2 molar DCCI in methylene chloride,
for two hours for the coupling of each additional residue. When
BOC-Arg(Tos) is being coupled, a mixture of 50% DMF and methylene
chloride is used. Bzl is used as the hydroxyl side-chain protecting
group for Ser and Thr. The amido group of Asn or Gln can be
protected by Xan but need not be. BOC-Asn or BOC-Gln is coupled
overnight using one equivalent of DCC and two equivalents of HOBt
in a 50% mixture of DMF and methylene chloride. Tos is used to
protect the guanidino group of Arg and the imidazole nitrogen of
His. The side chain carboxyl group of Glu or Asp is protected by
OChx. At the end of the synthesis, the following composition is
obtained:
[0104]
BOC-Asp(OChx)-Asp(OChx)-Pro-Pro-Leu-Ser(Bzl)-Ile-Asp(OChx)-Leu-Thr(-
Bzl)-Phe-His(Tos)-Leu-Leu-Arg(Tos)-Thr(Bzl)-Leu-Leu-Glu(OChx)-Leu-Ala-Arg(-
Tos)-Thr(Bzl)-Gln-Ser(Bzl)-Gln-Arg(Tos)-Glu(OChx)-Arg(Tos)-Ala-Glu(OChx)-G-
ln-Asn-Arg(TOS)-Ile-Ile-Phe-Asp(OChx)-Ser(Bzl)-Val-NH-resin
support.
[0105] In order to cleave and deprotect the resulting peptide, the
peptide-resin is treated with 1.5 ml. anisole, 0.5 ml. of
methylethylsulfide and 15 ml. hydrogen fluoride (HF) per gram of
peptide-resin, first at -20.degree. C. for 20 min. and then at
0.degree. C. for one and one-half hours. After elimination of HF
under high vacuum, the resin-peptide is washed with dry diethyl
ether, and the peptide amide is then extracted with de-gassed 2N
aqueous acetic acid or a 1:1 mixture of acetonitrile and water. The
extract is separated from the resin by filtration, and then
lyophilized.
[0106] The lyophilized peptide amide is purified by preparative or
semi-preparative HPLC as described in Rivier et al., J.
Chromatography, 288, 303-328 (1984); and Hoeger et al.,
Biochromatography, 2, 3, 134-142 (1987). The chromatographic
fractions are carefully monitored by HPLC, and only the fractions
showing substantial purity are pooled.
[0107] Specific optical rotation of the isolated and purified Ucn
peptide is measured on a Perkin Elmer Model 241 Polarimeter as
[.alpha.].sub.D=-62.5.degree..+-.1.0 (c=1 in 1% acetic acid,
without correction for the presence of H.sub.2O and TFA); it has a
purity of greater than about 95%. Purity is further confirmed by
mass spectroscopy and capillary zone electrophoresis (CZE). Liquid
secondary ion mass spectrometry (LSIMS) mass spectra are measured
with a JEOL model JMS-HX110 double-focusing mass spectrometer
fitted with a Cs.sup.+ gun. An accelerating voltage of 10 kV and
Cs.sup.+ gun voltage between 25 and 30 kV are employed. The
measured value of 4705.36 Da obtained using LSIMS is in agreement
with the calculated value of 4705.52 Da.
[0108] To verify the precise sequence, the Ucn peptide is
hydrolyzed in sealed evacuated tubes containing constant boiling
HCl, 3 .mu.l of thioglycol/ml. and 1 nmol of Nle (as an internal
standard) for 9 hours at 140.degree. C. Amino acid analyses of the
hydrolysates using a standard amino acid analyzer shows the
expected amino acid ratios, which confirms that a 40-residue
peptide structure is obtained with the expected amino acid residues
which constitute the intended sequence.
[0109] To provide a labelled Ucn peptide for use in assays,
including binding assays and the like, the synthesis is extended to
link Tyr to Asp at the N-terminus, producing the 41-residue peptide
referred to as [Tyr.sup..smallcircle.]-Ucn, which can be readily
iodinated with .sup.125I and then used in diagnostic assays and in
drug-screening assays.
[0110] To test the ability of purified Ucn or another candidate
Ucn-like peptide to function as a CRF agonist and to promote ACTH
production and/or secretion, cultures of dispersed primary murine
anterior pituitary cells (0.15.times.10.sup.6 cells/well are used),
as generally described in Vale et al. Meth. Enzym., 103, 565-577
(1983), and in Endocrinology, 91, 562 (1972). These cultures are
maintained in 0.5 ml/well of .beta.-PJ (a reagent available from
Kansas City Biochemicals) containing 2% fetal bovine serum. On the
morning of culture day 5, the cells are washed 3 times with
.beta.-PJ containing 0.1% bovine serum albumin and then incubated
for 1 hour at 37.degree. C. in the same medium. The medium is then
replaced with Ucn, or an analog thereof, diluted in .beta.-PJ
containing 0.1% bovine serum albumin. Incubations are terminated
after 3 hours, at which time the medium is removed and stored at
-20.degree. C. until ACTH radioimmunoassays are performed using a
suitable kit, such as that commercially available from Diagnostic
Products Corporation of Los Angeles, Calif. Secreted ACTH is
measured using Allfit computer program with results expressed as
the average.+-.s.e.m. of 3 replicate bioassays.
[0111] The rUcn peptide strongly stimulates the secretion of ACTH
and .beta.-endorphin in cultured rodent pituitary cells. It is more
biopotent than either r/hCRF or sauvagine, having an EC.sub.50 of
about 0.006.+-.0.003 nanomolar, compared to about 0.043.+-.0.012 nM
and about 0.033.+-.0.010 nM, respectively; it is also more potent
than oCRF. In addition, it is more potent than suckerfishUI (sfUI)
which has an EC.sub.50 of 0.017.+-.0.003. rUcn also stimulates ACTH
and .beta.-END-LI secretion in vivo in rats to a greater extent
than r/hCRF; in fact, at 30 minutes, a 1 .mu.g/kg dose of rUcn
elevates ACTH level in plasma to a substantially greater extent
(659.+-.53pg/ml) than does a 5 .mu.g/kg dose of r/hCRF (422.+-.66
pg/ml), using an assay as generally described in Science, 218, 377
(1982). Such a greater effect continues at 1 hour and at 2 hours.
The peptide when administered peripherally, e.g. iv, also causes a
marked fall in mean arterial blood pressure in rats at a dose as
low as 250 ng for a standard laboratory rat of about 250 to 275
grams. At a dose of about 3.77 .mu.g/kg, it lowers blood pressure
2-3 times as much as either sfUI or r/hCRF and for a longer
duration.
