U.S. patent application number 10/058053 was filed with the patent office on 2003-09-11 for beta-superfamily conotoxins.
This patent application is currently assigned to University of Utah Research Foundation. Invention is credited to Garrett, James E., Jones, Robert M., Olivera, Baldomero M., Watkins, Maren.
Application Number | 20030170222 10/058053 |
Document ID | / |
Family ID | 23005536 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170222 |
Kind Code |
A1 |
Jones, Robert M. ; et
al. |
September 11, 2003 |
Beta-superfamily conotoxins
Abstract
The present invention is directed to .beta.-superfamily
conotoxin peptides, derivatives or pharmaceutically acceptable
salts thereof. The present invention is further directed to the use
of this peptide, derivatives thereof and pharmaceutically
acceptable salts thereof for the treatment of disorders associated
with voltage-gated ion channels, ligand gated channels and other
receptors. The invention is further directed to nucleic acid
sequences encoding the .beta.-superfamily conotoxin peptides and
encoding .beta.-superfamily conotoxin propeptides, as well as the
.beta.-superfamily conotoxin propeptides.
Inventors: |
Jones, Robert M.; (Park
City, UT) ; Olivera, Baldomero M.; (Salt Lake City,
UT) ; Watkins, Maren; (Salt Lake City, UT) ;
Garrett, James E.; (Salt Lake City, UT) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
University of Utah Research
Foundation
Salt Lake City
UT
|
Family ID: |
23005536 |
Appl. No.: |
10/058053 |
Filed: |
January 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60264323 |
Jan 29, 2001 |
|
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|
Current U.S.
Class: |
424/94.1 ;
514/11.1; 514/12.5; 514/13.3; 514/13.9; 514/14.7; 514/16.1;
514/16.3; 514/17.4; 514/18.1; 514/18.9; 514/19.3; 514/19.5;
514/20.6; 514/6.9; 530/324 |
Current CPC
Class: |
C07K 14/43504
20130101 |
Class at
Publication: |
424/94.1 ;
514/12; 530/324 |
International
Class: |
A61K 038/17; A61K
038/43; C07K 014/435 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. PO1 GM48677 awarded by the National Institute of General
Medical Sciences, National Institutes of Health, Bethesda, Md. The
United States Government has certain rights in the invention.
Claims
What is claimed is:
1. An isolated peptide selected from the group consisting of: (a) a
peptide selected from the group consisting of peptides comprising
amino acid sequences set forth in SEQ ID NOs:3, 6, 9, 12, 15, 18,
21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69,
72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114,
117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153,
156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192,
195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228-287, 289-315,
319-321, 323-337 and 340; and (b) analogs and derivatives of the
peptide in (a).
2. The peptide of claim 1, wherein Xaa1 is Glu or
.gamma.-carboxy-Glu, Xaa2 is Gln or pyro-Glu, Xaa3 is Pro or
trans-4-hydroxy-Pro, Xaa4 is D or L Trp or D or L 6-bromo-Trp, and
Xaa5 is Tyr, mono-iodo-Tyr, .sup.125I -Tyr, di-iodo-Tyr,
O-sulpho-Tyr or O-phospho-Tyr.
3. The peptide derivative of claim 1, in which Arg residues may be
substituted by Lys, ornithine, homoarginine, nor-Lys, N-methyl-Lys,
N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino
acid; the Lys residues may be substituted by Arg, ornithine,
homoarginine, nor-Lys, or any synthetic basic amino acid; the Tyr
residues may be substituted with meta-Tyr, ortho-Tyr, nor-Tyr,
.sup.125I-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
O-phospho-Tyr, nitro-Tyr or any synthetic hydroxy containing amino
acid; the Ser residues may be substituted with Thr or any synthetic
hydroxylated amino acid; the Thr residues may be substituted with
Ser or any synthetic hydroxylated amino acid; the Phe residues may
be D or L, may be substituted at the ortho, meta, and/or para
positions with a halogen or may be substituted with any synthetic
aromatic amino acid; the Trp residues may be substituted with Trp
(D), neo-Trp, 6-halo-Trp (D or L), preferably 6-halo, or any
aromatic synthetic amino acid; the Asn, Ser, Thr or Hyp residues
may be substituted with a glycan; the halogen may be iodo, chloro,
fluoro or bromo; the Tyr residues may also be substituted with the
3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr,
respectively) and corresponding O-sulpho- and
O-phospho-derivatives; the acidic amino acid residues may be
substituted with any synthetic acidic amino acid, e.g., tetrazolyl
derivatives of Gly and Ala; the Leu may be substituted with Leu
(D); the Glu residues may be substituted with Gla or Asp; the
acidic amino acid residues may be substituted with any synthetic
acidic amino acid, e.g. tetrazolyl derivatives of Gly and Ala; the
N-terminal Gln may be substituted with pyro-glutamate (Z); the
aliphatic amino acids may be substituted by synthetic derivatives
bearing non-natural aliphatic branched or linear side chains
C.sub.nH.sub.2n+2 up to and including n=8; the Met residues may be
substituted with nor-leucine (Nle); the Cys residues may be in D or
L configuration and may optionally be substituted with homocysteine
(D or L); basic residues in the backbone may be D or L
configuration; the central Trp residue within the beta-turn is
preferably epimerized to the D-form; pairs of Cys residues may be
replaced pairwise with isoteric lactam or ester-thioether
replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu
or Asp) or Cys/Ala combinations; and individual Cys residues may be
replaced with homoCys, seleno-Cys or penicillamine, so that
disulfide bridges may be formed between Cys-homoCys or
Cys-penicillamine, or homoCys-penicillamine.
4. The peptide derivative of claim 3, wherein the glycan is any N-,
S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can
be attached to any hydroxy, amino or thiol group of natural or
modified amino, wherein the monosaccharides making up the glycan
can be unmodified or modified D-allose, D-altrose, D-glucose,
D-mannose, D-gulose, D-idose, D-galactose, D-talose,
D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),
D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose, wherein
the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3, and
wherein the linkage between the glycan and the amino acid may be
alpha or beta, preferably alpha and is 1-.
5. The peptide derivative of claim 4, wherein the modification may
include one or more O-sulfate, O-phosphate, O-acetyl or acidic
groups, and combinations thereof.
6. The peptide derivative of claim 4, wherein the gylcan may also
include similar polyhydroxy groups, such as D-penicillamine 2,5 and
halogenated derivatives thereof or polypropylene glycol
derivatives.
7. The derivative of the peptide of claim 1 in which the peptide is
truncated.
8. The peptide derivative of claim 3 in which the peptide
derivative is truncated.
9. The peptide derivative of claim 4 in which the peptide
derivative is truncated.
10. An peptide of claim 1 containing 4 cysteines which are bridged
as [1,4/2,3].
11. An peptide of claim 1 containing 4 cysteines which are bridged
as [1,3/2,4].
12. The peptide of claim 1, wherein the peptide is tagged with a
radiolabel.
13. The peptide derivative of claim 1 in which a basic or aromatic
amino acid in the beta turn is a D-isomer.
14. The peptide derivative of claim 13 in which the peptide
derivative is truncated.
15. The peptide of claim 13, wherein the peptide is tagged with a
radiolabel.
16. The peptide of claim 14, wherein the peptide is tagged with a
radiolabel.
17. An isolated nucleic acid encoding a .beta.-superfamily
conopeptide propeptide selected from the group of propeptides
comprising amino acid sequences set forth in SEQ ID NO:2, 5, 8, 11,
14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62,
65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110,
113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149,
152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188,
191, 194, 197, 200, 203, 206, 209, 215, 218, 221, 224, and 227.
18. The isolated nucleic acid of claim 17 wherein the nucleic acid
comprises a nucleotide sequence selected from the group of
nucleotide sequences set forth in SEQ ID NO:1, 4, 7, 10, 13, 16,
19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67,
70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112,
115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151,
154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190,
193, 196, 199, 202, 205, 208, 214, 217, 220, 223, and 226.
19. An isolated .beta.-superfamily conopeptide propeptide selected
from the group of propeptides comprising amino acid sequences set
forth in SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38,
41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89,
92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131,
134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170,
173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209,
215, 218, 221, 224, and 227.
20. A method for treating cancer which comprises administering an
active agent or a pharmaceutically acceptable salt thereof to an
individual having cancer, wherein said active agent is a peptide
tagged with a radionuclide, wherein said peptide is a
.beta.-superfamily conotoxin.
21. The method of claim 20, wherein said .beta.-superfamily
conotoxin is selected from the group consisting of: (a) a peptide
selected from the group consisting of peptides comprising amino
acid sequences set forth in SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24,
27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75,
78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120,
123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159,
162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198,
201, 204, 207, 210, 216, 219, 222, 225, 228-287, 289-315, 319-321,
323-337 and 340; and (b) analogs and derivatives of the peptide in
(a).
22. The method of claim 20, wherein the radionuclide is selected
from the group consisting of .sup.313iodine, .sup.123iodine,
.sup.99mtechnicium, .sup.111indium, .sup.188rhenium,
.sup.186rhenium, .sup.67gallium, .sup.90yttrium, .sup.105rhodium,
.sup.89strontium, .sup.153samarium, .sup.211astatine,
.sup.212bismuth, .sup.213bismuth, .sup.177 lutetium, .sup.64copper,
.sup.67copper, .sup.47scandium, .sup.109palladium.
23. The method of claim 21, wherein the radionuclide is selected
from the group consisting of .sup.131iodine, .sup.123iodine,
.sup.99mtechnicium, .sup.111indium, .sup.188rhenium,
.sup.186rhenium, .sup.67gallium, .sup.90yttrium, .sup.105rhodium,
.sup.89 strontium, .sup.153samarium, .sup.211astatine,
.sup.212bismuth, .sup.213bismuth, .sup.177lutetium, .sup.64copper,
.sup.67copper, .sup.47scandium, .sup.109palladium.
24. The method of claim 21, wherein the active agent a peptide
derivative in which Arg residues may be substituted by Lys,
ornithine, homoarginine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys,
N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys
residues may be substituted by Arg, ornithine, homoarginine,
nor-Lys, or any synthetic basic amino acid; the Tyr residues may be
substituted with meta-Tyr, ortho-Tyr, nor-Tyr, .sup.125I-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr
or any synthetic hydroxy containing amino acid; the Ser residues
may be substituted with Thr or any synthetic hydroxylated amino
acid; the Thr residues may be substituted with Ser or any synthetic
hydroxylated amino acid; the Phe residues may be D or L, may be
substituted at the ortho, meta, and/or para positions with a
halogen or may be substituted with any synthetic aromatic amino
acid; the Trp residues may be substituted with Trp (D), neo-Trp,
6-halo-Trp (D or L), preferably 6-halo, or any aromatic synthetic
amino acid; the Asn, Ser, Thr or Hyp residues may be substituted
with a glycan; the halogen may be iodo, chloro, fluoro or bromo;
the Tyr residues may also be substituted with the 3-hydroxyl or
2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and
corresponding O-sulpho- and O-phospho-derivatives; the acidic amino
acid residues may be substituted with any synthetic acidic amino
acid, e.g., tetrazolyl derivatives of Gly and Ala; the Leu may be
substituted with Leu (D); the Glu residues may be substituted with
Gla or Asp; the acidic amino acid residues may be substituted with
any synthetic acidic amino acid, e.g. tetrazolyl derivatives of Gly
and Ala; the N-terminal Gln may be substituted with pyro-glutamate
(Z); the aliphatic amino acids may be substituted by synthetic
derivatives bearing non-natural aliphatic branched or linear side
chains C.sub.nH.sub.2n+2 up to and including n=8; the Met residues
may be substituted with nor-leucine (Nle); the Cys residues may be
in D or L configuration and may optionally be substituted with
homocysteine (D or L); basic residues in the backbone may be D or L
configuration; the central Trp residue within the beta-turn is
preferably epimerized to the D-form; pairs of Cys residues may be
replaced pairwise with isoteric lactam or ester-thioether
replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu
or Asp) or Cys/Ala combinations; and individual Cys residues may be
replaced with homoCys, seleno-Cys or penicillamine, so that
disulfide bridges may be formed between Cys-homoCys or
Cys-penicillamine, or homoCys-penicillamine.
