U.S. patent application number 10/647519 was filed with the patent office on 2006-10-05 for gamma-conopeptides.
This patent application is currently assigned to Cognetix Inc.. Invention is credited to Alma L. Burlingame, Clark Colledge, Lourdes J. Cruz, Michael M. Fainzilber, Julita Imperial, Karel S. Kits, Baldomero M. Olivera, Reshma Shetty, Craig Walker, Maren Watkins.
Application Number | 20060223984 10/647519 |
Document ID | / |
Family ID | 22090703 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060223984 |
Kind Code |
A1 |
Fainzilber; Michael M. ; et
al. |
October 5, 2006 |
Gamma-conopeptides
Abstract
This invention relates to relatively short peptides about 25-40
residues in length, which are naturally available in minute amounts
in the venom of the cone snails or analogs to the naturally
available peptides, and which include three cyclizing disulfide
linkages and one or more .gamma.carboxyglutamate residues. More
specifically, the present invention is directed to
.gamma.-conopeptides having the general formula I:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Xaa.sub.5-Cys--
Xaa.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:1), as described herein; or
having the general formula II:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Xaa.sub.5-Xaa.sub-
.6-Cys-Xaa.sub.7-Cys-Xaa.sub.8 (SEQ ID NO:2), as defined herein; or
having the general formula III:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Cys-Cys-Ser-Asn-
-Ser-Cys-Asp-Xaa.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:3), as described
herein; or having the general formula IV:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Cys-Cys-Ser-Asn-
-Ser-Cys-Asp-Xaa.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:4), as described
herein; or having the general formula V:
Xaa.sub.1-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Phe-Xaa.sub.5-Cys-Thr-Xaa.sub-
.6-Ser-Xaa.sub.7-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa.sub.8-Leu-
-Xaa.sub.9 (SEQ ID NO:5), as described herein. The invention
further relates to specific .gamma.-conopeptides, specific
pro-.gamma.-conopeptides and nucleic acids encoding the
pro-.gamma.-conopeptides. The invention also includes
pharmaceutically acceptable salts of the conopeptides. These
conopeptides are useful as agonists of neuronal pacemaker calcium
channels.
Inventors: |
Fainzilber; Michael M.;
(Haifa, IL) ; Kits; Karel S.; (Amsterdam, NL)
; Burlingame; Alma L.; (Sausalito, CA) ; Olivera;
Baldomero M.; (Salt Lake City, UT) ; Walker;
Craig; (Salt Lake City, UT) ; Watkins; Maren;
(Salt Lake City, UT) ; Shetty; Reshma; (Salt Lake
City, UT) ; Cruz; Lourdes J.; (Manila, PH) ;
Imperial; Julita; (Salt Lake City, UT) ; Colledge;
Clark; (Draper, UT) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Cognetix Inc.
Salt Lake City
UT
University of Utah Research Foundation
Salt Lake City
UT
|
Family ID: |
22090703 |
Appl. No.: |
10/647519 |
Filed: |
August 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09210952 |
Dec 15, 1998 |
6624288 |
|
|
10647519 |
Aug 26, 2003 |
|
|
|
60069706 |
Dec 16, 1997 |
|
|
|
Current U.S.
Class: |
530/326 ;
530/327; 530/328 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 25/00 20180101; A61K 38/00 20130101; A61P 9/00 20180101; C07K
14/43504 20130101 |
Class at
Publication: |
530/326 ;
530/327; 530/328 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 14/435 20060101 C07K014/435 |
Goverment Interests
[0002] This invention was made in part with Government support
under Grant No. RR01614 and GM48677 awarded by the National
Institutes of Health, Bethesda, Md. and under Grant No. DIR8700766
awarded by the National Science Foundation, Washington, D.C. The
United States Government has certain rights in the invention.
Claims
1. A substantially pure conopeptide or pharmaceutically acceptable
salt thereof, said conopeptide having the general formula I:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Xaa.sub.5-Cys-Xaa-
.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:1), wherein Xaa.sub.1 is
des-Xaa.sub.1 or a peptide having 1-6 amino acids; Xaa.sub.2 is a
peptide having 5-6 amino acids; Xaa.sub.3 is a peptide having 4
amino acids; Xaa.sub.4 is Glu, .gamma.-carboxyglutamic acid
(.gamma.-Glu) or Gln; Xaa.sub.5 is a peptide having 3-4 amino
acids; Xaa.sub.6 is a peptide having 3-6 amino acids; and Xaa.sub.7
is des-Xaa.sub.7 or a peptide having 2-9 amino acids, with the
proviso that when Xaa.sub.1 is des-Xaa.sub.1, then Xaa.sub.5 is not
the tripeptide Ser-Asp-Asn.
2. The conopeptide of claim 1, wherein Xaa.sub.4 is
.gamma.-Glu.
3. The conopeptide of claim 1, wherein Xaa.sub.1 is
des-Xaa.sub.1.
4. The conopeptide of claim 1, wherein Xaa.sub.1 is a peptide
having 1-6 amino acids.
5. The conopeptide of claim 1, wherein Xaa.sub.7 is
des-Xaa.sub.7.
6. The conopeptide of claim 1, wherein Xaa.sub.7 is a peptide
having 2-9 amino acids.
7. A substantially pure conopeptide or pharmaceutically acceptable
salt thereof, said conopeptide having the general formula II:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Xaa.sub.5-Xaa.sub-
.6-Cys-Xaa.sub.7-Cys-Xaa.sub.8 (SEQ ID NO:2), wherein Xaa.sub.1 is
des-Xaa.sub.1 or a peptide having 1-6 amino acids; Xaa.sub.2 is a
peptide having 5-6 amino acids; Xaa.sub.3 is a peptide having 4
amino acids; Xaa.sub.4 is Glu, .gamma.-carboxyglutamic acid
(.gamma.-Glu) or Gln; Xaa.sub.5 is Ser or Thr; Xaa.sub.6 is a
peptide having 2-3 amino acids; Xaa.sub.7 is a peptide having 3-6
amino acids; and Xaa.sub.8 is des-Xaa.sub.8 or a peptide having 2-9
amino acids, with the proviso that when Xaa.sub.1 is des-Xaa.sub.1
and Xaa.sub.5 is Ser, then Xaa.sub.6 is not the dipeptide
Asp-Asn.
8. The conopeptide of claim 7, wherein Xaa.sub.4 is
.gamma.-Glu.
9. The conopeptide of claim 7, wherein Xaa.sub.1 is
des-Xaa.sub.1.
10. The conopeptide of claim 7, wherein Xaa.sub.1 is a peptide
having 1-6 amino acids.
11. The conopeptide of claim 7, wherein Xaa.sub.5 is Ser or
Thr.
12. The conopeptide of claim 7, wherein Xaa.sub.8 is
des-Xaa.sub.8.
13. The conopeptide of claim 1, wherein Xaa.sub.8 is a peptide
having 2-9 amino acids.
14. A substantially pure conopeptide or pharmaceutically acceptable
salt thereof, said conopeptide having the general formula III:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Ser-Asn-Ser-Cys-A-
sp-Xaa.sub.5-Cys-Xaa.sub.6 (SEQ ID NO:3), wherein Xaa.sub.1 is a
peptide having 1-6 amino acids; Xaa.sub.2 is a hexapeptide;
Xaa.sub.3 is a peptide having 4 amino acids; Xaa.sub.4 is Glu or
.gamma.-carboxyglutamic acid (.gamma.-Glu); Xaa.sub.5 is a
tripeptide; and Xaa.sub.6 is a peptide having 7-9 amino acids.
15. The conopeptide of claim 14, wherein Xaa.sub.4 is
.gamma.-Glu.
16. A substantially pure conopeptide or pharmaceutically acceptable
salt thereof, said conopeptide having the general formula IV:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Cys-Cys-Ser-Asn-
-Ser-Cys-Asp-Xaa.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:4), wherein
Xaa.sub.1 is a peptide having 1-6 amino acids; Xaa.sub.2 is a
hexapeptide; Xaa.sub.3 is Ser or Thr; Xaa.sub.4 is a tripeptide;
Xaa.sub.5 is Glu or .gamma.-carboxyglutamic acid (.gamma.-Glu);
Xaa.sub.6 is a tripeptide; and Xaa.sub.7 is a peptide having 7-9
amino acids.
17. The conopeptide of claim 16, wherein Xaa.sub.5 is
.gamma.-Glu.
18. A substantially pure conopeptide or pharmaceutically acceptable
salt thereof, said conopeptide having the general formula V:
Xaa.sub.1-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Phe-Xaa.sub.5-Cys-Thr-Xaa.sub-
.6-Ser-Xaa.sub.7-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa.sub.8-Leu-
-Xaa.sub.9 (SEQ ID NO:5), wherein Xaa.sub.1 is des-Xaa.sub.1 or a
dipeptide; Xaa.sub.2 is Asp, Glu or .gamma.-carboxyglutamic acid
(.gamma.-Glu); Xaa.sub.3 is a dipeptide; Xaa.sub.4 is Trp or
6-bromo-Trp; Xaa.sub.5 is a dipeptide; Xaa.sub.6 is a dipeptide;
Xaa.sub.7 is Glu or .gamma.-Glu; Xaa.sub.8 is any amino acid; and,
Xaa.sub.9 is a pentapeptide.
19. The conopeptide of claim 18, wherein Xaa.sub.7 is
.gamma.-Glu.
20. A substantially pure conopeptide selected from the group
consisting of: (a) PnVIIA:
Asp-Cys-Thr-Ser-Xaa.sub.1-Phe-Gly-Arg-Cys-Thr-Val-Asn-Ser-Xaa.sub.2-Cys-C-
ys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa.sub.2-Leu-Tyr-Ala-Phe-Xaa.sub.3-
-Ser (SEQ ID NO:6); (b) Tx6.4:
Xaa.sub.1-Leu-Xaa.sub.2-Cys-Ser-Val-Xaa.sub.1-Phe-Ser-His-Cys-Thr-Lys-Asp-
-Ser-Xaa.sub.2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Thr-Leu-Met-Xaa-
.sub.3-Xaa.sub.3-Asp-Xaa.sub.1 (SEQ ID NO:7); (c) Tx6.9:
Xaa.sub.1-Xaa.sub.1-Arg-Xaa.sub.1-Gly-Gly-Cys-Met-Ala-Xaa.sub.1-Phe-Gly-L-
eu-Cys-Ser-Arg-Asp-Ser-Xaa.sub.2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Val-Thr-Arg-C-
ys-Xaa.sub.2-Leu-Met-Xaa.sub.3-Phe-Xaa.sub.3-Xaa.sub.3-Asp-Xaa.sub.1
(SEQ ID NO:8); (d) J010:
Cys-Lys-Thr-Try-Ser-Lys-Try-Cys-Xaa.sub.2-Ala-Asp-Ser-Xaa.sub.2-Cys-Cys-T-
hr-Xaa.sub.2-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:9);
(e) Tx6.6:
Asp-Xaa.sub.1-Xaa.sub.1-Asp-Asp-Gly-Cys-Ser-Val-Xaa.sub.1-Gly-Xaa.-
sub.3-Cys-Thr-Val-Asn-Ala-Xaa.sub.2-Cys-Cys-Ser-Gly-Asp-Cys-His-Xaa.sub.2--
Thr-Cys-Ile-Phe-Gly-Xaa.sub.1-Xaa.sub.2-Val (SEQ ID NO:10); (f)
Tx6.5:
Gly-Met-Xaa.sub.1-Gly-Xaa.sub.2-Cys-Lys-Asp-Gly-Leu-Thr-Thr-Cys-Leu-Ala-X-
aa.sub.3-Ser-Xaa.sub.2-Cys-Cys-Ser-Xaa.sub.2-Asp-Cys-Xaa.sub.2-Gly-Ser-Cys-
-Thr-Met-Xaa.sub.1 (SEQ ID NO:11); (g) Gm6.7:
Xaa.sub.2-Cys-Arg-Ala-Xaa.sub.1-Tyr-Ala-Xaa.sub.3-Cys-Ser-Xaa.sub.3-Gly-A-
la-Gln-Cys-Cys-Ser-Leu-Leu-Met-Cys-Ser-Lys-Ala-Thr-Ser-Arg-Cys-Ile-Leu-Ala-
-Leu (SEQ ID NO:12); (h) Mr6.1:
Asn-Gly-Gln-Cys-Xaa.sub.2-Asp-Val-Xaa.sub.1-Met-Xaa.sub.3-Cys-Thr-Ser-Asn-
-Xaa.sub.1-Xaa.sub.2-Cys-Cys-Ser-Leu-Asp-Cys-Xaa.sub.2-Met-Tyr-Cys-Thr-Gln-
-Ile (SEQ ID NO:13); (i) Mr6.2:
Cys-Gly-Gly-Xaa.sub.1-Ser-Thr-Tyr-Cys-Xaa.sub.2-Val-Asp-Xaa.sub.2-Xaa.sub-
.2-Cys-Cys-Ser-Xaa.sub.2-Ser-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe
(SEQ ID NO:14); and (j) Mr6.3:
Asn-Gly-Gly-Cys-Lys-Ala-Thr-Xaa.sub.1-Met-Ser-Cys-Ser-Ser-Gly-Xaa.sub.1-X-
aa.sub.2 Cys-Cys-Ser-Met-Ser-Cys-Asp-Met-Try-Cys (SEQ ID NO:15),
wherein Xaa.sub.1 is Trp or 6-bromo-Trp; Xaa.sub.2 is Glu or
.gamma.-carboxyglutamic acid (.gamma.-Glu); and Xaa.sub.3 is Pro or
hydroxy-Pro (Hyp).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S.
application Ser. No. 09/210,952, filed 15 Dec. 1998, which claims
benefit of U.S. provisional patent application Ser. No. 60/069,706,
filed 16 Dec. 1997, each of which is incorporated herein by
reference, in its entirety.