[0112] Testing is also carried out for the ability of Ucn to cause
elevation of the level of intracellular cAMP in cells which express
murine CRF-R1 and also in cells which express murine CRF-R2.beta.
using an assay as generally described in Chen et al., Expression
Cloning of a Human Coritcotropin Releasing Factor (CRF) Receptor,
P.N.A.S., 90, 8967-8971 (1993). rUcn is slightly more potent than
either r/hCRF or sfUI in elevating cAMP levels in cells expressing
mCRF-R1. However, the effect is even more dramatic in assays
utilizing cells expressing mCRF-R2.beta. wherein the ED.sub.50 for
rUcn is 0.18.+-.0.04 nanomolar, compared to an ED.sub.50 for r/hCRF
of 1.7.+-.0.4 nM and an ED.sub.50 for urotensin of 0.74+0.1 nM. It
is also more potent than sauvagine which exhibits an ED.sub.50 of
0.5.+-.0.2.
[0113] Binding assays with cells expressing human CRF-R1 are
carried out as described in the Chen et al. P.N.A.S., supra. The
affinities of test peptides for CRF-R1 and CRF-R2.beta. stably
expressed in CHO cells were determined by competitive displacement
of .sup.125I-(Nle.sup.21, Tyr.sup.32) ovine CRF (for CRF-R1) or of
[.sup.125II-Tyr.sup..smallcircle- .]Ucn (for CRF-R2.beta.) as
described. Data from at lest 3 experiments were pooled and
inhibitory dissociation constant (K.sub.i) values (95% confidence
limits) were calculated using the LIGAND program of Munson and
Rodbard (1980), Anal. Biochem, 107:220-239. The cloned hCRF-R1
binds Ucn with high affinity as determined by the competitive
displacement of bound radioligand. The K.sub.i for rUcn was
determined to be about 0.16(0.08-0.32)nM, compared to r/hCRF of
about 0.95(0.47-2.0)nM, sfUI of about 0.43(0.23-0.81)nM, and
sauvagine of about 1.2(0.54-2.5)nM. Again, the difference is even
more dramatic for similar stably transfected CHO cells expressing
human CRF-R2.beta. where the respective results were
0.41(0.26-0.66)nM, 17(10-29)nM, 3.0(1.8-4.8)nM and 2.0(1.1-3.6)nM.
Testing also shows that rUcn binds more strongly than does r/hCRF
to human CRF binding protein (hCRF-BP) by a factor of about 2,
using a competitive hCRF-BP ligand binding assay with the
radioligand .sup.125I[Nle.sup.21, Tyr.sup.32]-oCRF, much more
strongly than does sauvagine, but less strongly than sfUI.
EXAMPLE IA
[0114] Human Ucn(1-40) having the formula (see SEQ ID NO: 15):
[0115]
H-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-L-
eu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-
-Ile-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner
described in Example I. LSIMS shows a value of 4694.31 Da which
agrees with the calculated value of 4694.51 Da. In vitro testing
for ACTH secretion using anterior pituitary cell cultures as set
forth in Example I shows that the peptide is about 3 times as
effective as r/hCRF, i.e. 3.10 (1.41-6.65). The peptide also has
significant mammalian vasodilatory-hypotensive activity, including
lowering systemic blood pressure and stimulating the secretion of
ACTH.
EXAMPLE IB
[0116] The peptide [Tyr.sup..smallcircle.]rUcn(1-40) having the
amino acid sequence (see SEQ ID NO: 8):
[0117]
H-Tyr-Asp-Asp-Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-T-
hr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-
-Ile-Ile-Phe-Asp-Ser-val-NH.sub.2 is synthesized in the manner
described in Example I. LSIMS shows a value of 4868.58 Da which
agrees perfectly with the calculated value of 4868.58 Da. In vitro
testing for ACTH secretion using anterior pituitary cell cultures
as set forth in Example I shows that the peptide is about twice as
effective as r/hCRF, i.e. 2.20 (1.28-3.88). The peptide also has
significant mammalian vasodilatory-hypotensive activity, including
lowering systemic blood pressure and stimulating the secretion of
ACTH.
EXAMPLE IC
[0118] The peptide [D-Tyr.sup..smallcircle.]hUcn(1-40) having the
formula:
[0119]
H-D-Tyr-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-
-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-A-
rg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner
described in Example I. LSIMS shows a value of 4857.37 Da which
agrees with the calculated value of 4857.58 Da. In vitro testing
for ACTH secretion using anterior pituitary cell cultures as set
forth in Example I shows that the peptide is about 1.25 times as
effective as r/hCRF, i.e. 1.23 (0.60-2.54). The peptide also has
significant mammalian vasodilatory-hypotensive activity, including
lowering systemic blood pressure and stimulating the secretion of
ACTH.
EXAMPLE II
[0120] The peptide [Ac-Pro.sup.3]-hUcn(3-40) having the amino acid
sequence (see SEQ ID NO: 15):
[0121]
Ac-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu--
Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Ph-
e-Asp-Ser-Val-NH.sub.2 is synthesized in the manner described in
Example I but, in addition, the N-terminus is subjected to
acetylation by treatment with acetic anhydride after removal of the
BOC-protecting group. The resultant peptide likewise stimulates the
secretion of ACTH and .beta.-END-LI and causes
vasodilatory-hypotensive activity, including lowering of systemic
blood pressure.
EXAMPLE IIA
[0122] The peptide (cyclo 29-32) [Ac-Pro.sup.3,D-Phe.sup.11,
Glu.sup.29, D-Glu.sup.31, Lys.sup.32]-hUcn(3-40) having the amino
acid sequence:
[0123]
Ac-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Le-
u-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Il-
e-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner as
generally described in Example I but, in addition, the N-terminus
is subjected to acetylation by treatment with acetic anhydride
after removal of the BOC-protecting group. The cyclizing lactam
bond is created as described in Example I of U.S. Pat. No.
5,064,939. LSIMS shows a value of 4562.36 Da which agrees with the
calculated value of 4462.42 Da. In vitro testing for ACTH secretion
using anterior pituitary cell cultures as set forth in Example I
shows that the peptide is about 6 times as effective as r/hCRF,
i.e. 6.14 (2.83-14.05). The resultant peptide likewise stimulates
the secretion of ACTH and .beta.-END-LI and causes
vasodilatory-hypotensive activity, including lowering of systemic
blood pressure.
EXAMPLE IIB
[0124] The peptide (cyclo 29-32)
[Ac-Pro.sup.3,D-Pro.sup.4,D-Phe.sup.11, Glu.sup.29,
Lys.sup.32]-hUcn(3-40) having the amino acid sequence:
[0125]
Ac-Pro-D-Pro-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu--
Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Il-
e-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner as
generally described in Example I but, in addition, the N-terminus
is subjected to acetylation by treatment with acetic anhydride
after removal of the BOC-protecting group. The cyclizing lactam
bond is created as described in Example I of U.S. Pat. No.
