U.S. patent application number 10/695516 was filed with the patent office on 2004-04-15 for contulakin-g, analogs thereof and uses therefor.
This patent application is currently assigned to Cognetix, Inc.. Invention is credited to Layer, Richard T., McCabe, R. Tyler, Wagstaff, John D..
Application Number | 20040072758 10/695516 |
Document ID | / |
Family ID | 27379856 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072758 |
Kind Code |
A1 |
Wagstaff, John D. ; et
al. |
April 15, 2004 |
Contulakin-G, analogs thereof and uses therefor
Abstract
The present invention is directed to contulakin-G (which is the
native glycosylated peptide), a des-glycosylated contulakin-G
(termed Thr.sub.10-contulakin-G), and derivatives thereof, to a
cDNA clone encoding a precursor of this mature peptide and to a
precursor peptide. The invention is further directed to the use of
this peptide as a therapeutic for anti-seizure, anti-inflammatory,
anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic,
Parkinson's disease, gastrointestinal disorders, depressive states,
cognitive dysfunction, anxiety, tardive dyskinesia, drug
dependency, panic attack, mania, irritable bowel syndrome,
diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's
chorea, vascular leakage, anti-arteriosclerosis, vascular and
vasodilation disorders, as well as neurological, neuropharmalogical
and neuropsychopharmacological disorders.
Inventors: |
Wagstaff, John D.; (Salt
Lake City, UT) ; Layer, Richard T.; (Sandy, UT)
; McCabe, R. Tyler; (Salt Lake City, UT) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Cognetix, Inc.
421 Wakara Way, Suite 201
Salt Lake City
UT
84108
|
Family ID: |
27379856 |
Appl. No.: |
10/695516 |
Filed: |
October 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10695516 |
Oct 29, 2003 |
|
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10067857 |
Feb 8, 2002 |
|
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|
10067857 |
Feb 8, 2002 |
|
|
|
09420797 |
Oct 19, 1999 |
|
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|
6369193 |
|
|
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|
60105015 |
Oct 20, 1998 |
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60128561 |
Apr 9, 1999 |
|
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60130661 |
Apr 23, 1999 |
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Current U.S.
Class: |
514/17.4 ;
514/1.9; 514/12.2; 514/15.6; 514/17.6; 514/18.1; 514/18.2;
514/18.3; 514/19.3 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
7/02 20180101; A61P 25/16 20180101; A61P 9/14 20180101; A61P 25/14
20180101; C07K 9/001 20130101; A61P 25/18 20180101; A61K 38/00
20130101; A61P 1/00 20180101; A61P 25/04 20180101; A61P 25/24
20180101; A61P 43/00 20180101; A61P 9/08 20180101; A61P 9/10
20180101; A61P 25/08 20180101; A61P 1/12 20180101; C07K 14/575
20130101; A61P 29/00 20180101; A61P 9/12 20180101; A61P 35/00
20180101; A61P 9/00 20180101; A61P 25/22 20180101; A61P 25/28
20180101; A61P 25/30 20180101; C07K 7/08 20130101; A61P 21/00
20180101 |
Class at
Publication: |
514/014 ;
514/008 |
International
Class: |
A61K 038/14 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. GM-48677 awarded by the National Institutes of Health,
Bethesda, Md. The United States Government has certain rights in
the invention.
Claims
What is claimed is:
1. A method for treating pain in an individual which comprises
administering an analgesic effective amount of an active agent to
an individual in need of pain treatment, said active agent
comprises contulakin-G which comprises the amino acid sequence
Xaa.sub.1-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Xaa.sub.2-Tyr-Ile-L-
eu (SEQ ID NO:1), where Xaa.sub.1 is pyro-Glu, Xaa.sub.2 is proline
or hydroxyproline and Thr.sub.10 is modified to contain an
O-glycan.
2. The method of claim 1, wherein said pain is acute pain.
3. The method of claim 2, wherein said acute pain is
post-trauma.
4. The method of claim 1, wherein said pain is chronic pain.
5. The method of claim 4, wherein said chronic pain results from
cancer.
6. The method of claim 4, wherein said chronic pain is neuropathic
pain.
7. The method of claim 4, wherein said chronic pain is
inflammatory.
8. The method of claim 1, wherein the active agent is administered
using a delivery system selected from the group consisting of
infusion, pump delivery, bioerodable polymer delivery,
microencapsulated cell delivery, injection and macroencapsulated
cell delivery.
9. The method of claim 8, wherein administration is into the
central nervous system.
10. The method of claim 9, wherein the central nervous system is
selected from the group consisting of the intrathecal space, the
brain ventricles and the brain parenchyma.
11. The method of claim 8, wherein the administration is selected
from the group consisting of subcutaneous, intravenous,
intra-arterial and intramuscular.
12. The method of claim 1, wherein the glycan is
Gal(.beta.1.fwdarw.3)GalN- Ac(.alpha.1.fwdarw.).
13. The method of claim 1, wherein the glycan has the structure
1wherein R.sub.1 is Thr; X is 0; R.sub.2 is OH, NH.sub.2,
NHSO.sub.3Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R.sub.3
is H, SO.sub.3, PO.sub.3, acetyl, sialic acid or monosaccharide;
R.sub.4 is H, SO.sub.3, PO.sub.3, acetyl or monosaccharide; R.sub.5
is OH, NH.sub.2, NHSO.sub.3Na, NHAc, O-sulphate, O-phosphate,
O-monosaccharide or, O-acetyl; R.sub.6 is H, SO.sub.3, PO.sub.3,
acetyl or monosaccharide; R.sub.7 is H, SO.sub.3, PO.sub.3, acetyl
or monosaccharide; R.sub.8 is H, SO.sub.3, PO.sub.3, acetyl or
monosaccharide; n is 0-4 and m is 1-4.
14. A method for treating pain in an individual which comprises
administering an analgesic effective amount of an active agent to
an individual in need of pain treatment, said active agent is
selected from the group consisting of (a) a generic contulakin-G
having the following general formula
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.3-Gly-Gly-Xaa.sub.2-
-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Xaa.sub.10-Il-
e-Leu (SEQ ID NO:2), where Xaa.sub.1 is pyro-Glu, Glu, Gln or
.gamma.-carboxy-Glu; Xaa.sub.2 is Ser, Thr or S-glycan modified
Cys; Xaa.sub.3 is Glu or .gamma.-carboxy-Glu; Xaa.sub.4 is Asn,
N-glycan modified Asn or S-glycan modified Cys; Xaa.sub.5 is Ala or
Gly; Xaa.sub.6 is Thr, Ser, S-glycan modified Cys, Tyr or any
hydroxy containing unnatural amino acid; Xaa.sub.7 is Lys,
N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg,
ornithine, homoarginine or any unnatural basic amino acid;
Xaa.sub.8 is Ala, Gly, Lys, N-methyl-Lys, N,N-dimethyl-Lys,
N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, any unnatural
basic amino acid or X-Lys where X is (CH.sub.2).sub.n, phenyl,
--(CH.sub.2).sub.m--(CH.dbd.CH)--(CH.sub.2).sub.mH or
--(CH.sub.2).sub.m--(C.ident.C)--(CH.sub.2).sub.mH in which n is
1-4 and m is 0-2; Xaa.sub.9 is Pro or hydroxy-Pro; and Xaa.sub.10
is Tyr, mono-iodo-Tyr, di-iodo-Tyr, O-sulpho-Tyr, O-phospho-Tyr,
nitro-Tyr, Trp, D-Trp, bromo-Trp, bromo-D-Trp, chloro-Trp,
chloro-D-Trp, Phe, L-neo-Trp, or any unnatural aromatic amino acid,
with the proviso that the generic contulakin-G is not
un-glycosylated contulakin-G; (b) a generic contulakin-G of (a)
which is modified to contain an O-glycan, an S-glycan or an
N-glycan; (c) a contulakin-G analog which comprises an N-terminal
truncation of from 1 to 9 amino acids of the generic contulakin-G
of (a); (d) a contulakin-G analog of (c), wherein a Ser-O-glycan,
Thr-O-glycan or Cys-S-glycan is substituted for the amino acid
residue at the truncated N-terminus; (e) a contulakin-G analog,
wherein a Ser-O-glycan, Thr-O-glycan or Cys-S-glycan is substituted
for a residue at positions 1-9 of the generic contulakin-G of (a);
and (f) a contulakin-G analog which comprises an N-terminal
truncation of 10 amino acids of the generic contulakin-G of (a)
which is further modified to contain a Lys-N-glycan at residue 11
of the generic contulakin-G.
15. The method of claim 14, wherein said pain is acute pain.
16. The method of claim 15, wherein said acute pain is
post-trauma.
17. The method of claim 14, wherein said pain is chronic pain.
18. The method of claim 17, wherein said chronic pain results from
cancer.
19. The method of claim 17, wherein said chronic pain is
neuropathic pain.
20. The method of claim 17, wherein said chronic pain is
inflammatory.
21. The method of claim 14, wherein the active agent is
administered using a delivery system selected from the group
consisting of infusion, pump delivery, bioerodable polymer
delivery, microencapsulated cell delivery, injection and
macroencapsulated cell delivery.
22. The method of claim 21, wherein administration is into the
central nervous system.
23. The method of claim 22, wherein the central nervous system is
selected from the group consisting of the intrathecal space, the
brain ventricles and the brain parenchyma.
24. The method of claim 21, wherein the administration is selected
from the group consisting of subcutaneous, intravenous,
intra-arterial and intramuscular.
25. The method of claim 14, wherein the glycan is
Gal(.beta.1.fwdarw.3)Gal- NAc(.alpha.1.fwdarw.).
26. The method of claim 14, wherein the glycan has the structure
2wherein R.sub.1 is Thr, Ser, Cys, Asn or Lys; X is 0 when R is Thr
or Ser, or X is S when R.sub.1 is Cys or X is N when R.sub.1 is Asn
or Lys; R.sub.2 is OH, NH.sub.2, NHSO.sub.3Na, NHAc, O-sulphate,
O-phosphate, or O-glycan; R.sub.3 is H, SO.sub.3, PO.sub.3, acetyl,
sialic acid or monosaccharide; R.sub.4 is H, SO.sub.3, PO.sub.3,
acetyl or monosaccharide; R.sub.5 is OH, NH.sub.2, NHSO.sub.3Na,
NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl;
R.sub.6 is H, SO.sub.3, PO.sub.3, acetyl or monosaccharide; R.sub.7
is H, SO.sub.3, PO.sub.3, acetyl or monosaccharide; R.sub.8 is H,
SO.sub.3, PO.sub.3, acetyl or monosaccharide; n is 0-4 and m is
1-4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 10/067,857, filed on Feb. 8, 2002,
which in turn is a continuation application of U.S. patent
application Ser. No. 09/420,797, filed on Oct. 19, 1999, now U.S.
Pat. No. 6,369,193, each incorporated herein by reference. The
present invention is related to and claims priority under 35 U.S.C.
.sctn.119(e) to U.S. provisional patent applications Serial No.
60/105,015, filed on Oct. 20, 1998, Serial No. 60/128,561, filed on
Apr. 9, 1999 and Serial No. 60/130,661, filed on Apr. 23, 1999,
each incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The present invention is directed to contulakin-G (which is
the native glycosylated peptide), a des-glycosylated contulakin-G
(termed Thr.sub.10-contulakin-G), and derivatives thereof, to a
cDNA clone encoding a precursor of this mature peptide and to a
precursor peptide. The invention is further directed to the use of
this peptide as a therapeutic for anti-seizure, anti-inflammatory,
anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic,
Parkinson's disease, gastrointestinal disorders, depressive states,
cognitive dysfunction, anxiety, tardive dyskinesia, drug
dependency, panic attack, mania, irritable bowel syndrome,
diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's
chorea, vascular leakage, anti-arteriosclerosis, vascular and
vasodilation disorders, as well as neurological, neuropharmalogical
and neuropsychopharmacological disorders.
[0004] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated by reference, and for convenience are numerically
referenced in the following text and respectively grouped in the
appended bibliography.
[0005] Mollusks of the genus Conus produce a venom that enables
them to carry out their unique predatory lifestyle. Prey are
immobilized by the venom that is injected by means of a highly
specialized venom apparatus, a disposable hollow tooth that
functions both in the manner of a harpoon and a hypodermic
needle.
[0006] Few interactions between organisms are more striking than
those between a venomous animal and its envenomated victim. Venom
may be used as a primary weapon to capture prey or as a defense
mechanism. Many of these venoms contain molecules directed to
receptors and ion channels of neuromuscular systems.
[0007] Several peptides isolated from Conus venoms have been
characterized. These include the .alpha.-, .mu.- and
.omega.-conotoxins which target nicotinic acetylcholine receptors,
muscle sodium channels, and neuronal calcium channels, respectively
(Olivera et al., 1985). Conopressins, which are vasopressin
analogs, have also been identified (Cruz et al. 1987). In addition,
peptides named conantokins have been isolated from Conus geographus
and Conus tulipa (Mena et al., 1990; Haack et al., 1990). These
peptides have unusual age-dependent physiological effects: they
induce a sleep-like state in mice younger than two weeks and
hyperactive behavior in mice older than 3 weeks (Haack et al.,
1990). The isolation, structure and activity of K-conotoxins are
described in U.S. Pat. No. 5,633,347. Recently, peptides named
contryphans containing D-tryptophan residues have been isolated
from Conus radiatus (U.S. Ser. No. 09/061,026), and
bromo-tryptophan conopeptides have been isolated from Conus
imperialis and Conus radiatus (U.S. Ser. No. 08/785,534).
[0008] It is desired to identify additional conopeptides having
activities of the above conopeptides, as well as conotoxin peptides
having additional activities.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to contulakin-G (which is
the native glycosylated peptide), a des-glycosylated contulakin-G
(termed Thr.sub.10-contulakin-G), and derivatives thereof, to a
cDNA clone encoding a precursor of this mature peptide and to a
precursor peptide. The invention is further directed to the use of
this peptide as a therapeutic for anti-seizure, anti-inflammatory,
anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic,
Parkinson's disease, gastrointestinal disorders, depressive states,
cognitive dysfunction, anxiety, tardive dyskinesia, drug
dependency, panic attack, mania, irritable bowel syndrome,
diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's
chorea, vascular leakage, anti-arteriosclerosis, vascular and
vasodilation disorders, as well as neurological,
neuropharmacological and neuropsychopharmacological disorders.
[0010] In one embodiment, the present invention is directed to
contulakin-G, contulakin-G propeptide and nucleic acids encoding
this peptide. The contulakin-G has the following formula:
Xaa.sub.1-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Xaa.sub.2-Tyr-Ile-Le-
u (SEQ ID NO: 1)
[0011] where Xaa.sub.1 is pyro-Glu, Xaa.sub.2 is proline or
hydroxyproline and Thr.sub.10 is modified to contain an O-glycan.
Xaa.sub.2 is preferably proline. In accordance with the present
invention, a glycan shall mean any N-, S- or O-linked mono-, di-,
tri-, poly- or oligosaccharide that can be attached to any hydroxy,
amino or thiol group of natural or modified amino acids by
synthetic or enzymatic methodologies known in the art. The
monosaccharides making up the glycan can include D-allose,
D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose,
D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine
(GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or
D-arabinose. These saccharides may be structurally modified as
described herein, e.g., with one or more O-sulfate, O-phosphate,
O-acetyl or acidic groups, such as sialic acid, including
combinations thereof. The gylcan may also include similar
polyhydroxy groups, such as D-penicillamine 2,5 and halogenated
derivatives thereof or polypropylene glycol derivatives. The
glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3. The
linkage between the glycan and the amino acid may be alpha or beta,
preferably alpha and is 1-. Preferred glycans are described further
herein, with the most preferred glycan being
Gal(.beta.1.fwdarw.3)GalNAc(.alpha.1.fwdarw.).