BACKGROUND OF THE INVENTION
[0003] This invention relates to relatively short peptides about
25-40 residues in length, which are naturally available in minute
amounts in the venom of the cone snails or analogs to the naturally
available peptides, and which include three cyclizing disulfide
linkages and one or more .gamma.-carboxyglutamate residues.
[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 referenced in
the following text by author and date and are listed alphabetically
by author 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] The predatory cone snails (Conus) have developed a unique
biological strategy. Their venom contains relatively small peptides
that are targeted to various neuromuscular receptors and may be
equivalent in their pharmacological diversity to the alkaloids of
plants or secondary metabolites of microorganisms. Many of these
peptides are among the smallest nucleic acid-encoded translation
products having defined conformations, and as such, they are
somewhat unusual. Peptides in this size range normally equilibrate
among many conformations. Proteins having a fixed conformation are
generally much larger.
[0008] The cone snails that produce these toxic peptides, which are
generally referred to as conotoxins or conotoxin peptides, are a
large genus of venomous gastropods comprising approximately 500
species. All cone snail species are predators that inject venom to
capture prey, and the spectrum of animals that the genus as a whole
can envenomate is broad. A wide variety of hunting strategies are
used, however, every Conus species uses fundamentally the same
basic pattern of envenomation.
[0009] 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). A conotoxin, TxVIIA, containing a
.gamma.-carboxyglutamate residued and three disulfide bonds has bee
isolated (Fainzilber et al., 1991). 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). Recently, peptides named contryphans containing
D-tryptophan or D-leucine 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).
[0010] Ion channels are integral plasma membrane proteins
responsible for electrical activity in excitable tissues. It has
been recognized that slow inward currents can influence neuronal
excitability via long-lasting depolarizations of the cell membrane
(Llinas, 1988). The role of slow inward currents in generating
endogenous bursting behavior has been recognized in molluscan
neurons (Wilson & Wachtel, 1974; Eckert & Lux, 1976;
Partridge et al., 1979), and more recently in some types of
mammalian neurons (Lanthorn et al., 1984; Stafstrom et al., 1985;
Llinas, 1988; Alonso & Llinas, 1989). Changes in the slow
inward currents carried by such nonspecific cation channels may
play a crucial role in bursting and pacemaker activities in a
variety of excitable systems, ranging from mammalian heart muscle
to molluscan neurons (Partridge & Swandulla, 1988; Hoehn et
al., 1993; Kits & Mansvelder, 1966; van Soest & Kits,
1997). Slow inward currents are also believed to be important in
generating epileptiform bursting in regions of the brain such as
the hippocampus.
[0011] It is desired to identify drugs which are useful for
modulating slow inward cation channels in vertebrates involved in
syndromes of clinical relevance, such as epileptic activity in
hippocampus (Hoehn et al., 1993) and pacemaker potentials in heart
muscle (Reuter, 1984).
SUMMARY OF THE INVENTION
[0012] This invention relates to relatively short peptides about
25-40 residues in length, which are naturally available in minute
amounts in the venom of the cone snails or analogs to the naturally
available peptides, and which include three cyclizing disulfide
linkages and one or more .gamma.-carboxyglutamate residues.
[0013] More specifically, the present invention is directed to
conopeptides having the general formula I: [0014]
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Xaa.sub.5-Cys-Xaa-
.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:1), wherein Xaa.sub.1 is
des-Xaa.sub.1 or a peptide having 1-6 amino acids; Xaa.sub.2 is a
peptide having 5-6 amino acids; Xaa.sub.3 is a peptide having 4
amino acids; Xaa.sub.4 is Glu, .gamma.-Glu (.gamma.-carboxyglutamic
acid; also referred to as Gla) or Gln; Xaa.sub.5 is a peptide
having 3-4 amino acids; Xaa.sub.6 is a peptide having 3-6 amino
acids; and Xaa.sub.7 is des-Xaa.sub.7 or a peptide having 2-9 amino
acids, with the proviso that when Xaa.sub.1 is des-Xaa.sub.1, then
Xaa.sub.5 is not the tripeptide Ser-Asp-Asn; general formula II:
[0015]
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Xaa.sub.5-Xaa.sub-
.6-Cys-Xaa.sub.7-Cys-Xaa.sub.8 (SEQ ID NO:2), wherein Xaa.sub.1 is
des-Xaa.sub.1 or a peptide having 1-6 amino acids; Xaa.sub.2 is a
peptide having 5-6 amino acids; Xaa.sub.3 is a peptide having 4
amino acids; Xaa.sub.4 is Glu, .gamma.-Glu or Gln; Xaa.sub.5 is Ser
or Thr; Xaa.sub.6 is a peptide having 2-3 amino acids; Xaa.sub.7 is
a peptide having 3-6 amino acids; and Xaa.sub.8 is des-Xaa.sub.8 or
a peptide having 2-9 amino acids, with the proviso that when
Xaa.sub.1 is des-Xaa.sub.1 and Xaa.sub.5 is Ser, then Xaa.sub.6 is
not the dipeptide Asp-Asn; general formula III:
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Cys-Cys-Ser-Asn-Ser-Cys-A-
sp-Xaa.sub.5-Cys-Xaa.sub.6 (SEQ ID NO:3), wherein Xaa.sub.1 is a
peptide having 1-6 amino acids; Xaa.sub.2 is a hexapeptide;
Xaa.sub.3 is a peptide having 4 amino acids; Xaa.sub.4 is Glu or
.gamma.-Glu; Xaa.sub.5 is a tripeptide; and Xaa.sub.6 is a peptide
having 7-9 amino acids; general formula IV: [0016]
Xaa.sub.1-Cys-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Cys-Cys-Ser-Asn-
-Ser-Cys-Asp-Xaa.sub.6-Cys-Xaa.sub.7 (SEQ ID NO:4), wherein
Xaa.sub.1 is a peptide having 1-6 amino acids; Xaa.sub.2 is a
hexapeptide; Xaa.sub.3 is Ser or Thr; Xaa.sub.4 is a tripeptide;
Xaa.sub.5 is Glu or .gamma.-Glu; Xaa.sub.6 is a tripeptide; and
Xaa.sub.7 is a peptide having 7-9 amino acids; or general formula
V: [0017]
Xaa.sub.1-Xaa.sub.2-Cys-Xaa.sub.3-Xaa.sub.4-Phe-Xaa.sub.5-Cys-Thr-Xaa.sub-
.6-Ser-Xaa.sub.7-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa.sub.8-Leu-
-Xaa.sub.9 (SEQ ID NO:5), wherein Xaa.sub.1 is des-Xaa.sub.1 or a
dipeptide; Xaa.sub.2 is Asp, Glu or .gamma.-Glu; Xaa.sub.3 is a
dipeptide; Xaa.sub.4 is Trp or 6-bromo-Trp; Xaa.sub.5 is a
dipeptide; Xaa.sub.6 is a dipeptide; Xaa.sub.7 is Glu or
.gamma.-Glu; Xaa.sub.8 is any amino acid; and, Xaa.sub.9 is a
pentapeptide
[0018] The amino acid or the amino acid residues of the peptides is
an amino acid selected from the group consisting of natural,
modified or non-natural amino acids. The disulfide bridges in the
conopeptides of general formulas I-V (as well as the specific
conopeptides described herein) are between the first and fourth
cysteine residues, between the second and fifth cysteine residues
and between the third and sixth cysteine residues. The C-terminal
end may contain a carboxyl or amide group. The invention also
includes pharmaceutically acceptable salts of the conopeptides.
These conopeptides are useful for modulating slow inward cation
channels in vertebrates involved in syndromes of clinical
relevance, such as epileptic activity in hippocampus (Hoehn et al,
1993) and pacemaker potentials in heart muscle (Reuter, 1984).
Thus, the conopeptides are useful as agonists of neuronal pacemaker
cation channels.