25. The method of claim 24, wherein the the glycan is any N-, S- or
O-linked mono-, di-, tri-, poly- or oligosaccharide that can be
attached to any hydroxy, amino or thiol group of natural or
modified amino, wherein the monosaccharides making up the glycan
can be unmodified or modified D-allose, D-altrose, D-glucose,
D-mannose, D-gulose, D-idose, D-galactose, D-talose,
D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),
D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose, wherein
the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3, and
wherein the linkage between the glycan and the amino acid may be
alpha or beta, preferably alpha and is 1-.
26. The method of claim 21, wherein the peptide has activity at
somatostatin receptors.
27. A method of alleviating pain in an individual which comprises
administering to an individual who is either exhibiting pain or is
about to be subjected to a pain-causing event a pain-alleviating
amount of an active agent or a pharmaceutically acceptible salt
thereof, wherein said active agent is a .beta.-superfamily
conotoxin.
28. The method of claim 27, wherein the .beta.superfamily conotoxin
is selected from the group consisting of: (a) a peptide selected
from the group consisting of peptides comprising amino acid
sequences set forth in SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24, 27,
30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78,
81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123,
126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162,
165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201,
204, 207, 210, 216, 219, 222, 225, 228-287, 289-315, 319-321,
323-337 and 340; and (b) analogs and derivatives of the peptide in
(a).
29. The method of claim 28, wherein the active agent a peptide
derivative in which Arg residues may be substituted by Lys,
ornithine, homoarginine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys,
N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys
residues may be substituted by Arg, ornithine, homoarginine,
nor-Lys, or any synthetic basic amino acid; the Tyr residues may be
substituted with meta-Tyr, ortho-Tyr, nor-Tyr, .sup.125I-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr
or any synthetic hydroxy containing amino acid; the Ser residues
may be substituted with Thr or any synthetic hydroxylated amino
acid; the Thr residues may be substituted with Ser or any synthetic
hydroxylated amino acid; the Phe residues may be D or L, may be
substituted at the ortho, meta, and/or para positions with a
halogen or may be substituted with any synthetic aromatic amino
acid; the Trp residues may be substituted with Trp (D), neo-Trp,
6-halo-Trp (D or L), preferably 6-halo, or any aromatic synthetic
amino acid; the Asn, Ser, Thr or Hyp residues may be substituted
with a glycan; the halogen may be iodo, chloro, fluoro or bromo;
the Tyr residues may also be substituted with the 3-hydroxyl or
2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and
corresponding O-sulpho- and O-phospho-derivatives; the acidic amino
acid residues may be substituted with any synthetic acidic amino
acid, e.g., tetrazolyl derivatives of Gly and Ala; the Leu may be
substituted with Leu (D); the Glu residues may be substituted with
Gla or Asp; the acidic amino acid residues may be substituted with
any synthetic acidic amino acid, e.g. tetrazolyl derivatives of Gly
and Ala; the N-terminal Gln may be substituted with pyro-glutamate
(Z); the aliphatic amino acids may be substituted by synthetic
derivatives bearing non-natural aliphatic branched or linear side
chains C.sub.nH.sub.2n+2 up to and including n=8; the Met residues
may be substituted with nor-leucine (Nle); the Cys residues may be
in D or L configuration and may optionally be substituted with
homocysteine (D or L); basic residues in the backbone may be D or L
configuration; the central Trp residue within the beta-turn is
preferably epimerized to the D-form; pairs of Cys residues may be
replaced pairwise with isoteric lactam or ester-thioether
replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu
or Asp) or Cys/Ala combinations; and individual Cys residues may be
replaced with homoCys, seleno-Cys or penicillamine, so that
disulfide bridges may be formed between Cys-homoCys or
Cys-penicillamine, or homoCys-penicillamine.
30. The method of claim 29, wherein the the glycan is any N-, S- or
O-linked mono-, di-, tri-, poly- or oligosaccharide that can be
attached to any hydroxy, amino or thiol group of natural or
modified amino, wherein the monosaccharides making up the glycan
can be unmodified or modified D-allose, D-altrose, D-glucose,
D-mannose, D-gulose, D-idose, D-galactose, D-talose,
D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),
D-N-acetyl-galactosamine (GalNAC), D-fucose or D-arabinose, wherein
the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3, and
wherein the linkage between the glycan and the amino acid may be
alpha or beta, preferably alpha and is 1-.
31. The method of claim 27, wherein the pain is visceral pain.
32. A method for treating or preventing disorders associated with a
disorder selected from the group consisting of voltage-gated ion
channel disorders, ligand-gated ion channel disorders and receptor
disorders, such as disorders of G-protein coupled receptors, in an
individual which comprises administering to an individual in need
thereof a therapeutically effective amount of an active agent or a
pharmaceutically acceptible salt thereof, wherein the active agent
is a .beta.-superfamily conotoxin.
33. The method of claim 32, wherein the .beta.-superfamily
conotoxin is selected from the group consisting of: (a) a peptide
selected from the group consisting of peptides comprising amino
acid sequences set forth in SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24,
27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75,
78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120,
123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159,
162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198,
201, 204, 207, 210, 216, 219, 222, 225, 228-287, 289-315, 319-321,
323-337 and 340; and (b) analogs and derivatives of the peptide in
(a).
34. The method of claim 33, wherein the active agent a peptide
derivative in which Arg residues may be substituted by Lys,
ornithine, homoarginine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys,
N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys
residues may be substituted by Arg, ornithine, homoarginine,
nor-Lys, or any synthetic basic amino acid; the Tyr residues may be
substituted with meta-Tyr, ortho-Tyr, nor-Tyr, .sup.125-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr
or any synthetic hydroxy containing amino acid; the Ser residues
may be substituted with Thr or any synthetic hydroxylated amino
acid; the Thr residues may be substituted with Ser or any synthetic
hydroxylated amino acid; the Phe residues may be D or L, may be
substituted at the ortho, meta, and/or para positions with a
halogen or may be substituted with any synthetic aromatic amino
acid; the Trp residues may be substituted with Trp (D), neo-Trp,
6-halo-Trp (D or L), preferably 6-halo, or any aromatic synthetic
amino acid; the Asn, Ser, Thr or Hyp residues may be substituted
with a glycan; the halogen may be iodo, chloro, fluoro or bromo;
the Tyr residues may also be substituted with the 3-hydroxyl or
2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and
corresponding O-sulpho- and O-phospho-derivatives; the acidic amino
acid residues may be substituted with any synthetic acidic amino
acid, e.g., tetrazolyl derivatives of Gly and Ala; the Leu may be
substituted with Leu (D); the Glu residues may be substituted with
Gla or Asp; the acidic amino acid residues may be substituted with
any synthetic acidic amino acid, e.g. tetrazolyl derivatives of Gly
and Ala; the N-terminal Gln may be substituted with pyro-glutamate
(Z); the aliphatic amino acids may be substituted by synthetic
derivatives bearing non-natural aliphatic branched or linear side
chains C.sub.nH.sub.2n+2 up to and including n=8; the Met residues
may be substituted with nor-leucine (Nle); the Cys residues may be
in D or L configuration and may optionally be substituted with
homocysteine (D or L); basic residues in the backbone may be D or L
configuration; the central Trp residue within the beta-turn is
preferably epimerized to the D-form; pairs of Cys residues may be
replaced pairwise with isoteric lactam or ester-thioether
replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu
or Asp) or Cys/Ala combinations; and individual Cys residues may be
replaced with homoCys, seleno-Cys or penicillamine, so that
disulfide bridges may be formed between Cys-homoCys or
Cys-penicillamine, or homoCys-penicillamine.
35. The method of claim 34, wherein the the glycan is any N-, S- or
O-linked mono-, di-, tri-, poly- or oligosaccharide that can be
attached to any hydroxy, amino or thiol group of natural or
modified amino, wherein the monosaccharides making up the glycan
can be unmodified or modified D-allose, D-altrose, D-glucose,
D-mannose, D-gulose, D-idose, D-galactose, D-talose,
D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),
D-N-acetyl-galactosamine (GalNac), D-fucose or D-arabinose, wherein
the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3, and
wherein the linkage between the glycan and the amino acid may be
alpha or beta, preferably alpha and is 1-.
36. The method of claim 32, wherein the disorder is a G-protein
coupled receptor disorder.
37. The method of claim 36, wherein the G-protein coupled receptor
is selected from the group consisting of sst, cortistatin (CST),
melanocortin (MC.sub.xR, wherein x=1, 2, 3, 4, 5), opioid (.mu.,
.delta., .kappa.), neurokinin, bradykinin, galanin, CCK.sub.A,
CCK.sub.B, endothelin, serotonin, adrenergic receptors,
angiotensin, neuropeptide-Y, sigmal, sigma2, oxytocin, CGRP, GRPR,
histamine, imidazoline, neurotensin, VIP, vasopressin, substance K,
chemokine receptors, CRF.sub.1, CRF.sub.2a, CRF.sub.2.beta.,
CRF.sub.2.gamma., CRF-BP, orexin, urotensin, glycoprotein IIb/IIIa,
thrombin receptors and orphan GPCRs.
38. The method of claim 36, wherein the GPCR is selected from the
group consisting of MCH.sub.2R/SLT, SP1999/P.sub.2Y.sub.12,
CRTH.sub.2, NPFF.sub.1, NPFF.sub.2, HH.sub.4R, h-GPR.sub.54,
CysLT.sub.2, neuromedin receptors, BLTR.sub.2, G.sub.2A, TA.sub.1,
LTB.sub.4, ghrelin, motilin MTL-R, purinergic receptors, muscarinic
receptors, ORL-1, apelin, CB.sub.1, CB.sub.2 and GPCRs of orphan
status having no known cognate ligand.
39. The method of claim 32, wherein the disorder is selected from
the group consisting of cancer, neoplasm, solid tumor, diabetic
nephropathy, fibrosis, hypophysis tumor, GI disease, IBS,
restinosis, angiogenesis disorder, diabetes mellitus, endocrine
tumor, diarrhea, pancreatic disease, prostate tumor, bleeding and
apoptosis.
40. The method of claim 39, wherein the peptide has activity at
somatostatin receptors.