5,064,939. LSIMS shows a value of 4472.40 Da which agrees with the
calculated value of 4472.44 Da. In vitro testing for ACTH secretion
using anterior pituitary cell cultures as set forth in Example I
shows that the peptide is about 10 times as effective as r/hCRF,
i.e. 9.90 (4.48-22.85). The resultant peptide likewise stimulates
the secretion of ACTH and .beta.-END-LI and causes
vasodilatory-hypotensive activity, including lowering of systemic
blood pressure.
EXAMPLE IIC
[0126] The peptide (cyclo 29-32)
[Ac-Pro.sup.3,D-Ser.sup.4,D-Phe.sup.11, Glu.sup.29,
Lys.sup.32]-hUcn(3-40) having the amino acid sequence:
[0127]
Ac-Pro-D-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu--
Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Il-
e-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner as
generally described in Example I but, in addition, the N-terminus
is subjected to acetylation by treatment with acetic anhydride
after removal of the BOC-protecting group. The cyclizing lactam
bond is created as described in Example I of U.S. Pat. No.
5,064,939. LSIMS shows a value of 4462.33 Da which agrees with the
calculated value of 4462.42. In vitro testing for ACTH secretion
using anterior pituitary cell cultures as set forth in Example I
shows that the peptide is about 5.75 times as effective as r/hCRF,
i.e. 5.69 (2.43-14.49). The resultant peptide likewise stimulates
the secretion of ACTH and .beta.-END-LI and causes
vasodilatory-hypotensive activity, including lowering of systemic
blood pressure.
EXAMPLE III
[0128] The peptide hUcn(2-40) having the amino acid sequence (see
SEQ ID NO: 15):
[0129]
H-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-L-
eu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-
-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner described in
Example I. The peptide has significant mammalian
vasodilatory-hypotensive activity, including lowering systemic
blood pressure and stimulating the secretion of ACTH.
EXAMPLE IV
[0130] The peptide [D-Phe.sup.11]-hUcn having the amino acid
sequence:
H-Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-L-
eu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-
-Phe-Asp-Ser-Val-NH.sub.2 is synthesized in the manner described in
Example I. The peptide has significant mammalian
vasodilatory-hypotensive activity, including lowering systemic
blood pressure and stimulating the secretion of ACTH.
EXAMPLE V
[0131] The peptide agonist analogs of hUcn as set forth
hereinafter, which are considered to have substantially the same
amino acid sequence as hUcn, are synthesized:
5 [Glu.sup.1]-hUcn [Leu.sup.36]-hUcn [Ile.sup.5]-hUcn
[Ile.sup.18]-hUcn [Val.sup.17]-hUcn [Ile.sup.20]-hUcn
[Lys.sup.22]-hUcn [Lys.sup.27]-hUcn [Leu.sup.35]-hUcn
[Leu.sup.37]-rUcn [Glu.sup.38]-hUcn [Ile.sup.40]-rUcn
[Ser.sup.10]-hUcn [Thr.sup.39]-rUcn [Leu.sup.11]-hUcn
[Leu.sup.7]-rUcn [Glu.sup.2]-hUcn [Leu.sup.7,11]-rUcn
[Gln.sup.29]-hUcn [Lys.sup.22,27]-rUcn [Asn.sup.32]-hUcn
[Ile.sup.5, Gln.sup.29]-rUcn
[0132] Each of the foregoing Ucn-like agonist peptides has
significant mammalian vasodilatory-hypotensive activity, including
lowering systemic blood pressure and stimulating the secretion of
ACTH.
EXAMPLE VI
[0133] A further group of Ucn-like agonist peptides are synthesized
which fall within the following amino acid sequence:
Y-Asp-Asn-Pro-Ser-Leu-Ser--
Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-R.sub.19-Leu-Ala-Arg-Thr-
-Gln-Ser-Gln-Arg-Glu-R.sub.29-Ala-Glu-R.sub.32-Asn-Arg-Ile-R.sub.36-Phe-R.-
sub.38-Ser-Val-NH.sub.2, wherein Y is an acyl group having 7 or
less carbon atoms or hydrogen; R.sub.19 is Glu or Ala; R.sub.29 is
Arg, Glu, Lys or Orn; R.sub.32 is Gln, Lys, Orn or Glu; R.sub.36 is
Ile, C.sup..alpha.MeIle or C.sup..alpha.MeLeu; R.sub.38 is Asp or
Ala; provided that when R.sub.29 is Glu, R.sub.32 is either Lys or
Orn and the side chains thereof are linked by an amide bond, and
that when R.sub.29 is either Lys or Orn, R.sub.32 is Glu, and the
side chains thereof are linked by an amide bond. In this group,
D-Phe.sup.11 can be replaced by D-Leu or by a D-isomer of another
natural .alpha.-amino acid; Glu in the 31-position can be replaced
by a D-isomer, e.g. D-Glu, D-Arg, imBzlD-His, etc.; and the
N-terminus can be shortened by 1 or 2 residues.
[0134] The specific peptides are as follows:
[0135] [Ac-Pro.sup.3,D-Phe.sup.11]-hUcn(3-40)
[0136] [D-Leu.sup.11, Ala.sup.19]-hUcn
[0137] [D-Phe.sup.11, Ala.sup.38]-hUcn
[0138] [D-Tyr.sup.11, C.sup..alpha.MeIle.sup.36]-hUcn
[0139] [D-Phe.sup.11, C.sup..alpha.MeLeu.sup.36]-hUcn
[0140] [Ac-Asp.sup.1, D-Phe.sup.11, Ala.sup.19,38]-hUcn
[0141] (cyclo 29-32)[D-Leu.sup.11, Glu.sup.29, Lys.sup.32]-hUcn
[0142] (cyclo 29-32)[D-Phe.sup.11, Glu.sup.29,
Orn.sup.32]-hUcn(2-40)
[0143] (cyclo 29-32)[Ac-Pro.sup.3,D-Phe.sup.11, Lys.sup.29,
Glu.sup.32]-hUcn(3-40)
[0144] (cyclo 29-32)[D-Tyr.sup.11, Orn.sup.29,
Glu.sup.32]-hUcn(3-40)
[0145] (cyclo 29-32)[D-Phe.sup.11, Glu.sup.29, D-Glu.sup.31,
Lys.sup.32]-hUcn
[0146] (cyclo 29-32)[D-Phe.sup.11, Glu.sup.29, D-Arg.sup.31,
Lys.sup.32]-hUcn
[0147] (cyclo 29-32)[D-Tyr.sup.11, Glu.sup.29, imBzD-His.sup.31,
Lys.sup.32]-hUcn
[0148] Each of the foregoing Ucn-like agonist peptides has
significant mammalian vasodilatory-hypotensive activity, including
lowering systemic blood pressure and stimulating the secretion of
ACTH.