[0019] The invention further relates to the specific peptides:
[0020]
Asp-Cys-Thr-Ser-Xaa.sub.1-Phe-Gly-Arg-Cys-Thr-Val-Asn-Ser-Xaa.sub.2-Cys-C-
ys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa.sub.2-Leu-Tyr-Ala-Phe-Xaa.sub.3-
-Ser (SEQ ID NO:6) (PnVIIA), wherein Xaa.sub.1 is Trp or
6-bromo-Trp; Xaa.sub.2 is Glu or .gamma.-Glu, preferably
.gamma.-Glu; Xaa.sub.3 is Pro or hydroxy-Pro (Hyp), preferably Hyp;
and the C-terminus is a free carboxyl group or is amidated,
preferably a free carboxyl group; [0021]
Xaa.sub.1-Leu-Xaa.sub.2-Cys-Ser-Val-Xaa.sub.1-Phe-Ser-His-Cys-Thr-Lys-Asp-
-Ser-Xaa.sub.2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Thr-Leu-Met-Xaa-
.sub.3-Xaa.sub.3-Asp-Xaa.sub.1 (SEQ ID NO:7) (Tx6.4), wherein
Xaa.sub.1 is Trp or 6-bromo-Trp; Xaa.sub.2 is Glu or .gamma.-Glu,
preferably .gamma.-Glu; Xaa.sub.3 is Pro or Hyp, preferably Hyp;
and the C-terminus is a free carboxyl group or is amidated,
preferably a free carboxyl group; [0022]
Xaa.sub.1-Xaa.sub.1-Arg-Xaa.sub.1-Gly-Gly-Cys-Met-Ala-Xaa.sub.1-Phe-Gly-L-
eu-Cys-Ser-Arg-Asp-Ser-Xaa.sub.2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Val-Thr-Arg-C-
ys-Xaa.sub.2-Leu-Met-Xaa.sub.3-Phe-Xaa.sub.3-Xaa.sub.3-Asp-Xaa.sub.1
(SEQ ID NO:8) (Tx6.9), wherein Xaa.sub.1 is Trp or 6-bromo-Trp;
Xaa.sub.2 is Glu or .gamma.-Glu, preferably .gamma.-Glu; Xaa.sub.3
is Pro or Hyp, preferably Hyp; and the C-terminus is a free
carboxyl group or is amidated, preferably a free carboxyl group;
[0023]
Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa.sub.2-Ala-Asp-Ser-Xaa.sub.2-Cys-Cys-T-
hr-Xaa.sub.2-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:9)
(J010), wherein Xaa.sub.2 is Glu or .gamma.-Glu, preferably
.gamma.-Glu; and the C-terminus is a free carboxyl group or is
amidated, preferably amidated; [0024]
Asp-Xaa.sub.1-Xaa.sub.1-Asp-Asp-Gly-Cys-Ser-Val-Xaa.sub.1-Gly-Xaa-
.sub.3-Cys-Thr-Val-Asn-Ala-Xaa.sub.2-Cys-Cys-Ser-Gly-Asp-Cys-His-Xaa.sub.2-
-Thr-Cys-Ile-Phe-Gly-Xaa.sub.1-Xaa.sub.2-Val (SEQ ID NO:10)
(Tx6.6), wherein Xaa.sub.1 is Trp or 6-bromo-Trp; Xaa.sub.2 is Glu
or .gamma.-Glu, preferably .gamma.-Glu; Xaa.sub.3 is Pro or Hyp,
preferably Hyp; and the C-terminus is a free carboxyl group or is
amidated, preferably a free carboxyl group; [0025]
Gly-Met-Xaa.sub.1-Gly-Xaa.sub.2-Cys-Lys-Asp-Gly-Leu-Thr-Thr-Cys-Leu-Ala-X-
aa.sub.3-Ser-Xaa.sub.2-Cys-Cys-Ser-Xaa.sub.2-Asp-Cys-Xaa.sub.2-Gly-Ser-Cys-
-Thr-Met-Xaa.sub.1 (SEQ ID NO:11) (Tx6.5), wherein Xaa.sub.1 is Trp
or 6-bromo-Trp; Xaa.sub.2 is Glu or .gamma.-Glu, preferably
.gamma.-Glu; Xaa.sub.3 is Pro or Hyp, preferably Hyp; and the
C-terminus is a free carboxyl group or is amidated, preferably a
free carboxyl group; [0026]
Xaa.sub.2-Cys-Arg-Ala-Xaa.sub.1-Tyr-Ala-Xaa.sub.3-Cys-Ser-Xaa.sub.3-Gly-A-
la-Gln-Cys-Cys-Ser-Leu-Leu-Met-Cys-Ser-Lys-Ala-Thr-Ser-Arg-Cys-Ile-Leu-Ala-
-Leu (SEQ ID NO:12) (Gm6.7), wherein Xaa.sub.1 is Trp or
6-bromo-Trp; Xaa.sub.2 is Glu or .gamma.-Glu, preferably
.gamma.-Glu; Xaa.sub.3 is Pro or Hyp, preferably Hyp; and the
C-terminus is a free carboxyl group or is amidated, preferably a
free carboxyl group; [0027]
Asn-Gly-Gln-Cys-Xaa.sub.2-Asp-Val-Xaa.sub.1-Met-Xaa.sub.3-Cys-Thr-Ser-Asn-
-Xaa.sub.1-Xaa.sub.2-Cys-Cys-Ser-Leu-Asp-Cys-Xaa.sub.2-Met-Tyr-Cys-Thr-Gln-
-Ile (SEQ ID NO:13) (Mr6.1), wherein Xaa.sub.1 is Trp or
6-bromo-Trp; Xaa.sub.2 is Glu or .gamma.-Glu, preferably
.gamma.-Glu; Xaa.sub.3 is Pro or Hyp, preferably Hyp; and the
C-terminus is a free carboxyl group or is amidated, preferably
amidated; [0028]
Cys-Gly-Gly-Xaa.sub.1-Ser-Thr-Tyr-Cys-Xaa.sub.2-Val-Asp-Xaa.sub.2-Xaa.sub-
.2-Cys-Cys-Ser-Xaa.sub.2-Ser-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe
(SEQ ID NO:14) (Mr6.2), wherein Xaa.sub.1 is Trp or 6-bromo-Trp;
Xaa.sub.2 is Glu or .gamma.-Glu, preferably .gamma.-Glu; and the
C-terminus is a free carboxyl group or is amidated, preferably
amidated; [0029]
Asn-Gly-Gly-Cys-Lys-Ala-Thr-Xaa.sub.1-Met-Ser-Cys-Ser-Ser-Gly-Xaa.sub.1-X-
aa.sub.2 Cys-Cys-Ser-Met-Ser-Cys-Asp-Met-Try-Cys (SEQ ID NO:15)
(Mr6.3), wherein Xaa.sub.1 is Trp or 6-bromo-Trp; Xaa.sub.2 is Glu
or .gamma.-Glu, preferably .gamma.-Glu; and the C-terminus is a
free carboxyl group or is amidated, preferably amidated.
[0030] Finally, the invention further relates to the propeptide
sequences for the above peptides and the DNA sequences coding for
these propeptide sequences as described in further detail
herein.
SEQUENCE SUMMARY
[0031] SEQ ID NO:1=.gamma.-conopeptides of general formula I; SEQ
ID NO:2=.gamma.-conopeptides of general formula II; SEQ ID
NO:3=.gamma.-conopeptides of general formula III; SEQ ID
NO:4=.gamma.-conopeptides of general formula IV; SEQ ID
NO:5=.gamma.-conopeptides of general formula V; SEQ ID
NO:6=.gamma.-conopeptide corresponding to PnVIIA; SEQ ID
NO:7=.gamma.-conopeptide corresponding to Tx6.4; SEQ ID
NO:8=.gamma.-conopeptide corresponding to Tx6.9; SEQ ID
NO:9=.gamma.-conopeptide corresponding to J010; SEQ ID
NO:10=.gamma.-conopeptide corresponding to Tx6.6; SEQ ID
NO:11=.gamma.-conopeptide corresponding to Tx6.5; SEQ ID
NO:12=.gamma.-conopeptide corresponding to Gm6.7; SEQ ID
NO:13=.gamma.-conopeptide corresponding to Mr6.1; SEQ ID
NO:14=.gamma.-conopeptide corresponding to Mr6.2; SEQ ID
NO:15=.gamma.-conopeptide corresponding to Mr6.3; SEQ ID NO:16=DNA
encoding propeptide of Tx6.4; SEQ ID NO:17=propeptide of Tx6.4; SEQ
ID NO:18=DNA encoding propeptide of Tx6.9; SEQ ID NO:19=propeptide
of Tx6.9; SEQ ID NO:20=DNA encoding propeptide of J010; SEQ ID
NO:21=propeptide of J010; SEQ ID NO:22=DNA encoding propeptide of
Tx6.6; SEQ ID NO:23=propeptide of Tx6.6; SEQ ID NO:24=DNA encoding
propeptide of Tx6.5; SEQ ID NO:25=propeptide of Tx6.5; SEQ ID
NO:26=DNA encoding propeptide of Gm6.7; SEQ ID NO:27=propeptide of
Gm6.7; SEQ ID NO:28=DNA encoding propeptide of Mr6.1; SEQ ID
NO:29=propeptide of Mr6.1; SEQ ID NO:30=DNA encoding propeptide of
Mr6.2; SEQ ID NO:31=propeptide of Mr6.2; SEQ ID NO:32=DNA encoding
propeptide of Mr6.3; SEQ ID NO:33=propeptide of Mr6.3; SEQ ID
NO:34=DNA encoding propeptide of Tx6.1; SEQ ID NO:35=propeptide of
Tx6.1; SEQ ID NO:36=.gamma.-conopeptide corresponding to Tx6.1; SEQ
ID NO:37=consensus sequence of .gamma.-conopeptides PnVIIA and
Tx6.4; SEQ ID NO:38=degenerate probe for consensus sequence of
.gamma.-conopeptides; SEQ ID NO:39=degenerate probe for consensus
sequence of .gamma.-conopeptides; SEQ ID NO:40=consensus sequence
of pro-.gamma.-conopeptides; SEQ ID NO:41=degenerate probe for
consensus sequence of pro-.gamma.-conopeptides; SEQ ID
NO:42=.gamma.-conopeptide PnVIIA; SEQ ID NO:43=.gamma.-conopeptide
TxVIIA; SEQ ID NO:44=N-terminal tryptic peptide of
.gamma.-conopeptide PnVIIA; SEQ ID NO:45=C-terminal tryptic peptide
of .gamma.-conopeptide PnVIIA; SEQ ID NO:46=primer for isolating
conopeptides from Conus textile cDNA library; SEQ ID NO:47=primer
for isolating conopeptides from Conus textile cDNA library.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0032] This invention relates to relatively short peptides about
25-40 residues in length, which are naturally available in minute
amounts in the venom of the cone snails or analogs to the naturally
available peptides, and which include three cyclizing disulfide
linkages and one or more .gamma.-carboxyglutamate residues.
[0033] More specifically, the present invention is directed to
conopeptides having the general formulas I-V described above. The
invention is also directed to the specific .gamma.-conopeptides
PnVIIA, Tx6.4, Tx6.9, J010, Tx6.6, Tx6.5, Gm6.7, Mr6.1, Mr6.2 and
Mr6.3, the sequences of which are described above.
[0034] The invention is further directed to isolated nucleic acids
which encode .gamma.-conopeptides, including the above and
.gamma.-conopeptide Tx6.1, and to isolated propeptides encoded by
the nucleic acids. This aspect of the present invention is set
forth in Table 1. TABLE-US-00001 TABLE 1 Nucleic Acids and
Propeptides of .gamma.-Conopeptides Nucleic Acid Propeptide SEQ
.gamma.-Conopeptide SEQ ID NO: ID NO: Tx6.4 16 17 Tx6.9 18 19 J010
20 21 Tx6.6 22 23 Tx6.5 24 25 Gm6.7 26 27 Mr6.1 28 29 Mr6.2 30 31
Mr6.3 32 33 Tx6.1 34 35 The mature peptide sequence for Tx6.1 is
LCX.sub.3DYTX.sub.2X.sub.3CSHAHX.sub.2CCSX.sub.1NCYNGHCT (SEQ ID
NO: 36), wherein X.sub.1, X.sub.2 and X.sub.3 are as described for
Xaa.sub.1, Xaa.sub.2 and Xaa.sub.3, respectively. The C-terminus is
preferably amidated.
[0035] The conopeptides of the present invention are useful for
modulating slow inward cation channels in vertebrates involved in
syndromes of clinical relevance, such as epileptic activity in
hippocampus (Hoehn et al., 1993) and pacemaker potentials in heart
muscle (Reuter, 1984). Thus, the conopeptides are useful as
agonists of neuronal pacemaker cation channels.
[0036] The .gamma.-conopeptides of the present invention are
identified by isolation from Conus venom. Alternatively, the
.gamma.-conopeptides of the present invention are identified using
recombinant DNA techniques. According to this method of
identification, cDNA libraries of various Conus species are
screened using conventional techniques with degenerate probes for
the peptide consensus sequence Xaa-Cys-Cys-Ser (SEQ ID NO:37),
wherein Xaa is Glu or Gln. Suitable probes are 5' SARTGYTGYAGY 3'
(SEQ ID NO:38) or 5' SARTGYTGYTCN 3' (SEQ ID NO:39). Alternatively,
cDNA libraries are screened with degenerate probes for the
propeptide consensus sequence Ile-Leu-Leu-Val-Ala-Ala-Val-Leu (SEQ
ID NO:40). Suitable probes for this sequence are 5'
ATHYTNYTNGTNGCNGCNGTNYTN 3' (SEQ ID NO:41). 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 .gamma.-conopeptides, such as
the presence of a Glu residue which could be modified to a
.gamma.-Glu and 6 cysteine residues. The biological activity of the
peptides identified by this method is tested as described
herein.
[0037] These peptides are sufficiently small to be chemically
synthesized. General chemical syntheses for preparing the foregoing
conopeptides peptides 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.
[0038] 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.
[0039] The conopeptides can also be produced by recombinant DNA
techniques well known in the art. Such techniques are described by
Sambrook et al. (1979). 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.
[0040] 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.
[0041] 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.
[0042] 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., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole,
various active esters, e.g., esters of N-hydroxyphthalimide or
N-hydroxy-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).
[0043] 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.
[0044] 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.
[0045] 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).
[0046] 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).
[0047] 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).
[0048] 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'-diisopropylcarbodiimide 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).
[0049] 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).
[0050] 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.
[0051] 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).