41. The method of claim 32, wherein the disorder is selected from
the group consisting of inflammation, pain, diabetes, obesity,
sexual dysfunction, acromegaly, glaucoma, cardiovascular, diabetic,
retinopathy, depression, myocardial infarction, stroke, epilepsy,
anorexia, wasting diseases, seborrheic dermatitis, schizophrenia,
mood disorders, chemotherapeutic induced emesis, disorders
associated with changes in blood pressure, immune disorders, nerve
damage, acne, GI infections, myocardial infarction, angina,
thromboembolism and cardiovascular disease.
42. The method of claim 41, wherein the peptide has activity at
somatostatin receptors or at melanocortin receptors.
43. The method of claim 32, wherein the receptor is LHRH.
44. The method of claim 43, wherein the disorder is
osteoporosis.
45. The method of claim 32, wherein the disorder is associated with
a melanocortin system or MCR dysfunction.
46. The method of claim 45, wherein the disorder is selected from
the group consisting of erectile dysfunction, obesity inflammation
and melanoma.
47. The method of claim 45, wherein the peptide contains a
.beta.-turn.
48. The method of claim 47, wherein the peptide is a .beta.-Ge14.1
analogue, derivative thereof or pharmaceutically acceptable salt
thereof, wherein the .beta.-Ge14.1 analogue is selected from the
group of peptides comprising an amino acid sequence set forth in
SEQ ID NOs:334-337,.
49. A method for identifying drug candidates for use as treating or
preventing disorders associated with a disorder selected from the
group consisting of voltage-gated ion channel disorders,
ligand-gated ion channel disorders and receptor disorders, such as
disorders of G-protein coupled receptors which comprises screening
a drug candidate for its action at or partially at the same
functional site as a .beta.-superfamily conotoxin and capable of
elucidation of similar functional response as said conotoxin.
50. The method of claim 49, wherein the displacement of a labeled
.beta.-superfamily conotoxin from its receptor or other complex by
a candidate drug agent is used to identify suitable candidate
drugs.
51. The method of claim 49, wherein a biological assay on a test
compound to determine the therapeutic activity is conducted and
compared to the results obtained from the biological assay of a
.beta.-superfamily conotoxin.
52. The method of claim 49, wherein the binding affinity of a small
molecule to the receptor of a .beta.-conotoxin is measured and
compared to the binding affinity of a .beta.-superfamily conotoxin
to its receptor.
53. A method of identifying compounds that mimic the therapeutic
activity of a .beta.-superfamily conotoxin, comprising the steps
of: (a) conducting a biological assay on a test compound to
determine the therapeutic activity; and (b) comparing the results
obtained from the biological assay of the test compound to the
results obtained from the biological assay of a .beta.-superfamily
conotoxin.
54. A method for characterizing a new site on a voltage-gated ion
channel, a ligand-gated ion channel and a receptor, such as a
G-protein coupled receptorswhich comprises contacting a peptide of
claim 1 with a channel or receptor and measuring the binding of the
peptide with the channel or receptor or by measuring a functional
in vitro parameters such as fluorescence, phosphorescence and
illuminescence.
55. The method of claim 54, wherein the receptor is a G-protein
coupled receptor.
56. The method of claim 54, wherein the peptide is
radiolabeled.
57. A method for designing a .beta.-beta turn mimetic of a
.beta.-superfamily conotoxin containing a .beta.-turn motif
selected from the group consistinf of (i) a -CX1X2KX1C- (SEQ ID
NO:338) motif, wherein X1 is any amino acid and X2 is D or L Trp or
D or L 6-bromo Trp and (ii) a -CPX3RVC- (SEQ ID NO:339) motif,
wherein X3 is D or L Phe, which comprises replacing this motif with
a non-peptide turn mimetic .beta.-turn scaffold and then attaching
receptor binding domains contained within the N and C-terminal
sequences of a .beta.-superfamily conotoxin to the .beta.-turn
scaffold to mimic the 3D spatial array within the native
.beta.-superfamily conotoxin.
58. A method for identifying a ligand which binds to an orphan
G-protein coupled receptor (orphan GPCR) which comprises contacting
a peptide of claim 1 or a radiolabeled derivative of the peptide
with an orphan GPCR and measuring the amount of binding of the
peptide to the orphan GPCR.
59. The method of claim 58, wherein the peptide is
radiolabeled.
60. The method of claim 59, wherein the radiolabel is selected from
the group consisting of .sup.3H and .sup.125I.
61. The method of claim 58, which further comprises performing a
homology search for the peptide which binds to the orphan GPCR to
identify other candidate ligands for testing.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is related to U.S. provisional
patent application Serial No. 60/264,323 filed on Jan. 29, 2001,
incorporated herein by reference, and claims priority thereto under
35 USC .sctn.119(e).
BACKGROUND OF THE INVENTION
[0003] The present invention is directed to .beta.-superfamily
conotoxin peptides (also referred to as .beta.-conotoxins),
derivatives or pharmaceutically acceptable salts thereof. The
present invention is further directed to the use of this peptide,
derivatives thereof and pharmaceutically acceptable salts thereof
for the treatment of disorders associated with voltage-gated ion
channels, ligand-gated ion channels and/or receptors. The invention
is further directed to nucleic acid sequences encoding the
conotoxin peptides and encoding propeptides, as well as the
propeptides.
[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] Conus is a genus of predatory marine gastropods (snails)
which envenomate their prey. Venomous cone snails use a highly
developed projectile apparatus to deliver their cocktail of toxic
conotoxins into their prey. In fish-eating species such as Conus
magus the cone detects the presence of the fish using chemosensors
in its siphon. When close enough it extends its proboscis and
impales the fish hollow harpoon-like tooth containing venom. This
immobilizes the fish and enables the cone snail to wind it into its
mouth via the tooth at the end of the proboscis. For general
information on Conus and their venom see the website address
http://grimwade.biochem.unimelb.edu.au/cone/referenc.html- . Prey
capture is accomplished through a sophisticated arsenal of peptides
which target specific ion channel and receptor subtypes. Each Conus
species venom appears to contain a unique set of 50-200 peptides.
The composition of the venom differs greatly between species and
between individual snails within each species, each optimally
evolved to paralyse it's prey. The active components of the venom
are small peptides toxins, typically 10-30 amino acid residues in
length and are typically highly constrained peptides due to their
high density of disulphide bonds.
[0006] The venoms consist of a large number of different peptide
components that when separated exhibit a range of biological
activities: when injected into mice they elicit a range of
physiological responses from shaking to depression. The paralytic
components of the venom that have been the focus of recent
investigation are the .alpha.-, .omega.- and .mu.-conotoxins. All
of these conotoxins act by preventing neuronal communication, but
each targets a different aspect of the process to achieve this. The
.alpha.-conotoxins target nicotinic ligand gated channels, the
.mu.-conotoxins target the voltage-gated sodium channels and the
.omega.-conotoxins target the voltage-gated calcium channels
(Olivera et al., 1985; Olivera et al., 1990). For example a linkage
has been established between .alpha., .alpha.A- &
.psi.-conotoxins and the nicotinic ligand-gated ion channel;
.omega.-conotoxins and the voltage-gated calcium channel;
.mu.-conotoxins and the voltage-gated sodium channel;
.delta.-conotoxins and the voltage-gated sodium channel;
.kappa.-conotoxins and the voltage-gated potassium channel;
conantokins and the ligand-gated glutamate (NMDA) channel.
[0007] However, the structure and function of only a small minority
of these peptides have been determined to date. For peptides where
function has been determined, three classes of targets have been
elucidated: voltage-gated ion channels; ligand-gated ion channels,
and G-protein-linked receptors.
[0008] Conus peptides which target voltage-gated ion channels
include those that delay the inactivation of sodium channels, as
well as blockers specific for sodium channels, calcium channels and
potassium channels. Peptides that target ligand-gated ion channels
include antagonists of NMDA and serotonin receptors, as well as
competitive and noncompetitive nicotinic receptor antagonists.
Peptides which act on G-protein receptors include neurotensin and
vasopressin receptor agonists. The unprecedented pharmaceutical
selectivity of conotoxins is at least in part defined by a specific
disulfide bond frameworks combined with hypervariable amino acids
within disulfide loops (for a review see McIntosh et al.,
1998).
[0009] There are drugs used in the treatment of pain, which are
known in the literature and to the skilled artisan. See, for
example, Merck Manual, 16th Ed. (1992). However, there is a demand
for more active analgesic agents with diminished side effects and
toxicity and which are non-addictive. The ideal analgesic would
reduce the awareness of pain, produce analgesia over a wide range
of pain types, act satisfactorily whether given orally or
parenterally, produce minimal or no side effects, be free from
tendency to produce tolerance and drug dependence.
[0010] Due to the high potency and exquisite selectivity of the
conopeptides, several are in various stages of clinical development
for treatment of human disorders. For example, two Conus peptides
are being developed for the treatment of pain. The most advanced is
.omega.-conotoxin MVIIA (ziconotide), an N-type calcium channel
blocker (see Heading, C., 1999; U.S. Pat. No. 5,859,186).
.omega.-Conotoxin MVIIA, isolated from Conus magus, is
approximately 1000 times more potent than morphine, yet does not
produce the tolerance or addictive properties of opiates.
.omega.-Conotoxin MVIIA has completed Phase III (final stages) of
human clinical trials and has been approved as a therapeutic agent.
.omega.-Conotoxin MVIIA is introduced into human patients by means
of an implantable, programmable pump with a catheter threaded into
the intrathecal space. Preclinical testing for use in post-surgical
pain is being carried out on another Conus peptide, contulakin-G,
isolated from Conus geographus (Craig et al. 1999). Contulakin-G is
a 16 amino acid O-linked glycopeptide whose C-terminus resembles
neurotensin. It is an agonist of neurotensin receptors, but appears
significantly more potent than neurotensin in inhibiting pain in in
vivo assays.
[0011] In view of a large number of biologically active substances
in Conus species it is desirable to further characterize them and
to identify peptides capable of treating disorders voltage-gated
ion channels, ligand-gated ion channels and/or receptors.
Surprisingly, and in accordance with this invention, Applicants
have discovered novel conotoxins that can be useful for the
treatment of disorders involving voltage-gated ion channels,
ligand-gated ion channels and/or receptors and could address a long
felt need for a safe and effective treatment.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to conotoxin peptides,
derivatives or pharmaceutically acceptable salts thereof. The
present invention is further directed to the use of this peptide,
derivatives thereof and pharmaceutically acceptable salts thereof
for the treatment of disorders associated with voltage-gated ion
channels, ligand-gated ion channels and/or G-protein coupled
receptors (GPCRs). The invention is further directed to nucleic
acid sequences encoding the conotoxin peptides and encoding
propeptides, as well as the propeptides.
[0013] More specifically, the present invention is directed to
conotoxin peptides, having the amino acid sequences set forth in
Tables 1-3 below. In the .beta.-superfamily conotoxins containing
4-Cys residues (e.g., Ge14.1), the peptide may be bridged [1,4/2,3]
or [1,3/2,4].