[0012] In a second embodiment, the present invention is directed to
a generic contulakin-G having the following general formula,
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.3-Gly-Gly-Xaa.sub.2-Xaa.sub.4-Xaa.su-
b.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Xaa.sub.10-Ile-Leu (SEQ
ID NO:2),
[0013] where Xaa.sub.1 is pyro-Glu, Glu, Gln or
.gamma.-carboxy-Glu; Xaa.sub.2 is Ser, Thr or S-glycan modified
Cys; Xaa.sub.3 is Glu or .gamma.-carboxy-Glu; Xaa.sub.4 is Asn,
N-glycan modified Asn or S-glycan modified Cys; Xaa.sub.5 is Ala or
Gly; Xaa.sub.6 is Thr, Ser, S-glycan modified Cys, Tyr or any
unnatural hydroxy containing amino acid (such as
4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr,
3-nitro-Tyr and 5-amino-Tyr); Xaa.sub.7 is Lys, N-methyl-Lys,
N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine
or any unnatural basic amino acid (such as
N-1-(2-pyrazolinyl)-Arg); Xaa.sub.8 is Ala, Gly, Lys, N-methyl-Lys,
N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine,
homoarginine, any unnatural basic amino acid (such as
N-1-(2-pyrazolinyl)-Arg) or X-Lys where X is (CH.sub.2).sub.n,
phenyl, --(CH.sub.2).sub.m--(CH.dbd.CH)--(CH.sub.2).sub.mH or
--(CH.sub.2).sub.m--(C.ident.C)--(CH.sub.2).sub.mH in which n is
1-4 and m is 0-2; Xaa.sub.9 is Pro or hydroxy-Pro; and Xaa.sub.10
is Tyr, mono-iodo-Tyr, di-iodo-Tyr, O-sulpho-Tyr, O-phospho-Tyr,
nitro-Tyr, Trp, D-Trp, bromo-Trp, bromo-D-Trp, chloro-Trp,
chloro-D-Trp, Phe, L-neo-Trp, any unnatural aromatic amino acid
(such as nitro-Phe, 4-substituted-Phe wherein the substituent is
C.sub.1-C.sub.3 alkyl, carboxyl, hyrdroxymethyl, sulphomethyl,
halo, phenyl, --CHO, --CN, --SO.sub.3H and --NHAc, 2,6-dimethyl-Tyr
and 5-amino-Tyr). The C-terminus contains a free carboxyl group, is
amidated is acylated, contains a glycan or contains an aldehyde. It
is preferred that the C-terminus contains a free carboxyl. This
peptide may further contain one or more glycans as described above.
The glycans may occur at residues 2, 7, 8, 10 and 16. The above and
other unnatural basic amino acids, unnatural hydroxy containing
amino acids or unnatural aromatic amino acids are described in
Building Block Index, Version 2.2, incorporated herein by
reference, by and available from RSP Amino Acid Analogues, Inc.,
Worcester, Mass.
[0014] In a third embodiment, the present invention is directed to
analogs of contulakin-G or the generic contulakin-G. These analogs
include N-terminal truncations of contulakin-G or the generic
contulakin-G up to and including Thr.sub.10. When the N-terminal
truncation is through Thr.sub.10, Lys.sub.11 is N-glycosylated
using a carboxylated modified linker. This N-glycosylated
Lys.sub.11 can be represented as shown in FIG. 1 (Toth et al.,
1999), in which R.sub.2, R.sub.3 and R.sub.4 are as described
herein. In these truncations, it is preferred that the residue
proximal to the truncation is substituted with a glycosylated
serine. Additional analogs include peptides in which Ser-O-glycan,
Thr-O-glycan or Cys-S-glycan is substituted for a residue at
position 1-9.
[0015] In a fourth embodiment, the present invention is directed to
uses of the peptides described herein as a therapeutic for
anti-seizure, anti-inflammatory, anti-shock, anti-thrombus,
hypotensive, analgesia, anti-psychotic, Parkinson's disease,
gastrointestinal disorders, depressive states, cognitive
dysfunction, anxiety, tardive dyskinesia, drug dependency, panic
attack, mania, irritable bowel syndrome, diarrhea, ulcer, GI
tumors, Tourette's syndrome, Huntington's chorea, vascular leakage,
anti-arteriosclerosis, vascular and vasodilation disorders, as well
as neurological, neuropharmacological and
neuropsychopharmacological disorders. In one aspect of this
embodiment, analgesia is induced in a mammal using one of the
peptides described herein. In a second aspect of this embodiment,
epilepsy or convulsions are treated in a mammal. In a third aspect
of this embodiment, schizophrenia is treated in an mammal. In a
fourth aspect of this embodiment, tardive dyskinesia and acute
dystonic reactions are treated in a mammal. In a fifth aspect of
this embodiment, inflammation is treated in a mammal.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows the structure of an N-glycosylation of Lys
using a carboxylated modified linker.
[0017] FIG. 2 shows the native O-glycan attached to Thr.sub.10 of
contulakin-G.
[0018] FIG. 3 shows analogs of the glycan which can be attached to
one or more residues of contulakin-G.
[0019] FIG. 4 shows the preferred core O-glycans (Van de Steen et
al., 1998). Mucin type O-linked oligosaccharides are attached to
Ser or Thr (or other hydroxylated residues of the present peptides)
by a GalNAc residue. The monosaccharide building blocks and the
linkage attached to this first GalNAc residue define the "core
glycans," of which eight have been identified. The type of
glycosidic linkage (orientation and connectivities) are defined for
each core glycan.
[0020] FIG. 5 shows the purification of Contulakin-G. One gram of
crude lyophilized venom from Conus geographus was extracted and
applied on a Sephadex G-25 column as previously described (Olivera
et al., 1984). Three successive fractions containing paralytic and
sleeper activities (Ve/Vo=1.37 to 1.41) were pooled, applied on a
preparative reversed phase Vydac C.sub.18 column and eluted with a
gradient of acetonitrile in 0.1% trifluroacetic acid. The component
indicated by an arrow in panel A caused wobbling and death when
administered icv in mice.
[0021] FIG. 6 shows a nano-ESI MS/MS spectrum (m/z 1035 precursor)
of native contulakin-G (286-1886 Da) (the MS/MS experiment is
denoted using a suggested shorthand (Mcluckey et al., 1991) where
the closed circle represents m/z 1035 [M+2H].sup.2+ precursor and
the arrows are directed towards the open circles which represent
the fragments generated from the precursor). Above the spectrum,
the structure of the glycoamino acid is represented where the
arrows indicate 2 sites which lead to major fragment ions observed
in the MS/MS spectrum (Craig et al., 1993).
[0022] FIGS. 7A-7C show dose-response of CGX-1063
(Thr.sub.10-contulakin-G- ) on spinally mediated (limb withdrawal)
and supraspinally mediated (hindlimb lick) nociceptive behaviors
elicited by noxious heat. Data are expressed as seconds to response
(FIGS. 7A and 7B) or to first fall (FIG. 7C). In FIG. 7A, latency
to the first observable response after placement on a 50.degree. C.
hotplate is shown. FIG. 7B shows latency to the first hindpaw lick.
FIG. 7C shows latency to first fall after placement on the
accelerating rotorod (in FIGS. 7A-7C, n=3-10).
[0023] FIGS. 8A-8B show the effect of CGX-1063 on the nociceptive
response to persistent pain. In FIG. 8A, data are presented as the
amount of time animals spent licking the formalin-injected hindpaw
(n=7-10 animals/treatment group). Intrathecal CGX-1063
dose-dependently decreased the phase 2 nociceptive response in the
formalin test compared to intrathecal saline injected controls.
FIG. 8B shows the latency to first fall from an accelerated rotorod
immediately following the formalin test.
[0024] FIG. 9 shows paw withdrawal threshold to mechanical
stimulation one week following partial sciatic nerve ligation. Data
are presented as the 50% withdrawal threshold in grams determined
with calibrated von Frey filaments (n=3-9 animals per group).
[0025] FIGS. 10A-10B show a comparison of CGX-1160 (contulakin-G),
CGX-1063 and NT in the tail-flick test. Dose-response of the three
compounds is shown in FIG. 10A. FIG. 10B shows the duration of
effect at the highest doses tested for each compound (CGX-1160=100
pmol; CGX-1063=100 pmol; NT=10 nmol).
[0026] FIGS. 11A-11B show the effect of CGX-1160, CGX-1063 and NT
on phase 1 (FIG. 11A) and phase 2 (FIG. 11B) of the formalin test.
All three of the compounds dose-dependently reduced nociceptive
behavior following i.pl. formalin. In phase 2 (FIG. 11B), CGX-1160
was 10 times more potent than CGX-1063, and 600-700 times more
potent than NT.
[0027] FIGS. 12A-12C show effect of CGX-1160, CGX-1063 and NT on
chronic inflammation-induced mechanical allodynia. Numbers in
parentheses indicate percentage of each corresponding control
value. In FIG. 12A, CGX-1160 potently and dose-dependently reversed
CFA-induced allodynia. In FIG. 12B, CGX-1063 reversed CFA-induced
allodynia, but was approximately 100-fold less potent in this model
than CGX-1160. In FIG. 12C, NT reversed CFA-induced allodynia at
1,000 pmol, but not 100 pmol, approximately 10,000-fold less potent
than CGX-1160.
[0028] FIGS. 13A-13B show locomotor impairing effects of CGX-1160,
CGX-1063 and NT. FIG. 13A shows time to peak effect and duration of
effect of the three compounds at the highest doses tested
(approximately 100 times the ED.sub.50 in phase 2 of the formalin
test). FIG. 13B shows dose-response of each compound on locomotor
impairment.
[0029] FIGS. 14A-14C show dose-effect and time to peak effect and
duration of locomotor impairment of CGX-1160, CGX-1063 and NT. FIG.
14A shows that CGX-1160 caused long-lasting motor impairment only
at doses 100-fold or greater than its ED.sub.50. FIG. 14B shows
that CGX-1063 caused long-last motor impairment at doses 10-fold or
greater than its ED.sub.50. FIG. 14C shows that NT caused long-last
motor impairment at doses 100-fold greater than its ED.sub.50.
[0030] FIGS. 15A-15B show a comparison of CGX-1160, CGX-1063 and NT
on change in body temperature. FIG. 15A shows time to peak effect
and duration of each compound, and FIG. 15B shows dose-response of
each compound.
[0031] FIGS. 16A-16C show hypothermic dose-effect and duration of
CGX-1160, CGX-1063 and NT. In FIG. 16A, CGX-1160 caused hypothermia
only at doses 100-500 times greater than ED.sub.50. FIG. 16B shows
the long-lasting hypothermic effect of CGX-1063 at doses 10-fold
higher than ED.sub.50.(100 pmol). In FIG. 16C, NT had a
hypothalamic effect at doses 10-100 times higher than its
ED.sub.50.
[0032] FIG. 17 shows effects of Thr.sub.10-g Contulakin-G
(CGX-1160; 100 pmol i.c.v.) on D-amphetamine-stimulated locomotor
activity as measured by distance traveled. Abbreviations: sal-sal:
i.p. treatment was saline, i.c.v. treatment was saline; amphet (3
mg/kg)-sal: i.p. treatment was D-amphetamine sulphate (3 mg/kg),
i.c.v. treatment was saline; amphet (10 mg/kg)-sal: i.p. treatment
was D-amphetamine sulphate (10 mg/kg), i.c.v. treatment was saline;
sal-ctl: i.p. treatment was saline, i.c.v. treatment was
Thr.sub.10-g contulakin-G (100 pmol); amphet (3 mg/kg)-ctl: i.p.
treatment was D-amphetamine sulphate (3 mg/kg), i.c.v. treatment
was Thr.sub.10-g contulakin-G (100 pmol). Each bar shows the
mean.+-.SEM of 3-7 mice per group. a: P<0.05 vs saline-saline
treated group (sal-sal); b: P<0.05 vs D-amphetamine-saline group
(amphet (3 mg/kg)-sal).
[0033] FIG. 18 shows the effects of Thr.sub.10-g Contulakin-G
(CGX-1160; 100 pmol i.c.v.) on D-amphetamine-stimulated locomotor
activity as measured by time spent ambulatory (s). Abbreviations:
sal-sal: i.p. treatment was saline, i.c.v. treatment was saline;
amphet (3 mg/kg)-sal: i.p. treatment was D-amphetamine sulphate (3
mg/kg), i.c.v. treatment was saline; amphet (10 mg/kg)-sal: i.p.
treatment was D-amphetamine sulphate (10 mg/kg), i.c.v. treatment
was saline; sal-ctl: i.p. treatment was saline, i.c.v. treatment
was Thr .sub.10-g contulakin-G (100 pmol); amphet (3 mg/kg)-ctl:
i.p. treatment was D-amphetamine sulphate (3 mg/kg), i.c.v.
treatment was Thr.sub.10-g contulakin-G (100 pmol). Each bar shows
the mean.+-.SEM of 3-7 mice per group. a: P<0.05 vs
saline-saline treated group (sal-sal); b: P<0.05 vs
D-amphetamine-saline group (amphet (3 mg/kg)-sal).
[0034] FIG. 19 shows CGX-1160 and CGX-1063 dose-dependently protect
against audiogenic seizures following i.c.v. administration in
Frings mice, at doses well below minimal motor impairing doses.
Each point represents the percent protection (toxic in groups of at
least four mice).
[0035] FIG. 20 shows CGX-1160's long-lasting efficacy in blocking
audiogenic seizures following i.c.v. administration in Frings mice.
Neurotensin is only 50% effective following i.c.v. administration
of up to 5 nmol. Each point represents the percent protection in a
group of four mice.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] The present invention is directed to contulakin-G (which is
the native glycosylated peptide), a des-glycosylated contulakin-G
(termed Thr.sub.10-contulakin-G), and derivatives thereof, to a
cDNA clone encoding a precursor of this mature peptide and to a
precursor peptide. The invention is further directed to the use of
this peptide as a therapeutic for anti-seizure, anti-inflammatory,
anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic,
Parkinson's disease, gastrointestinal disorders, depressive states,
cognitive dysfunction, anxiety, tardive dyskinesia, drug
dependency, panic attack, mania, irritable bowel syndrome,
diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's
chorea, vascular leakage, anti-arteriosclerosis, vascular and
vasodilation disorders, as well as neurological, neuropharmalogical
and neuropsychopharmacological disorders.
[0037] The present invention is directed to contulakin-G and
contulakin-G analogues as described above. These peptides may
contain single or multiple glycan post-translational modifications
at one or more, up to all, of the hydroxyl sites of the peptides.