[0052] The present .gamma.-conotoxins are useful for modulating
slow inward cation channels in vertebrates involved in syndromes of
clinical relevance, such as epileptic activity in hippocampus
(Hoehn et al., 1993) and pacemaker potentials in heart muscle
(Reuter, 1984). Thus, the conopeptides are useful as agonists of
neuronal pacemaker cation channels.
[0053] 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 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. The
compositions may further contain antioxidizing agents, stabilizing
agents, preservatives and the like.
[0054] 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. The
active agent can be encapsulated to make it stable to passage
through the gastrointestinal tract while at the same time allowing
for passage across the blood brain barrier. See for example, WO
96/11698.
[0055] For parenteral administration, the compound may dissolved in
a pharmaceutical carrier and administered as either a solution of 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.
[0056] The conopeptides are administered in an amount sufficient to
agonize the neuronal pacemaker calcium channels. The dosage range
at which the conopeptides exhibit this agonistic effect can vary
widely depending upon the particular condition being treated, the
severity of the patient's condition, the patient, the specific
conopeptide being administered, the route of administration and the
presence of other underlying disease states within the patient.
Typically, the conopeptides of the present invention exhibit their
therapeutic effect at a dosage range from about 0.05 mg/kg to about
250 mg/kg, and preferably from about 0.1 mg/kg to about 100 mg/kg
of the active ingredient. A suitable dose can be administered in
multiple sub-doses per day. Typically, a dose or sub-dose may
contain from about 0.1 mg to about 500 mg of the active ingredient
per unit dosage form. A more preferred dosage will contain from
about 0.5 mg to about 100 mg of active ingredient per unit dosage
form. Dosages are generally initiated at lower levels and increased
until desired effects are achieved.
EXAMPLES
[0057] The present invention is described by reference to 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 were utilized.
Example 1
Experimental Procedures
[0058] Toxins and Bioassays. Venom of Conus pennaceus was obtained
from specimens collected in the Northern Red Sea. Conotoxin-TxVIIA
was from venom-purified aliquots (Fainzilber et al., 1991). Assays
for paralysis in limpet snails (Patella caerulea), bivalves
(Mytilus edulis), and fish (Gambusia affinis) were performed as
previously described (Fainzilber et al., 1995).
[0059] Column Chromatography. Conus pennaceus venom was extracted
and fractionated on Sephadex G-50 (Pharmacia) and semipreparative
C18 (Vydac) columns as previously described (Fainzilber et al.,
1994). Final purification of the active peptides was on wide pore
reverse-phase phenyl (Vydac, 25.times.0.46 cm, 0.5 .mu.m particle
size) as described in FIG. 1, with on-line spectral analysis of
peak purity utilizing a Hewlett-Packard 1040A Diode Array Detector
coupled with HP 300 Chemstation Software.
[0060] Amino Acid Analysis. Analysis of amino acid composition
after acid hydrolysis and 9-fluorenylmethyl-oxycabonyl-chloride
(FMOC) derivatization was performed on a Merck-Hitachi
reverse-phase HPLC system, according to Betner & Foldi, 1988.
The system was calibrated prior to each analysis with FMOC-amino
acid standards.
[0061] Reduction and alkylation. Dried purified peptides were
dissolved in 50 .mu.l of 0.1M NH.sub.4HCO.sub.3 (pH 8) containing
6M guanidine.about.HCl and 10 .mu.M EDTA, and reduced with 200
.mu.g of D11 at 37.degree. C. for 2 hrs under argon. 4
vinylpyridine, or iodoacetic acid, or iodoacetamide were added and
the mixture incubated at 37.degree. C. for 1.5 hrs under argon. The
alkylated peptide sample was purified on reverse-phase HPLC
immediately after derivatization
[0062] Edman Degradation Analyses. Reverse-phase purified peptides
were applied to PVDF or glass fiber filters, and sequenced by
automated Edman degradation on an Applied Biosystems 475A gas-phase
protein sequencing system.
[0063] Proteolytic digest. HPLC purified sample of reduced and
alkylated peptide was digested with TPCK-trypsin (Pierce, Rockford,
Ill.) for 20 hrs at 37.degree. C. A portion of the digest was
directly analyzed by LC/ESI/MS, and the remainder purified by
reverse-phase HPLC pH of the digest was adjusted to 3.0 prior to
loading on the HPLC, in order to minimize the possibility of
.gamma.-carboxyglutamate decomposition in extremely acidic
conditions. Purified C-terminal peptide fragments were further
digested by Endoproteinase Asp-N (Boehringer-Mannheim,
Indianapolis, Ind.) for 20 hrs at 37.degree. C., and immediately
purified on reverse-phase HPLC. A portion of the purified Asp N
peptide was then methylated for LSI CID mass spectrometry.
[0064] Mass spectrometry. Microbore LC/ESI/MS experiments were
carried out on a VG/Fisons Manchester, U.K.) platform mass
spectrometer using a C18 column (macrosphere C18, 5 .mu.m particle
size, 1.times.250 mm, Alltech Deerfield Ill.) with a linear
gradient of 2-62% acetonitrile in 0.1% TFA in 60 min. A post column
addition of make up solvent, 2-propanol/2-methoxyethanol (1:1) was
used to optimize spraying and ionization performance (Medzihradszky
et al., 1994). High energy CID mass spectra were obtained with a
Kratos (Manchester, U.K.) Concept IIHH tandem mass spectrometer
equipped with a continuous flow liquid secondary ionization source
and a scanning charge-coupled device array detector (Burlingame,
1994).
[0065] Electrophysiology. Isolated Lymnaea caudodorsal neurons were
kept in Petri dishes (Costar) and bathed in Hepes buffered saline
(in mM: NaCl 30, NaCH.sub.3SO.sub.4 10, NaHCO.sub.3 5, KCl 1.7,
CaCl.sub.2 4, MgCl.sub.2 1.5, HEPES 10; pH 7.8 set with NaOH). To
record calcium, sodium or potassium currents, HBS was replaced
under continuous perfusion by the appropriate saline. The
compositions of extracellular and pipette solutions used to
selectively record specific currents were as follows (in mM):
Extracellular I.sub.Ca saline: TEACl 40, CaCl.sub.2 4, HEPES 10,
4aminopyridine 2, pH 7.8 set with TEAOH; Extracellular I.sub.Na
saline: NaCl 47.5, CaCl.sub.2 4, MgCl.sub.2 1, HEPES 10, CdCl 0.1,
4-aminopyridine 1, pH 7.8 set with NaOH; Pipette saline (I.sub.Ca
and I.sub.Na): CsCl 29, CaCl.sub.2 2.3, HEPES 10, EGTA 11, ATPMg 2,
GTPtris 0.1, pH 7.4 adjusted with CsOH; Pipette saline
(non-selective): KCl 29, CaCl.sub.2 2.3, HEPES 10, EGTA 11, ATPMg
2, GTPtris 0.1, pH 7.4 adjusted with KOH. Toxin was administered by
means of a laboratory-built pressure ejection system through a
small glass pipette (tip diameter 20 .mu.M) placed at -100 .mu.M
from the recorded cell. This enabled rapid application of toxins,
which were applied continuously during voltage ramps or series of
depolarizing voltage steps.
[0066] Membrane potential measurements were performed using sharp
microelectrodes filled with 0.5 M KCl (40 M.OMEGA.) using an
Axoclamp 2A (Axon Instr., Foster City, Iowa) amplifier in the
bridge balance mode. Whole-cell voltage-clamp experiments were
performed using the Axoclamp 2A amplifier in the continuous single
electrode voltage clamp mode. Pipettes (2-6 M.OMEGA.) were pulled
on a Flaming/Brown P-87 (Sutter Instruments, CO) horizontal
micro-electrode puller from Clark GC-150T glass (Clark
Electromedical Instruments, U.K.) (seal resistance >1 G.OMEGA.).
After disrupture of the patch membrane series resistance (<10
M.OMEGA.) was compensated for--80%. With current amplitudes of
<5 nA, the maximal voltage error is estimated to be <10 mV.
Cell capacitance (.about.100 pF) was not compensated. Measurements
of calcium or sodium currents were commenced 20 mins after access
to the cell, in order to allow equilibration with the pipette
solution. Data acquisition was controlled by a CED AD/DA converter
(Cambridge Electronics Design, Cambridge, U.K.) connected to an
Intel 80486-based computer, run with voltage-clamp software
developed in our laboratory. The current recordings were filtered
at 1-5 kHz, sampled at 1 kHz (calcium currents and K+ currents) or
3 kHz (Na+ currents) and stored on-line. This system allowed
simultaneous application of voltage-steps, acquisition of current
recordings and timed application of toxins.
Example 2
Purification of .gamma.-Conotoxin PnVIIA
[0067] Conus pennaceus venom was fractionated as described under
Methods, and reverse-phase peptide containing fractions were
assayed for .gamma.Glu content using a comparison of positive ion
versus negative ion modes of MALDI mass spectrometry (Nakamura et
al., 1996). The positive fraction indicated as PnVII in FIG. 1B of
Fainzilber et al. (1994) was repurified by reverse phase phenyl
chromatography and conotoxin-PnVIIA was obtained as the major
component. On-line spectral analyses of the final chromatographic
step suggested homogeneity of the purified toxin. ESI/MS
measurements of the purified peptide revealed a single mass of
3718.4, further confirming homogeneity of PnVIIA.
Example 3
Chemical Characterization of .gamma.-Conotoxin PnVIIA
[0068] Automated Edman sequencing of PnVIIA after alkylation with
4-vinylpyridine revealed a 32 amino acid sequence, allowing
unambiguous assignments of 30 residues (Table 2). The extremely low
yields of Glu at steps 14 and 26 further suggested the presence of
.gamma.-carboxyglutamate residues at these positions. Amino acid
composition analysis (Table 3) was consistent with the proposed
sequence (Table 4), and the ESI/MS measurement fits that predicted
from the sequence assuming two .gamma.-carboxyglutamate residues,
three disulfide bridges and a free carboxy terminus (measured mass
3718.4, predicted 3719.0) TABLE-US-00002 TABLE 2 Edman Degradation
of PnVIIA Assigned Yield Cycle # Residue (pmoles) 1 Asp 185 2 Cys
170 3 Thr 180 4 Ser 190 5 Trp 170 6 Phe 140 7 Gly 210 8 Arg 85 9
Cys 93 10 Thr 150 11 Val 170 12 Asn 85 13 Ser 110 14 Glu 9 15 Cys
50 16 Cys 56 17 Ser 18 18 Asn 35 19 Ser 15 20 Cys 11 21 Asp 23 22
Gln 26 23 Thr 22 24 Tyr 17 25 Cys 14 26 Glu 3 27 Leu 17 28 Tyr 14
29 Ala 11 30 Phe 13 31 Hyp 8 32 Ser 9
[0069] TABLE-US-00003 TABLE 3 Amino Acid Composition Analysis of
Conotoxin-PnVIIA Amino Acid Mole Ratio Asx 3.9 (4) Ser 4.7 (5) Glx
3.0 (3) Cys 5.2 (6) Thr 2.8 (3) Gly 1.1 (1) Arg 1.0 (1) Hyp 0.8 (1)
Ala 1.2 (1) Tyr 2.0 (2) Val 1.2 (1) Phe 2.0 (2) Leu 1.2 (1) Trp n.d
(1) Molar ratios of amino acids determined after acid hydrolysis
and FMOC derivatization. Values in brackets are those predicted
from the amino acid sequence.
[0070] TABLE-US-00004 TABLE 4 Amino Acid Sequence of PnVIIA and
TxVIIA PnVIIA (SEQ ID NO:42): D C TSWFGR C T V N S.gamma.CCS N S C
DQT YC .gamma. L YAFOS-COOH TxVIIA (SEQ ID NO:43): C GGYSTY C
.gamma. V D S.gamma.CCSD N C VRS YC T L F-NH.sub.2 Sequence
identities are underlined; similarities are in bold type; and
spaces inserted to maximize homologies.