[0014] The present invention is also directed to derivatives or
pharmaceutically acceptable salts of the conotoxin peptides or the
derivatives. Examples of derivatives include peptides in which the
Arg residues may be substituted by Lys, ornithine, homoarginine,
nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any
synthetic basic amino acid. The Lys residues may be substituted by
Arg, ornithine, homoarginine, nor-Lys, or any synthetic basic amino
acid. The Tyr residues may be substituted with meta-Tyr, ortho-Tyr,
nor-Tyr, .sup.125I-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
O-phospho-Tyr, nitro-Tyr or any synthetic hydroxy containing amino
acid. The Ser residues may be substituted with Thr or any synthetic
hydroxylated amino acid. The Thr residues may be substituted with
Ser or any synthetic hydroxylated amino acid. The Phe residues may
be D or L, may be substituted at the ortho, meta, and/or para
positions with a halogen or may be substituted with any synthetic
aromatic amino acid. The Trp residues may be substituted with Trp
(D), neo-Trp, 6-halo-Trp (D or L), preferably 6-halo, or any
aromatic synthetic amino acid; and the Asn, Ser, Thr or Hyp
residues may be glycosylated. The halogen may be iodo, chloro,
fluoro or bromo; preferably iodo for halogen substituted-Tyr and
bromo for halogen-substituted Trp. The Tyr residues may also be
substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or
ortho-Tyr, respectively) and corresponding O-sulpho- and
O-phospho-derivatives. The acidic amino acid residues may be
substituted with any synthetic acidic amino acid, e.g., tetrazolyl
derivatives of Gly and Ala. The Leu may be substituted with Leu
(D). The Glu residues may be substituted with Gla or Asp. The Gla
residues may be substituted with Glu or Asp. The acidic amino acid
residues may be substituted with any synthetic acidic amino acid,
e.g. tetrazolyl derivatives of Gly and Ala. The N-terminal Gln may
be substituted with pyro-glutamate (Z). The aliphatic amino acids
may be substituted by synthetic derivatives bearing non-natural
aliphatic branched or linear side chains C.sub.nH.sub.2n+2 up to
and including n=8. The Met residues may be substituted with
nor-leucine (Nle). The Cys residues may be in D or L configuration
and may optionally be substituted with homocysteine (D or L). Basic
residues in the backbone may be D or L configuration. The central
Trp residue within the beta-turn is preferably epimerized to the
D-form.
[0015] Examples of synthetic aromatic amino acid include, but are
not limited to, nitro-Phe, 4-substituted-Phe wherein the
substituent is C.sub.1-C.sub.3 alkyl, carboxyl, hyrdroxymethyl,
sulphomethyl, halo, phenyl, --CHO, --CN, --SO.sub.3H and --NHAc.
Examples of synthetic hydroxy containing amino acid, include, but
are not limited to, such as 4-hydroxymethyl-Phe,
4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples of
synthetic basic amino acids include, but are not limited to,
N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala,
2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala. These
and other synthetic basic amino acids, synthetic hydroxy containing
amino acids or synthetic aromatic amino acids are described in
Building Block Index, Version 3.0 (1999 Catalog, pages 4-47 for
hydroxy containing amino acids and aromatic amino acids and pages
66-87 for basic amino acids; see also http://www.amino-acids.co-
m), incorporated herein by reference, by and available from RSP
Amino Acid Analogues, Inc., Worcester, Mass. Examples of synthetic
acid amino acids include those derivatives bearing acidic
functionality, including carboxyl, phosphate, sulfonate and
synthetic tetrazolyl derivatives such as described by Ornstein et
al. (1993) and in U.S. Pat. No. 5,331,001, each incorporated herein
by reference, and such as shown in the following schemes 1-3. 1 2
3
[0016] Additional derivatives are peptides in which the Asn
residues may be modified to contain an N-glycan and the Ser, Thr
and Hyp residues may be modified to contain an O-glycan (e.g., g-N,
g-S, g-T and g-Hyp). In accordance with the present invention, a
glycan shall mean any N-, S- or O-linked mono-, di-, tri-, poly- or
oligosaccharide that can be attached to any hydroxy, amino or thiol
group of natural or modified amino acids by synthetic or enzymatic
methodologies known in the art. The monosaccharides making up the
glycan can include D-allose, D-altrose, D-glucose, D-mannose,
D-gulose, D-idose, D-galactose, D-talose, D-galactosamine,
D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),
D-N-acetyl-galactosamine (GalNac), D-fucose or D-arabinose. These
saccharides may be structurally modified, e.g., with one or more
O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic
acid, including combinations thereof. The gylcan may also include
similar polyhydroxy groups, such as D-penicillamine 2,5 and
halogenated derivatives thereof or polypropylene glycol
derivatives. The glycosidic linkage is beta and 1-4 or 1-3,
preferably 1-3. The linkage between the glycan and the amino acid
may be alpha or beta, preferably alpha and is 1-.
[0017] Core O-glycans have been described by Van de Steen et al.
(1998), incorporated herein by reference. Mucin type O-linked
oligosaccharides are attached to Ser or Thr (or other hydroxylated
residues of the present peptides) by a GalNAc residue. The
monosaccharide building blocks and the linkage attached to this
first GalNac residue define the "core glycans," of which eight have
been identified. The type of glycosidic linkage (orientation and
connectivities) are defined for each core glycan. Suitable glycans
and glycan analogs are described further in U.S. Ser. No.
09/420,797 filed Oct. 19, 1999 and in PCT Application No.
PCT/US99/24380 filed Oct. 19, 1999 (PCT Published Application No.
WO 00/23092), each incorporated herein by reference. A preferred
glycan is Gal(.beta.1.fwdarw.3)GalNac(.alpha.1.fwdarw.).
[0018] Derivatives also include peptides in which pairs of Cys
residues may be replaced pairwise with isosteric lactam or
ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or
Asp), Cys/(Glu or Asp) or Cys/Ala combinations. Sequential coupling
by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et
al., 1997) allows replacement of native Cys bridges with lactam
bridges. Thioether analogs may be readily synthesized using
halo-Ala residues commercially available from RSP Amino Acid
Analogues. In addition, individual Cys residues may be replaced
with homoCys, seleno-Cys or penicillamine, so that disulfide
bridges may be formed between Cys-homoCys or Cys-penicillamine, or
homoCys-penicillamine and the like.
[0019] Derivatives and analogs also include truncations of the
peptides disclosed herein. As used herein "truncations" are used to
refer to peptides in which the sequence has been shortened from the
mature conotoxin sequence that is predicted by the prepropeptide
cleavage site with significant retention of activity of the native
conotoxin. These truncations can be shortened from the N-terminus,
the C-terminus, or both. As used herein significant retention of
activity is used to refer to an activity of the truncated conotoxin
which is less that 100-fold loss of activity and specificity.
[0020] Derivatives also include radiometal and chelated anti-tumor
peptides. The incorporation of the radiometal eg .sup.99mTc,
.sup.111In, .sup.90Y, .sup.188re, .sup.105RhS.sub.4,
.sup.188Re-tisuccin, .sup.89Sr, .sup.153Sm, .sup.186Re, .sup.67Ga,
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.177Lu, .sup.67Cu,
.sup.64Cu, .sup.105Rh, .sup.47Sc, .sup.109Pd] in to the conotoxin
generally involves use of a chelate, specific to the particular
metal, and a linker group to covalently attach the chelate to the
conotoxin [the bifunctional chelate approach]. The design of useful
chelates is dependent on the coordination requirements of the
specific radiometal. DTPA, DOTA, P.sub.2S.sub.2--COOH BFCA
requirement for kinetic TETA, NOTA are common egs. The requirement
for kinetic stability of the metal complex is often achieved
through the use of multidentate chelate ligands with a
functionalised arm for covalent bonding to some part of the
conotoxin ie the amino lysine group. Hence, the conotoxins of the
present invention may be tagged to produce radiopharmaceuticals. In
relation to radioligand probes of .beta.-conotoxins for screening
of small molecules, acting at unique allosteric sites, synthesis of
such screening tools is not restricted to radioiodinated tyrosine
derivatives. Incorporation of standard commercially available
tritiated amino acid residues can also be utilized.
[0021] The present invention is further directed to a method of
treating disorders associated with voltage-gated ion channels,
ligand-gated ion channels and/or receptor disorders in a subject
comprising administering to the subject an effective amount of the
pharmaceutical composition comprising a therapeutically effective
amount of a conotoxin peptide described herein or a
pharmaceutically acceptable salt or solvate thereof. The present
invention is also directed to a pharmaceutical composition
comprising a therapeutically effective amount of a conotoxin
peptide described herein or a pharmaceutically acceptable salt or
solvate thereof and a pharmaceutically acceptable carrier.
[0022] More specifically, the present invention is also directed to
nucleic acids which encode conotoxin peptides of the present
invention or which encodes precursor peptides for these conotoxin
peptides, as well as the precursor peptide. The nucleic acid
sequences encoding the precursor peptides of other conotoxin
peptides of the present invention are set forth in Table 1. Table 1
also sets forth the amino acid sequences of these precursor
peptides.
[0023] Another embodiment of the invention contemplates a method of
identifying compounds that mimic the therapeutic activity of the
instant peptide, comprising the steps of: (a) conducting a
biological assay on a test compound to determine the therapeutic
activity; and (b) comparing the results obtained from the
biological assay of the test compound to the results obtained from
the biological assay of the peptide.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0024] The present invention is directed to .beta.-superfamily
conotoxin peptides (also referred to as .beta.-conotoxins),
derivatives or pharmaceutically acceptable salts thereof. The
present invention is further directed to the use of this peptide,
derivatives thereof and pharmaceutically acceptable salts thereof
for the treatment of disorders associated with voltage-gated ion
channels, ligand-gated ion channels and/or receptors, such as
G-protein coupled receptors (GPCRs). The invention is further
directed to nucleic acid sequences encoding the .beta.-superfamily
conotoxin peptides and encoding propeptides, as well as the
propeptides.
[0025] The present invention, in another aspect, relates to a
pharmaceutical composition comprising an effective amount of a
.beta.-superfamily conotoxin peptide, a mutein thereof, an analog
thereof, a derivative thereof, an active fragment thereof or
pharmaceutically acceptable salts or solvates. Such a
pharmaceutical composition has the capability of acting at
voltage-gated ion channels, ligand-gated ion channels and/or
receptors (such as G-protein coupled receptors (GPCRs)), and are
thus useful for treating a disorder or disease of a living animal
body, including a human, which disorder or disease is responsive to
the partial or complete blockade of such channels or receptors
comprising the step of administering to such a living animal body,
including a human, in need thereof a therapeutically effective
amount of a pharmaceutical composition of the present
invention.
[0026] G-protein-coupled receptors (GPCRs) are a large, upwards of
1000, and functionally diverse protein superfamily, which form a
seven transmembrane (TM) helices bundle with alternating
extra-cellular and intracellular loops. GPCRs are considered to be
one of the most important groups of drug targets because they are
involved in a broad range of body functions and processes and are
related to major diseases. Over the last decades distinct members
of the G Protein-Coupled Receptor (GPCR) family emerged as
prominent drug targets within pharmaceutical research, since
approximately 60% of marketed prescription drugs act by selectively
addressing representatives of that class of transmembrane signal
transduction systems. It is noteworthy that the majority of
GPCR-targeted drugs elicit their biological activity by selective
agonism or antagonism of biogenic monoamine receptors, while the
development status of peptide-binding GPCR-addressing compounds is
still in its infancy.