The glycans are as described herein. The native O-glycan attached
to contulakin-G is shown in FIG. 2. FIG. 3 shows analogs of the
glycan which can be attached to one or more residues of
contulakin-G. In this figure, R.sub.1 is an amino capable of being
derivatized with a gylcan either chemically or enzymatically;
R.sub.2 is OH, NH.sub.2, NHSO.sub.3Na, NHAc, O-sulphate,
O-phosphate, or O-glycan; R.sub.3 is H, SO.sub.3, PO.sub.3, acetyl,
sialic acid or monosaccharide; R.sub.4 is H, SO.sub.3, PO.sub.3,
acetyl or monosaccharide; R.sub.5 is OH, NH.sub.2, NHSO.sub.3Na,
NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl;
R.sub.6 is H, SO.sub.3, PO.sub.3, acetyl or monosaccharide; R.sub.7
is H, SO.sub.3, PO.sub.3, acetyl or monosaccharide; R.sub.8 is H,
SO.sub.3, PO.sub.3, acetyl or monosaccharide; n is 0-4 and m is
1-4.
[0038] The preferred core glycans which can be used to modify
contulakin-G or analogs disclosed herein are shown in FIG. 4.
Further branching from these cores using the monosaccharides
described herein may also be made. Preferred glycosidic linkages
are specified by cores 5 and 7 of FIG. 4 with further homolgation
of the glycan at positions 3, 4 and 6 of the GalNAc template using
the monosaccharides described herein Any free hydroxy function may
be O-sulphated, O-phosphorylated or O-aceylated.
[0039] The glycosylated conopeptide (contulakin-G or CGX-1160) has
higher in vivo potency than the unglycosylated conopeptide
(Thr.sub.10-contulakin-G or CGX-1063), although their in vitro
potencies are about the same. The glycosylation may be important
for better binding with the receptor, and/or enhanced delivery of
the conopeptide to its site of action, and/or inhibition of
degradation of the conopeptide.
[0040] The present invention is further directed to DNA sequence
coding for contulakin-G as described in further herein. The
invention is further directed to the propeptide for contulakin-G as
described in further detail herein.
[0041] The present invention relates to a novel linear glycosylated
contulakin-G, and derivatives thereof that are useful as
pharmaceutical agents, to methods for their production, to
pharmaceutical compositions which include these compounds and a
pharmaceutically acceptable carrier, and to pharmaceutical methods
of treatment. The novel compounds of the present invention are
central nervous system agents and their biological actions are
effected at a novel "Contulakin-G binding site on the neurotensin
receptor". More particularly, the novel compounds of the present
invention are analgesics, anti-inflammatory agents, antipsychotic
agents for treating psychoses such as schizophrenia and display
potent anti-seizure properties in established animal models of
epilepsy.
[0042] PAIN: Chronic or intractable pain, such as may occur in
conditions such as bone degenerative diseases and cancer, is a
debilitating condition which is treated with a variety of analgesic
agents, and often opioid compounds, such as morphine.
[0043] In general, brain pathways governing the perception of pain
are still incompletely understood, sensory afferent synaptic
connections to the spinal cord, termed "nociceptive pathways" have
been documented in some detail. In the first leg of such pathways,
C- and A-fibers which project from peripheral sites to the spinal
cord carry nociceptive signals. Polysynaptic junctions in the
dorsal horn of the spinal cord are involved in the relay and
modulation of sensations of pain to various regions of the brain,
including the periaqueductal grey region. Analgesia, or the
reduction of pain perception, can be effected directly by
decreasing transmission along such nociceptive pathways. Analgesic
opiates are thought to act by mimicking the effects of endorphin or
enkephalin peptide-containing neurons, which synapse
presynaptically at the C- or A-fiber terminal and which, when they
fire, inhibit release of neurotransmitters, including substance P.
Descending pathways from the brain are also inhibitory on C- and
A-fiber firing.
[0044] Certain types of pain have complex etiologies. For example,
neuropathic pain is generally a chronic condition attributable to
injury or partial transection of a peripheral nerve. This type of
pain is characterized by hyperesthesia, or enhanced sensitivity to
external noxious stimuli. The hyperesthetic component of
neuropathic pain does not respond to the same pharmaceutical
interventions as does more generalized and acute forms of pain.
[0045] Opioid compounds such as morphine, while effective in
producing analgesia for many types of pain, are not always
effective, and may induce tolerance in patients. When a subject is
tolerant to opioid narcotics, increased doses are required to
achieve a satisfactory analgesic effect. These compounds can
produce side effects, such as respiratory depression, which can be
life threatening. In addition, opioids frequently produce physical
dependence in patients. Dependence appears to be related to the
dose of opioid taken and the period of time over which it is taken
by the subject. For this reason, alternate therapies for the
management of chronic pain are widely sought after. In addition,
compounds which serve as either a replacement for or as an adjunct
to opioid treatment in order to decrease the dosage of analgesic
compound required, have utility in the treatment of pain,
particularly pain of the chronic, intractable type.
[0046] Since contulakin-G has been shown to act at a site on
certain neurotensin receptors, and neurotensin has been shown to
have analgesic actions (Clineschmidt et al. 1979), then
contulakin-G like conopeptides are useful for the treatment of pain
and related disorders.
[0047] SCHIZOPHRENIA: Schizophrenia is a neurogenic disorder that
is currently treated primarily with neuroleptic compounds such as
phenothiazines and butyrophenones, which block dopamine receptors.
Since contulakin-G has been shown to act at a site on certain
neurotensin receptors, and neurotensin actions are implicated in
the etiology of schizophrenia (Nemeroff et al. 1992), then
contulakin-G like conopeptides are useful for the treatment of
schizophrenia and related disorders.
[0048] The in vitro selection criteria for conopeptides useful in
treating schizophrenia, include: a) activation of Contulakin-G
sites; b) high affinity reversible binding to a Contulakin-G
binding site localized to the limbic region of the brain, and c)
inhibition of dopamine release from brain regions, particularly
limbic brain regions.
[0049] Compounds exhibiting sufficiently high activities in the
above in vitro screening assays are then tested in an animal model
used in screening anti-psychotic compounds.
[0050] TARDIVE DYSKINESIA AND OTHER ACUTE DYSTONIC REACTIONS:
Tardive dyskinesia and acute dystonic reactions are movement
disorders that are commonly produced as side effects of
anti-psychotic therapy employing dopamine antagonists, such as
haloperidol. These disorders are characterized by supersensitivity
of dopamine receptors in certain regions of the brain associated
with control of movement, particularly the basal ganglia.
Currently, intermittent antipsychotic therapy is used in attempt to
avoid onset of the disorder, and such disorders are treated by
withdrawal of therapy.
[0051] Criteria for selection of a conopeptide for treatment of
tardive dyskinesia include: a) activation of Contulakin-G sites; b)
high affinity reversible binding to the Contulakin-G site; c)
inhibition of dopamine release from striatal brain regions, and
other regions of the basal ganglia, and d) a ratio of inhibition of
dopamine release in the basal ganglia to inhibition of dopamine
release in the limbic regions.
[0052] Compounds showing sufficiently high activities in in vitro
screening assays are then tested in the rat striatal turning model,
described above. Compounds useful in the method of treating such
movement disorders, when injected to the striatum on the side of
the brain contralateral to the lesion, correct the turning
behavior.
[0053] INFLAMMATION: A neurogenic component of inflammation has
been described, in that blockade of the sympathetic nervous system,
and particularly blockade of beta-adrenergic receptors, is helpful
in reducing inflammatory joint damage. Compounds useful in the
treatment of inflammation would be expected to have the following
in vitro properties: a) activation of novel Contulakin-G sites; b)
high affinity binding to the Contulakin-G binding sites, and c)
inhibition of norepinephrine release from nervous tissue. Compounds
exhibiting sufficiently high activities in such in vitro screening
assays are tested in an animal model of rheumatoid arthritis.
[0054] EPILEPSY: Epilepsy is a general term which describes
disorders of the central nervous system characterized by repeated
episodes of seizures. Such seizures may involve the sensory,
autonomic or motor nervous systems and are recognized
electrophysiologically by the presence of abnormal electric
discharges in the brain. The pathophysiology of such abnormal
discharge activity is not well understood; however, there is
evidence that loss of inhibitory neural input, such as GABA input,
is involved in at least some epileptic seizures.
[0055] The ability of certain of the benzodiazepines (e.g.,
diazepam) to repress or inhibit epileptic episodes is considered by
some to be evidence of a GABAergic pathophysiology in seizure
activity, since these drugs are known to potentiate GABAergic
neural inhibition via an effect on the GABA receptor-associated
chloride ion channel. Biochemical effects of other anti-epileptic
compounds include stabilization of excitable membranes by
inhibition of voltage-sensitive sodium or potassium channels
(phenytoin), and general depression of neuronal function
characterized by facilitation of GABAergic transmission, inhibition
of the effects of excitatory (glutaminergic) neurotransmission and
depression of neurotransmitter release (phenobarbital).
[0056] Compounds useful in the treatment of epilepsy would be
expected to have the following in vitro properties: a) activation
of novel Contulakin-G sites; b) high affinity binding to the
contulakin-G conopeptide binding sites, and c) inhibition of
excitatory neurotransmitter release from nervous tissue. Compounds
exhibiting sufficiently high activities in such in vitro screening
assays fare tested in an established animal model of epilepsy.
[0057] In addition to the above specific disorders, since the
peptides, derivatives and analogs of the present invention have
been found to bind to the neurotensin receptor, these compounds are
also useful in connection with conditions associated with the
neurotensin receptor and for which neurotensin-like compounds or
other compounds have been shown to be active. These activities
include: methamphetamine antagonists, antipsychotic agents,
cerebral medicaments, analgesic agents, anti-endotoxin shock
effect, protease inhibition action (an anti-thrombin action, an
anti-plasmin action), a hypotensive action, an anti-DIC action, an
anti-allergic action, a wound healing action, cerebral edema, an
edema of the lung, an edema of the trachea, a thrombus, an
arteriosclerosis, a burn, and a hypertension, allergic diseases
(such as a bronchial asthma and a pollenosis), reducing hemorrhage
from a sharp trauma such as an injured tissue portion at the time
of surgical operation, a lacerated wound of a brain or other
tissues caused by a traffic accident and the like, and for relaxing
and curing swelling, pain inflammation caused by trauma,
suppressing internal hemorrhage caused by a dull trauma, edemata
and inflammation which are accompanied with the internal
hemorrhage, suppression and improvement of cerebral edemata by
suppressing a leakage of blood components to a tissue matrix found
in cerebral ischemetic diseases which include cerebral infarctions
(e.g., a cerebral thrombus and a cerebral embolism), intracranial
hemorrhages (e.g., a cerebral hemorrhage and a subarachnoidal
hemorrhage), a transient cerebral ischemic attack, acute cerebral
blood vessel disorders in a hypertensive encephalopathy,
suppression and improvement of burns, chilblains, other skin
inflammations and swelling, an upper tracheal inflammation, an
asthma, nasal congestion, a pulmonary edema, and inflammable
disorders caused by endogenous and exogenous factors, which
directly damage vascular endothelia and mucous membranes, such as
an environmental chemical substance, chemotherapeutics of cancer,
an endotoxin, and an inflammation mediator.
[0058] The conopeptides of the present invention are identified by
isolation from Conus venom. Alternatively, the conopeptides of the
present invention are identified using recombinant DNA techniques
by screening cDNA libraries of various Conus species using
conventional techniques with degenerate probes. Clones which
hybridize to these probes are analyzed to identify those which meet
minimal size requirements, i.e., clones having approximately 300
nucleotides (for a propeptide), as determined using PCR primers
which flank the cDNA cloning sites for the specific cDNA library
being examined. These minimal-sized clones are then sequenced. The
sequences are then examined for the presence of a peptide having
the characteristics noted above for conopeptides. The biological
activity of the peptides identified by this method is tested as
described herein, in U.S. Pat. No. 5,635,347 or conventionally in
the art.
[0059] These peptides are sufficiently small to be chemically
synthesized. General chemical syntheses for preparing the foregoing
conopeptides are described hereinafter, along with specific
chemical synthesis of conopeptides and indications of biological
activities of these synthetic products. Various ones of these
conopeptides can also be obtained by isolation and purification
from specific Conus species using the techniques described in U.S.
Pat. No. 4,447,356 (Olivera et al., 1984), U.S. Pat. No. 5,514,774
(Olivera et al., 1996) and U.S. Pat. No. 5,591,821 (Olivera et al.,
1997), the disclosures of which are incorporated herein by
reference.
[0060] Although the conopeptides of the present invention can be
obtained by purification from cone snails, because the amounts of
conopeptides obtainable from individual snails are very small, the
desired substantially pure conopeptides are best practically
obtained in commercially valuable amounts by chemical synthesis
using solid-phase strategy. For example, the yield from a single
cone snail may be about 10 micrograms or less of conopeptide. By
"substantially pure" is meant that the peptide is present in the
substantial absence of other biological molecules of the same type;
it is preferably present in an amount of at least about 85% purity
and preferably at least about 95% purity. Chemical synthesis of
biologically active conopeptides depends of course upon correct
determination of the amino acid sequence. Thus, the conopeptides of
the present invention may be isolated, synthesized and/or
substantially pure.
[0061] The conopeptides can also be produced by recombinant DNA
techniques well known in the art. Such techniques are described by
Sambrook et al. (1989). The peptides produced in this manner are
isolated, reduced if necessary, and oxidized to form the correct
disulfide bonds, if present in the final molecule.
[0062] One method of forming disulfide bonds in the conopeptides of
the present invention is the air oxidation of the linear peptides
for prolonged periods under cold room temperatures or at room
temperature. This procedure results in the creation of a
substantial amount of the bioactive, disulfide-linked peptides. The
oxidized peptides are fractionated using reverse-phase high
performance liquid chromatography (HPLC) or the like, to separate
peptides having different linked configurations. Thereafter, either
by comparing these fractions with the elution of the native
material or by using a simple assay, the particular fraction having
the correct linkage for maximum biological potency is easily
determined. It is also found that the linear peptide, or the
oxidized product having more than one fraction, can sometimes be
used for in vivo administration because the cross-linking and/or
rearrangement which occurs in vivo has been found to create the
biologically potent conopeptide molecule. However, because of the
dilution resulting from the presence of other fractions of less
biopotency, a somewhat higher dosage may be required.
[0063] The peptides are synthesized by a suitable method, such as
by exclusively solid-phase techniques, by partial solid-phase
techniques, by fragment condensation or by classical solution
couplings.
[0064] In conventional solution phase peptide synthesis, the
peptide chain can be prepared by a series of coupling reactions in
which constituent amino acids are added to the growing peptide
chain in the desired sequence. Use of various coupling reagents,
e.g., dicyclohexylcarbodiimid- e or diisopropyl-carbonyldimidazole,
various active esters, e.g., esters of N-hydroxyphthalimide or
Nhydroxy-succinimide, and the various cleavage reagents, to carry
out reaction in solution, with subsequent isolation and
purification of intermediates, is well known classical peptide
methodology. Classical solution synthesis is described in detail in
the treatise, "Methoden der Organischen Chemie (Houben-Weyl):
Synthese von Peptiden," (1974). Techniques of exclusively
solid-phase synthesis are set forth in the textbook, "Solid-Phase
Peptide Synthesis," (Stewart and Young, 1969), and are exemplified
by the disclosure of U.S. Pat. No. 4,105,603 (Vale et al., 1978).
The fragment condensation method of synthesis is exemplified in
U.S. Pat. No. 3,972,859 (1976). Other available syntheses are
exemplified by U.S. Pat. No. 3,842,067 (1974) and U.S. Pat. No.
3,862,925 (1975). The synthesis of peptides containing
.gamma.-carboxyglutamic acid residues is exemplified by Rivier et
al. (1987), Nishiuchi et al. (1993) and Zhou et al. (1996).
Synthesis of conopeptides have been described in U.S. Pat. No.