[0071] In order to verify the presence of .gamma.-carboxyglutamate,
and to determine the C-terminus, the peptide was further analyzed
by mass spectrometry. A tryptic digest of reduced and
carboxymethylated PnVIIA gave two peptides, T1 and T2, whose
average molecular masses by ESI/MS were 1029.0 and 3062.6,
respectively. These masses fit those predicted for the two PnVIIA
tryptic peptides, namely 1029.1 for the sequence DXTSWFGR (SEQ ID
NO:44), where X is carboxymethylcys, and 3062.2 for the sequence
XTVNSX.sub.1XXSNSXDQTYXX.sub.1LYAFX.sub.2S (SEQ ID NO:45), where
X.sub.1 is .gamma.-carboxyglutamate and X.sub.2 is
4-trans-hydroxyproline. Asp-N digest of the C-terminal tryptic
peptide T2 gave two products, AN1 and AN2. ESI/MS average mass for
AN1 was 1525.4, fitting the predicted mass of the Asp-N fragment
XTVNSX.sub.1XXSNSX (residues 1-12 of SEQ ID NO:45; predicted
1525.6). The monoisotopic LSI/MS measured mass for the C-terminal
fragment AN2 was 1553.7, in agreement with the calculated value
assuming the C-terminal is a free acid. An attempt to further
confirm the C-terminal sequence of PnVIIA by LSI tandem MS failed,
perhaps due to poor ionization efficiency of AN2. Therefore, PnVIIA
was reduced and alkylated with iodoacetamide, a procedure expected
to generate derivatives with better CID spectra than
carboxymethylated peptides. After trypsin followed by Asp-N
digests, the C-terminal carbamolmethylated peptide AN2u was
isolated. Methylation with HCl/MeOH gave a tetra ester, with
monoisotopic LSI/MS mass of 1608.9. This mass fits a peptide with
incorporation of four methyl groups--one at the side chain of Asp,
two at the carboxyl groups of the .gamma.-carboxyglutamate, and the
fourth at the presumed C-terminal free carboxyl (predicted
monoisotopic mass 1608.7). The protonated tetra-methylated AN2u was
further analyzed by CID mass spectrometry, giving a spectrum
confining all details of the C-terminal sequence. The
.gamma.-carboxyglutamate residue is clearly indicated by the
immonium ion at m/z 174, and its position revealed by the b5 and b6
molecular ions. The y2 ion confirms a C-terminal structure of
-Hyp-Ser-OMe, derived from the free carboxy terminal of PnVIIA.
Thus, the sequence of the peptide including the modified residues
.gamma.-carboxyglutamate and Hyp was confirmed; and the free
carboxy terminus established by mass spectrometry.
[0072] PnVIIA belongs to the large group of conotoxins with the
cysteine framework of .omega. and .delta. conotoxins, however, the
sequence is most homologous to conotoxin-TxVIIA (Table 4). These
homologies comprise approximately 48% amino acid identity and 63%
similarity, including positioning of most hydrophobic and some
charged residues, as well as one of the
.gamma.carboxyglutamates.
Example 4
Biological Activity of .gamma.-Conotoxin PnVIIA
[0073] Paralytic Activity of PnVllA. Initial injections of PnVIIA
to limpet snails (Patella) did not reveal the contractile paralysis
previously observed for TxVIIA and other conotoxins in this
bioassay (Fainzilber et al., 1991), however at doses above 50
pmoles/100 mg body weight some flaccidity of the foot musculature
could be observed. Flaccid or relaxation paralytic effects are more
easily observed in bioassays on bivalve molluscs, hence toxicity of
PnVIIA was quantified in bioassays in freshwater mussels (Mytilus),
as previously done for conotoxins PnIVA and PnIVB (Fainzilber et
al., 1995). The ED.sub.50 for Mytilus paralysis was 63.2 pmoles/100
mg body weight. No toxic or other effects could be observed upon
injection of 1 nmole PnVEA (15-fold higher than the Mytilus
ED.sub.50) per 100 mg body weight in Gambusia fish or blowfly
(Sarcophaga) larvae. Interestingly, decarboxylated PnVIIA had no
observable effects on Mytilus at doses of up to five-fold the
ED.sub.50 of the native peptide.
[0074] Electrophysiological Effects of PnVIIA on Lymnaea
Neuroendocrine Cells. Effects of PnVIIA were first screened in a
number of mollusc or vertebrate electrophysiological preparations.
Consistent effects were observed on caudodorsal neurons from the
snail Lymnaea stagnalis, and this system was therefore used for
detailed investigations on toxin activity. The caudodorsal neurons
are typical rythmic bursting cells responsible for production of
egg laying hormone, and their ionic currents have been
characterized exhaustively (Brussaard et al., 1991; Dreijer &
Kits, 1995; Kits & Mansvelder, 1996). In the first series of
experiments, PnVIIA was applied to caudodorsal neurons recorded
under current clamp and the effects on membrane potential and
action potential firing were investigated. It was found that PnVIIA
enhances the excitability of these cells in a dose-dependent way.
Thus, a dose-dependent increase in excitability of caudodorsal
cells (CDCs), inducing depolarization and repetitive spiking upon
application of micromolar doses of PnVIIA was seen. Cells that were
silent responded to low doses (.ltoreq.1 .mu.M) of the toxin by
depolarization, while doses of 10 .mu.M or more induced trains of
action potentials. The number of action potentials increased with
increasing doses. The duration of PnVIIA application also markedly
influenced the response. In silent cells, responding with a burst
of action potentials, the number of action potentials and the
duration of the burst increased with increasing duration of the
PnVIIA pulse. Thus, a time dependence of the excitatory effect of
PnVIIA in silent CDCs, showing increased duration of spiking with
increased duration of application was seen. Cells that were
spontaneously active responded by a temporary increase in firing
frequency, followed by an afterburst hyperpolarization during which
the cell stops firing for a short period. Increasing the duration
of PnVIIA application under these circumstances led to an increase
in the duration of the burst, but even more so in the duration of
the afterburst silent refractory period. Thus, a time dependence of
the excitatory effect of PnVIIA in spontaneously active CDCs,
showing that not only spiking increases but also the duration of
silent period after the afterburst increases with longer
applications was seen. The latter effect is possibly indirect, as a
natural consequence from the increased firing frequency induced by
the toxin.
[0075] Whether the effect was due to closure (blockade) or opening
(activation) of ion channels was investigated by measuring input
resistance of the cell membrane upon injection of hyperpolarizing
current pulses (30 .mu.A). The amplitude of the resulting
hyperpolarization is a direct measure of the membrane resistance.
In this experiment, pulses of hyperpolarizing current were injected
into CDCs, giving rise to hyperpolarizations of the membrane
potential. While the injected current is constant, the
hyperpolarizing response decreases upon application of PnVIIA,
showing that the membrane resistance decreases or, in other words,
the membrane conductance increases. It was seen that during PnVIIA
application hyperpolarization amplitude is strongly decreased
(.about.50% attenuation), thus revealing a marked decrease in
membrane resistance. Thus, PnVIIA induces an increase in
conduction, i.e., leads to the opening of ion channels, and
therefore acts primarily as a channel agonist or activator, rather
than as a channel blocker.
[0076] In a further series of experiments, the identity of the
channel(s) activated by PnVIIA was investigated. To this end, whole
cell voltage clamp experiments were performed on caudodorsal
neurons, however, no consistent effects of the toxin could be
observed on fast voltage gated sodium or calcium currents, nor on
the potassium currents that are activated in a standard voltage
step protocol. A slow ramp protocol was then applied to investigate
possible effects on slow voltage gated currents (also designated as
pacemaker currents) that are believed to underlie spontaneous
firing. In this experiment current responses to a voltage ramp
protocol in standard HBS during which the membrane potentials go
from -80 to +20 mV at a rate of mV/s (control) were measured. This
protocol will only reveal slow, voltage-gated currents, as fast
currents will inactivate during the slow voltage ramp. An inward
current is activated at .about.-30 mV and more positive. Most
likely, this represents a pacemaker current. With 10 .mu.M PnVIIA
(10 .mu.M) the voltage dependence shifts to the left (i.e., the
current activates already at more hyperpolarized potentials).
Furthermore, an increase in outward current at >.about.0 mV
occurs. Thus, the experiments indicated that a noninactivating
inward current is activated at voltages above .about.30 mV to the
voltage ramp protocol. Preliminary experiments indicate that this
inward current is a nonspecific cation current that is reduced in
Na.sup.+ free selective saline and completely blocked by 1 mM
Ni.sup.2+. Thus, most likely, Na.sup.+ and Ca.sup.2+ carry the
inward current. In voltage dependence and ion selectivity, this
current strongly resembles a pacemaker current in other Lymnaea
neurons elaborately described by van Soest and Kits (1997). In the
presence of 10 .mu.M PnVIIA, a dual effect was observed. First, the
activation range of the slow inward current shifted by .about.10 mV
to a more negative potential, thus accounting for the enhanced
excitability of the cells. Second, we saw an increase in
noninactivating outward current at potentials above .about.0 mV.
Whether the latter is a direct effect of PnVIIA, or an indirect
effect due to the increased inward current, remains to be
determined. It is, however, in line with the previously observed
prolongation of afterburst hyperpolarization under current clamp
conditions. These data show that the primary event mediating the
excitatory effects of PnVIIA on Lymnaea caudodorsal neurons is an
enhancement of a slow, voltage-activated inward cation channel.
Example 5
Isolation of a .gamma.-Conotoxin Tx6.4 from Conus textile
[0077] A Conus textile cDNA library was prepared from venom duct
using conventional techniques. DNA from single clones was amplified
by conventional techniques using primers which correspond
approximately to the M13 universal priming site and the M13 reverse
universal priming site. The primers which were used are:
TABLE-US-00005 5'-TTTCCCAGTCACGACGTT-3' (SEQ ID NO:46) and
5'-CACACAGGAAACAGCTATG-3'. (SEQ ID NO:47)
Clones having a size of approximately 300 nucleotides were
sequenced and screened for similarity in sequence to PnVIIA and
TxVIIA. A DNA was isolated having the sequence set forth in SEQ ID
NO:16, which encoded the propeptide sequence set forth in SEQ ID
NO:17. This new .gamma.-conotoxin has the sequence described above
and set forth in SEQ ID NO:7. Preferably, Xaa.sub.1 is Trp,
Xaa.sub.2 is .gamma.-Glu and Xaa.sub.3 is Hyp. The C-terminus
preferably contains a free hydroxyl group.
Example 6
Isolation of .gamma.-Conopeptides
[0078] The procedure of Example 5 was followed to isolate
additional nucleic acids encoding .gamma.-conopeptides. The nucleic
acids which were isolated have the nucleotide sequences set forth
in SEQ ID NOs:18, 20, 22, 24, 26, 28, 30, 32 and 34. These nucleic
acids encode the propeptides having the amino acid sequences set
forth in SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33 and 35,
respectively. The mature peptide sequences are set forth in SEQ ID
NOs:8-15 and 36.
Example 7
Biological Activity of .gamma.-Conotoxin TxVIIA
[0079] Isolated medial neurons from Aplysia oculifera pleuropedal
ganglia (Kehoe, 1972) were cultured as previously described
(Schacher & Proshansky, 1983). The neurons were cultured at
very low densities to prevent any possible synaptic interactions
among them. Passive and active membrane properties of the cultured
neurons were studied using conventional intracellular recording and
stimulation techniques. Briefly, the cell body of a cultured neuron
was impaled by two microelectrodes filled with 2 M KCl (5-10
M.OMEGA. resistance), one for current injection and the other for
voltage recording. Analysis of the resting potential, input
resistance, and action potential amplitude and shape was carried
out in artificial sea water composed of 460 nM NaCl, 10 mM KCl, 11
mM CaCl.sub.2, 55 mM MgCl.sub.2 and 10 mM Hepes, pH 7.6. Venom
fractions for electrophysiological experiments were dissolved in
artificial sea water containing 10 mg/ml bovine serum albumin. The
Sephadex G-50 fraction was applied at 100-200 .mu.g/ml and purified
toxin at final concentrations of 0.25-0.5 .mu.M.