[0027] The .beta.-conotoxins may function as allosteric modulators
(i.e., agonists, partial agonists, neutral antagonists, inverse
agonists) of GPCRs including, but not limited to, sst (sst.sub.1,
sst.sub.2, sst.sub.3, sst.sub.2a, sst.sub.2b, sst.sub.4,
sst.sub.5), cortistatin (CST), melanocortin (MC.sub.xR, wherein
x=1, 2, 4, 5), opioid (.mu., .delta., .kappa.), neurokinin
(NK.sub.1, NK.sub.2, NK.sub.3), bradykinin (B.sub.1, B.sub.2),
galanin (Gal.sub.1, Gal.sub.2, Gal.sub.3), CCK.sub.A, CCK.sub.B,
endothelin, serotonin, adrenergic receptors, angiotensin (AT.sub.1,
AT.sub.2), neuropeptide-Y, sigmal, sigma2, oxytocin, CGRP, GRPR,
histamine, imidazoline, neurotensin (NT.sub.1, NT.sub.2, NT.sub.3),
VIP, vasopressin (V1a, V1b, V2), substance K, chemokine receptors
(CXCR.sub.1, CXCR.sub.2, CXCR.sub.3, CXCR.sub.4, CXCR.sub.5,
CCR.sub.1, CCR.sub.2b, CCR.sub.3, CCR.sub.4, CCR.sub.5, CCR.sub.6,
CCR.sub.7, CCR.sub.8, CX.sub.3Cr.sub.1), CRF.sub.1, CRF.sub.2a,
CRF.sub.2b, CRF.sub.2g, CRF-BP orexin (Ox.sub.1, Ox.sub.2),
urotensin (UT-II), glycoprotein IIb/IIIa, thrombin receptors,
orphan GPCRs (eg. MCH.sub.2R/SLT, SP1999/P.sub.2Y.sub.12,
CRTH.sub.2, NPFF.sub.1, NPFF.sub.2, HH.sub.4R, h-GPR.sub.54,
CysLT.sub.2, neuromedin receptors, BLTR.sub.2, G.sub.2A, TA.sub.1,
LTB.sub.4, ghrelin, motilin MTL-R, purinergic receptors, muscarinic
receptors, ORL-1, apelin, CB.sub.1, CB.sub.2 and the like). For an
extensive list of GPCRs see
http://www.gpcr.org/7tm/htmls/entries.html. For additional orphan
GPCR references see Shaaban (2001) and Civelli et. al. (2001). This
beta turn toxin template may also be used to characterize new
functional allosteric sites on known GPCRs. Radiolabelled
dervatives serve as screening tools for such sites and will allow
for identification of new small molecule modulators. The reverse
beta turn motif serves as a template for beta turn peptidomimetic
design in which the turn template contains the cone snail WK
recognition "fingerprint", examples of such templates can be found
in Golebiowski et al. (2001), Horwell (1996) and Beeley (2000).
[0028] Somatostatin Receptors (SSTRs): Somatostatin (SRIF), was
first identified as a peptide that inhibits growth hormone release.
Later it was shown to have other physiological activities,
including the inhibition of the release of many endocrine
secretions such as prolactin, TSH and insulin. The peptide also
exhibits neuromodulatory functions and may act as a
neurotransmitter. Somatostatin has two major actions; inhibition of
hormone and cytokine (IFN-.gamma., IL-6, IL-8, IL-1) release and
inhibition of cellular proliferation. SRIF can inhibit the release
of hormones in the brain and almost all hormone release in the gut,
together with many cytokines and growth factors eg PDGF. SRIF can
be produced in cells from neuroendocrine and immune systems. It is
produced in two forms SRIF-28 and SRIF-14. Additionally a novel
SST-like peptide called cortistatin (CST) has been isolated from
human. CST-14 shares 11 of the 14 amino acids with SRIF. One
measurable difference between SRIF and CST is that SRIF increases
the duration of REM sleep in rats whereas CST decreases it. It is
thus possible to postulate that specific CST receptors exist.
[0029] Five sst receptors have been identified sst1, 2, 3, 4, 5.
All are GPCRs and are encoded on a different chromosome. Both
SRIF-14 and SRIF-28 bind to all receptors but SRIF-28 has higher
affinity for sst.sub.5. It must be noted that 2 isoforms of
sst.sub.2 exist namely, sst.sub.2A and sst.sub.2B. SRIF receptors
are widely expressed. Cells from the CNS, gut, pancreas, kidney,
thyroid, lung and the immune system express the receptors in
varying proportions.
[0030] Overproduction of growth factors from tumours can result in
systemic effects, as seen in acromegaly, a chronic debilitating
disease caused by excessive production of GH. SRIF analogs suppress
the release of GH and thus can benefit patients. A majority of
tumours originating from tissues naturally targeted by SRIF express
sst receptors at high levels.
[0031] Synthetic peptides such as, lanreotide, octreotide and
vapreotide bind sst.sub.2 and 5 with high affinity and sst.sub.3 to
a lesser extent. Not only have peptide analogs of SRIF been used to
reduce tumour growth directly through somatostatin receptor
signalling but also medicine finds considerable use for
somatostatin receptors in tumour detection and imaging and now in
tumour targeting (Slooter et al., 2001).
[0032] The high level expression of somatostatin receptors (SSTR)
on various tumor cells has provided the molecular basis for
successful use of radiolabeled octreotide/lanreotide analogs as
tumor tracers in nuclear medicine, similar chemical; modifications
of conotoxins bT2, bM1, bG1 may be achieved. The vast majority of
human tumors seem to overexpress the one or the other of five
distinct h-SSTR sub-type receptors. Whereas neuroendocrine tumors
frequently overexpress h-SSTR.sub.2, intestinal adenocarcinomas
seem to over-express more often h-SSTR.sub.3 or h-SSTR.sub.4, or
both of these h-SSTR. In contrast to
.sup.111In-DTPA-DPhe1-octreotide (OCTREOSCAN) which binds to
h-SSTR.sub.2 and .sub.5 with high affinity (K.sub.d 0.1-5 nM), to
h-SSTR.sub.3 with moderate affinity (Kd 10-100 nM) and does not
bind to h-SSTR.sub.1 and h-SSTR.sub.4,
.sup.111In/.sup.90Y-DOTA-lanreotide was found to bind to
h-SSTR.sub.2, .sub.3, .sub.4, and .sub.5 with high affinity, and to
h-SSTR.sub.1 with lower affinity (K.sub.d 200 nM). Based on its
unique h-SSTR binding profile, .sup.111In-DOTA-lanreotide was
suggested to be a potential radioligand for tumor diagnosis, and
.sup.90Y-DOTA-lanreotide suitable for receptor-mediated
radionuclide therapy. As opposed to
.sup.111In-DTPA-DPhe1-octreotide and
.sup.111In-DOTA-DPhe1-Tyr3-octreotid- e, discrepancies in the
scintigraphic results were seen in about one third of
(neuroendocrine) tumor patients concerning both the tumor uptake as
well as detection of tumor lesions. On a molecular level, these
discrepancies seem to be based on a "higher" high-affinity binding
of .sup.111In-DOTA-DPhe1-Tyr3-octreotide to h-SSTR.sub.2. Other
somatostatin analogs with divergent affinity to the five known
h-SSTR subtype receptors have also found their way into the
clinics, including .sup.99mTc-HYNIC-octreotide or
.sup.99mTc-depreotide (NEOSPECT; NEOTECT).
[0033] Most of the imaging results are reported for neuroendocrine
tumors (octreotide analogs) or non-small cell lung cancer
(.sup.99mTc-depreotide), indicating high diagnostic capability of
this type of receptor tracers. Consequently to their use as
receptor imaging agents, h-SSTR recognizing radioligands have also
been implemented for experimental receptor-targeted radionuclide
therapy. The study "MAURITIUS" (MulticenterAnalysis of a Universal
Receptor Imaging and Treatment Initiative, a European Study), a
Phase IIa study, showed in patients with a calculated tumor dose
>10 Gy/GBq .sup.90Y-DOTA-lanreotide, the proof-of-principle for
treating tumor patients with receptor imaging agents. Overall
treatment results in >60 patients indicated stable tumor disease
in roughly 35% of patients and regressive disease in 15% of tumor
patients with different tumor entities. No acute or chronic severe
hematological toxicity, change in renal or liver function
parameters due to .sup.90Y-DOTA-lanreotide, was reported.
.sup.90In-DOTA-DPhe1-Tyr3-octreotide may show a higher tumor uptake
in neuroendocrine tumor lesions and may therefore provide even
better treatment results in tumor patients, but there is only
limited excess to long-term and survival data at present. Besides
newer approaches and recent developments of .sup.188Re-labeled
radioligands no clinical results on the treatment response is
available yet. In conclusion, several radioligands have been
implemented on the basis of peptide receptor recognition throughout
the last decade. A plentitude of preclinical data and clinical
studies confirm "proof-of-principle" for their use in diagnosis as
well as therapy of cancer patients. However, an optimal
radiopeptide formulation does not yet exist for receptor-targeted
radionuclide therapy (Virgolini, 2001).
[0034] During the last decade five different subtypes of
melanocortin receptors have been identified and cloned, all of them
are possible as new targets for drugs in the treatment of a number
of clinical important conditions such as inflammatory diseases
(MC.sub.1-receptor agonists), MC.sub.3/MC.sub.4-receptors in the
treatment of feeding disorder, agonists for treatment of obesity
and antagonists for anorectic conditions.
MC.sub.3/MC.sub.4-agonists or also assumed to be useful for
treating sexual dysfunction. In the treatment of seborrheic
dermatitis the MC.sub.5 receptor is considered as a target. A
number of peptide or peptide like ligands, agonists and/or
antagonist has been discovered, however, most of them have a large
similarity with the endogenous ligand .alpha.-MSH.
[0035] Melanocortins: The major source of melanocortins is the
pituitary, where ACTH and .beta.-lipotropin are the main products
from the anterior pituitary, and .alpha.-MSH and .beta.-lipotropin
are major products from the intermediate lobe. All melanocortins,
i.e. .alpha.-Melanocyte stimulating hormone (.alpha.-MSH),
.beta.-MSH, .gamma.-MSH and the endogenous opioid .beta.-endorphin,
are cleaved from POMC, but .beta.-MSH and .beta.-endorphin emanate
from the C-terminal part of POMC, i.e. the .beta.-lipotropin.
.gamma.-MSH is cleaved from the N-terminal part of POMC. While
.alpha.-MSH is a tri-decapeptide proteolytically cleaved from
proopiomelanocortin (POMC) comprising of the N-terminal part of
ACTH and is considered as the endogenous ligand to the melanocortin
receptors.
[0036] .beta.-MSH is found in the hypothalamus, whereas .gamma.-MSH
is found in different areas of the CNS, adrenal medulla and neurons
of the intestine. .alpha.-MSH has been demonstrated in the
pituitary, but also in other parts of the CNS, as well as in
peripheral parts of the body. Only low circulating concentrations
of .alpha.-MSH have been detected in humans in normal situations,
whereas the concentration is increased in several diseases.
[0037] Melanocortin Receptors MCRs: Melanocortin receptors belong
to the family of G-protein coupled, 7-TM receptors, and have been
identified in several tissues of the body. Today, 5 different
subtypes of receptors, MC1-5, have been described. The MC.sub.2
receptor binds only ACTH, and is present in the adrenal cortex and
also in white adipose tissue of rodents, but not in man or
primates. The MC.sub.1, MC.sub.2, MC.sub.3, MC.sub.4 and MC.sub.5
receptors are distributed in different areas/organs of the body.