4,447,356 (Olivera et al., 1984), U.S. Pat. No. 5,514,774 (Olivera
et al., 1996) and U.S. Pat. No. 5,591,821 (Olivera et al.,
1997).
[0065] Common to such chemical syntheses 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.
Usually also common is the protection of an .alpha.-amino group on
an amino acid or a fragment while that entity reacts at the
carboxyl group, 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 such a synthesis, an intermediate compound is produced which
includes each of the amino acid residues located in its desired
sequence in the peptide chain with appropriate side-chain
protecting groups linked to various ones of the residues having
labile side chains.
[0066] As far as the selection of a side chain amino protecting
group is concerned, generally one is chosen which is not removed
during deprotection of the .alpha.-amino groups during the
synthesis. However, for some amino acids, e.g., His, protection is
not generally necessary. In selecting a particular side chain
protecting group to be used in the synthesis of the peptides, the
following general rules are followed: (a) the protecting group
preferably retains its protecting properties and is not split off
under coupling conditions, (b) the protecting group should be
stable under the reaction conditions selected for removing the
.alpha.-amino protecting group at each step of the synthesis, and
(c) the side chain protecting group must be removable, upon the
completion of the synthesis containing the desired amino acid
sequence, under reaction conditions that will not undesirably alter
the peptide chain.
[0067] It should be possible to prepare many, or even all, of these
peptides using recombinant DNA technology. However, when peptides
are not so prepared, they are preferably prepared using the
Merrifield solid-phase synthesis, although other equivalent
chemical syntheses known in the art can also be used as previously
mentioned. Solid-phase synthesis is commenced from the C-terminus
of the peptide by coupling a protected .alpha.-amino acid to a
suitable resin. Such a starting material can be prepared by
attaching an .alpha.-amino-protected amino acid by an ester linkage
to a chloromethylated resin or a hydroxymethyl resin, or by an
amide bond to a benzhydrylamine (BHA) resin or
paramethylbenzhydrylamine (MBHA) resin. Preparation of the
hydroxymethyl resin is described by Bodansky et al. (1966).
Chloromethylated resins are commercially available from Bio Rad
Laboratories (Richmond, Calif.) and from Lab. Systems, Inc. The
preparation of such a resin is described by Stewart and Young
(1969). BHA and MBHA resin supports are commercially available, and
are generally used when the desired polypeptide being synthesized
has an unsubstituted amide at the C-terminus. Thus, solid resin
supports may be any of those known in the art, such as one having
the formulae --O--CH.sub.2-resin support, --NH BHA resin support,
or --NH--MBHA resin support. When the unsubstituted amide is
desired, use of a BHA or MBHA resin is preferred, because cleavage
directly gives the amide. In case the N-methyl amide is desired, it
can be generated from an N-methyl BHA resin. Should other
substituted amides be desired, the teaching of U.S. Pat. No.
4,569,967 (Kornreich et al., 1986) can be used, or should still
other groups than the free acid be desired at the C-terminus, it
may be preferable to synthesize the peptide using classical methods
as set forth in the Houben-Weyl text (1974).
[0068] The C-terminal amino acid, protected by Boc or Fmoc and by a
side-chain protecting group, if appropriate, can be first coupled
to a chloromethylated resin according to the procedure set forth in
Horiki et al. (1978), using KF in DMF at about 60.degree. C. for 24
hours with stirring, when a peptide having free acid at the
C-terminus is to be synthesized. Following the coupling of the
BOC-protected amino acid to the resin support, the .alpha.-amino
protecting group is removed, as by using trifluoroacetic acid (TFA)
in methylene chloride or TFA alone. The deprotection is carried out
at a temperature between about 0.degree. C. and room temperature.
Other standard cleaving reagents, such as HCl in dioxane, and
conditions for removal of specific .alpha.-amino protecting groups
may be used as described in Schroder and Lubke (1965).
[0069] After removal of the .alpha.-amino-protecting group, the
remaining .alpha.-amino- and side chain-protected amino acids are
coupled step-wise in the desired order to obtain the intermediate
compound defined hereinbefore, or 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.
Selection of an appropriate coupling reagent is within the skill of
the art. Particularly suitable as a coupling reagent is
N,N'-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the
presence of HoBt or HoAt).
[0070] The activating reagents used in the solid phase synthesis of
the peptides are well known in the peptide art. Examples of
suitable activating reagents are carbodiimides, such as
N,N'-diisopropylcarbodiimi- de and
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activating
reagents and their use in peptide coupling are described by
Schroder and Lubke (1965) and Kapoor (1970).
[0071] Each protected amino acid or amino acid sequence is
introduced into the solid-phase reactor in about a twofold or more
excess, and the coupling may be 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 cases where intermediate 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 success of the coupling reaction at each stage of
the synthesis, if performed manually, is preferably monitored by
the ninhydrin reaction, as described by Kaiser et al. (1970).
Coupling reactions can be performed automatically, as on a Beckman
990 automatic synthesizer, using a program such as that reported in
Rivier et al. (1978).
[0072] After the desired amino acid sequence has been completed,
the intermediate peptide can be removed from the resin support by
treatment with a reagent, such as liquid hydrogen fluoride or TFA
(if using Fmoc chemistry), which not only cleaves the peptide from
the resin but also cleaves all remaining side chain protecting
groups and also the .alpha.-amino protecting group at the
N-terminus if it was not previously removed to obtain the peptide
in the form of the free acid. If Met is present in the sequence,
the Boc protecting group is preferably first removed using
trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the
peptide from the resin with HF to eliminate potential S-alkylation.
When using hydrogen fluoride or TFA for cleaving, one or more
scavengers such as anisole, cresol, dimethyl sulfide and
methylethyl sulfide are included in the reaction vessel.
[0073] Cyclization of the linear peptide is preferably affected, as
opposed to cyclizing the peptide while a part of the peptido-resin,
to create bonds between Cys residues. To effect such a disulfide
cyclizing linkage, fully protected peptide can be cleaved from a
hydroxymethylated resin or a chloromethylated resin support by
ammonolysis, as is well known in the art, to yield the fully
protected amide intermediate, which is thereafter suitably cyclized
and deprotected. Alternatively, deprotection, as well as cleavage
of the peptide from the above resins or a benzhydrylamine (BHA)
resin or a methylbenzhydrylamine (MBHA), can take place at
0.degree. C. with hydrofluoric acid (HF) or TFA, followed by
oxidation as described above. A suitable method for cyclization is
the method described by Cartier et al. (1996).
[0074] Muteins, analogs or active fragments, of the foregoing
contulakin-G or Thr.sub.10-g contulakin-G are also contemplated
here. See, e.g., Hammerland et al (1992). Derivative muteins,
analogs or active fragments of the conotoxin peptides may be
synthesized according to known techniques, including conservative
amino acid substitutions, such as outlined in U.S. Pat. No.
5,545,723 (see particularly col. 2, line 50 to col. 3, line 8);
U.S. Pat. No. 5,534,615 (see particularly col. 19, line 45 to col.
22, line 33); and U.S. Pat. No. 5,364,769 (see particularly col. 4,
line 55 to col. 7, line 26), each incorporated herein by
reference.
[0075] Pharmaceutical compositions containing a compound of the
present invention as the active ingredient can be prepared
according to conventional pharmaceutical compounding techniques.
See, for example, Remington's Pharmaceutical Sciences, 18th Ed.
(1990, Mack Publishing Co., Easton, Pa.). Typically, an
antagonistic amount of the active ingredient will be admixed with a
pharmaceutically acceptable carrier. The carrier may take a wide
variety of forms depending on the form of preparation desired for
administration, e.g., intravenous, oral or parenteral.
[0076] For oral administration, the compounds can be formulated
into solid or liquid preparations such as capsules, pills, tablets,
lozenges, melts, powders, suspensions or emulsions. In preparing
the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed, such as, for example, water,
glycols, oils, alcohols, flavoring agents, preservatives, coloring
agents, suspending agents, and the like in the case of oral liquid
preparations (such as, for example, suspensions, elixirs and
solutions); or carriers such as starches, sugars, diluents,
granulating agents, lubricants, binders, disintegrating agents and
the like in the case of oral solid preparations (such as, for
example, powders, capsules and tablets). Because of their ease in
administration, tablets and capsules represent the most
advantageous oral dosage unit form, in which case solid
pharmaceutical carriers are obviously employed. If desired, tablets
may be sugar-coated or enteric-coated by standard techniques.
[0077] For parenteral administration, the compound may be dissolved
in a pharmaceutical carrier and administered as either a solution
or a suspension. Illustrative of suitable carriers are water,
saline, dextrose solutions, fructose solutions, ethanol, or oils of
animal, vegetative or synthetic origin. The carrier may also
contain other ingredients, for example, preservatives, suspending
agents, solubilizing agents, buffers and the like. When the
compounds are being administered intrathecally, they may also be
dissolved in cerebrospinal fluid.
[0078] Administration of the active agent according to this
invention may be achieved using any suitable delivery means,
including:
[0079] (a) pump (see, e.g., Annals of Pharmacotherapy, 27:912
(1993); Cancer, 41:1270 (1993); Cancer Research, 44:1698
(1984));
[0080] (b), microencapsulation (see, e.g., U.S. Pat. Nos.
4,352,883; 4,353,888; and 5,084,350);
[0081] (c) continuous release polymer implants (see, e.g., U.S.
Pat. No. 4,883,666);
[0082] (d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761,
5,158,881, 4,976,859 and 4,968,733 and published PCT patent
applications WO92/19195, WO 95/05452);
[0083] (e) naked or unencapsulated cell grafts to the CNS (see,
e.g., U.S. Pat. Nos. 5,082,670 and 5,618,531);
[0084] (f) injection, either subcutaneously, intravenously,
intra-arterially, intramuscularly, or to other suitable site;
or
[0085] (g) oral administration, in capsule, liquid, tablet, pill,
or prolonged release formulation.
[0086] In one embodiment of this invention, an active agent is
delivered directly into the CNS, preferably to the brain
ventricles, brain parenchyma, the intrathecal space or other
suitable CNS location, most preferably intrathecally.
[0087] Alternatively, targeting therapies may be used to deliver
the active agent more specifically to certain types of cells, by
the use of targeting systems such as antibodies or cell-specific
ligands. Targeting may be desirable for a variety of reasons, e.g.
if the agent is unacceptably toxic, if it would otherwise require
too high a dosage, or if it would not otherwise be able to enter
target cells.
[0088] The active agents, which are peptides, can also be
administered in a cell based delivery system in which a DNA
sequence encoding an active agent is introduced into cells designed
for implantation in the body of the patient, especially in the
spinal cord region. Suitable delivery systems are described in U.S.
Pat. No. 5,550,050 and published PCT Application Nos. WO 92/19195,
WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO
96/40871, WO 96/40959 and WO 97/12635. Suitable DNA sequences can
be prepared synthetically for each active agent on the basis of the
developed sequences and the known genetic code.
[0089] The active agent is preferably administered in a
therapeutically effective amount. The actual amount administered,
and the rate and time-course of administration, will depend on the
nature and severity of the condition being treated. Prescription of
treatment, e.g. decisions on dosage, timing, etc., is within the
responsibility of general practitioners or specialists, and
typically takes into account the disorder to be treated, the
condition of the individual patient, the site of delivery, the
method of administration and other factors known to practitioners.
Examples of techniques and protocols can be found in Remington's
Pharmaceutical Sciences. Typically, the active agents of the
present invention exhibit their effect at a dosage range of from
about 0.001 .mu.g/kg to about 500 .mu.g/kg, preferably from about
0.01 .mu.g/kg to about 100 .mu.g/kg, of the active ingredient, more
preferably, from about 0.10 .mu.g/kg to about 50 .mu.g/kg, and most
preferably, from about 1 .mu.g/kg to about 10 .mu.g/kg. A suitable
dose can be administered in multiple sub-doses per day. Typically,
a dose or sub-dose may contain from about 0.1 .mu.g to about 500
.mu.g of the active ingredient per unit dosage form. A more
preferred dosage will contain from about 0.5 .mu.g to about 100
.mu.g of active ingredient per unit dosage form. Dosages are
generally initiated at lower levels and increased until desired
effects are achieved.
EXAMPLES
[0090] The present invention is further detailed in the following
examples, which are offered by way of illustration and are not
intended to limit the invention in any manner. Standard techniques
well known in the art or the techniques specifically described
below are utilized. The abbreviations used are: Bop,
benzotriazoyloxy-tris (dimethyl amino) phosphonium
hexafluorophosphate; Boc, tert butyloxycarbonyl; Fmoc,
9-fluoroenylmethoxy carbonyl; Gal, galactose; GalNAc, N-acetyl
galactosamine; hNTR1, human neurotensin type 1 receptor; Hex,
hexose; HexNAc, N-acetyl hexosamine; icv, intra cerebroventricular;
LSI, liquid secondary ionization; MALD, matrix assisted laser
desorption; MS, mass spectrometry; mNTR3, mouse neurotensin type 3
receptor; nano-ESI, nano-electrospray; NMP, N-methylpyrrolidone;
NMR, nuclear magnetic resonance; ppm, parts per million; rNTR1, rat
neurotensin type 1 receptor; rNTR2, rat neurotensin type 2
receptor; RP-HPLC, reverse phase-high performance liquid
chromatography. Amino acids are indicated by the standard three or
one letter abbreviations.
Example 1
Experimental Procedures for Initial Analysis of Contulakin-G
[0091] 1. Crude venom. Conus geographus specimens were collected
from Marinduque Is. in the Philippines. The crude venom was
obtained by dissection of the venom duct gland and then freeze
dried and stored at -70.degree. C.
[0092] 2. Peptide purification. Freeze dried C. geographus venom (1
g) was extracted with 1.1% acetic acid and chromatographed on a
Sephadex G-25 column eluted with 1.1% acetic acid as previously
described (Olivera et al., 1984). A peptide that makes mice
sluggish and unresponsive was purified by a series of RP-HPLC
purifications on preparative and semi-preparative and analytical
reverse phase C.sub.18 columns. A gradient of acetonitrile in 0.1%
trifluoroacetic acid was used to elute the peptide from the
columns. The major species was re-purified prior to further
characterization. Briefly, one gram of crude lyophilized venom from
Conus geographus was extracted and applied on a Sephadex G-25
column as previously described (Olivera et al., 1984). Three
successive fractions containing paralytic and sleeper activities
(Ve/Vo=1.37 to 1.41) were pooled, applied on a preparative reversed
phase Vydac C.sub.18 column and eluted with a gradient of
acetonitrile in 0.1% trifluroacetic acid (FIG. 1). The component
indicated by an arrow in FIG. 1 caused wobbling and death when
administered icv in mice. This was applied on a semipreparative
C.sub.18 column, eluted with 12-42% acetonitrile gradient in 0.1%
trifluroacetic acid. The component which made mice unresponsive
when administered icv, was further purified with an isocratic
elution at 20.4% acetonitrile in 0.1% trifluroacetic acid. A mouse
injected icv with an aliquot of the component had trouble righting
itself in 5 min and became very sluggish within 12 min. In
approximately 25-30 min, the mouse was stretched out and laid on
its stomach.
[0093] 3. Bioactivity. Typically, mice injected icv with the
partially purified native peptide initially had trouble righting
after 5 min, became sluggish after 12 min and then rested on their
stomachs after 30 min. These signs were used as an assay to
identify the biologically active peptide during purification.