[0080] The effects of venom fractions and purified toxin on
isolated Aplysia neurons were characterized by measuring the
resting potential, input resistance and action potential amplitude
and shape. The Vt fraction from the Sephadex.TM. G-50 column
(G-50-Vt) (Fainzilber et al., 1991) and the purified toxin revealed
significant effects at concentrations of 100 .mu.g/ml and 0.25-0.5
.mu.M, respectively. The effects of G-50-Vt, TxIA and TxIB were
essentially similar Fainzilber et al., 1991). These fractions
induced a transient membrane depolarization of 5-12 mV for 40-120
s. Within 3-30 s. after bath application of the toxin, the
quiescent neurons fired spontaneously. Concomitantly, the action
potential duration increased by one to two orders of magnitude,
extending in many experiments to over 1 s. The prolonged action
potentials are typically composed of an initial spike with a
prolonged shoulder. In the continuous presence of the toxin in the
bathing solution, the action potential duration gradually recovers.
20-30 min. after toxin application, the action potential duration
was only 50-100% longer than in the control. Throughout this
period, the threshold for action potential initiation was reduced.
The changes in membrane excitability and action potential duration
induced by the toxins were completely reversible upon washing of
the neuron with artificial sea water. TxI-induced prolongation of
the action potential duration was observed also when Ca.sup.2+ and
K.sup.+ conductances were blocked (Ca.sup.2+ free artificial sea
water, 16 mM Ca.sup.2+ and 50 mM tetraethylammonium, 150 .mu.M
3,4-diaminopyridine and 10 nM Cs.sup.+). Addition of tetrodotoxin
(10 .mu.M) under these conditions reduced the TxI-induced spike
prolongation. TxVIIA induced similar effects on the membrane
properties of isolated neurons, including membrane depolarization
and repetitive firing. However, TxVIIA did not cause any increase
in action potential duration.
[0081] The amino acid sequence of PnVIIA conserves the
six-cysteine, four-loop framework C...C...CC...C...C typical of
.omega. and .delta. conotoxins, and as shown in Table 4, is most
homologous to the sequence of conotoxin-TxVIIA, an excitatory toxin
from Conus textile venom (Fainziber et al., 1991; Nakamura et al.,
1996). Both of these toxins have an identical, extremely acidic net
charge (-5) and are similar in their surface
hydrophobic/hydrophilic interaction properties, as evidenced by
comparable elusion properties in reverse-phase chromatography.
Furthermore, the grow effects of these toxins in their respective
sensitive systems (Aplysia versus Lymaea neurons) are very similar,
comprising an enhancement of excitability decreased membrane
resistance, and increased repetitive firing. PnVIIA and TxVIIA may
therefore represent closely related members of the same family, or
convergent evolution to closely related receptor/channel
targets.
[0082] The paralytic activities of both TxVIIA and PnVIIA in their
respective bioassays are markedly decreased upon decarboxylation of
the .gamma.Glu residues. Although the primarily structural
importance of .gamma.Glu-metal chelates in mammalian vitamin
K-dependent blood coagulation proteins and in mollusc conantokins
is well established (Freedman et al., 1995; Skjaerbaek et al.,
1997), there is also evidence, for example, in prothrombin of a
functional role of individual .gamma.Glu residues in membrane
binding (Ratcliffe et al., 1993). Although the 3-D structures of
conotokins TxVIIA and .gamma.PnVIIA are most likely directed and
stabilized by the three disulfide bonds, as in conotoxins in
general, we cannot rule out at this stage a secondary
microstructural role of the .gamma.Glu residues. However, an
attractive hypothesis is that the .gamma.Glu residues in these
peptides form part of a membrane or receptor recognition patch,
with other variable residues (Table 4) providing specific
recognition for channel isoforms or subtypes. Hypervariability in
structurally related conotoxins is a well established mediator of
the exquisite selectivity of these peptides for receptor subtypes
(Myers et al., 1993; Nielsen et al., 1996).
[0083] 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, the described
embodiments are illustrative and should not be construed as
restrictive.
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(1996). U.S. Pat. No. 5,514,774. [0115] Olivera, B. M. et al.
(1997). U.S. Pat. No. 5,591,821. [0116] Partridge, L. D. &
Swandulla, D. (1988). Trends Neurosci. 11:69-72. [0117] Ratcliffe,
J. V., Furie, B., Furie, B. C. (1993). J. Biol. Chem.
268:24339-24345. [0118] Remington's Pharmaceutical Sciences, 17th
Ed., Mack Publishing Co., Easton, Pa. (1985). [0119] Rivier, J. R.
et al. (1978). Biopolymers 17:1927-38. [0120] Rivier, J. R. et al.
(1987). Total synthesis and further characterization of the
gamma-carboxy-glutamate-containing `sleeper` peptide from Conus
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The Peptides 5:342-429. [0122] Reuter, H. (1984). Annual Rev.
Physiol. 46:473-484. [0123] Sambrook, J. et al. (1979). Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. [0124] Schroder & Lubke
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D. J. (1997). J. Biol. Chem. 272:291-2299. [0126] Stewart and
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U.S. Pat. No. 3,862,925 (1975). [0134] PCT Published Application WO
96/11698
Sequence CWU 1
1
47 1 42 PRT Artificial Sequence Description of Artificial Sequence
generic formula of gamma-conopeptides 1 Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Cys Cys
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 2 42 PRT Artificial Sequence
Description of Artificial Sequence generic sequence of
gamma-conopeptides. 2 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 3 39 PRT Artificial Sequence Description of
Artificial Sequence generic formula of gamma-conopeptides 3 Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Cys Cys Ser Asn Ser Cys Asp Xaa Xaa Xaa Cys Xaa Xaa 20
25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 4 39 PRT Artificial Sequence
Description of Artificial Sequence generic sequence of
gamma-conopeptides. 4 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Cys Cys Ser Asn Ser Cys
Asp Xaa Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35
5 34 PRT Artificial Sequence Description of Artificial Sequence
generic sequence of gamma-conopeptides. 5 Xaa Xaa Xaa Cys Xaa Xaa
Xaa Phe Xaa Xaa Cys Thr Xaa Xaa Ser Xaa 1 5 10 15 Cys Cys Ser Asn
Ser Cys Asp Gln Thr Tyr Cys Xaa Leu Xaa Xaa Xaa 20 25 30 Xaa Xaa 6
32 PRT Conus pennaceus PEPTIDE (1)..(31) Xaa at residue 5 is Trp or
6-bromo-Trp; Xaa at residues 14 and 26 are Glu or
gamma-carboxyglutamate; Xaa at residue 31 is Pro or hydroxy-Pro. 6
Asp Cys Thr Ser Xaa Phe Gly Arg Cys Thr Val Asn Ser Xaa Cys Cys 1 5
10 15 Ser Asn Ser Cys Asp Gln Thr Tyr Cys Xaa Leu Tyr Ala Phe Xaa
Ser 20 25 30 7 34 PRT Conus textile PEPTIDE (1)..(34) Xaa at
residues 1, 7 and 34 are Trp or 6-bromo-Trp; Xaa at residues 3 and
16 are Glu or gamma-carboxyglutamate; Xaa at residues 31 and 32 are
Pro or hydroxy-Pro. 7 Xaa Leu Xaa Cys Ser Val Xaa Phe Ser His Cys
Thr Lys Asp Ser Xaa 1 5 10 15 Cys Cys Ser Asn Ser Cys Asp Gln Thr
Tyr Cys Thr Leu Met Xaa Xaa 20 25 30 Asp Xaa 8 39 PRT Conus textile
PEPTIDE (1)..(39) Xaa at residues 1, 2, 4, 10 and 39 are Trp or
6-bromo-Trp ; Xaa at residues 19 and 31 are Glu or
gammacarboxyglutamate; Xaa at residues 34, 36 and 37 are Pro or
hydroxy-Pro. 8 Xaa Xaa Arg Xaa Gly Gly Cys Met Ala Xaa Phe Gly Leu
Cys Ser Arg 1 5 10 15 Asp Ser Xaa Cys Cys Ser Asn Ser Cys Asp Val
Thr Arg Cys Xaa Leu 20 25 30 Met Xaa Phe Xaa Xaa Asp Xaa 35 9 27
PRT Conus textile PEPTIDE (1)..(27) Xaa at residues 9, 13 and 17
are Glu or gamma-carboxyglutamate. 9 Cys Lys Thr Tyr Ser Lys Tyr
Cys Xaa Ala Asp Ser Xaa Cys Cys Thr 1 5 10 15 Xaa Gln Cys Val Arg
Ser Tyr Cys Thr Leu Phe 20 25 10 34 PRT Conus textile PEPTIDE
(1)..(34) Xaa at residues 2, 3, 10 and 32 are Trp or 6-bromo-Trp;
Xaa at residues 18, 26 and 33 are Glu or gamma-carboxyglutamate;
Xaa at residue 12 is Pro or hydroxy-Pro. 10 Asp Xaa Xaa Asp Asp Gly
Cys Ser Val Xaa Gly Xaa Cys Thr Tyr Asn 1 5 10 15 Ala Xaa Cys Cys
Ser Gly Asp Cys His Xaa Thr Cys Ile Phe Gly Xaa 20 25 30 Xaa Val 11
31 PRT Conus textile PEPTIDE (1)..(31) Xaa at residues 3 and 31 are
Trp of 6-bromo-Trp; Xaa at residues 5, 18, 22 and 25 are Glu or
gamma-carboxyglutamate; Xaa at residue 16 is Pro or hydroxy-Pro. 11
Gly Met Xaa Gly Xaa Cys Lys Asp Gly Leu Thr Thr Cys Leu Ala Xaa 1 5
10 15 Ser Xaa Cys Cys Ser Xaa Asp Cys Xaa Gly Ser Cys Thr Met Xaa
20 25 30 12 32 PRT Conus gloriamaris PEPTIDE (1)..(32) Xaa at
residue 5 is Trp or 6-bromo-Trp; Xaa at residue 1 is Glu or
gamma-carboxyglutamate; Xaa at residues 8 and 11 are Pro or
hydroxy-Pro. 12 Xaa Cys Arg Ala Xaa Tyr Ala Xaa Cys Ser Xaa Gly Ala
Gln Cys Cys 1 5 10 15 Ser Leu Leu Met Cys Ser Lys Ala Thr Ser Arg
Cys Ile Leu Ala Leu 20 25 30 13 29 PRT Conus marmoreus PEPTIDE
(1)..(29) Xaa at residues 8 and 15 are Trp or 6-bromo-Trp; Xaa at
residues 5, 16 and 23 are Glu or gamma-carboxyglutamate; Xaa at
residue 10 is Pro or hydroxy-Pro. 13 Asn Gly Gln Cys Xaa Asp Val
Xaa Met Xaa Cys Thr Ser Asn Xaa Xaa 1 5 10 15 Cys Cys Ser Leu Asp
Cys Xaa Met Tyr Cys Thr Gln Ile 20 25 14 27 PRT Conus marmoreus
PEPTIDE (1)..(27) Xaa at residue 4 is Trp or 6-bromo-Trp; Xaa at
residues 9, 12, 13 and 17 are Glu or gamma-carboxyglutamate. 14 Cys
Gly Gly Xaa Ser Thr Tyr Cys Xaa Val Asp Xaa Xaa Cys Cys Ser 1 5 10
15 Xaa Ser Cys Val Arg Ser Tyr Cys Thr Leu Phe 20 25 15 26 PRT
Conus marmoreus PEPTIDE (1)..(26) Xaa at residues 8 and 15 are Trp
or 6-bromo-Trp; Xaa at residue 16 is Glu or gamma-carboxyglutamate.