EXAMPLE VII
[0149] A still further group of Ucn-like agonist peptides are
synthesized which fall within the following amino acid sequence
(SEQ ID NO: 14):
Y-Xaa.sub.1-Xaa.sub.2-Pro-Xaa.sub.4-Xaa.sub.5-Ser-Xaa.sub.7-Asp-Leu-Xaa.s-
ub.10-Xaa.sub.11-Xaa.sub.12-Xaa.sub.13-Leu-Arg-Xaa.sub.16-Xaa.sub.17-Xaa.s-
ub.18-Xaa.sub.19-Xaa.sub.20-Xaa.sub.21-Xaa.sub.22-Xaa.sub.23-Xaa.sub.24-Xa-
a.sub.25-Xaa.sub.26-Xaa.sub.27-Xaa.sub.28-Xaa.sub.29-Ala-Xaa.sub.31-Xaa.su-
b.32-Asn-Arg-Xaa.sub.35-Xaa.sub.36-Xaa.sub.37-Xaa.sub.38-Xaa.sub.39-Xaa.su-
b.40-NH.sub.2, wherein Y is an acyl group having 7 or less carbon
atoms or hydrogen; Xaa, is Asp, Glu or Gln; Xaa.sub.2 is Asn, Asp,
Glu or Gly; Xaa.sub.4 is Ser or Pro; Xaa.sub.5 is Leu, Ile or Met;
Xaa.sub.7 is Ile or Leu; Xaa.sub.10 is Thr or Ser; Xaa.sub.11 is
Phe or Leu; Xaa.sub.12 is His or Glu; Xaa.sub.13 is Leu or Met;
Xaa.sub.16 is Thr, Asn, Glu, or Lys; Xaa.sub.17 is Leu, Met or Val;
Xaa.sub.18 is Leu or Ile; Xaa.sub.19 is Glu or His; Xaa.sub.20 is
Leu, Met, Ile or Arg; Xaa.sub.21 is Ala, Glu or Thr; Xaa.sub.22 is
Arg or Lys; Xaa.sub.23 is any natural amino acid and preferably
Thr, Ser, Ala, Ile, Met, Val, Asn, Gin, Gly, Lys, His, Leu, Glu or
Asp; Xaa.sub.24 is Gin, Glu or Asp; Xaa.sub.25 is any natural amino
acid and preferably Ser, Thr, Ala, Ile, Met, Val, Asn, Gln, Gly,
Lys, His, Leu, Glu or Asp; Xaa.sub.26 is Gin, Leu or Glu;
Xaa.sub.27 is Arg, Ala or Lys; Xaa.sub.28 is Glu or Gln; Xaa.sub.29
is Arg or Gln; Xaa.sub.31 is any natural amino acid and preferably
Ala, Ile, Met, Val, Asn, Gin, Gly, Lys, His, Leu, Glu or Asp;
Xaa.sub.32 is any natural amino acid and preferably Ala, Ile, Met,
Val, Asn, Gin, Gly, Lys, His, Leu, Glu or Asp; Xaa.sub.35 is Ile,
Lys, Leu or Asn; Xaa.sub.36 is Ile, Tyr, Met or Leu; Xaa.sub.37 is
Phe, Leu or Met; Xaa.sub.38 is Asp or Glu; Xaa.sub.39 is Ser, Ile,
Glu or Thr; and Xaa.sub.40 is Val, Ile, Phe or Ala; provided that
there are no more than 3 residues different from Ucn, and that the
N-terminus may be shortened by 1 or 2 residues.
[0150] The following specific peptides are synthesized:
6 [Ac-Pro.sup.3, Met.sup.5]-hUcn(3-40) [Glu.sup.12]-hUcn
[Met.sup.13]-hUcn [Asn.sup.16, Ala.sup.23]-hUcn [Glu.sup.16,
Ala.sup.25, 31]-hUcn [Lys.sup.16, Ile.sup.23, Ala.sup.32]-hUcn
[Val.sup.17, Ile.sup.25, Met.sup.31]-hUcn [Met.sup.17, Ile.sup.31,
32]-hUcn [Ser.sup.10, Ile.sup.18, His.sup.19]-hUcn [Glu.sup.1,
Met.sup.20, Lys.sup.35]-hUcn [Ile.sup.20, Met.sup.32,
Glu.sup.38]-hUcn [Arg.sup.20, Asn.sup.23, Thr.sup.25]-hUcn
[Thr.sup.21, Gly.sup.23, 32]-hUcn [Glu.sup.21, Lys.sup.23,
Gln.sup.28]-hUcn [Lys.sup.22, His.sup.23, Leu.sup.31]-hUcn
[Val.sup.23, Glu.sup.24, Met.sup.36]-hUcn [Gln.sup.23, Asp.sup.25,
Glu.sup.39]-hUcn [Met.sup.23, Gly.sup.25, Leu.sup.35]-hUcn
[Glu.sup.23, Tyr.sup.36, Phe.sup.40]-hUcn [Asp.sup.23, Lys.sup.27,
Leu.sup.32]-hUcn [Ser.sup.23, Asp.sup.24, Met.sup.25]-hUcn
[Ac-Pro.sup.3, Leu.sup.23, 25, Gln.sup.29]-hUcn(3-40) [
[Asn.sup.25, 32, Ile.sup.39]-hUcn [Gln.sup.25, 31, Asn.sup.35]-hUcn
[Lys.sup.25, Val.sup.31, Ala.sup.32]-hUcn [Glu.sup.2, Asn.sup.31,
His.sup.32]-hUcn [Gln.sup.1, Ile.sup.5, Val.sup.32]-hUcn
[Gly.sup.2, Leu.sup.11, Thr.sup.31]-hUcn [Glu.sup.25,
Leu.sup.37]-hUcn [Ile.sup.5, His.sup.25, Ala.sup.40]-hUcn
[Leu.sup.7, His.sup.31, Asp.sup.32]-hUcn [Gln.sup.32, Leu.sup.36,
Ala.sup.40]-hUcn [His.sup.25, Ala.sup.31, Thr.sup.39]-hUcn
[Leu.sup.26, Lys.sup.31, Thr.sup.32]-hUcn [Glu.sup.26, Ala.sup.27,
Lys.sup.32]-hUcn [Lys.sup.27, Asp.sup.31, Met.sup.37]-hUcn
[Ac-Pro.sup.3, Val.sup.25, Leu.sup.36]-hUcn(3-40) [Ala.sup.25,
Asp.sup.31, Ile.sup.40]-hUcn [Val.sup.32, Leu.sup.37,
Ile.sup.39]-hUcn [Leu.sup.11, Ile.sup.18, Thr.sup.39]-hUcn
[0151] Each of the foregoing Ucn-like agonist peptides has
significant mammalian vasodilatory-hypotensive activity, including
lowering systemic blood pressure and stimulating the production of
ACTH.