[0094] 4. Enzyme hydrolysis. Approximately 180 pmol of the peptide
(6 .mu.L) was incubated with 7 mU .beta.-Galactosidase (bovine
testes) (2 .mu.L) in 50 of .mu.L 50 mM citrate/phosphate buffer (pH
4.5) for 53 hr at 32.degree. C. Approximately 60 pmol of the
peptide (2 .mu.L) was incubated with 2 mU O-glycosidase
(Diplococcus pneumoniae) (2 .mu.L) in 50 .mu.L of 20 mM cacodylic
acid (pH 6.0) for 19 hr at 32.degree. C.
[0095] 5. Chemical sequence and amino acid analysis. Automated
chemical sequence analysis was performed on a 477A Protein
Sequencer (Applied Biosystems, Foster City, Calif.). Amino acid
analysis was carried out using pre-column derivatization.
Approximately 500 pmol of the contulakin-G was sealed under vacuum
with concentrated HCl, hydrolyzed at 110.degree. C. for 24 hr,
lyophilized and then derivatized with o-phthalaldehyde. The
derivatized amino acids were then analyzed with RP-HPLC.
[0096] 6. Mass spectrometry. Matrix assisted laser desorption
(MALD) (Hillenkamp et al., 1993) mass spectra were measured using a
`Bruker REFLEX` (Bruker Daltonics, Billerica, Mass.) time-of-flight
(Cotter, 1989) mass spectrometer fitted with a gridless reflectron,
an N.sub.2 laser and a 100 MHz digitizer. An accelerating voltage
of +31 kV and a reflector voltage between 1.16 and 30 kV were
employed for the post source decay (Spengler et al., 1992)
measurements. The sample (in 0.1% aqueous trifluoroacetic acid) was
applied with .alpha.-cyano-4-hydroxycin- namic acid. Liquid
secondary ionization (LSI) (Barber et al., 1982) mass spectra were
measured using a Jeol HX110 (Jeol, Tokyo, Japan) double focusing
mass spectrometer operated at 10 kV accelerating voltage, 1000 or
3000 resolution. The sample (in 0.1% aqueous trifluoroacetic acid
and 25% acetonitrile) was mixed in a thioglycerol and
dithiothreitol matrix. Nano-electrospray (nano-ESI) mass spectra
were measured using an Esquire ion trap mass spectrometer (Bruker
Daltonics, Billerica, Mass.). The RP-HPLC purified sample,
collected in 0.1% aqueous trifluoro-acetic acid and acetonitrile
was diluted in methanol 1% acetic acid, transferred to a nanospray
capillary and analyzed. The mass accuracy was typically better than
1000 ppm for the time-of-flight instrument, 200 ppm for the ion
trap instrument and 20-100 ppm for the double focusing mass
spectrometer depending on the resolving power settings of the
magnetic sector instrument employed.
[0097] 7. Synthesis of contulakin-G. The solid-phase glycopeptide
synthesis was carried out manually using Fmoc chemistry, with
t-butyl ether side chain protection for tyrosine and serine,
N-t-Boc side chain protection for lysine, and t-butyl ester side
chain protection for glutamic acid (protected amino acids were
obtained from Bachem, Torrance, Calif.). Starting with a Wang
resin, the amino acids were coupled with
Bop/diisopropylethylamine/N-methylpyrrolidone/dichloromethane
(Stewart et al., 1984; LeNguyen et al., 1986) and the
N-deprotections were done with N-methylpyrrolidone/piperidine
(Stewart et al., 1984; LeNguyen et al., 1986). The Wang resin was
prepared at The Salk Institute with a substitution of 0.2 nmol/g.
After coupling of the first six amino acids, the resin was coupled
with peracetylated Fmoc-O.beta.-D-Galp-(1.fwdarw.3)-
-.alpha.-D-GalpNAc-(1.fwdarw.O)-threonine, synthesized as described
elsewhere (Luning et al., 1989), followed by single coupling of the
remaining nine amino acids in the sequence. Care was taken to
remove acetic acid and acetate impurities from the glycosylated
amino acids; this included chromatographic purification on silica
gel using dichloromethane-ethyl acetate 4:1 as eluant,
concentration and final lyophilization of the product from benzene.
Non-glycosylated peptide was similarly synthesized using
Fmoc-threonine (Bachem, Torrance, Calif.). The resin was subjected
to cleavage conditions (95% trifluoroacetic acid/5% anisole
(Stewart et al., 1984)), and in the case of the glycopeptide, the
resulting peracetylated glycopeptide was isolated with RP-HPLC, the
major component m/z 2322.3 (MALD analysis) corresponding to the
desired product (2322.0 Da). After lyophilization, the
peracetylated glycopeptide was treated with 20 .mu.L of sodium
methoxide (Sigma, St Louis, Mo.) (50 mM) in dry methanol for 1
minute (to remove O-acetyl groups on the sugar (Norberg et al.,
1994)) and lyophilized at -20.degree. C. The deacetylated sample
was loaded onto a Waters Prep LC/System 500A equipped with gradient
controller, Waters Model 450 Variable Wavelength Detector and
Waters 1000 PrepPack cartridge chamber column (65.5.times.320 mm)
packed with Vydac C.sub.18 15-20 .mu.m particles. Flow conditions:
wavelength 230 nm, AUFS 2.0, flow 100 L/mmin., gradient 20-60% B/60
min; (where the A buffer was 0.1% trifluoroacetic acid in water and
the B buffer was 0.1% trifluoroacetic acid in 60% aqueous
acetonitrile). The fractions (200 mL) were collected manually. The
major component, m/z 2069.9 (LSI analysis), corresponded to the
desired product (2069.98 Da). After preparative RP-HPLC
purification, sufficient purified contulakin-G was obtained for
analytical characterization and biological studies. A more
extensive characterization of the synthetic contulakin-G including
.sup.1H NMR data will be presented elsewhere.
[0098] 8. Co-elution. The native and synthetic contulakin-G were
analyzed separately and co-eluted with RP-HPLC, using a
2.1.times.150 mm Vydac C.sub.18 column and a 0.5%/min gradient from
0% B to 40% B (where the A buffer was 0.55% trifluoroacetic acid in
water and the B buffer was 0.05% trifluoroacetic acid in 90%
aqueous acetonitrile).
[0099] 9. Binding studies. The non-glycosylated
Thr.sub.10-contulakin-G and synthetic contulakin-G were assayed
with the human neurotensin type 1 receptor (hNTR1) using a Biomek
1000 robotic workstation for all pipetting steps in the radioligand
binding assays, as previously described (Cusack et al., 1993).
Competition binding assays with [.sup.3H] neurotensin.sub.1-13 (1
nM) and varying concentrations of unlabeled neurotensin.sub.1-13,
non-glycosylated Thr.sub.10-contulakin-G or synthetic contulakin-G
were carried out with membrane preparations from HEK-293 cell line.
Nonspecific binding was determined with 11 M unlabeled
neurotensin.sub.1-13 in assay tubes with a total volume of 1 mL.
Incubation was at 20.degree. C. for 30 min. The assay was routinely
terminated by addition of cold 0.9% NaCl (5.times.1.5 mL), followed
by rapid filtration through a GF/B filter strip that had been
pretreated with 0.2% polyethylenimine. Details of binding assays
have been described before (Cusack et al., 1991). The data were
analyzed using the LIGAND program (Munson et al., 1980).
[0100] The non-glycosylated Thr.sub.10-contulakin-G and synthetic
contulakin-G were separately assayed with the rat neurotensin type
1 and type 2 receptors (rNTR1 and rNTR2) and mouse neurotensin type
3 receptor (mNTR3). [.sup.125I-Tyr3] neurotensin.sub.1-13 was
prepared and purified as previously described (Saadoul et al.,
1984). Stable transfected CHO cells expressing either the rNTR1
(Tanaka et al., 1990) or the rNTR2 (cloned in the laboratory of J.
Mazella by screening a rat brain cDNA library (Stratagene)) were
grown in DMEM containing 10% fetal calf serum and 0.25 mg/mL G418
(Sigma, France). Cell membrane homogenates were prepared as
initially described (Chabry et al., 1994). Protein concentration
was determined by the Bio-Rad procedure with ovalbumin as the
standard.
[0101] 10. Binding experiments on cell membranes. Membranes (25
.mu.g for NTR2 and 10 .mu.g for NTR1) were incubated with 0.4 nM
[.sup.125I-Tyr.sup.3] neurotensin.sub.1-13 (2000 Ci/mmol) and
increasing concentrations of Neurotensin.sub.1-13, non-glycosylated
Thr.sub.10-contulakin-G or synthetic contulakin-G for 20 min at
25.degree. C. in 250 .mu.l of 50 mM Tris-HCl (pH 7.5) containing
0.1% bovine serum albumin and 0.8 mM 1-10-phenanthroline. Binding
experiments were terminated by the addition of 2 mL of ice-cold
buffer followed by filtration through cellulose acetate filters
(Sartorius) and washing twice. Radioactivity retained on filters
was counted with a y-counter.
[0102] 11. Binding experiments on solubilized extracts.
CHAPS-solubilized extracts (100 .mu.g) were incubated with 0.2 nM
[.sup.125I-Tyr.sup.3] neurotensin.sub.1-.sub.13 for 1 hr at
0.degree. C. in 250 .mu.L of Tris-glycerol buffer containing 0.1%
CHAPS. Bound ligand was separated from free ligand by filtration on
GF/B filters pretreated with 0.3% polyethylenimine. Filters were
rapidly washed twice with 3 mL of ice cold buffer and counted for
radioactivity.
[0103] For binding experiments on mNTR3, membrane homogenates from
mouse brain were re-suspended in 25 mM Tris-HCl buffer (pH 7.5)
containing 10% (w/v) glycerol, 0.1 mM phenylmethylsulfonyl
fluoride, 1 .mu.M pepstatin, 1 mM iodoacetamide, and 5 mM EDTA
(Tris-glycerol buffer). Solubilization was carried out by
incubating homogenates at a concentration of 10 mg/mL in the
Tris-glycerol buffer with 0.625% CHAPS containing 0.125% CHS
(Mazella et al., 1988). Solubilized extracts were recovered by
centrifugation at 100,000.times.g during 30 min at 4.degree. C. and
used either immediately or stored at -20.degree. C.
[0104] 12. Phosphoinositides determination. Cells expressing the
rNTR1 or NTR2 were grown in 12-well plates for 15-18 hr in the
presence of 1 .mu.Ci of myo-[.sup.3H]inositol (ICN) in a serum-free
HAM's-F-10 medium. Cells were washed with Earle buffer, pH 7.5, (25
mM Hepes, 25 mM Tris, 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl.sub.2, 0.8
mM MgCl.sub.2, 5 mM glucose) containing 0.1% bovine serum albumin,
and incubated for 15 min at 37.degree. C. in 900 .mu.l of 30 mM
LiCl in Earle buffer. Neurotensin.sub.1-13 was then added at the
indicated concentrations for 15 min. The reaction was stopped by
750 .mu.L of ice cold 10 mM HCOOH, pH 5.5. After 30 min at
4.degree. C., the supernatant was collected and neutralized by 2.5
mL of 5 mM NH.sub.4OH. Total [.sup.3H]phosphoinositide- s (PIs)
were separated from free [.sup.3H]inositol on Dowex AG-X8 (Bio-Rad)
(Van Renterghem et al., 1988) chromatography by eluting
successively with 5 mL of water and 4 mL of 40 mM and 1 M ammonium
formate, pH 5.5. The radioactivity contained in the 1 M fraction
was counted after addition of 5 mL of Ecolume (ICN).
[0105] 13. Identification of a cDNA clone encoding contulakin-G.
Contulakin-G encoding clones were selected from a size-fractionated
cDNA library constructed using mRNA obtained from a Conus
geographus venom duct as previously described (Colledge et al.,
1992). The library was screened using a specific probe
corresponding to amino acids #10-15 of the peptide (5'-ATR ATN GGY
TTY TTN GT-3'; SEQ ID NO:3). The oligonucleotide was end-labeled,
hybridized and a secondary screening by polymerase chain reaction
was performed on 10 clones that hybridized to this probe as
previously described (Jimenez et al., 1996). Clones identified in
the secondary screen were prepared for DNA sequencing as previously
described (Monje et al., 1993). The nucleic acid sequence was
determined according to the standard protocol for Sequenase version
2.0 DNA sequencing kit as previously described (Jimenez et al.,
1996).
Example 2
Purification of Contulakin-G
[0106] A fraction of Conus geographus venom was detected which made
mice exceedingly sluggish. Normally, when mice that are sitting
down are poked with a rod, they immediately get up and run a
considerable distance. Upon i.c.v. injection of the fraction from
Conus geographus indicated in FIG. 5, the mice had to be poked with
much more force before they got up at all, and after getting up,
they would walk one or two steps and immediately sit down again.
This "sluggish behavior" was followed through several steps of
purification, and the apparently homogeneous peptide was further
analyzed. This peptide was designated contulakin-G (the Filipino
woilakin means "has to be pushed or prodded," from the root word
tulak, to push). The "G" indicates that the peptide is from Conus
geographus.
Example 3
Biochemical Characterization of the Purified Contulakin-G
[0107] Attempted amino acid sequence analysis of the purified
peptide revealed that the peptide was blocked at the N-terminus.
Since most N-terminally blocked Conus peptides have a pyroglutamate
residue at position 1, the peptide was treated with pyroglutamate
aminopeptidase. This resulted in a shift in retention time
suggesting removal of a pyroglutamate residue. After enzyme
treatment, the sequence
Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Xaa-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ ID
NO:4) was obtained by standard Edman methods confirming removal of
the pyroglutamate residue, where Xaa indicates no amino acid was
assigned in the 9th cycle (at position 10) although a very low
signal for threonine was obtained. Amino acid analyses were
consistent with the presence of one threonine residue in the
peptide.
[0108] In order to confirm the nature of the amino acid residue in
position 10, a cDNA clone encoding the peptide was isolated. The
nucleotide sequence and presumed amino acid sequence revealed by
the clone are shown in Table 1 and in SEQ ID NO:5 and SEQ ID NO:6,
respectively. The amino acid sequence of contulakin-G obtained by
direct Edman sequencing is found encoded towards the C-terminal end
of the only significant open reading frame in the clone (at
residues 51-66); the predicted amino acid sequence reveals that
position 10 of the mature peptide (residue 60 of the precursor) is
encoded by a codon for threonine. Thus, the Edman sequencing,
together with cloning results, suggested that a modified threonine
residue was present in position 10.