The MC.sub.2 receptor is not further discussed since it is
considered as the ACTH receptor. Interestingly, the MC.sub.3
receptor is expressed in low abundance during fetal life and
expression increases to adult levels after birth, as demonstrated
in rats. The opposite is true for the MC4 receptor, which is
predominant during fetal life. However, both receptors seem to be
important for different physiological functions postnatally.
[0038] The MC receptors and .alpha.-MSH are involved in several
physiological functions besides affecting skin pigmentation. They
have effects on learning, memory, behaviour, including sexual
behaviour, regeneration in the neuromuscular system and protection
from central nerve injury, cardiovascular functions, feeding and
weight homeostasis, fever and immunomodulation/inflammation,
exocrine functions and interact with opioids and dopamine. They are
also ascribed effects such as regulation of the release of
pituitary and peripheral hormone.
[0039] Examples of voltage-gated ion channels include the
voltage-gated calcium channel, the voltage-gated sodium channel,
the voltage-gated potassium channel and the proton-gated ion
channel. Examples of ligand-gated channels include the nicotinic
ligand-gated ion channel, ligand-gated glutamate (NMDA) channel and
the ligand-gated 5HT.sub.3 (serotonin) channel. Examples of
receptors include the G-protein receptors. Activity of
.psi.-conotoxins is described in U.S. Pat. No. 5,969,096 and in
Shon et al. (1997). Activity of bromosleeper conotoxins is
described in U.S. Pat. No. 5,889,147 and in Craig et al. (1997).
Activity of .sigma.-conotoxins is described in U.S. Pat. No.
5,889,147. Activity of contryphan conotoxins is described in U.S.
Pat. No. 6,077,934 and in Jimenez et al. (1996). Activity of
conopressins is described in Cruz et al. (1987) and in Kruszynski
et al. (1990). Activity of .gamma.-conotoxins is described in
Fainzilber et al. (1998). Activity of .alpha.A-conotoxins is
described in Jacobsen et al. (1997) and in Hopkins et al. (1995).
Activity of .gamma.-conotoxins is described in U.S. Ser. No.
09/497,491 (PCT/US00/03021, PCT published application WO 00/46371)
as an antagonist for acetylcholine receptors and as analgesic
agents for the treatment of pain (whether acute or chronic),
including migraine, chronic pain, and neuropathic pain, without
undesirable side effects. Activity of contulakins is described in
U.S. Ser. No. 09/420,797 (PCT/US99/24380, PCT published application
WO 00/23092). Each of these references is incorporated herein by
reference.
[0040] Since .sigma.-conotoxins are antagonists of the 5HT.sub.3
receptor, they are also useful in treating irritable bowel syndrome
(IBS) and visceral pain. Visceral pain is a common experience in
health and disease. Chronic visceral hyperalgesia in the absence of
detectable organic disease has been implicated in many common
functional bowel disorders (FDB), such as IBS, non-ulcer dyspepsia
(NUD) and non-cardiac chest pain (NCCP).
[0041] Pain in IBS cannot be explained by normal perception of
abnormal motility. In the majority of patients, sensory perception
itself is abnormal. Most visceral afferent information is part of
the reflex activity of digestion and does not reach concious
perception. Increasing evidence suggests that long term changes in
the thresholds and gain of the visceral afferent pathways are
present in patients with FDBs. This has been referred to as
visceral hyperalgesia (Mayer et al., 1994).
[0042] It has been proposed that FDBs are a result of increased
excitability of spinal neurones. According to their model, many
inputs can result in transient, short term, or life long
sensitization of afferent pathways involved in visceral reflexes
and sensations from the gut. The increased sensory input to
interneurons and/or dorsal horn neurons in the spinal cord will
result in secondary hyperalgesia, in which adjacent, undamaged
viscera develop sensitivity to normal innocuous stimuli
(allodynia), and central hyperexcitability as a consequence of
changes in the circuitary of the dorsal horn. This central
sensitization may subsequently extend to supraspinal centers
also.
[0043] Altered spinal processing of visceral sensory information
can explain altered sensory thresholds and altered referral
patterns, the perception of visceral sensations without stimulation
of visceral mechnoreceptors (sensation of incomplete evacuation),
and the symptomatic involvement of multiple sites in the GI tract,
including extra intestinal sites. Increased excitability of dorsal
horn neurones, resulting in the recruitment of previously
sub-threshold inputs, may explain cutaneous allodynia in some
patients with IBS, burning sensations referred to different parts
of the body, cold hypersensitivity and pain referral to upper and
lower extremities.
[0044] A number of compounds have been shown to modulate visceral
sensitivity in IBS patients. These include octreotide (sst.sub.2;
Novartis), the 5-HT.sub.3 antgonists odansetron (Glaxo) and
granisetron (SKB) and the peripheral kappa opioid agonist,
fedotozine (Jouveinal SA). The 5-HT.sub.3 antagonist alosteron
(Glaxo), cuurrently in development for IBS, is active in modifying
the perception of colonic distension and gut compliance in IBS
patients. New drugs in development for the treatment of IBS that
are targeted at pain control as well as dysmotility include
5-HT.sub.3 and 5-HT.sub.4 receptor antagonists. 5-HT.sub.3
receptors are located throughout the central and peripheral nervous
system--their role in modulating the activity of visceral afferent
and enteric neurones has led to the proposal that 5-HT acts as a
sensitizing agent via these receptors on visceral afferent
neurones. 5-HT.sub.3 receptor antagonists have been widely reported
to attenuate blood pressure responses to intestinal distension.
5-HT.sub.3 antagonists in development for IBS include Alosteron
(phase III), which is reported to reduce abdominal pain, slow
colonic transit and increase colon compliance in IBS patients.
Other compounds with positive effects include the antiemetic
Ramosteron (Yamanouchi), Cilansteron (Solvay) and YM-114
(Yamanouchi). An animal model for dysmotility of the GI tract has
been described by Maric et al. (1989).
[0045] In addition to the above uses, the peptides of the present
invention are also useful (i) for treating or diagnosis of cancer,
neoplasm, solid tumor, diabetic nephropathy, fibrosis, hypophysis
tumor, GI disease, IBS, restinosis, angiogenesis disorder, diabetes
mellitus, endocrine tumor, diarrhea, pancreatic disease, prostate
tumor, bleeding, apoptosis, inflammation, pain, diabetes, obesity,
sexual dysfunction, acromegaly, glaucoma, cardiovascular, diabetic,
retinopathy, depression, myocardial infarction, stroke, epilepsy,
anorexia, wasting diseases, seborrheic dermatitis, schizophrenia,
mood disorders, chemotherapeutic induced emesis, disorders
associated with changes in blood pressure, immune disorders, nerve
damage, acne, GI infections, myocardial infarction, angina,
thromboembolism, cardiovascular disease, (ii) as templates for
small molecule design and (iii) as screening tools.
[0046] The superfamily of seven-transmembrane-domain
G-protein-coupled receptors (GPCRs) is the largest and most diverse
group of transmembrane proteins involved in signal transduction.
Each of the approximately 1000 family members found in vertebrates
responds to stimuli as diverse as hormones, neurotransmitters,
odorants and light, which selectively activate intracellular
signaling events mediated by heterotrimeric G proteins. Because
GPCRs are centrally positioned in the plasma membrane to initiate a
cascade of cellular responses by diverse extracellular mediators,
it is not surprising that modulation of GPCR function has been
successful in the development of many marketed therapeutic agents.
It has become clear that GPCRs for which a natural activating
ligand has not yet been identified (orphan GPCRs) might provide a
path to discovering new cellular substances that are important in
human physiology. The process of `de-orphanizing` these novel
proteins has accelerated significantly and opened up new avenues
for research in human physiology and pharmacology.
[0047] In most cases the extent of sequence homology is
insufficient to assign these `orphan` receptors to a particular
receptor subfamily. Consequently, reverse molecular pharmacological
and functional genomic strategies are being employed to identify
the activating ligands of the cloned receptors. Briefly, the
reverse molecular pharmacological methodology includes cloning and
expression of orphan GPCRs in mammalian cells and screening these
cells for a functional response to cognate or surrogate agonists
present in biological extract preparations, peptide libraries, and
complex compound collections. The functional genomics approach
involves the use of "humanized" yeast cells, where the yeast GPCR
transduction system is engineered to permit functional expression
and coupling of human GPCRs to the endogenous signalling machinery.
Both systems provide an excellent platform for identifying novel
receptor ligands. Once activating ligands are identified they can
be used as pharmacological tools to explore receptor function and
relationship to disease.
[0048] The .beta.-superfamily conotoxins can also be used to design
a .beta.-turn mimetic of the .beta.-superfamily conotoxins
containing a .beta.-turn motif, e.g., the -CX1X2KX1C- (SEQ ID
NO:338) motif where X1 is any amino acid and X2 is Trp in the D or
L orientation (or halogenated at position 6 of the indole) or the
-CPX3RVC- (SEQ ID NO:339) motif where X3 is Phe in the D or L
configuration. Other .beta.-turn motifs are also present in the
.beta.-superfamily conotoxins as evident from the peptide sequences
disclosed in Tables 2 and 3. This hairpin turn would be replaced by
a non-peptide turn mimetic, preferably an orally available mimetic.
The unique receptor binding domains contained within the N and
C-terminal regions of the .beta.-superfamily conotoxin would then
be attached to the .beta.-turn scaffold, in such a way as to mimic
the 3D spatial array within the native toxin. As an example of the
.beta.-turn motif and a .beta.-turn mimetic, see Scheme 4. 4
[0049] The .beta.-superfamily conotoxins of the present invention
are also useful for characterizing sites on GPCRs and for
identifying novel receptor ligands for GPCRS, especially orphan
GCPRs. For example, the .beta.-beta turn toxin template may also be
used to characterize new functional allosteric sites on known
GPCRs. Radiolabelled derivatives serve as screening tools for such
sites and will allow for identification of new small molecule
modulators. The reverse beta turn motif serves as a template for
beta turn peptidomimetic design in which the turn template contains
the cone snail WK recognition "fingerprint", examples of such
templates can be found in Golebiowski et al. (2001) and Horwell
(2000). In addition, a ligand which binds to an orphan G-protein
coupled receptor (orphan GPCR) can be identified by contacting a
.beta.-superfamily conotoxin with an orphan GPCR and measuring the
amount of binding of the conotoxin to the orphan GPCR by methods
that are well known in the art (Murphy et al., 1998). A homology
search to identify other candidate ligands for testing can then be
done on the basis of any peptide which binds to the orphan GPCR.
The candidate ligands may be peptides or peptide mimetics.
[0050] The conotoxin peptides described herein are sufficiently
small to be chemically synthesized. General chemical syntheses for
preparing the foregoing conotoxin peptides are described
hereinafter. Various ones of the conotoxin peptides can also be
obtained by isolation and purification from specific Conus species
using the technique described in U.S. Pat. No. 4,447,356 (Olivera
et al., 1984); U.S. Pat. Nos. 5,514,774; 5,719,264; and 5,591,821,
as well as in PCT published application WO 98/03189, the
disclosures of which are incorporated herein by reference.