[0152] The following group of Examples are directed to N-terminally
shortened versions (e.g. shortened by 7-10 residues) of the
Ucn-like peptides which have antagonistic properties. All of the
statements made hereinbefore with respect to the chemical character
and/or the synthesis of Ucn analogs are considered to apply equally
to the antagonists and are not thus repeated; the antagonists are
merely N-terminally shortened versions of the agonists. The
specific peptides set forth in the following Examples exhibit
antagonistic biological properties with respect to the effect of
Ucn on at least the CRF receptors, CRF-R1, and CRF-R2. In this
respect these Ucn-antagonists are considered to generally at least
have characteristics and uses similar to those described for CRF
antagonists in U.S. Pat. No. 5,245,009.
EXAMPLE VIII
[0153] The peptide, Ucn(11-40), having the amino acid sequence (see
SEQ ID NO: 8):
H-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-
-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2
is synthesized in the manner described in Example I. To evaluate
the biological activity of a candidate peptide as a Ucn antagonist
(which will be indicative of its effective binding to CRF
receptors), the previously mentioned assay from Endocrinology, 91,
supra, is run in the presence of a challenge dose of ovine CRF. The
performance of such candidate in this assay is routinely compared
to the performance of a highly potent linear CRF antagonist, i.e.
[D-Phe.sup.12, Nle.sup.21,38]-r/hCRF(12-41) which is hereinafter
referred to as the Standard Antagonist. An in vivo test measuring
elevation of mean arterial blood pressure as a result of iv
injection is also simply and straightforwardly performed. The
peptide Ucn(11-40) exhibits significant mammalian vasoconstrictive
activity causing elevation of mean arterial blood pressure, as do
known CRF antagonists.
[0154] The synthesis is repeated twice to produce Ucn (10-40) and
[Ac-Thr.sup.10]Ucn(10-40); both show bioactivity as Ucn
antagonists.
EXAMPLE IX
[0155] The peptide, [D-Phe.sup.11]-Ucn(11-40), having the amino
acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-
-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2
is synthesized in the manner described in Example I. Its specific
optical rotation is measured under the conditions set forth
hereinbefore as [.alpha.].sub.D=-62.degree..+-.1.0. LSIMS shows a
value of 3638.88 Da which agrees with the calculated value of
3639.00 Da. In vitro testing is carried out as described in Example
VIII, which demonstrates the peptide is bioactive, exhibiting a
value of 0.551 (0.333-0.857) compared to this highly biopotent
Standard Antagonist. The peptide has significant mammalian
vasoconstrictive activity, causing elevation of mean arterial blood
pressure, indicative of its being a Ucn antagonist.
EXAMPLE X
[0156] The peptide, [D-Tyr.sup.11]-Ucn(11-40), having the amino
acid sequence:
H-D-Tyr-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-
-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2
is synthesized in the manner described in Example I. The peptide
has significant mammalian vasoconstrictive activity causing
elevation of mean arterial blood pressure, indicative of its being
a Ucn antagonist. It is also effectively iodinated to provide
[.sup.125I-D-Tyr.sup.11]-Ucn(11-40) for use in screening assays and
the like.
EXAMPLE XI
[0157] The peptide, (cyclo 29-32)[D-Phe.sup.11, Glu.sup.29,
Lys.sup.32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939, issued Nov. 12, 1991. Its specific optical
rotation is measured under the conditions set forth hereinbefore as
[.alpha.]D =-49.degree..+-.1.0. LSIMS shows a value of 3593.89 Da
which agrees with the calculated value of 3593.97 Da. In vitro
testing for ACTH secretion using anterior pituitary cell cultures
as set forth in Example VIII shows that the peptide is about 10
times as effective as the Standard Antagonist, i.e. 10.34
(4.27-25.58). The peptide also has significant mammalian
vasoconstrictive activity causing elevation of mean arterial blood
pressure, indicative of its being a Ucn antagonist.
EXAMPLE XII
[0158] The peptide, (cyclo 29-32) [D-Tyr.sup.11, Glu.sup.29,
Lys.sup.32]-Ucn(11-40), having the amino acid sequence:
H-D-Tyr-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Glu-Ala-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. LSIMS shows a value of 3609.82 Da which
agrees with the calculated value of 3609.96 Da. In vitro testing
for ACTH secretion using anterior pituitary cell cultures as set
forth in Example VIII shows that the peptide is about 4 times as
effective as the Standard Antagonist, i.e. 4.01 (2.32-7.05). The
peptide has significant mammalian vasoconstrictive activity causing
elevation of mean arterial blood pressure, indicative of its being
a Ucn antagonist. It is also iodinated to provide
.sup.125I-D-Tyr.sup.11 cyclic analog for use in screening assays
and the like.
EXAMPLE XIIA
[0159] The peptide, (cyclo 29-32)[D-Tyr.sup.11, Glu.sup.29,
D-Glu.sup.31, Lys.sup.32]-Ucn(11-40), having the amino acid
sequence:
H-D-Tyr-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. The peptide has significant mammalian
vasoconstrictive activity causing elevation of mean arterial blood
pressure, indicative of its being a Ucn antagonist. It is also
iodinated to provide .sup.125I-D-Tyr.sup.11 cyclic analog for use
in screening assays and the like.
EXAMPLE XIII
[0160] The peptide, (cyclo 29-32) [D-Phe.sup.11, Glu.sup.29,
D-Glu.sup.31, Lys.sup.32]-Ucn(11-40), having the amino acid
sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. LSIMS shows a value of 3593.80 Da which
agrees with the calculated value of 3593.97. In vitro testing for
ACTH secretion using anterior pituitary cell cultures as set forth
in Example VIII shows that the peptide is about 4.75 times as
effective as the Standard Antagonist, i.e. 4.72 (2.19-10.00). The
peptide also has significant mammalian vasoconstrictive activity
causing elevation of mean arterial blood pressure, indicative of
its being a Ucn antagonist.
[0161] The synthesis is repeated twice to substitute D-Leu and
D-His for D-Phe. The peptides show similar bioactivity as Ucn
antagonists.
EXAMPLE XIIIA
[0162] The peptide, (cyclo 29-32) [Pro.sup.10,D-Phe.sup.11,
Glu.sup.29,D-Glu.sup.31, Lys.sup.32]-Ucn(10-40), having the amino
acid sequence:
H-Pro-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-
-Ser-Gln-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.-
2 is synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. LSIMS shows a value of 3690.91 Da which
agrees with the calculated value of 3691.02 Da. In vitro testing
for ACTH secretion using anterior pituitary cell cultures as set
forth in Example VIII shows that the peptide is about 2.75 times as
effective as the Standard Antagonist, i.e. 2.74 (1.02-8.02). The
peptide also has significant mammalian vasoconstrictive activity
causing elevation of mean arterial blood pressure, indicative of
its being a Ucn antagonist. This synthesis and testing show that
the inclusion of an additional L-isomer at the N-terminus does not
significantly alter its bioactivity as a Ucn antagonist.