1TABLE 1 DNA (SEQ ID NO:5) and Peptide (SEQ ID NO:6) Sequences of
Contulakin-G atg cag acg gcc tac tgg gtg atg gtg atg atg atg Met
Gln Thr Ala Tyr Trp Val Met Val Met Met Met gtg tgg att gca gcc cct
ctg tct gaa ggt ggt aaa Val Trp Ile Ala Ala Pro Leu Ser Glu Gly Gly
Lys ctg aac gat gta att cgg ggt ttg gtg cca gac gac Leu Asn Asp Val
Ile Arg Gly Leu Val Pro Asp Asp ata acc cca cag ctc atg ttg gga agt
ctg att tcc Ile Thr Pro Gln Leu Met Leu Gly Ser Leu Ile Ser cgt cgt
caa tcg gaa gag ggt ggt tca aat gca acc Arg Arg Gln Ser Glu Glu Gly
Gly Ser Asn Ala Thr aag aaa ccc tat att cta agg gcc agc gac cag gtt
Lys Lys Pro Tyr Ile Leu Arg Ala Ser Asp Gln Val gca tct ggg cca tag
Ala Ser Gly Pro
[0109] Mass spectrometric analyses (MALD, LSI and nano-ESI) of the
purified contulakin-G fraction revealed a variety of intact species
as summarized in Table 2. Some variation in the intensity of the
different species was observed with different ionization
techniques, which was ascribed to differences in the bias (Craig et
al., 1994) with each ionization technique. In the following
analysis, we have concentrated on the major glycoform with intact
mass M.sub.1=2069 observed with all of the ionization techniques
investigated. The difference between the observed mass (2069 Da)
and the mass calculated for the sequence assuming Thr at residue 10
(1703.83 Da) was 365 Da. Because one possible modification of
threonine is O-glycosylation, we proposed, based on this mass
difference, that the unidentified residue was
hexose-N-acetyl-hexosaminethreonine (Hex-HexNAc-Thr) which would
result in the addition of 365.13 Da. The observed masses (Table 2)
are consistent with the calculated monoisotopic mass of the
[M.sub.1+H].sup.+ or [M.sub.1+2H].sup.2+ of the proposed
disaccharide-linked peptide (2069.98 or 1035.5 Da respectively).
Intense fragment ions were observed in the nano-ESI MS/MS mass
spectrum of the doubly charged [M.sub.1+2H].sup.2+ intact molecule
ion of contulakin-G (FIG. 6) corresponding to the loss of the
complete Hex-HexNAc glycan (denoted p(.chi..sub.3).sub.10 (Craig et
al., 1993) or loss of the terminal hexose residue
(P(.chi..sub.8).sub.10).
2TABLE 2 Species Observed with MALD, LSI and nano-ESI Analysis of
Purified Contulakin-G Molecule M.sub.1 (Da) M.sub.2 (Da) M.sub.3
(Da) M.sub.4 (Da) Species Proposed HexHexNAc SO.sub.4HexHexNAc
Hex.sub.3 Hex.sub.2HexNAc.sub.2 glycan MALD/TOF 2069.sup.a --
2186.sup.b -- LSI/ 2068.7 2149.6 -- 2436.5 Magnetic nano-ESI/IT
2068.6.sup.c 2148.6.sup.c -- -- Mono.sup.d 2068.97 2148.92 2189.94
2434.10 [M + H].sup.+ Average.sup.e 2070.19 2150.25 2191.27 2435.53
[M + H].sup.+ .sup.am/z 2091 and 2107 corresponding with [M.sub.1 +
Na].sup.+ and [M.sub.1 + K].sup.+ were also observed. .sup.bm/z
2210 and 2225 corresponding with [M.sub.3 + Na].sup.+ and [M.sub.3
+ K].sup.+ were also observed. .sup.cm/z 1035.3 and 1075.3 doubly
charged ions were observed. .sup.dmonoisotopic [M + H].sup.+
calculated on based on proposed glycan and contulakin-G sequence.
.sup.eaverage [M + H].sup.+ masses were calculated based on
proposed glycan and contulakin-G sequence.
Example 4
[0110] Evidence that Thr-10 is O-glycosylated
[0111] Native contulakin-G was treated with .beta.-galactosidase
isolated from bovine testes. This enzyme preferentially hydrolyzes
terminal .beta.1->3 galactopyranosyl residues from the
non-reducing end of glycoconjugates. After .beta.-galactosidase
treatment of the native sample a new component was observed on
RP-HPLC. This component was collected and analyzed with MALD-MS in
which a species was observed at m/z 1907. The difference in mass
and the specificity of the enzyme are consistent with a terminal
galactose residue being released. Based on the .beta.-galactosidase
hydrolysis results we reasoned that the glycan moiety might be
susceptible to O-glycosidase treatment, which liberates the
disaccharide Gal (.beta.1->3) GalNAc (.alpha.1->) bound to
serine or threonine as a core unit of glycopeptides. O-glycosidase
treatment of the native contulakin-G did in fact result in a new
species after the enzyme hydrolysis mixture was analyzed on
RP-HPLC. The new component was collected and analyzed with MALD-MS
where an m/z 1704 species was observed consistent with loss of
Hex-HexNAc (i.e., the mass was consistent with that predicted for
the peptide with an unmodified threonine residue at position 10).
The enzyme hydrolysis results are consistent with the presence of a
Gal (.beta.1->3) GalNAc (.alpha.1->) glycan. Based on the
O-glycosidase and the P-galactosidase hydrolysis results, the
structure of the most abundant glycopeptide is:
3 Gal (.beta.1->3) GalNAc (.alpha.1->) (SEQ ID NO:1)
.vertline.
pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH
Example 5
Synthesis of the Non-Glycosylated and Glycosylated Contulakin-G
[0112] The 16-amino acid non-glycosylated peptide was chemically
synthesized. The synthetic material was found to have the same
retention time as the enzymatically des-glycosylated contulakin-G
on RP-HPLC. The 16-amino acid glycosylated contulakin-G containing
Gal(.beta.1->3) GalNAc (.alpha.1->) attached to Thr.sub.10
was also synthesized. This synthetic glycosylated contulakin-G
co-eluted with the native contulakin-G on RP-HPLC. The post source
decay fragmentation spectra observed for both native and synthetic
contulakin-G showed very similar fragmentation patterns.
Example 6
Biological Potency of Synthetic Glycosylated and Non-Glycosylated
Contulakin-G
[0113] The loss of motor control for which the native contulakin-G
was originally isolated, together with gut contraction, absence of
preening/grooming, and reduced sensitivity to tail depression were
signs observed when Neurotensin.sub.1-13, non-glycosylated
Thr.sub.10-contulakin-G or synthetic contulakin-G were administered
icv. In order to investigate these observations in more detail, a
dose response comparison was performed as detailed in Table 3.
While the non-glycosylated Thr.sub.10-contulakin-G analog was
active at doses of 1 nmol and higher, it was inactive at 300 pmol
doses. In contrast, contulakin-G was found to elicit loss of motor
control at doses of 30 pmol or approximately 5 pmol/g.
4TABLE 3 Effect of icv administration of Neurotensin.sub.1-13,
Thr.sub.10-Contulakin-G and Contulakin-G in 14-18 day old mice Av.
Time of symptom dose Number age weight A.sup.c B.sup.c R.sup.c
Compound (pmol) of mice.sup.a (days) (g).sup.b (min) (min) (min)
NSS 0 8 16 6.2 --.sup.d --.sup.d --.sup.d neuro- 1000 2 14 7.1 9 23
>120 tensin.sub.1-13 Thr.sub.10-contu- 1000 7 16.6 6.5 9 99 159
lakin-G Thr.sub.10-contu- 300 6 15.7 6.1 --.sup.d --.sup.d --.sup.d
lakin-G contulakin-G 1000 6 18 7.1 1.0 120 187 contulakin-G 300 8
15.5 6.6 2.9 42 151 contulakin-G 100 8 15.9 6.6 2.8 40 136
contulakin-G 30 7/9 15 6.3 5.3 23 114 .sup.anumber of mice
affected. .sup.baverage age (days) and weight (g). .sup.caverage
time after which specific behavior observed (observations were made
every 2 min for first 15 min and every 15 min thereafter). Symptoms
A and B were observed # when a mouse was placed onto a bench top
after lifting it by the tail for a second. Symptom A: The mouse
moved at most a few steps and rested with the hind legs spread out.
# Mouse remained stationary unless pushed or lifted. Symptom B: The
mouse remained sluggish but the position of the hind part of the
body when at rest resembled that of the NSS # controls. Symptom R:
Recovery, the mouse moved freely when released. .sup.dno symptom
observed.
[0114] The six C-terminal amino acids of contulakin-G show
significant similarity to the sequences of neurotensin.sub.1-13,
neuromedin, xenin and the C-terminus of xenopsin (see Table 4).
Because of the similar signs observed when either contulakin-G or
Neurotensin.sub.1-13 were administered icv and the significant
homology between contulakin-G and Neurotensin.sub.1-13, we tested
the affinity of contulakin-G for a number of the cloned neurotensin
receptors. As shown in Table 5, the non-glycosylated
Thr.sub.10-contulakin-G analog was found to bind the human
neurotensin type I receptor (hNTR1) with 10 fold lower affinity
than Neurotensin.sub.1-13, and even lower affinities for the other
NTR's. Contulakin-G exhibited significantly lower affinity than the
non-glycosylated Thr.sub.10-ocontulakin-G analog for all of the
NTR's tested.
[0115] Both contulakin-G and the non-glycosylated
Thr.sub.10-contulakin-G analog acted as agonists when tested on CHO
cells expressing the rNTR1. No response was observed with CHO cells
expressing the rNTR2. The non-glycosylated Thr.sub.10-contulakin-G
analog resulted in slightly lower potency (0.6 nM) but with similar
efficacy as compared with Neurotensin.sub.1-13. The synthetic
glycosylated contulakin-G potency was significantly lower (20-30
nM) and the agonistic efficacy was approximately half that observed
for Neurotensin.sub.1-13.
5TABLE 4 Sequence Comparison of Contulakin-G and Members of the
Neurotensin Family of Peptides Name Sequence (SEQ ID NO:) Id.sup.a
Si.sup.b Source Ref Contulakin- <ESEEGGSNAT*KKPYIL-OH -- -- C.
geographus G (7) neurotensin <ELYENKPRRPYIL-OH 66 33 bovine (1)
(8) hypothalamus neuromedin KIPYIL-OH 83 0 porcine spinal cord (2)
N (9) xenopsin QGKRPWIL-OH 66 16 Xenopus laevis (3) (10) xenin
MLTKFETKSARVKGLSFHPKRPWIL-OH 66 16 human gastric (4) (12) mucosa
*indicates an O-linked glycosylated threonine/serine residue.
.sup.apercentage identity of the 6 C-terminal amino acids compared
to contulakin-G.sub.11-16. .sup.bpercentage similarity of the 6
C-terminal amino acids compared to contulakin-G.sub.11-16.
[0116] References (1) Carraway et al., 1973; (2) Minamino et al.,
1984; (3) Araki et al., 1973; (4) Feurle et al., 1992.
6TABLE 5 Comparison of Binding Affinity of Neurotensin.sub.1--13,
Thr.sub.10-Contulakin-G and Contulakin-G for the Cloned Human and
Rat Neurotensin Type 1 Receptor (NTR1), the Rat Neurotensin Type 2
Receptor (rNTR2), and the Solubilized Mouse Neurotensin Type 3
Receptor (mNTR3) IC.sub.50 (nM) Receptor Compound hNTR1 rNTR1 rNTR2
mNTR3 neurotensin.sub.1--13 1.4 3.2 6.0 1.4 Thr.sub.10-contulakin-G
23 79 170 71 contulakin-G 960 524 730 250
Example 7
Biological Activity of Contulakin-G Analogs
[0117] The biological activity of several peptide analogs of
contulakin-G was tested in a similar manner as described above by
icv injection in mice. These peptides were synthesized as described
herein, and include the following analogs:
[0118] Ser.sub.10-contulakin-G containing the native glycosylation
on Ser.sub.10 (analog A); and
[0119] .DELTA.1-9-Ser.sub.10-contulakin-G containing the native
glycosylation on Ser.sub.10 (analog B)
[0120] It was found that analog A was slightly more active than the
native contulakin-G. It was also found that analog B had the same
activity, i.e., onset and recovery time than analog A when tested
in two week old mice at a dose of 100 pmole. In this test, the mice
were still not able to right themselves after 75 minutes. When
tested in three week old mice at doses of 1 nmole and 300 pmole,
the same activity was seen between the analogs and these mice were
drowsy for 100 minutes. These experiments demonstrate that
glycosylated contulakin-G analogs in which N-terminal amino acids
residues have been removed, retain activity. Similar results are
achieved for other analogs, such as
.DELTA.1-5-Ser.sub.6-contulakin-G containing the native
glycosylation on Ser.sub.6 with or without the native glycosylation
on Thr.sub.10. These results show that the placement of a
glycosylated serine residue proximal to the site of truncation
yields active analogs.
[0121] The Conus peptide characterized above, contulakin-G, has a
novel biochemical feature: a post-translationally O-glycosylated
threonine not previously found in Conus peptides. Using mass
spectrometry and specific enzymatic hydrolyses, it was found that
Thr.sub.10 was modified with the disaccharide Gal (.beta.1->3)
GalNAc (.alpha.1->). The corresponding glycosylated and
non-glycosylated forms of contulakin-G were synthesized which
confirmed the molecular structure of this major glycosylated form
of the native molecule based on RP-HPLC co-elution and MS
fragmentation criteria. The masses of the other more minor molecule
species observed with mass spectrometry are consistent with glycan
structural variations at peripheral sites on the characterized
oligosaccharide core unit (Baenziger, 1994).
[0122] An analysis of a cDNA clone encoding contulakin-G reveals
that the prepropeptide organization of the contulakin-G precursor
is similar to that of other Conus peptide precursors (Olivera et
al., 1997). A typical signal sequence is found, and immediately
N-terminal to the contulakin-G sequence are two basic amino acids
which presumably signal a proteolytic cleavage to generate the
N-terminus of the mature peptide (the glutamine residue would
cyclize to pyroglutamate either spontaneously or due to the action
of glutaminyl cyclase (Fischer et al., 1987)). Although in most
respects the contulakin-G precursor has the same organization as
all other Conus venom peptide precursors and would be predicted to
be processed in the same way, the ten C-terminal amino acids
predicted by the clone are not present in contulakin-G purified
from venom. One possibility is that the clone represents a
different variant, for example one which was alternatively spliced.
Alternatively, furtherproteolytic processing at the C-terminus
maybe required to generate mature contulakin-G.
[0123] Over the last 20 years an increasing number of biologically
important glycopeptides and glycoproteins have been identified.
Vespulakinin 1, first identified by Pisano et al. (Yoshida et al.,
1976), is, to our knowledge, the only other O-glycosylated peptide
toxin which has been isolated from venom other than Conus.
Vespulakinin 1 was extracted from the venom sacs of the yellow
jacket wasp, Vespula maculifrons. The peptide
(TAT*T*RRRGRPPGFSPFR-OH (SEQ ID NO: 12) where the asterisk
indicates an O-linked glycosylated threonine residue) contains two
sequential sites of O-linked glycosylation. The C-terminus of
Vespulakinin is identical to the sequence of Bradykinin
(RPPGFSPFR-OH (SEQ ID NO: 13)) and the peptide was found to elicit
a number of signs also elicited by Bradykinin. Vespulakinin is
therefore another example of an O-linked glycosylated peptide toxin
in which the C-terminus appears to target a mammalian
neurotransmitter receptor. Thus, both contulakin-G and Vespulakinin
I contain glycosylated N-terminal extensions to sequences with very
high homology to mammalian neuropeptides. .kappa.A-conotoxin SIVA,
a K.sup.+ channel inhibitor is unusual among disulfide-rich Conus
peptides in having a long N-terminal tail, which has an
O-glycosylated residue (Craig et al., 1998).