[0051] Although the conotoxin peptides of the present invention can
be obtained by purification from cone snails, because the amounts
of conotoxin peptides obtainable from individual snails are very
small, the desired substantially pure conotoxin peptides 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 conotoxin
peptides peptide. 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 conotoxin peptides peptides
depends of course upon correct determination of the amino acid
sequence.
[0052] The conotoxin peptides can also be produced by recombinant
DNA techniques well known in the art. Such techniques are described
by Sambrook et al. (1989). A gene of interest (i.e., a gene that
encodes a suitable conotoxin peptides) can be inserted into a
cloning site of a suitable expression vector by using standard
techniques. These techniques are well known to those skilled in the
art. The expression vector containing the gene of interest may then
be used to transfect the desired cell line. Standard transfection
techniques such as calcium phosphate co-precipitation, DEAE-dextran
transfection or electroporation may be utilized. A wide variety of
host/expression vector combinations may be used to express a gene
encoding a conotoxin peptide of interest. Such combinations are
well known to a skilled artisan. The peptides produced in this
manner are isolated, reduced if necessary, and oxidized to form the
correct disulfide bonds.
[0053] One method of forming disulfide bonds in the conotoxin
peptides 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. However, because of the dilution resulting from the
presence of other fractions of less biopotency, a somewhat higher
dosage may be required.
[0054] 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.
[0055] In conventional solution phase peptide synthesis, the
peptide chain can be prepared by a series of coupling reactions in
which constituent amino acids are added to the growing peptide
chain in the desired sequence. Use of various coupling reagents,
e.g., dicyclohexylcarbodiimid- e or 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).
[0056] 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.
[0057] 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.
[0058] 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).
[0059] 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
K. 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 & Lubke (1965).
[0060] 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).
[0061] The activating reagents used in the solid phase synthesis of
the peptides are well known in the peptide art. Examples of
suitable activating reagents are carbodiimides, such as
N,N'-diisopropylcarbodiimi- de and
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activating
reagents and their use in peptide coupling are described by
Schroder & Lubke (1965) and Kapoor (1970).
[0062] 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).
[0063] 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 -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.
[0064] 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.
[0065] The peptides are also synthesized using an automatic
synthesizer. Amino acids are sequentially coupled to an MBHA Rink
resin (typically 100 mg of resin) beginning at the C-terminus using
an Advanced Chemtech 357 Automatic Peptide Synthesizer. Couplings
are carried out using 1,3-diisopropylcarbodimide in
N-methylpyrrolidinone (NMP) or by
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) and diethylisopropylethylamine (DIEA).
The FMOC protecting group is removed by treatment with a 20%
solution of piperidine in dimethylformamide(DMF). Resins are
subsequently washed with DMF (twice), followed by methanol and
NMP.
[0066] Muteins, analogs or active fragments, of the foregoing
conotoxin peptides are also contemplated here. See, e.g.,
Hammerland et al. (1992). Derivative muteins, analogs or active
fragments of the conotoxin peptides may be synthesized according to
known techniques, including conservative amino acid substitutions,
such as outlined in U.S. Pat. Nos. 5,545,723 (see particularly col.
2, line 50--col. 3, line 8); U.S. Pat. No. 5,534,615 (see
particularly col. 19, line 45--col. 22, line 33); and U.S. Pat. No.
5,364,769 (see particularly col. 4, line 55--col. 7, line 26), each
herein incorporated by reference.
[0067] Radiolabeled peptides have been used to show that perhaps
only the amino acid sequence actually involved in binding to the
receptor is essential for achieving tumor uptake.
.sup.111In-radiolabelled octreotide, which is used to image
somatostatin positive tumors, is a prime example of this. In
addition, advances in tumor biology have demonstrated metabolic
pathways to deliver the nuclide within the cell cytoplasm through
internalization mechanisms. Some classes of tumors have been shown
to overexpress certain receptors, e.g., glutamate, AMPA, NMDA,
somatostatin, melanocortin and the like, and whenever these
substances can be radiolabeled and introduced to the system, they
can become the lethal magic bullet by working inside the cell.
[0068] Specificity is the paramount goal in radionuclide therapy
because with specificity comes safety and efficacy. The strategy in
biologically targeted therapy is to chemically package the
radionuclide to take advantage of metabolic pathways or tumor
characteristics so that the radionuclide is localized in the target
organ or tissue while the nuclear energy is discharged with minimal
exposure to healthy tissue. The concentration differential of the
therapeutic radio-pharmaceutical must be orders of magnitude
between target and non-target tissues. Radiation doses of 4000-6000
rads are desirable in the target tissue, while only a few tens of
radiation units can be functionally tolerated by some
radiosensitive tissues. The short range of the emitted particles in
the tissues makes them very damaging over the range in which their
decay energy is deposited. The specificity of certain of the
.beta.-superfamily conotoxins for the somatostatin receptor provide
the necessary specificity for the treatment and diagnosis of
tumors.
[0069] Where the aim is to provide an image of the tumor, one will
desire to use a diagnostic agent that is detectable upon imaging,
such as a paramagnetic, radioactive or fluorogenic agent. Many
diagnostic agents are known in the art to be useful for imaging
purposes, as are methods for their attachment to peptides (see,
e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated
herein by reference). In the case of paramagnetic ions, one might
mention by way of example ions such as chromium (III), manganese
(II), iron (III), iron (II), cobalt (II), nickel (II), copper (II),
neodymium (III), samarium (III), ytterbium (III), gadolinium (III),
vanadium (II), terbium (III), dysprosium (III), holmium (II) and
erbium (III), with gadolinium being particularly preferred. Ions
useful in other contexts, such as X-ray imaging, include but are
not limited to lanthanum (III), gold (III), lead (II), and
especially bismuth (III). Moreover, in the case of radioactive
isotopes for therapeutic and/or diagnostic application, one might
mention .sup.131iodine, .sup.123iodine, .sup.99mtechnicium,
.sup.111indium, .sup.188rhenium, .sup.186rhenium, .sup.67gallium,
.sup.67copper, .sup.90yttrium, .sup.125iodine, or .sup.211astatine.
Short-lived positron emission tomography (PET) isotopes, such as
.sup.18flourine, can also be used for labeling peptides for use in
tumor diagnosis (Okarvi, 2001).
[0070] Where the aim is to treat the tumor, one will desire to use
a radionuclide that will irradiate the tumor. Suitable
radionuclides include .sup.131iodine, .sup.123iodine,
.sup.99mtechnicium, .sup.111indium, .sup.188rhenium,
.sup.186rhenium, .sup.67gallium, .sup.90yttrium, .sup.105rhodium,
.sup.89strontium, .sup.153samarium, .sup.211astatine,
.sup.212bismuth .sup.213bismuth, .sup.177lutetium .sup.67copper,
.sup.47scandium, .sup.109palladium. Optimally, radionuclides are
chosen for the specific application on the basis of physical and
chemical properties such that (a) their decay mode and emitted
energy are matched to the delivery site, (b) their half life and
chemical properties are complementary to the biological processing
and (c) production methods can yield the radionuclide at the
necessary level of specific activity and radionuclide purity.
[0071] The incorporation of the radiometal into the
.beta.-superfamily conotoxins generally involves use of a chelate,
specific to the particular metal, and a linker group to covalently
attach the chelate to the conotoxin, i.e., a the bifunctional
chelate approach. The design of useful chelates is dependent on the
coordination requirements of the specific radiometal. DTPA, DOTA,
P.sub.2S.sub.2--COOH BFCA requirement for kinetic TETA, NOTA are
common examples. The requirement for kinetic stability of the metal
complex is often achieved through the use of multidentate chelate
ligands with a functionalized arm for covalent bonding to some part
of the conantokin or .gamma.-carboxyglutamate containing
conopeptide, i.e., the lysine amino group. Techniques for chelating
radioonuclides with proteins are well known in the art, such as
demonstrated by interantional patent application publication No. WO
91/01144, incorporated herein by reference.
[0072] In some embodiments, the .beta.-superfamily conotoxins are
used in combination with one or more potentiators and/or
chemotherapeutic agents for the treatment of cancer or tumors. An
exemplary potentiator is triprolidine (U.S. Pat. No. 5,114,951) or
its cis-isomer which are used in combination with chemotherapeutic
agents. Another potentiator is procodazole, which is a non-specific
immunoprotective agent active against viral and bacterial
infections. Other potentiators which can be used with conantokins
or .gamma.-carboxyglutamate containing peptides and optionally
another chemotherapeutic agent to treat or inhibit the growth of
cancer include monensin, an anti-sense inhibitor of the RAD51 gene,
bromodeoxyuridine, dipyridamole, indomethacin, a monoclonal
antibody, an anti-transferrin receptor immunotoxin, metoclopramide,
7-thia-8-oxoguanosine,
N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylened- iamine,
leucovorin, heparin, N-[4-[(4-fluorphenyl)sulfonly]phenyl]acetamid-
e, heparin sulfate, cimetidine, a radiosensitizer, a
chemosensitizer, a hypoxic cell cytotoxic agent, muramyl dipeptide,
vitamin A, 2'-deoxycoformycin, a bis-diketopiperazine derivative,
and dimethyl sulfoxide.
[0073] The chemotherapeutic agents which can be used with
conantokins or .gamma.-carboxyglutamate containing peptides and an
optional potentiator are generally grouped as DNA-interactive
agents, antimetabolites, tubulin-interactive agents, hormonal
agents and others such as asparaginase or hydroxyurea. Each of the
groups of chemotherapeutic agents can be further divided by type of
activity or compound. The chemotherapeutic agents used in
combination with .gamma.-carboxy-glutamat- e containing peptides
include members of all of these groups. For a detailed discussion
of chemotherapeutic agents and their method of administration, see
Dorr et al. (1994) and U.S. Pat. No. 6,290,929.
[0074] DNA-interactive agents include the alkylating agents, e.g.
cisplatin, cyclophosphamide, altretamine; the DNA strand-breakage
agents, such as bleomycin; the intercalating topoisomerase II
inhibitors, e.g., dactinomycin and doxorubicin; the
nonintercalating topoisomerase II inhibitors such as, etoposide and
teniposde; and the DNA minor groove binder plicamydin. The
alkylating agents form covalent chemical adducts with cellular DNA,
RNA, and protein molecules and with smaller amino acids,
glutathione and similar chemicals. Generally, these alkylating
agents react with a nucleophilic atom in a cellular constituent,
such as an amino, carboxyl, phosphate, sulfhydryl group in nucleic
acids, proteins, amino acids, or glutathione.
[0075] The antimetabolites interfere with the production of nucleic
acids by one or the other of two major mechanisms. Some of the
drugs inhibit production of the deoxyribonucleoside triphosphates
that are the immediate precursors for DNA synthesis, thus
inhibiting DNA replication. Some of the compounds are sufficiently
like purines or pyrimidines to be able to substitute for them in
the anabolic nucleotide pathways. These analogs can then be
substituted into the DNA and RNA instead of their normal
counterparts.
[0076] Tubulin interactive agents act by binding to specific sites
on tubulin, a protein that polymerizes to form cellular
microtubules. Microtubules are critical cell structure units. When
the interactive agents bind on the protein, the cell cannot form
microtubules. Tubulin interactive agents include colchicine,
vincristine and vinblastine, both alkaloids and paclitaxel and
cytoxan.