EXAMPLE XIIIB
[0163] The peptide, (cyclo 29-32) [D-Pro.sup.10,D-Phe.sup.11,
Glu.sup.29, D-Glu.sup.31, Lys.sup.32]-Ucn(10-40), having the amino
acid sequence:
H-D-Pro-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-
-Arg-Glu-Glu-Ala-D-Glu-Lys-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2
is synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. LSIMS shows a value of 3691.00 Da which
agrees with the calculated value of 3691.02 Da. In vitro testing
for ACTH secretion using anterior pituitary cell cultures as set
forth in Example VIII shows that the peptide is about 5 times as
effective as the Standard Antagonist, i.e. 4.99 (2.26-11.55). The
peptide also has significant mammalian vasoconstrictive activity
causing elevation of mean arterial blood pressure, indicative of
its being a Ucn antagonist. This synthesis and testing show that
the inclusion of an additional D-isomer at the N-terminus does not
significantly alter the bioactivity of a Ucn antagonist, as by
comparison to Peptide XIII.
EXAMPLE XIV
[0164] The peptide, (cyclo 29-32)[D-Phe.sup.11, Glu.sup.29,
D-Arg.sup.31, Orn.sup.32]-Ucn(11-40), having the amino acid
sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Glu-Ala-D-Arg-Orn-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. The peptide has significant mammalian
vasoconstrictive activity causing elevation of mean arterial blood
pressure, indicative of its being a Ucn antagonist.
[0165] The synthesis is repeated twice substituting imBzlD-His and
D-2Nal for D-Arg and twice again to also add Ac-Thr at the
N-terminus with these substitutions. All four peptides show good
bioactivity as Ucn antagonists.
EXAMPLE XV
[0166] The peptide, (cyclo 29-32) [D-Phe.sup.11, Lys.sup.29,
Glu.sup.32]-Ucn(11-40), having the amino acid sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Lys-Ala-Glu-Glu-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. The peptide has significant mammalian
vasoconstrictive activity causing elevation of mean arterial blood
pressure, indicative of its being a Ucn antagonist.
EXAMPLE XVI
[0167] The peptide, (cyclo 29-32) [D-Phe.sup.11, Lys.sup.29,
D-Glu.sup.31, Glu.sup.32]-Ucn(11-40), having the amino acid
sequence:
H-D-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-G-
lu-Lys-Ala-D-Glu-Glu-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH.sub.2 is
synthesized in the manner described in Example I, with the
cyclizing lactam bond being created as described in Example I of
U.S. Pat. No. 5,064,939. The peptide has significant mammalian
vasoconstrictive activity causing elevation of mean arterial blood
pressure, indicative of its being a Ucn antagonist.
EXAMPLE XVII
[0168] Additional Ucn antagonist peptides, as set forth as follows,
many of which are considered to be substantially the same as
Ucn(11-40), are synthesized in the manner described in Example
I:
7 [Val.sup.17]-rUcn(11-40) [C.sup..alpha.MeLeu.sup.36]-rUcn(11-40)
[Lys.sup.22]-rUcn(8-40) [D-Phe.sup.11, Ile.sup.18]-rUcn(11-40)
[Ac-Asp.sup.8, Lys.sup.22]-Ucn(8-40) [Leu.sup.35]-rUcn(11-40)
[Ile.sup.20]-rUcn(10-40) [Ac-Thr.sup.10, Ile.sup.20]-Ucn(10-40)
[Glu.sup.38]-rUcn(11-40) [D-Phe.sup.11, Lys.sup.27]-rUcn(11-40)
[Ser.sup.10]-rUcn(10-40) [C.sup..alpha.MeIle.sup.36,
Leu.sup.37]-rUcn(11-40) [D-Leu.sup.11]-rUcn(11-40) [Ala.sup.38,
Ile.sup.40]-rUcn(11-40) [Gln.sup.29]-rUcn(9-40) [Ala.sup.19,
Thr.sup.39]-rUcn(11-40) [Asn.sup.32]-rUcn(11-40) [D-Phe.sup.11,
Lys.sup.22, 27]-rUcn(11-40) [Ac-Leu.sup.9, Gln.sup.29]-Ucn(9-40)
[Lys.sup.22, Gln.sup.29]-rUcn(11-40)
[0169] Each of the foregoing Ucn-like antagonist peptides has
significant mammalian vasoconstrictive activity causing elevation
of mean arterial blood pressure, indicative of its being a Ucn
antagonist.
[0170] The following group of Examples are directed to the
synthesis of CRF-BP blockers which increase the available amount of
endogenous CRF and Ucn by complexing with CRF-BP.
EXAMPLE XVIII
[0171] A peptide, Ucn(5-32), having the amino acid sequence (see
SEQ ID NO: 8):
Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-A-
la-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln is synthesized in
the manner described in Example I.
[0172] A prospective peptide blocker is evaluated using a
competitive hCRF-BP ligand binding assay. Binding of
[125I-DTyr.sup..smallcircle.]hCR- F to soluble hCRF-BP is performed
in phosphate-buffered saline (PBS) containing 25 mM EDTA, 0.25%
bovine serum albumin, and 0.01% Triton X-100, using medium enriched
by recombinant CHO cells as a source of hCRF-BP. Reactions are
performed in a total volume of 400 .mu.l including 50,000
CPM[.sup.125I-DTyr.sup..smallcircle.]hCRF. A constant amount of
radioactive hCRF and hCRF-BP and varying amounts of the sample
peptide are used to carry out competitive binding assays. After an
overnight incubation at room temperature, precipitation is
accomplished using rabbit anti-hCRF-BP antiserum (1:9000 final
dilution) and 200 .mu.l of sheep anti-rabbit IgG solution. After
incubating with the primary and secondary antisera for 30 minutes
each, 1 ml of saline wash is added, and the test tubes are
centrifuged at 2000.times. g for 20 minutes at 4.degree. C.
Precipitates are counted in a gamma counter. Inhibitory binding
affinity constant (K.sub.i) values are determined using parameters
calculated by the LIGAND computer program, Munson et al., Anal.
Biochem., 107, 220 (1980), and a Vax/VMS computer system.
[0173] The CRF-BP blocker Ucn(5-32) has a K.sub.i lower than that
of hCRF(9-33) and thus is a potentially superior blocking agent for
increasing the available amount of CRF and/or Ucn.
EXAMPLE XIX
[0174] A peptide, Ucn(8-32), having the amino acid sequence (see
SEQ ID NO: 8):
Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-G-
ln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln is synthesized in the manner
described in Example I. LDMS shows a value of 3023.48 Da which
agrees with the calculated value of 3023.65 Da. Testing shows that
the peptide has a K.sub.i lower than that of hCRF(9-33) and thus is
a potentially superior blocking agent for increasing the available
amount of CRF and/or Ucn.