[0124] For most Conus peptides, a specific conformation appears to
be stabilized either by multiple disulfide linkages or by the
appropriate spacing of .gamma.-carboxyglutamateresidues to promote
formation of .alpha.-helices (Olivera et al., 1990). Conus peptides
without multiple disulfides comprise a most eclectic set of
families, including the conopressins, conantokins, contryphans and
now contulakin-G. The conopressins are probably endogenous
molluscan peptides, clearly homologous to the vassopressin/oxytocin
family of peptides; these are more widely distributed in molluscan
tissues than in Conus venom ducts. However, the other
non-disulfide-rich peptides (conantokins, contryphans and
contulakin-G) may be specialized venom peptides exhibiting unusual
post-translational modifications. In addition to the O-glycosylated
threonine moiety of contulakin-G described here,
.gamma.-carboxylation of glutamate residues and the
post-translational epimerization and bromination of tryptophan
residues were discovered in conantokins and contryphans.
[0125] Several lines of evidence are consistent with contulakin-G
being the first member of the neurotensin family of peptides to be
isolated from an invertebrate source. First, the C-terminal region
of contulakin-G exhibits a striking degree of similarity to other
members of the neurotensin family (all from vertebrates), as shown
in Table 4. Furthermore, it was shown above that contulakin-G
competes for binding to three known neurotensin receptor subtypes;
evidence that contulakin-G acts as an agonist on a cloned
neurotensin receptor is also presented above. Most convincingly
however, when contulakin-G is injected into mice, the same
behavioral signs are elicited with administration of neurotensin.
Thus, structural data, binding data and in vivo behavioral
symptomatology are all consistent with the assignment of
contulakin-G to the neurotensin family of peptides.
[0126] Clearly, both contulakin-G and the non-glycosylated
Thr.sub.10-contulakin-G are rNTR1 agonists at physiologically
relevant concentrations (20-30 and 0.6 nM, respectively). The
observed agonistic effects of both contulakin-G and the
non-glycosylated analog, as well as the absence of any agonistic
effect of these ligands on CHO cells expressing rNTR2 using the IP
accumulation assay does not correlate with the in vitro binding
data; both peptides are agonists at concentrations significantly
below their IC.sub.50 binding affinity (524 and 79 nM,
respectively). Most unexpected therefore, given its apparently
lower binding affinity, is the increased potency of glycosylated
contulakin-G compared with the non-glycosylated analog after icv
administration.
[0127] Thus, the role of the glycan is somewhat paradoxical. In
vitro, the glycan neither increases the binding affinity, the
agonistic potency nor agonistic efficacy. In contrast, in vivo, the
glycan significantly increases the potency of the peptide. One
simple explanation is that the increased potency of contulakin-G
compared with the non-glycosylated analog is due to increased
stability. An alternative mechanism for the increased potency is
transport to the site of action facilitated by the glycan.
Additionally, the glycosylated peptide may act with high affinity
on an as-yet-undefined neurotensin receptor subtype (Tyler et al.,
1998), or may be a selective high affinity ligand for a particular
state of a neurotensin receptor subtype. Yet another possibility is
that the relevant targeted neurotensin receptors may be closely
co-localized with carbohydrate binding sites, and that the glycan
may serve as an "address label", a mechanism postulated for certain
opiate peptides. Preliminary data supporting the increased
stability hypothesis has been obtained--proteolytic degradation of
contulakin-G is inhibited by the presence of the glycan moiety. The
increased stability may well result in an enhanced supply of the
glycopeptide at the receptor. However, the increased in vivo
potency of contulakin-G conferred by O-glycosylation clearly
requires a more balanced evaluation of the possibilities outlined
above.
Example 8
Materials and Methods for Assessing
[0128] 1. Analgesic Activity of Thr.sub.10-Contulakin-G
[0129] 1. Acute pain (hotplate). Thr.sub.10-contulakin-G (CGX-1063)
or vehicle was administered via intracerebroventricular (icv) in a
volume of 5 .mu.l. Fifteen minutes after injection, animals were
placed on a 55.degree. C. hotplate. The latency to the first
response (flinch), a spinally mediated behavioral response, and the
first hindlimb lick, a centrally organized motor response to acute
pain, were recorded. Mice were removed from the hotplate after 60
seconds if no response was observed. Immediately prior to being
placed on the hotplate, motor function was tested by determining
the latency to first fall from an accelerating rotarod.
[0130] 2. Persistentpain (formalin test). Intrathecal (it) drug
injections were performed as described by Hyldon and Wilcox (1980).
CGX-1063 (10 or 100 pmol) or vehicle was administered in a volume
of 5 .mu.l. Fifteen minutes after the it injection, the right
hindpaw was injected with 20 .mu.l of 5% formalin. Animals were
placed in clear plexiglass cylinders backed by mirrors to
facilitate observation. Animals were closely observed for 2 minutes
per 5 minute period, and the amount of time the animal spent
licking the injected paw was recorded in this manner for a total of
45-50 minutes. Results are expressed as licking time in seconds per
five minutes. At the end of the experiment, all animals were placed
on an accelerating rotorod and the latency to first fall was
recorded.
[0131] 2. Neuropathic pain. The partial sciatic nerve ligation
model was used to assess the efficacy of CGX-1063 in neuropathic
pain. Nerve injury was produced according to the methods of
Malmberg and Basbaum (1998). Animals were anesthetized with a
ketamine/xylazine solution, the sciatic nerve was exposed and
tightly ligated with 8-0 silk suture around 1/3 to 1/2 of the
nerve. In sham-operated mice the nerve was exposed, but not
ligated. Animals were allowed to recover for at least 1 week before
testing was performed. On the testing day, mice were placed in
plexiglass cylinders on a wire mesh frame and allowed to habituate
for at least 60 minutes. Mechanical allodynia was assessed with
calibrated von Frey filaments using the up-down method as described
by Chaplan et al. (1994), and the 50% withdrawal threshold was
calculated. Animals that did not respond to any of the filaments in
the series were assigned a maximal value of 3.6 grams, which is the
filament that typically lifted the hindlimb without bending, and
corresponds to approximately {fraction (1/10)} the animal's body
weight.
Example 9
Analgesic Activity of Thr.sub.10-Contulakin-G
[0132] CGX-1063(10 fmol-10 nmol, icv) dose-dependently increased
the latency to the first hindpaw lick and first response elicited
by the hotplate (FIGS. 7A-7B). Of interest is the difference in
potency of CGX-1063 in increasing the latency to the first hindpaw
lick compared to the latency to first response. CGX-1063 also
dose-dependently decreased the latency to first fall on the rotarod
(FIG. 7C). However, this apparent motor impairment did not appear
to be the result of the loss of motor function, since animals were
capable of normal locomotor activity when stimulated. Thus, the
effect of CGX-1063 on the hotplate unequivocally was an analgesic
effect.
[0133] CGX-1063(10 or 100 pmol, it) dose-dependently and
significantly decreased the second phase of the formalin test (FIG.
8A). Interestingly, the lower dose (10 pmol) was more effective in
decreasing the first phase response time than was the higher dose.
This will be examined in more detail in future experiments. After
it administration, CGX-1063 treated animals showed no motor
impairment compared to vehicle treated animals (FIG. 8B),
indicating that the effect of icv CGX-1063 (observed in the
hotplate test above) on motor impairment is mediated at higher
brain regions, not spinally, and that the analgesic effects of
CGX-1063 can be separated from the motor toxicity by using this
route (it) of administration. The downward shift in the rotorod
scores compared to those from animals used in the hotplate test
reflects an overall impairment in these animals due to
formalin-induced allodynia and inflammation of the hindpaw.
[0134] One week after partial sciatic nerve ligation, animals
showed a marked decrease in the paw withdrawal threshold on the
operated side (ipsilateral) relative to the unoperated side
(contralateral), indicating an increase in sensitivity to
mechanical stimuli (FIG. 9). Intrathecal administration of CGX-1063
(100 pmol) dramatically increased the withdrawal threshold on the
ligated side (an approximate six fold increase). Interestingly, the
mechanical threshold on the contralateral side was not
significantly altered. In sham-operated animals, there was no
difference in withdrawal threshold between operated and un-operated
sides. After intrathecal CGX-1063, the withdrawal threshold was
uniformly increased in both hindpaws of these animals.
[0135] The present data demonstrate that CGX-1063 has potent
analgesic properties in three commonly used models of pain: acute,
persistent/inflammatory and neuropathic pain models. CGX-1063
administered centrally (icv) dose-dependently reduced the response
latency in the hot plate model of acute pain, and was effective in
the low picomole to high femtomole range. Preliminary data indicate
that the analgesic effect of CGX-1063 in this model is not mediated
through an opioid mechanism. CGX-1063 was also effective in
reducing nociceptive activity in the formalin model of
persistent/inflammatory pain. CGX-1063 dose-dependently reduced the
second (inflammatory) phase of the formalin test, while at the
lower dose, reduced phase one activity. Finally, CGX-1063 showed
profound analgesic activity in a model of neuropathic pain.
Mechanical withdrawal thresholds in this model were increased
nearly six fold compared to pre-treatment values, while not
altering sensitivity in the non-injured paw, possibly indicating
that CGX-1063 reduces neuropathic allodynia while not affecting
normal sensory transmission.
Example 10
P Materials and Methods for Assessing Analgesic Activity of
Contulakin-G
[0136] 1. Acute pain (tail-flick). Drug (contulakin-G (CGX-1160) or
Thr.sub.10-contulakin-G (CGX-1063)) or saline was administered
intrathecally (i.t.) according to the method of Hylden and Wilcox
(Hylden and Wilcox, 1980) in a constant volume of 5 .mu.l. Mice
were gently wrapped in a towel with the tail exposed. At various
time-points following the i.t. injection, the tail was dipped in a
water bath maintained at 54.degree. C. and the time to a vigorous
tail withdrawal was recorded. If there was no withdrawal by 8
seconds, the tail was removed to avoid tissue damage.
[0137] 2. Persistent pain formalin test). CGX-1160, CGX-1063 (1, 10
or 100 pmol), neurotensin (NT) (1, 10, 100 or 10000 pmol), or
vehicle was administered i.t. in a volume of 5 .mu.l. Fifteen
minutes after the i.t. injection, the right hindpaw was injected
with 20 .mu.l of 5% formalin. Animals were placed in clear
plexiglass cylinders backed by mirrors to facilitate observation.
Animals were closely observed for two minutes per five minute
period, and the amount of time the animal spent licking the
injected paw was recorded in this manner for a total of 45-50
minutes. Results are expressed as licking time in seconds per five
minutes. At the end of the experiment, all animals were placed on
an accelerating rotorod and the latency to first fall was
recorded.
[0138] 3. Chronic inflammatory allodynia (CFA model). Mice were
given intraplantar (i.pl.) injections of 20 .mu.l of CFA into the
right hindpaw and returned to their home cage. Three days later
mice were placed in plexiglass cylinders on a wire mesh frame and
allowed to habituate for at least 60 minutes. Mechanical allodynia
was assessed with calibrated von Frey filaments using the up-down
method as described (Chaplan et al., 1994), and the 50% withdrawal
threshold was calculated. Animals that did not respond to any of
the filaments in the series were assigned a maximal value of 3.6
grams, which is the filament that typically lifted the hindlimb
without bending, and corresponds to approximately {fraction (1/10)}
of the body weight.
[0139] 4. Toxicity testing. To accurately assess the motor
impairing effects of CGX-1160, CGX-1063, and NT, 50 mice were
divided into groups receiving i.t. CGX-1160 or CGX-1063 (1, 10,
100, 500 and 1000 pmol), NT (0.1, 1, 10, and 100 nmol), or saline
(n=5 per group except for the highest dose of each compound where
n=3). Starting at 15 minutes post injection animals were place on
an accelerating rotorod and the latency to first fall was recorded.
Animals were retested at 30, 60, 120, 240 and 300 minutes (or until
the latency to fall had returned to control values). Rectal
temperature was also recorded in these animals at the same time
points.
Example 11
Analgesic Activity of Contulakin-G
[0140] CGX-1160 dose-dependently increased the tail-flick latency
(FIG. 10A) with a time to peak effect of .ltoreq.30 minutes (the
earliest time tested, FIG. 10B). Furthermore, the increase in
latency was long-lasting with elevated withdrawal times at 5 hour
post injection that returned to baseline at 24 hours post injection
(FIG. 10B). CGX-1063 also showed a dose-dependent, though more
variable increase in withdrawal latency, and showed only modest
antinociceptive efficacy in this model relative to CGX-1160 (FIGS.
10A-10B). In comparison, NT did not significantly elevate
withdrawal latency in the tail-flick assay (FIGS. 10A-10B).
[0141] All of the compounds tested dose-dependently showed
antinociceptive properties in both phases of the formalin test, but
with different potencies. CGX-1160 was the most potent of the three
compounds. In phase 1 of the formalin test (FIG. 1A), CGX-1160 had
an ED.sub.50 of approximately 30-40 pmol while NT had an ED.sub.50
of .apprxeq.1 nmol. CGX-1063 did not reach the 50% antinociception
threshold in phase 1, however, the irregular dose-response in this
test warrants repeating the 100 pmol dose in this assay. In phase 2
of the formalin test, all three compounds dose-dependently reduced
the paw licking time (indicated in the figures as an increase in
the percent antinociception; FIG. 1B). Again, CGX-1160 was more
potent than the other compounds with an estimated ED.sub.50 of 1
pmol. Lower doses of this compound will be assessed in the future
to complete the dose response curve necessary to calculate a more
precise ED.sub.50. CGX-1063 was also effective in reducing
nociceptive behavior in phase 2, with an estimated ED.sub.50 of
10-20 pmol. NT was dramatically less potent than either of the
contulakins with an estimated ED.sub.50 of 600-700 pmol (FIG.
11B).
[0142] CGX-1160 showed extremely potent and dose-dependent reversal
of CFA-induced mechanical allodynia (FIG. 12A). One-hundred (100)
fmol of CGX-1160 given i.t. completely reversed the CFA-induced
mechanical allodynia. Interestingly, at this dose, the
contralateral sensitivity to mechanical pressure was unaltered
indicating a potential unilateral alteration in NT receptors in
chronic inflammation. At higher doses of CGX-1160, the mechanical
withdrawal threshold in both the CFA-injected paw and the
contralateral uninjected paw was dramatically elevated. In FIGS.
12A and 12B, the numbers over the bars indicate the percent
increase in mechanical threshold relative to the pre-drug level. As
indicated, at all doses tested, CGX-1160 had a much greater
antiallodynic effect on the CFA injected side relative to the
uninjected side. CGX-1063 was less potent than CGX-1160, but also
completely reversed the CFA-induced allodynia (FIG. 12B). The
minimally effective dose was 10 pmol, however, at this dose, unlike
CGX-1160, the contralateral side was also elevated relative to
pre-drug baseline measurements. Consistent with the other models
examined in this study, NT showed efficacy in the CFA model at 1
mol, but not at 100 pmol (FIG. 12C). Other doses of CGX-1160 and NT
will be examined in the future to determine accurate ED.sub.50s for
these compounds.
[0143] CGX-1160, -1063, and NT all showed dose-dependent effects on
locomotor impairment and body temperature. For all three compounds,
maximal impairment was at 15 minutes post i.t. injection (locomotor
impairment, FIG. 13A) or 30 minutes (hypothermic effects, FIG.
14A). CGX-1063 had no motor toxicity at the lowest dose tested (1
pmol, FIG. 13B), but at higher doses animals showed significant
motor toxicity (estimated TD.sub.50 of 10 pmol, FIGS. 13B and 15A).