15 Asn Gly Gly Cys Lys Ala Thr Xaa Met Ser Cys Ser Ser Gly Xaa Xaa
1 5 10 15 Cys Cys Ser Met Ser Cys Asp Met Tyr Cys 20 25 16 323 DNA
Conus textile CDS (1)..(153) 16 gaa cgg gct aag atc aac ttg ctt cca
aag aga aag cca cct gct gag 48 Glu Arg Ala Lys Ile Asn Leu Leu Pro
Lys Arg Lys Pro Pro Ala Glu 1 5 10 15 cgt tgg ttg gaa tgc agt gtt
tgg ttt tca cat tgt acg aag gac tcg 96 Arg Trp Leu Glu Cys Ser Val
Trp Phe Ser His Cys Thr Lys Asp Ser 20 25 30 gaa tgt tgt tct aat
agt tgt gac caa acg tac tgc acg tta atg cca 144 Glu Cys Cys Ser Asn
Ser Cys Asp Gln Thr Tyr Cys Thr Leu Met Pro 35 40 45 ccg gac tgg
tgacatcgcc actctcctgt tcagagtctt caaggctttt 193 Pro Asp Trp 50
gttctctttt gaagaatttt aacgagtgaa caaaaaagtg gactagcatg tttccttttc
253 cctttgcaaa atcaatgatg gaggtaaaag cctcccattt tgtcttcatc
aataaagaac 313 ttatcatcat 323 17 51 PRT Conus textile 17 Glu Arg
Ala Lys Ile Asn Leu Leu Pro Lys Arg Lys Pro Pro Ala Glu 1 5 10 15
Arg Trp Leu Glu Cys Ser Val Trp Phe Ser His Cys Thr Lys Asp Ser 20
25 30 Glu Cys Cys Ser Asn Ser Cys Asp Gln Thr Tyr Cys Thr Leu Met
Pro 35 40 45 Pro Asp Trp 50 18 510 DNA Conus textile CDS
(95)..(337) 18 tgactcgcca tctcctctct cagtctccct gacagctgcc
ttcagtcgac cctgccgtca 60 tctcaacgca cacttgaagt gaaaaacctt tatc atg
gag aaa ctg aca att ctg 115 Met Glu Lys Leu Thr Ile Leu 1 5 ctt ctt
gtt gct gct gta ctg ttg tcg atc cag gcc cta aat caa gaa 163 Leu Leu
Val Ala Ala Val Leu Leu Ser Ile Gln Ala Leu Asn Gln Glu 10 15 20
aaa cac caa cgg gca aag atc aac ttg ctt tca aag aga aag cca cct 211
Lys His Gln Arg Ala Lys Ile Asn Leu Leu Ser Lys Arg Lys Pro Pro 25
30 35 gct gag cgt tgg tgg cgg tgg gga gga tgc atg gct tgg ttt ggg
ctt 259 Ala Glu Arg Trp Trp Arg Trp Gly Gly Cys Met Ala Trp Phe Gly
Leu 40 45 50 55 tgt tcg agg gac tcg gaa tgt tgt tct aat agt tgt gac
gta acg cgc 307 Cys Ser Arg Asp Ser Glu Cys Cys Ser Asn Ser Cys Asp
Val Thr Arg 60 65 70 tgc gag tta atg cca ttc cca cca gac tgg
tgacatcgac actctcctct 357 Cys Glu Leu Met Pro Phe Pro Pro Asp Trp
75 80 tcagagtctt caaggctttt gttctctttt gaagaatttt tacgagtgaa
caaaaacgtg 417 gactagcacg tttccttttc cctttgcaaa atcaatgatg
gaggtaaaag tgtcccattt 477 tgtcttcatc aataaagaac ttatcatcat aat 510
19 81 PRT Conus textile 19 Met Glu Lys Leu Thr Ile Leu Leu Leu Val
Ala Ala Val Leu Leu Ser 1 5 10 15 Ile Gln Ala Leu Asn Gln Glu Lys
His Gln Arg Ala Lys Ile Asn Leu 20 25 30 Leu Ser Lys Arg Lys Pro
Pro Ala Glu Arg Trp Trp Arg Trp Gly Gly 35 40 45 Cys Met Ala Trp
Phe Gly Leu Cys Ser Arg Asp Ser Glu Cys Cys Ser 50 55 60 Asn Ser
Cys Asp Val Thr Arg Cys Glu Leu Met Pro Phe Pro Pro Asp 65 70 75 80
Trp 20 441 DNA Conus textile CDS (16)..(243) 20 ggaaaaactt ttatc
atg gag aaa ctg aca atc ctg ctc ctt gtt gct gct 51 Met Glu Lys Leu
Thr Ile Leu Leu Leu Val Ala Ala 1 5 10 gta ctg atg tcg acc cag gcc
atg ttt caa ggt gat gga gaa aaa tcc 99 Val Leu Met Ser Thr Gln Ala
Met Phe Gln Gly Asp Gly Glu Lys Ser 15 20 25 cgg aag gcg gag atc
aac ttt tct gaa aca aga aag ttg gcg aga aac 147 Arg Lys Ala Glu Ile
Asn Phe Ser Glu Thr Arg Lys Leu Ala Arg Asn 30 35 40 aag cag aaa
cgc tgc aaa act tat tca aag tat tgt gaa gct gac tcg 195 Lys Gln Lys
Arg Cys Lys Thr Tyr Ser Lys Tyr Cys Glu Ala Asp Ser 45 50 55 60 gaa
tgc tgt acc gaa cag tgt gta agg tct tac tgc acg ttg ttt gga 243 Glu
Cys Cys Thr Glu Gln Cys Val Arg Ser Tyr Cys Thr Leu Phe Gly 65 70
75 tgaattcgga ccacaagcca tccgatatca cccctctcct cttcagaggc
ttcaaggctt 303 ttgttatcct tttgaagaat ctttatcgag taaacataag
tagacaagct ttttttttcc 363 tttgcaaaat gaagaatgat ggcaaaaagc
cccccatttt gtcttcatca ataaagaact 423 cgctatcaga ataaaaaa 441 21 76
PRT Conus textile 21 Met Glu Lys Leu Thr Ile Leu Leu Leu Val Ala
Ala Val Leu Met Ser 1 5 10 15 Thr Gln Ala Met Phe Gln Gly Asp Gly
Glu Lys Ser Arg Lys Ala Glu 20 25 30 Ile Asn Phe Ser Glu Thr Arg
Lys Leu Ala Arg Asn Lys Gln Lys Arg 35 40 45 Cys Lys Thr Tyr Ser
Lys Tyr Cys Glu Ala Asp Ser Glu Cys Cys Thr 50 55 60 Glu Gln Cys
Val Arg Ser Tyr Cys Thr Leu Phe Gly 65 70 75 22 460 DNA Conus
textile CDS (49)..(273) 22 ctgccgtcat ctcagcgcac acttggtaag
aagtgaaaaa ccttgatc atg gag aaa 57 Met Glu Lys 1 ctg aca att ctg
ctt ctt gtt gct gct gtg ctg atg tcg acc cag gcc 105 Leu Thr Ile Leu
Leu Leu Val Ala Ala Val Leu Met Ser Thr Gln Ala 5 10 15 cta att caa
gat caa cgc caa aag gca aag atc aac ttg ttt tca aag 153 Leu Ile Gln
Asp Gln Arg Gln Lys Ala Lys Ile Asn Leu Phe Ser Lys 20 25 30 35 aga
cag gca tat gct cgt gat tgg tgg gac gat ggc tgc agt gtg tgg 201 Arg
Gln Ala Tyr Ala Arg Asp Trp Trp Asp Asp Gly Cys Ser Val Trp 40 45
50 ggg cct tgt acg gtg aac gca gaa tgt tgt tct ggt gat tgt cat gaa
249 Gly Pro Cys Thr Val Asn Ala Glu Cys Cys Ser Gly Asp Cys His Glu
55 60 65 acg tgc att ttc ggg tgg gaa gtc tgaccacaaa ccatccgaca
tcgccactct 303 Thr Cys Ile Phe Gly Trp Glu Val 70 75 cctcttcaga
gacttcaagg cttttgttct cttttgaaga attttacgag tgagcaaaaa 363
ggtagactag cacgtttctt tttccctttg caaaatcaat gatggaggta aaagcctccc
423 attttgtcct catcaataaa gaacttatca tcataat 460 23 75 PRT Conus
textile 23 Met Glu Lys Leu Thr Ile Leu Leu Leu Val Ala Ala Val Leu
Met Ser 1 5 10 15 Thr Gln Ala Leu Ile Gln Asp Gln Arg Gln Lys Ala
Lys Ile Asn Leu 20 25 30 Phe Ser Lys Arg Gln Ala Tyr Ala Arg Asp
Trp Trp Asp Asp Gly Cys 35 40 45 Ser Val Trp Gly Pro Cys Thr Val
Asn Ala Glu Cys Cys Ser Gly Asp 50 55 60 Cys His Glu Thr Cys Ile
Phe Gly Trp Glu Val 65 70 75 24 533 DNA Conus textile CDS
(110)..(337) misc_feature (1)..(533) n is an unknown nucleotide 24
ctctgccggt tgacacntca tctactctct cagtctccct gacagctgcc ttcagtcgac
60 cctgccgtca tctcagcgca gacttgataa gaagtgaaaa acctttatc atg gag
aaa 118 Met Glu Lys 1 ctg aca atc ctg ctt ctt gtt gct gct gta ctg
atg tcg acc cag gcc 166 Leu Thr Ile Leu Leu Leu Val Ala Ala Val Leu
Met Ser Thr Gln Ala 5 10 15 ctg gtt gaa cgt gct gga gaa aac cac tca
aag gag aac atc aat ttt 214 Leu Val Glu Arg Ala Gly Glu Asn His Ser
Lys Glu Asn Ile Asn Phe 20 25 30 35 tta tta aaa aga aag aga gct gct
gac agg ggg atg tgg ggc gaa tgc 262 Leu Leu Lys Arg Lys Arg Ala Ala
Asp Arg Gly Met Trp Gly Glu Cys 40 45 50 aaa gat ggg tta acg aca
tgt ttg gcg ccc tca gag tgt tgt tct gag 310 Lys Asp Gly Leu Thr Thr
Cys Leu Ala Pro Ser Glu Cys Cys Ser Glu 55 60 65 gat tgt gaa ggg
agc tgc acg atg tgg tgatgaattc tgaccacaag 357 Asp Cys Glu Gly Ser
Cys Thr Met Trp 70 75 ccatctgaca tcaccactct cctcttcaga ggcttcaagg
cttttgtttt ccttttgaat 417 aatctttacg agtaaacaaa taagtagact
agcgcgtttt tttccctttg agaaatcaat 477 gatggaggta aatagcttcc
tattttgtct tattcaataa agaacttatc ataata 533 25 76 PRT Conus textile
25 Met Glu Lys Leu Thr Ile Leu Leu Leu Val Ala Ala Val Leu Met Ser
1 5 10 15 Thr Gln Ala Leu Val Glu Arg Ala Gly Glu Asn His Ser Lys
Glu Asn 20 25 30 Ile Asn Phe Leu Leu Lys Arg Lys Arg Ala Ala Asp
Arg Gly Met Trp 35 40 45 Gly Glu Cys Lys Asp Gly Leu Thr Thr Cys
Leu Ala Pro Ser Glu Cys 50 55 60 Cys Ser Glu Asp Cys Glu Gly Ser
Cys Thr Met Trp 65 70 75 26 408 DNA Conus gloriamaris CDS
(2)..(211) 26 g ctg aca atc ctg ctt ctt gtt gct gct gta ctg atg tcg
acc cag gcc 49 Leu Thr Ile Leu Leu Leu Val Ala Ala Val Leu Met Ser
Thr Gln Ala 1 5 10 15 ctg att caa ggt ggt ggt gac aaa cgt caa aag
gca aac atc aac ttt 97 Leu Ile Gln Gly Gly Gly Asp Lys Arg Gln Lys
Ala Asn Ile Asn Phe 20 25 30 ctt tca agg tgg gac cgt gag tgc agg
gct tgg tat gcg ccg tgt agc 145 Leu Ser Arg Trp Asp Arg Glu Cys Arg
Ala Trp Tyr Ala Pro Cys Ser 35 40 45 cct ggc gcg caa tgt tgt agt
ttg ctg atg tgt tca aaa gcg acc agc 193 Pro Gly Ala Gln Cys Cys Ser
Leu Leu Met Cys Ser Lys Ala Thr Ser 50 55 60 cgc tgc ata ttg gcg
tta tgaactctga ccacaagcca tccgacatca 241 Arg Cys Ile Leu Ala Leu 65
70 ccactctcct cttcagaggc ttcaaggctt tttgtttttc ttttgaagaa