EXAMPLE XX
[0175] A peptide, Ucn(3-27), having the amino acid sequence (see
SEQ ID NO: 8):
Pro-Pro-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-25
Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg is synthesized in the
manner described in Example I. The peptide has a K.sup.i lower than
that of hCRF(9-33) and thus is a potentially superior blocking
agent for increasing the available amount of CRF and/or Ucn.
EXAMPLE XXI
[0176] A peptide, [Ile.sup.18]-Ucn(6-29), having the amino acid
sequence (see SEQ ID NO: 8):
Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Ile-G-
lu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg is synthesized in the
manner described in Example I. The peptide has a K.sub.i lower than
that of hCRF(9-33) and thus is a potentially superior blocking
agent for increasing the available amount of CRF and/or Ucn.
[0177] Ucn profoundly stimulates the pituitary-adrenalcortical axis
and is considered to be useful to stimulate the functions of this
axis in some types of patients with low endogenous glucocorticoid
production. For example, Ucn and its analogs should be useful in
restoring pituitary-adrenal function in patients having received
exogenous glucocorticoid therapy whose pituitary-adrenalcortical
functions remain suppressed.
[0178] Most other regulatory peptides have been found to have
effects upon the central nervous system and upon the
gastrointestinal tract. Because ACTH and sympathetic nervous system
activation secretion is the "sine qua non" of mammal's response to
stress, it was not surprising that CRFs have significant effects on
the brain as a mediator of the body's stress response. Accordingly,
Ucn and its analogs are considered to also find application in
modifying the mood, learning, memory and behavior of normal and
mentally disordered individuals. Because Ucn elevates the levels of
ACTH, .beta.-END, .beta.-lipotropin, other pro-opiomelanocortin
gene products and corticosterone, its administration can be used to
induce the effects of the foregoing POMC-derived peptides on the
brain to thereby influence memory, mood, pain appreciation, etc.,
and more specifically, alertness, depression and/or anxiety, and
also their effects peripherally. For example, when administered
directly into the ventricles, CRFs increase physical activity and
improve learning performance in rats and thus may function as a
natural stimulant; because Ucn similarly activates the CRF
receptors, it will function similarly.
[0179] Because CRF-R2 has been found to be abundantly expressed in
the heart, especially in association with blood vessels, and
because it is known that the addition of CRF into the left atrium
of an isolated perfused heart induces a prolonged dilatory effect
on coronary arteries, transiently produces a positive inotropic
effect and stimulates the secretion of atrial natriuretic peptide,
it is now believed that Ucn is responsible, at least in part, for
regulating cardiac perfusion because of its particularly high
affinity for the CRF-R2s. It is also expected that other vascular
beds, such as the superior mesenteric, will be dilated by Ucn and
its analogs. Because of these biological effects in the heart, Ucn
and agonists/antagonists thereof (as well as anti-Ucn antibodies),
can be effectively used to selectively modulate cardiac
perfusion.
[0180] Moreover, because of the localization of CRF-R2 on blood
vessels, it is considered that Ucn-like agonist and antagonist
peptides of the invention are therapeutically useful to modulate
blood flow in many various vascular beds, and particularly in
desired tissues and organs. Ucn and its agonist analogs are
considered to be of use for increasing blood flow to the
gastrointestinal tract of animals, particularly humans and other
mammals, because all CRF-related peptides have been shown to dilate
the mesenteric vascular bed. CRF and certain fragments have been
shown to modulate vascular permeability (Wei E. T. et al.,
"Peripheral anti-inflammatory actions of corticotropin-releasing
factor", pp. 258-276, Corticotropin-Releasing Factor (Ciba
Foundation Symposium 172) John Wiley & Sons, 1993). Ucn and its
fragments will also reduce vascular leakage and have a salutary
effect on injury- or surgery-induced tissue swelling and
inflammation. Therefore, Ucn and its analogs and fragments that are
agonists can be administered parenterally to decrease inflammation,
swelling and oedema and to reduce fluid loss following heat
injury.
[0181] oCRF, r/hCRF, urotensin I and sauvagine have been shown to
inhibit gastric acid production, and Ucn and its analogs are
considered to also be effective in the treatment of gastric ulcers
by reducing gastric acid production and/or inhibiting certain
gastrointestinal functions in a mammal. Ucn and its analogs will be
effective in increasing intestinal transit rate and useful in the
treatment of acute constipation.
[0182] A number of direct stimulatory effects of CRF on the GI
tract have earlier been described. For example, CRF acts on the gut
in vitro to depolarize myenteric neurons in the small intestine.
The results of in vivo studies with intravenously administered CRF
and CRF antagonists have been consistent with the observed effect
of CRF to control gastric emptying and intestinal motility. The
Ucn-like peptides of the invention are considered useful in
treating intestinal and gastrointestinal disorders, such as
irritable bowel syndrome. CRF antagonists have previously been used
to therapeutically treat irritable bowel syndrome, and antagonists
based upon Ucn (which would be selective for CRF-R2) are considered
to be even more useful. These antagonists may also be used to treat
spastic colon and Crohn's disease.
[0183] These Ucn-like peptides may also be used to evaluate
hypothalamic pituitary adrenal function in mammals with suspected
endocrine or central nervous system pathology by suitable
administration followed by monitoring bodily functions. For
example, administration may be used as a diagnostic tool to
evaluate Cushing's disease and affective disorders, such as
depressive illness.
[0184] Ucn, an analog or a nontoxic salt thereof, combined with a
pharmaceutically or veterinarily acceptable carrier to form a
pharmaceutical composition, may be administered to animals,
including humans and other mammals, either intravenously,
subcutaneously, intramuscularly, percutaneously, e.g. intranasally,
intracerebrospinally or orally. The isolated peptides should be at
least about 90% pure and preferably should have a purity of at
least about 98%; however, lower purities are effective and may well
be used with mammals other than humans. This purity means that the
intended peptide constitutes the stated weight % of all like
peptides and peptide fragments present. Administration to humans
may be employed by a physician to lower blood pressure or to
stimulate endogenous gluco-corticoid production. The required
dosage will vary with the particular condition being treated, with
the severity of the condition and with the duration of desired
treatment. Ucn or Ucn analogs can also be administered, e.g., icv,
to cause an increase of Ucn in the brain and thereby cause (a)
improvement in short to medium term memory in a subject afflicted
with Alzheimer's disease; (b) relief from chronic fatigue syndrome;
(c) suppression of appetite; (d) stimulation of the respiratory
system, (e) improvement in learning performance; (f) improvement in
memory; (g) improvement in alertness; (h) reduction of depression
and/or (i) lessening of anxiety. Particular effectiveness is shown
in appetite suppression.