At 10 pmol this toxicity lasted for 30 minutes, but resolved by 60
minutes. When 100 pmol or 1 nmol was administered, animals were
motor impaired for 2-3 hours (FIG. 14A). CGX-1160 was equipotent to
CGX-1063 in causing motor impairment (estimated TD.sub.50 of 10-20
pmol, FIG. 13B). Similar to CGX-1063, at higher doses
(100-500.times. its ED.sub.50) CGX-1160 showed motor impairment
that resolved after 5 hours (FIGS. 13A and 14B). The estimated
TD.sub.50 for NT-induced motor impairment was 3 nmol (FIG. 13B).
Similar to the contulakins, at high doses, NT-induced motor
impairment that lasted 2-4 hours (FIGS. 13A and 14C).
[0144] The hypothermic effects of these compounds were similar to
motor toxicity. All three caused a dose-dependent decrease in body
temperature. CGX-1160 and -1063 were equipotent with an estimated
TD.sub.50 of 100 pmol (FIG. 15B). However, at this dose CGX-1063
induced a drop in body temperature lasting 2-3 hours (FIGS. 15A and
16A), while the hypothermic effect caused by CGX-1160 resolved by
60 minutes (FIGS. 15A and 16B). At the highest dose of CGX-1160
(500 pmol, 500.times. the ED.sub.50), the hypothermic effect had
not resolved by six hours post-injection (FIG. 16B). NT showed a
very similar dose-response and time course to the contulakins. At
the lower doses, NT had no effect or showed a short lasting
hypothermic effect (FIG. 16C). At the highest dose, however (100
nmol), NT caused a dramatic and long-lasting hypothermia that had
not resolved by three hours (FIGS. 15A and 16C).
[0145] The present data show that CGX-1160 and CGX-1063 are potent,
broad-spectrum analgesic agents effective in several animal models
of acute and chronic pain. CGX-1160 is typically 10 fold more
potent than CGX-1063, and 1000 times more potent than NT (Table 6).
CGX-1160 is particularly potent in the model of chronic
inflammatory pain where CGX-1160 selectively increases the
mechanical withdrawal threshold only in the paw receiving the CFA
injection, while not altering the threshold of the uninjected paw.
This finding indicates that chronic inflammation may lead to a
reorganization of NT receptors in nociceptive pathways
corresponding to the inflamed paw. Since CGX-1160 was the only
compound in these experiments to show an increased potency, this
may indicate an upregulation of a receptor subtype for which
CGX-1160 may have particular selectivity and specificity. In
support of this hypothesis of CGX-1160 subtype selectivity are the
findings that this compound shows antinociception at doses 10-100
fold less than for either locomotor impairment or hypothermia,
whereas CGX-1063 and NT cause antinociception, locomotor
impairment, and hypothermia at approximately equal doses when
administered i.t. Particularly interesting is the long-lasting
hypothermic effect of CGX-1063. When given i.t. at 100 pmol
(approximately 10 times its ED.sub.50 in phase 2 of the formalin
test, see FIG. 16A), CGX-1063 caused long-lasting hypothermia
relative to comparable antinociceptive doses of CGX-1160 (compare
the 10 pmol dose in FIG. 16B) and NT (compare the 10 nmol dose in
FIG. 16C). This potentially indicates that CGX-1063 is selective
for the NT receptor subtype involved in the hypothermic effect of
NT analogs. Thus the O-glycosylation of Thr.sub.10 in CGX-1160 may
impart selectivity for the antinociceptive NTR subtype, currently
thought to be NTR2, as well as metabolic resistance to
peptidases.
7TABLE 6 Comparison of the Antinociceptive Effects, Motor
Impairment Effects, and Protective Index of CGX-1160, CGX-1063, and
NT in the Formalin Test (phase 2) and CFA-Induced Allodynia Test
Compound ED.sub.50, pmol TD.sub.50, pmol PI Formalin Test (phase 2)
CGX-1160 1 10-20 10-20 CGX-1063 10-20 10-20 1-2 NT 600-700 3000
5-4.3 CFA-Induced Allodynia Test CGX-1160 <0.1 10-20 >100
CGX-1063 <10 10-20 1-5 NT .apprxeq.500-600 3000 5-6 ED.sub.50,
pmol, estimated from antinociceptive tests TD.sub.50, pmol,
estimated from rotorod test of minimal motor impairment Protective
Index, PI = (TD.sub.50/ED.sub.50)
Example 12
Materials and Methods for Assessing Antipsychotic Activity of
Contulakin-G
[0146] 1. Materials. D-amphetamine was obtained from Sigma (St.
Louis, Mo.). Contulakin-G (CGX-1160; a synthetic 16 amino acid
O-linked glycopeptide) was synthesized as described above.
[0147] 2. Animals. Male CF-1 mice (30-35 g; Charles River
Laboratories) were used. All animals were housed in a temperature
controlled (23.degree..+-.3.degree. C.) room with a 12 hour
light-dark cycle with free access to food and water. All animals
were euthanized in accordance with Public Health Service policies
on the humane care of laboratory animals.
[0148] 3. Locomotor Activity. Animals were placed in clear plastic
cages (40 cm.times.22 cm, 20 cm deep) and allowed to acclimate for
30 minutes. Animals then received either contulakin-G (100 pmol) or
saline (vehicle) by freehand intracerebroventricular (i.c.v.)
injection (5 .mu.l volume) through a 10 .mu.l Hamilton syringe.
After 5 minutes, animals received saline or D-amphetamine sulphate
(3 mg/kg) via intraperitoneal (i.p.) administration. Distance
traveled (cm) and time spent ambulatory (s) were monitored for 30
minutes using a Videomex-V tracking system (Columbus Instruments,
Columbus, Ohio). All testing was done in an isolated, dimly lit
behavioral room.
[0149] 4. Statistics. Data were analyzed using one-way analysis of
variance (ANOVA) with drug treatment as the only factor, followed
by a Newman-Keuls multiple comparison test for comparison of
individual groups, with P<0.05 accepted as statistically
significant. Statistical analyses were performed with GraphPad
PRISM software (Version 2.01, GraphPad, San Diego, Calif.).
Example 13
Antipsychotic Activity of Contulakin-G
[0150] A significant effect of drug treatment on locomotor activity
as measured by both distance traveled [F(4,21)=7.87, P<0.05] and
time spent ambulating [F(4,21)=6.17, P<0.05] was found in the
present study. Administration of D-amphetamine resulted in a dose
dependent increase in both distance traveled and time spent
ambulating (FIGS. 17-18). Pretreatment of mice with contulakin-G
(100 pmol i.c.v.) significantly reduced amphetamine-stimulated (3
mg/kg i.p.) increases in distance traveled and time spent
ambulating. A reduction in basal locomotor activity (both distance
traveled and time spent ambulating) was seen after pretreatment
with contulakin-G (100 pmol i.c.v.), however, this reduction did
not reach statistical significance.
[0151] Converging lines of evidence imply that neurotensin may have
antipsychotic properties without the associated adverse side effect
profiles of standard neuroleptic drugs (reviewed in (Nemeroff et
al., 1992)). Subsequently, many groups have focused on neurotensin
analogs as novel antipsychotic drugs. Since contulakin-G shares
C-terminal homology with neurotensin, and resembles neurotensin in
both in vivo and in vitro assays, the ability of contulakin-G to
inhibit D-amphetamine-stimulated locomotor activity, a preclinical
screen predictive of antipsychotic efficacy, was assessed. This
example demonstrates that pretreatment of mice with contulakin-G
significantly reduced amphetamine-stimulated increases in locomotor
activity. These data indicate that contulakin-G has similar
antipsychotic activity as neurotensin. However as shown above,
while neurotensin was far more potent than contulakin-G at the rat
neurotensin receptors rNTR1 (IC.sub.50: 3.2 nM for neurotensin; 524
nM for contulakin-G) and rNTR2 (IC.sub.50: 6.0 nM for neurotensin;
730 nM for contulakin-G), and the mouse neurotensin receptor mNTR3
(C.sub.50: 1.4 nM for neurotensin; 250 nM for contulakin-G),
contulakin-G was 1 to 2 orders of magnitude more potent in an in
vivo assay (a visually rated assessment of locomotor activity)
following i.c.v. administration. These results indicate that
contulakin-G and neurotensin may interact with overlapping but
distinct populations of neurotensin receptor subtypes or activation
states. Thus, contulakin-G would not share the limiting side
effects of neurotensin.
Example 14
Materials and Methods for Assessing Anticonvulsant Activity of
Contulakin-G
[0152] 1. Animals. Male Frings (20-25 g) were housed in a
temperature controlled (23.degree..+-.1.degree. C.) room with a 12
hour light-dark cycle with free access to food and water. Mice were
housed, fed, and handled in a manner consistent with the
recommendations in HEW publication (NIH) No. 8623, "Guide for the
Care and Use of Laboratory Animals." All mice were euthanized in
accordance with Public Health Service policies on the humane care
of laboratory animals.
[0153] 2. Anticonvulsant Assessment. Frings mice were placed in a
round, plexiglass jar (diameter 15 cm, height 18 cm) and exposed to
a sound stimulus of 110 decibels (11 KHz). Mice were then observed
for 25 sec for the presence or absence of hindlimb tonic extension.
Animals not displaying hindlimb tonic extension were considered
protected.
[0154] 3. Rotorod Test. Motor impairment was assessed at time of
peak effect by placing mice on a rotorod turning at 6 rpm. Animals
falling three times in one minute were considered impaired.
Example 15
Anticonvulsant Activity of Contulakin-G
[0155] Contulakin-G (CGX-1160) and Thr.sub.10-contulakin-G
(CGX-1063) potently and dose-dependently blocked audiogenic
seizures in Frings mice following i.c.v. administration (FIG. 19).
Similar to the efficacy in pain models, CGX-1160 was more potent
than CGX-1063 with ED.sub.50s of 7.1 pmol and 27.0 pmol,
respectively (Table 7). Also consistent with previous studies, NT
was dramatically less potent than CGX-1160 or -1063. Although a
dose-response curve for NT has not yet been completed, NT showed
50% protection following 1 nmol administered i.c.v. When tested for
motor toxicity, CGX-1160 did not reach the 50% toxic level at doses
up to 200 pmol (FIG. 19), whereas the TD.sub.50 for CGX-1063 is
estimated to be approximately 375 pmol resulting in an estimated PI
of 14 for the doses tested.
8TABLE 7 Anticonvulsant Profile of CGX-1160 and CGX-1063 in Frings
AGS Mice Following i.c.v. Administration Time of test Compound
(min.).sup.a TD.sub.50(pmol) ED.sub.50(pmol) P.I..sup.b X more
potent than NT CGX-1160 15, 60 >200 7.1 >28 .apprxeq.140
(4.9-8.5) CGX-1063 15, 60 .apprxeq.375 27.0 .apprxeq.14 .apprxeq.37
(18.6-34.9) Neurotensin 15, 60 not yet .apprxeq.1000 N.D.
determined .sup.aFirst time, TD.sub.50; second time, ED.sub.50
.sup.bProtective index = TD.sub.50/ED.sub.50 ( )95% confidence
interval
[0156] In a separate experiment, the time to peak effect and
duration of action of CGX-1160 was examined. I.c.v. administration
of 100 pmol (approximately 14.times.ED.sub.50) of CGX-1160 showed
no activity at 30 minutes, but was 100% protective at 60 minutes,
and still showed 50% protection in animals tested 4 hours following
i.c.v. injection (FIG. 20).
[0157] It will be appreciated that the methods and compositions of
the instant invention can be incorporated in the form of a variety
of embodiments, only a few of which are disclosed herein. It will
be apparent to the artisan that other embodiments exist and do not
depart from the spirit of the invention. Thus, embodiments
described are illustrative and should not be construed as
restrictive.
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[0253]
Sequence CWU 1
1
13 1 16 PRT Conus geographus PEPTIDE (1)..(13) Xaa at residue 1 is
pyro-Glu; Xaa at residue 13 is Pro or hydroxy-Pro; Thr at residue
10 is modified to contain an O-glycan. 1 Xaa Ser Glu Glu Gly Gly
Ser Asn Ala Thr Lys Lys Xaa Tyr Ile Leu 1 5 10 15 2 16 PRT
Artificial Sequence Description of Artificial SequenceGeneric
Contulakin-G formula 2 Xaa Xaa Xaa Xaa Gly Gly Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Ile Leu 1 5 10 15 3 17 DNA Conus geographus
misc_feature (1)..(17) n is any nucleotide 3 atratnggyt tyttngt 17
4 15 PRT Conus geographus PEPTIDE (9) Xaa at residue 9 is unknown 4
Ser Glu Glu Gly Gly Ser Asn Ala Xaa Lys Lys Pro Tyr Ile Leu 1 5 10
15 5 231 DNA Conus geographus CDS (1)..(228) 5 atg cag acg gcc tac
tgg gtg atg gtg atg atg atg gtg tgg att gca 48 Met Gln Thr Ala Tyr
Trp Val Met Val Met Met Met Val Trp Ile Ala 1 5 10 15 gcc cct ctg
tct gaa ggt ggt aaa ctg aac gat gta att cgg ggt ttg 96 Ala Pro Leu
Ser Glu Gly Gly Lys Leu Asn Asp Val Ile Arg Gly Leu 20 25 30 gtg
cca gac gac ata acc cca cag ctc atg ttg gga agt ctg att tcc 144 Val
Pro Asp Asp Ile Thr Pro Gln Leu Met Leu Gly Ser Leu Ile Ser 35 40
45 cgt cgt caa tcg gaa gag ggt ggt tca aat gca acc aag aaa ccc tat
192 Arg Arg Gln Ser Glu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr
50 55 60 att cta agg gcc agc gac cag gtt gca tct ggg cca tag 231
Ile Leu Arg Ala Ser Asp Gln Val Ala Ser Gly Pro 65 70 75 6 76 PRT
Conus geographus 6 Met Gln Thr Ala Tyr Trp Val Met Val Met Met Met
Val Trp Ile Ala 1 5 10 15 Ala Pro Leu Ser Glu Gly Gly Lys Leu Asn
Asp Val Ile Arg Gly Leu 20 25 30 Val Pro Asp Asp Ile Thr Pro Gln
Leu Met Leu Gly Ser Leu Ile Ser 35 40 45 Arg Arg Gln Ser Glu Glu
Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr 50 55 60 Ile Leu Arg Ala
Ser Asp Gln Val Ala Ser Gly Pro 65 70 75 7 16 PRT Conus geographus
PEPTIDE (1)..(10) Xaa at residue 1 is pyro-Glu; Thr at residue 10
contains an O-glycan. 7 Xaa Ser Glu Glu Gly Gly Glu Asn Ala Thr Lys
Lys Pro Tyr Ile Leu 1 5 10 15 8 13 PRT Bos sp. PEPTIDE (1) Xaa at
residue 1 is pyro-Glu. 8 Xaa Leu Tyr Glu Asn Lys Pro Arg Arg Pro
Tyr Ile Leu 1 5 10 9 6 PRT porcine 9 Lys Ile Pro Tyr Ile Leu 1 5 10
8 PRT Xenopus laevis 10 Gln Gly Lys Arg Pro Trp Ile Leu 1 5 11 25
PRT Homo sapiens 11 Met Leu Thr Lys Phe Glu Thr Lys Ser Ala Arg Val
Lys Gly Leu Ser 1 5 10 15 Phe His Pro Lys Arg Pro Trp Ile Leu 20 25
12 17 PRT Vespula maculifrons 12 Thr Ala Thr Thr Arg Arg Arg Gly
Arg Pro Pro Gly Phe Ser Pro Phe 1 5 10 15 Arg 13 9 PRT Homo sapiens
13 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5
* * * * *