tctttacgag 301 tgaacaaata agtagaatag cacgtttttc cccctttgca
aaatcaataa tggaggttaa 361 aaaaaaactt ctgtcttctt caataaagaa
gttatcataa taaaaaa 408 27 70 PRT Conus gloriamaris 27 Leu Thr Ile
Leu Leu Leu Val Ala Ala Val Leu Met Ser Thr Gln Ala 1 5 10 15 Leu
Ile Gln Gly Gly Gly Asp Lys Arg Gln Lys Ala Asn Ile Asn Phe 20 25
30 Leu Ser Arg Trp Asp Arg Glu Cys Arg Ala Trp Tyr Ala Pro Cys Ser
35 40 45 Pro Gly Ala Gln Cys Cys Ser Leu Leu Met Cys Ser Lys Ala
Thr Ser 50 55 60 Arg Cys Ile Leu Ala Leu 65 70 28 278 DNA Conus
marmoreus CDS (4)..(222) 28 atc atg cag aaa ctg ata atc ctg ctt ctt
gtt gct gct gtg ctg ctg 48 Met Gln Lys Leu Ile Ile Leu Leu Leu Val
Ala Ala Val Leu Leu 1 5 10 15 tcg acc cag gcc cta aat caa gaa aaa
cgc cca aag gag atg atc aat 96 Ser Thr Gln Ala Leu Asn Gln Glu Lys
Arg Pro Lys Glu Met Ile Asn 20 25 30 ttt tta tca aaa gga aag aca
aat gct gag agg cgg aac ggc caa tgc 144 Phe Leu Ser Lys Gly Lys Thr
Asn Ala Glu Arg Arg Asn Gly Gln Cys 35 40 45 gag gat gtt tgg atg
cct tgt aca tcg aac tgg gaa tgc tgt tct ttg 192 Glu Asp Val Trp Met
Pro Cys Thr Ser Asn Trp Glu Cys Cys Ser Leu 50 55
60 gat tgt gaa atg tac tgc aca cag ata gga tgaactctga ccacaagcca
242 Asp Cys Glu Met Tyr Cys Thr Gln Ile Gly 65 70 tccgacatca
ccactctcct cttcagagtc ttcaag 278 29 73 PRT Conus marmoreus 29 Met
Gln Lys Leu Ile Ile Leu Leu Leu Val Ala Ala Val Leu Leu Ser 1 5 10
15 Thr Gln Ala Leu Asn Gln Glu Lys Arg Pro Lys Glu Met Ile Asn Phe
20 25 30 Leu Ser Lys Gly Lys Thr Asn Ala Glu Arg Arg Asn Gly Gln
Cys Glu 35 40 45 Asp Val Trp Met Pro Cys Thr Ser Asn Trp Glu Cys
Cys Ser Leu Asp 50 55 60 Cys Glu Met Tyr Cys Thr Gln Ile Gly 65 70
30 287 DNA Conus marmoreus CDS (4)..(231) 30 atc atg gag aaa ctg
aca atc ctg ctt ctt gtt gct gct gta ctg ata 48 Met Glu Lys Leu Thr
Ile Leu Leu Leu Val Ala Ala Val Leu Ile 1 5 10 15 ccg acc cag gcc
ctt ttt caa ggt gat gac gga aaa tcc cag aag gcg 96 Pro Thr Gln Ala
Leu Phe Gln Gly Asp Asp Gly Lys Ser Gln Lys Ala 20 25 30 gag atc
aag tct ttt gaa aca aga aag tta gcg aga aac aag cag gta 144 Glu Ile
Lys Ser Phe Glu Thr Arg Lys Leu Ala Arg Asn Lys Gln Val 35 40 45
cgc tgc ggt ggt tgg tca acg tat tgt gaa gtt gac gag gaa tgc tgt 192
Arg Cys Gly Gly Trp Ser Thr Tyr Cys Glu Val Asp Glu Glu Cys Cys 50
55 60 tcg gaa tca tgt gta agg tct tac tgc acg ctg ttt gga
tgaactcgga 241 Ser Glu Ser Cys Val Arg Ser Tyr Cys Thr Leu Phe Gly
65 70 75 ccacaagcca tccgatatca ccactctcct gttcagagtc ttcaag 287 31
76 PRT Conus marmoreus 31 Met Glu Lys Leu Thr Ile Leu Leu Leu Val
Ala Ala Val Leu Ile Pro 1 5 10 15 Thr Gln Ala Leu Phe Gln Gly Asp
Asp Gly Lys Ser Gln Lys Ala Glu 20 25 30 Ile Lys Ser Phe Glu Thr
Arg Lys Leu Ala Arg Asn Lys Gln Val Arg 35 40 45 Cys Gly Gly Trp
Ser Thr Tyr Cys Glu Val Asp Glu Glu Cys Cys Ser 50 55 60 Glu Ser
Cys Val Arg Ser Tyr Cys Thr Leu Phe Gly 65 70 75 32 278 DNA Conus
marmoreus CDS (4)..(213) 32 atc atg cag aaa ctg ata att ctg ctt ctt
gtt gct gct gtg ctg atg 48 Met Gln Lys Leu Ile Ile Leu Leu Leu Val
Ala Ala Val Leu Met 1 5 10 15 acg acc cag gcc cta tat caa gaa aaa
cgc cga aag gag atg atc aat 96 Thr Thr Gln Ala Leu Tyr Gln Glu Lys
Arg Arg Lys Glu Met Ile Asn 20 25 30 ttt tta tca aaa gga aag ata
aat gct gag agg cgg aac ggc gga tgc 144 Phe Leu Ser Lys Gly Lys Ile
Asn Ala Glu Arg Arg Asn Gly Gly Cys 35 40 45 aaa gct act tgg atg
tct tgt tca tcg ggc tgg gaa tgc tgt tct atg 192 Lys Ala Thr Trp Met
Ser Cys Ser Ser Gly Trp Glu Cys Cys Ser Met 50 55 60 agt tgt gac
atg tac tgc gga tagataggat gaactctgac cacaagccat 243 Ser Cys Asp
Met Tyr Cys Gly 65 70 ccgacatcac cactctcctc ttcagagtct tcaag 278 33
70 PRT Conus marmoreus 33 Met Gln Lys Leu Ile Ile Leu Leu Leu Val
Ala Ala Val Leu Met Thr 1 5 10 15 Thr Gln Ala Leu Tyr Gln Glu Lys
Arg Arg Lys Glu Met Ile Asn Phe 20 25 30 Leu Ser Lys Gly Lys Ile
Asn Ala Glu Arg Arg Asn Gly Gly Cys Lys 35 40 45 Ala Thr Trp Met
Ser Cys Ser Ser Gly Trp Glu Cys Cys Ser Met Ser 50 55 60 Cys Asp
Met Tyr Cys Gly 65 70 34 528 DNA Conus textile CDS (98)..(316) 34
gcacgtcatc ttctctctca gtctgcctga cagctgcctt cagtcaaccc tgccgtcatc
60 tcagcgtaga cttggtaaga agtgaaaaac atttatc atg cag aaa ctg ata atc
115 Met Gln Lys Leu Ile Ile 1 5 ctg ctt ctt gtt gct gct gtg ctg atg
tcg acc cag gcc gtg ctt caa 163 Leu Leu Leu Val Ala Ala Val Leu Met
Ser Thr Gln Ala Val Leu Gln 10 15 20 gaa aaa cgc cca aag gag aag
atc aag ctt tta tca aag aga aag aca 211 Glu Lys Arg Pro Lys Glu Lys
Ile Lys Leu Leu Ser Lys Arg Lys Thr 25 30 35 gat gct gag aag cag
cag aag cgc ctt tgc ccg gat tac acg gag cct 259 Asp Ala Glu Lys Gln
Gln Lys Arg Leu Cys Pro Asp Tyr Thr Glu Pro 40 45 50 tgt tca cat
gcc cat gaa tgc tgt tca tgg aat tgt tat aat ggg cac 307 Cys Ser His
Ala His Glu Cys Cys Ser Trp Asn Cys Tyr Asn Gly His 55 60 65 70 tgt
acg gga tgaactcgga ccacaagcca tccgacatca ccactctcct 356 Cys Thr Gly
cttcagaggc ttcaagactt ttgttctgat tttggacaat ctttacgagt aaacaaataa
416 ttagactagc actttttttc ccctttgcaa aatcaatgat ggaggtaaaa
agcctcccat 476 tttgtcttca tcaataaaga acttatcatc aaaaaaaaaa
aaaaaaaaaa aa 528 35 73 PRT Conus textile 35 Met Gln Lys Leu Ile
Ile Leu Leu Leu Val Ala Ala Val Leu Met Ser 1 5 10 15 Thr Gln Ala
Val Leu Gln Glu Lys Arg Pro Lys Glu Lys Ile Lys Leu 20 25 30 Leu
Ser Lys Arg Lys Thr Asp Ala Glu Lys Gln Gln Lys Arg Leu Cys 35 40
45 Pro Asp Tyr Thr Glu Pro Cys Ser His Ala His Glu Cys Cys Ser Trp
50 55 60 Asn Cys Tyr Asn Gly His Cys Thr Gly 65 70 36 26 PRT Conus
textile PEPTIDE (1)..(26) Xaa at residue 18 is Trp or 6-bromo-Trp;
Xaa at residues 7 and 14 are Glu or gamma-carboxyglutamate; Xaa at
residues 3 and 8 are Pro or hydroxy-Pro. 36 Leu Cys Xaa Asp Tyr Thr
Xaa Xaa Cys Ser His Ala His Xaa Cys Cys 1 5 10 15 Ser Xaa Asn Cys
Tyr Asn Gly His Cys Thr 20 25 37 4 PRT Artificial Sequence
Description of Artificial Sequence consensus gamma-conopeptide
sequence for probe 37 Xaa Cys Cys Ser 1 38 12 DNA Artificial
Sequence Description of Artificial Sequence degenerate probe for
consensus gamma-conopeptide sequence. 38 sartgytgya gy 12 39 12 DNA
Artificial Sequence Description of Artificial Sequence degenerate
probe for consensus gamma-conopeptide sequence. 39 sartgytgyt cn 12
40 8 PRT Artificial Sequence Description of Artificial Sequence
consensus pro-gamma-conopeptide sequence for probe. 40 Ile Leu Leu
Val Ala Ala Val Leu 1 5 41 24 DNA Artificial Sequence Description
of Artificial Sequence degenerate probe for consensus
pro-gamma-conopeptide sequence. 41 athytnytng tngcngcngt nytn 24 42
32 PRT Conus pennaceus PEPTIDE (1)..(31) Xaa at residues 14 and 26
are gamma-carboxyglutamate; Xaa at residue 31 is hdroxy-Pro. 42 Asp
Cys Thr Ser Trp Phe Gly Arg Cys Thr Val Asn Ser Xaa Cys Cys 1 5 10
15 Ser Asn Ser Cys Asp Gln Thr Tyr Cys Xaa Leu Tyr Ala Phe Xaa Ser
20 25 30 43 27 PRT Conus textile PEPTIDE (1)..(27) Xaa at residues
9 and 13 are gamma-carboxyglutamate. 43 Cys Gly Gly Tyr Ser Thr Tyr
Cys Xaa Val Asp Ser Xaa Cys Cys Ser 1 5 10 15 Asp Asn Cys Val Arg
Ser Tyr Cys Thr Leu Phe 20 25 44 8 PRT Conus pennaceus MOD_RES (2)
Xaa at residue 2 is carboxymethylCys 44 Asp Xaa Thr Ser Trp Phe Gly
Arg 1 5 45 24 PRT Conus pennaceus PEPTIDE (1)..(24) Xaa at residues
6 and 18 are gamma-carboxyglutamate; Xaa at residue 23 is
hydroxy-Pro. 45 Xaa Thr Val Asn Ser Xaa Xaa Xaa Ser Asn Ser Xaa Asp
Gln Thr Tyr 1 5 10 15 Xaa Xaa Leu Tyr Ala Phe Xaa Ser 20 46 18 DNA
Artificial Sequence Description of Artificial Sequence primer for
M13 universal priming site. 46 tttcccagtc acgacgtt 18 47 19 DNA
Artificial Sequence Description of Artificial Sequence primer for
M13 reverse priming site. 47 cacacaggaa acagctatg 19
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