[0185] Such peptides are often administered in the form of
pharmaceutically or veterinarily acceptable nontoxic salts, such as
acid addition salts or metal complexes, e.g., with zinc, iron,
calcium, barium, magnesium, aluminum or the like. Illustrative of
such nontoxic salts are hydrochloride, hydrobromide, sulphate,
phosphate, tannate, oxalate, fumarate, gluconate, alginate,
maleate, acetate, citrate, benzoate, succinate, malate, ascorbate,
tartrate and the like. If the active ingredient is to be
administered in tablet form, the tablet may contain a binder, such
as tragacanth, corn starch or gelatin; a disintegrating agent, such
as alginic acid; and a lubricant, such as magnesium stearate. If
administration in liquid form is desired, sweetening and/or
flavoring may be used, and intravenous administration in isotonic
saline, phosphate buffer solutions or the like may be effected.
[0186] It may also be desirable to deliver Ucn or analogs thereof
over prolonged periods of time, for example, for periods of one
week to one year from a single administration, and slow release,
depot or implant dosage forms may be utilized as well known in this
art. For example, a dosage form may contain a pharmaceutically
acceptable non-toxic salt of the compound which has a low degree of
solubility in body fluids, for example, an acid addition salt with
a polybasic acid; a salt with a polyvalent metal cation; or
combination of the two salts. A relatively insoluble salt may also
be formulated in a gel, for example, an aluminum stearate gel. A
suitable, slow-release depot formulation for injection may also
contain Ucn or an analog or a salt thereof dispersed or
encapsulated in a slow degrading, non-toxic or non-antigenic
polymer such as a polylactic acid/polyglycolic acid polymer, for
example, as described in U.S. Pat. No. 3,773,919.
[0187] Therapeutically effective amounts of the peptides should be
administered under the guidance of a physician, and pharmaceutical
compositions will usually contain the peptide in conjunction with a
conventional, pharmaceutically or veterinarily-acceptable carrier.
A therapeutically effective amount is considered to be a
predetermined amount calculated to achieve the desired effect, e.g.
to increase or decrease the amount of ACTH, in a patient. The
required dosage will vary with the particular treatment and with
the duration of desired treatment; however, it is anticipated that
dosages between about 10 micrograms and about 1 milligram per
kilogram of body weight per day will be used for therapeutic
treatment. It may be particularly advantageous to administer such
compounds in depot or long-lasting form as earlier described. A
therapeutically effective amount is typically an amount of a Ucn or
an analog thereof that, when administered peripherally in a
physiologically acceptable composition, is sufficient to achieve a
plasma concentration thereof from about 0.1 .mu.g/ml to about 100
.mu.g/ml, preferably from about 1 .mu.g/ml to about 50 .mu.g/ml,
more preferably at least about 2 .mu.g/ml and usually 5 to 10
.mu.g/ml. Antibodies or antisense polynucleotides are also
administered in proportionately appropriate amounts in accordance
with known practices in this art. The level of ACTH present in a
patient, particularly in the plasma, can be readily determined by
routine clinical analysis. Changes in ACTH levels can be monitored
during a treatment regimen to determine the effectiveness of the
administered Ucn-like peptide over time. In some instances,
treatment of subjects with these peptides can be carried out in
lieu of the administration of ACTH or corticosteroids, in such
instances a dosage as low as about 10 ng/Kg of body weight may be
employed.
[0188] Although the invention has been described with regard to its
preferred embodiments, which constitute the best mode presently
known to the inventors, it should be understood that various
changes and modifications as would be obvious to one having the
ordinary skill in this art may be made without departing from the
scope of the invention which is set forth in the claims appended
hereto. Although the claims variously define the invention in terms
of a peptide sequence, it should be understood that such is
intended to include nontoxic salts thereof which are well known to
be the full equivalent thereof and which are most frequently
administered. Instead of the simple amide at the C-terminus, a
lower alkyl-substituted amide, e.g. methylamide, ethylamide, etc,
may be incorporated or the C-terminus may be otherwise blocked as
well known in the peptide art. Polypeptides having an amino acid
residue sequence substantially identical to the sequence of Ucn
specifically shown herein, in which one or more residues have been
conservatively substituted with a functionally similar residue, are
considered to be equivalents of Ucn so long as they mimic a
biological function of CRF. Such peptides and salts thereof are
considered as being within the scope of the claimed invention.
[0189] The disclosures of all patents and publications set forth
hereinbefore, as well as of the two priority applications, are
expressly incorporated herein by reference. As used herein, all
temperatures are .degree. C., and all ratios and percentages of
liquid materials are by volume.
Sequence Listing Summary
[0190] SEQ ID NO: 1, when the C-terminus is amidated, is the amino
acid sequence of ovine CRF.
[0191] SEQ ID NO: 2, when pGlu is at the N-terminus and the
C-terminus is amidated, is the amino acid sequence of frog
sauvagine.
[0192] SEQ ID NO: 3, when the C-terminus is amidated, is the amino
acid sequence of rat/human CRF.
[0193] SEQ ID NO: 4, when the C-terminus is amidated, is the amino
acid sequence of suckerfish urotensin.
[0194] SEQ ID NO: 5, when the C-terminus is amidated, is the amino
acid sequence of carp urotensin.
[0195] SEQ ID NO: 6, when the C-terminus is amidated, is the amino
acid sequence of flathead sole (Maggy).
[0196] SEQ ID NO: 7, when the C-terminus is amidated, is the amino
acid sequence of fish CRF.
[0197] SEQ ID NO: 8, when the C-terminus is amidated, is the amino
acid sequence of rat-derived urocortin(Ucn).
[0198] SEQ ID NO: 9 is the nucleic acid sequence from which SEQ ID
NO: 8 was deduced.
[0199] SEQ ID NO: 10 is the amino acid sequence of the rat-derived
CRF receptor referred to as "rCRF-R1 ".
[0200] SEQ ID NO: 11 is the amino acid sequence of a mouse-derived
CRF receptor referred to as "mCRF-R2.beta.".
[0201] SEQ ID NO: 12 is the amino acid sequence of a rat-derived
CRF receptor referred to as "rCRF-R2.alpha.".
[0202] SEQ ID NO: 13 is the amino acid sequence of a rat-derived
CRF receptor referred to as "rCRF-R2.beta.".
[0203] SEQ ID NO: 14 is the amino acid sequence of a 40-residue
peptide defining certain analogs of Ucn.
[0204] SEQ ID NO: 15 is the amino acid sequence of the precursor
plus the mature human Ucn peptide.
[0205] SEQ ID NO: 16 is the nucleic acid sequence from which SEQ ID
NO: 15 was deduced.
[0206] SEQ ID NO: 17 is the amino acid sequence SEQ ID NO: 8 with
Tyr added at the N-terminus.
[0207] SEQ ID NO: 18 is an oligo of 27 bases.
[0208] SEQ ID NO: 19 is an oligo of 28 bases.
Sequence CWU 1
1
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