[0077] Hormonal agents are also useful in the treatment of cancers
and tumors. They are used in hormonally susceptible tumors and are
usually derived from natural sources. These include: estrogens,
conjugated estrogens and ethinyl estradiol and diethylstilbesterol,
chlortrianisen and idenestrol; progestins such as
hydroxyprogesterone caproate, medroxyprogesterone, and megestrol;
and androgens such as testosterone, testosterone propionate;
fluoxymesterone, methyltestosterone. Adrenal corticosteroids are
derived from natural adrenal cortisol or hydrocortisone. They are
used because of their anti inflammatory benefits as well as the
ability of some to inhibit mitotic divisions and to halt DNA
synthesis. These compounds include, prednisone, dexamethasone,
methylprednisolone, and prednisolone. Leutinizing hormone releasing
hormone agents or gonadotropin-releasing hormone antagonists are
used primarily the treatment of prostate cancer. These include
leuprolide acetate and goserelin acetate. They prevent the
biosynthesis of steroids in the testes. Antihormonal antigens
include: antiestrogenic agents such as tamoxifen, antiandrogen
agents such as flutamide; and antiadrenal agents such as mitotane
and aminoglutethimide.
[0078] 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, parenteral or
intrathecally. For examples of delivery methods see U.S. Pat. No.
5,844,077, incorporated herein by reference.
[0079] "Pharmaceutical composition" means physically discrete
coherent portions suitable for medical administration.
"Pharmaceutical composition in dosage unit form" means physically
discrete coherent units suitable for medical administration, each
containing a daily dose or a multiple (up to four times) or a
sub-multiple (down to a fortieth) of a daily dose of the active
compound in association with a carrier and/or enclosed within an
envelope. Whether the composition contains a daily dose, or for
example, a half, a third or a quarter of a daily dose, will depend
on whether the pharmaceutical composition is to be administered
once or, for example, twice, three times or four times a day,
respectively.
[0080] The term "salt", as used herein, denotes acidic and/or basic
salts, formed with inorganic or organic acids and/or bases,
preferably basic salts. While pharmaceutically acceptable salts are
preferred, particularly when employing the compounds of the
invention as medicaments, other salts find utility, for example, in
processing these compounds, or where non-medicament-type uses are
contemplated. Salts of these compounds may be prepared by
art-recognized techniques.
[0081] Examples of such pharmaceutically acceptable salts include,
but are not limited to, inorganic and organic addition salts, such
as hydrochloride, sulphates, nitrates or phosphates and acetates,
trifluoroacetates, propionates, succinates, benzoates, citrates,
tartrates, fumarates, maleates, methane-sulfonates, isothionates,
theophylline acetates, salicylates, respectively, or the like.
Lower alkyl quaternary ammonium salts and the like are suitable, as
well.
[0082] As used herein, the term "pharmaceutically acceptable"
carrier means a non-toxic, inert solid, semi-solid liquid filler,
diluent, encapsulating material, formulation auxiliary of any type,
or simply a sterile aqueous medium, such as saline. Some examples
of the materials that can serve as pharmaceutically acceptable
carriers are sugars, such as lactose, glucose and sucrose, starches
such as corn starch and potato starch, cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose acetate; powdered tragacanth; malt, gelatin, talc;
excipients such as cocoa butter and suppository waxes; oils such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
corn oil and soybean oil; glycols, such as propylene glycol,
polyols such as glycerin, sorbitol, mannitol and polyethylene
glycol; esters such as ethyl oleate and ethyl laurate, agar;
buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen-free water; isotonic saline,
Ringer's solution; ethyl alcohol and phosphate buffer solutions, as
well as other non-toxic compatible substances used in
pharmaceutical formulations.
[0083] Wetting agents, emulsifiers and lubricants such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator. Examples of pharmaceutically acceptable antioxidants
include, but are not limited to, water soluble antioxidants such as
ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
metabisulfite, sodium sulfite, and the like; oil soluble
antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
aloha-tocopherol and the like; and the metal chelating agents such
as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid and the like.
[0084] 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.
[0085] For parenteral administration, the compound may be dissolved
in a pharmaceutical carrier and administered as either a solution
or a suspension. Illustrative of suitable carriers are water,
saline, dextrose solutions, fructose solutions, ethanol, or oils of
animal, vegetative or synthetic origin. The carrier may also
contain other ingredients, for example, preservatives, suspending
agents, solubilizing agents, buffers and the like. When the
compounds are being administered intrathecally, they may also be
dissolved in cerebrospinal fluid.
[0086] A variety of administration routes are available. The
particular mode selected will depend of course, upon the particular
drug selected, the severity of the disease state being treated and
the dosage required for therapeutic efficacy. The methods of this
invention, generally speaking, may be practiced using any mode of
administration that is medically acceptable, meaning any mode that
produces effective levels of the active compounds without causing
clinically unacceptable adverse effects. Such modes of
administration include oral, rectal, sublingual, topical, nasal,
transdermal or parenteral routes. The term "parenteral" includes
subcutaneous, intravenous, epidural, irrigation, intramuscular,
release pumps, or infusion.
[0087] For example, administration of the active agent according to
this invention may be achieved using any suitable delivery means,
including:
[0088] (a) pump (see, e.g., Luer & Hatton (1993), Zimm et al.
(1984) and Ettinger et al. (1978));
[0089] (b), microencapsulation (see, e.g., U.S. Pat. Nos.
4,352,883; 4,353,888; and 5,084,350);
[0090] (c) continuous release polymer implants (see, e.g., U.S.
Pat. No. 4,883,666);
[0091] (d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761,
5,158,881, 4,976,859 and 4,968,733 and published PCT patent
applications W092/19195, WO 95/05452);
[0092] (e) naked or unencapsulated cell grafts to the CNS (see,
e.g., U.S. Pat. Nos. 5,082,670 and 5,618,531);
[0093] (f) injection, either subcutaneously, intravenously,
intra-arterially, intramuscularly, or to other suitable site;
or
[0094] (g) oral administration, in capsule, liquid, tablet, pill,
or prolonged release formulation.
[0095] In one embodiment of this invention, an active agent is
delivered directly into the CNS, preferably to the brain
ventricles, brain parenchyma, the intrathecal space or other
suitable CNS location, most preferably intrathecally.
[0096] Alternatively, targeting therapies may be used to deliver
the active agent more specifically to certain types of cell, by the
use of targeting systems such as antibodies or cell specific
ligands. Targeting may be desirable for a variety of reasons, e.g.
if the agent is unacceptably toxic, or if it would otherwise
require too high a dosage, or if it would not otherwise be able to
enter the target cells.
[0097] The active agents, which are peptides, can also be
administered in a cell based delivery system in which a DNA
sequence encoding an active agent is introduced into cells designed
for implantation in the body of the patient, especially in the
spinal cord region. Suitable delivery systems are described in U.S.
Pat. No. 5,550,050 and published PCT Application Nos. WO 92/19195,
WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO
96/40871, WO 96/40959 and WO 97/12635. Suitable DNA sequences can
be prepared synthetically for each active agent on the basis of the
developed sequences and the known genetic code.
[0098] Exemplary methods for administering compounds (e.g., so as
to achieve sterile or aseptic conditions) will be apparent to the
skilled artisan. Certain methods suitable for administering
compounds useful according to the present invention are set forth
in Goodman and Gilman's The Pharmacological Basis of Therapeutics,
7th Ed. (1985). The administration to the patient can be
intermittent; or at a gradual, continuous, constant or controlled
rate. Administration can be to a warm-blooded animal (e.g. a
mammal, such as a mouse, rat, cat, rabbit, dog, pig, cow or
monkey); but advantageously is administered to a human being.
Administration occurs after general anesthesia is administered. The
frequency of administration normally is determined by an
anesthesiologist, and typically varies from patient to patient.
[0099] The active agent is preferably administered in an
therapeutically effective amount. By a "therapeutically effective
amount" or simply "effective amount" of an active compound is meant
a sufficient amount of the compound to treat the desired condition
at a reasonable benefit/risk ratio applicable to any medical
treatment. The actual amount administered, and the rate and
time-course of administration, will depend on the nature and
severity of the condition being treated. Prescription of treatment,
e.g. decisions on dosage, timing, etc., is within the
responsibility of general practitioners or spealists, and typically
takes account of the disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners. Examples
of techniques and protocols can be found in Remington's
Pharmaceutical Sciences.
[0100] Dosage may be adjusted appropriately to achieve desired
levels, locally or systemically, and depending on use as a
diagnostic agent or a therapeutic agent. For therapeutic uses, the
active agents of the present invention typically exhibit their
effect at a dosage range from about 0.001 mg/kg to about 250 mg/kg,
preferably from about 0.01 mg/kg to about 100 mg/kg of the active
ingredient, more preferably from a bout 0.05 mg/kg to about 75
mg/kg. A suitable dose can be administered in multiple sub-doses
per day. Typically, a dose or sub-dose may contain from about 0.1
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. In the event that the response in a subject is
insufficient at such doses, even higher doses (or effective higher
doses by a different, more localized delivery route) may be
employed to the extent that patient tolerance permits. Continuous
dosing over, for example 24 hours or multiple doses per day are
contemplated to achieve appropriate systemic levels of
compounds.
[0101] For diagnostic uses, an appropriate dosage will depend on
the peptide and the detectable label. A suitable dose to be
injected is in the range to enable imaging by scanning procedures
known in the art. When a radiolabeled conantokin is used, it may be
administered in a dose having a radioactivity of form 0.1 to 50
mCi, preferably, 0.1 to 30 mCi and more preferably, 0.1 to mCi. For
therpeutic uses, an appropriate dosage will depend on the peptide,
the radionuculide, the size and location of the tumor and the half
life of the active agent in the tumor. In general, the dose is
calculated on the basis of of the radioactivity distribution to
each organ and on observed target uptake. For example, the active
agent may be administered at a daily dosage range having a
radioactivity of from 0.1 to 3 mCi/kg, preferably 1 to 3 mCi/kg,
more preferably 1 to 1.5 mCi/kg.
[0102] Advantageously, the compositions are formulated as dosage
units, each unit being adapted to supply a fixed dose of active
ingredients. Tablets, coated tablets, capsules, ampoules and
suppositories are examples of dosage forms according to the
invention.
[0103] It is only necessary that the active ingredient constitute
an effective amount, i.e., such that a suitable effective dosage
will be consistent with the dosage form employed in single or
multiple unit doses. The exact individual dosages, as well as daily
dosages, are determined according to standard medical principles
under the direction of a physician or veterinarian for use humans
or animals.
[0104] The pharmaceutical compositions will generally contain from
about 0.0001 to 99 wt. %, preferably about 0.001 to 50 wt. %, more
preferably about 0.01 to 10 wt. % of the active ingredient by
weight of the total composition. In addition to the active agent,
the pharmaceutical compositions and medicaments can also contain
other pharmaceutically active compounds. Examples of other
pharmaceutically active compounds include, but are not limited to,
analgesic agents, cytokines and therapeutic agents in all of the
major areas of clinical medicine. When used with other
pharmaceutically active compounds, the conopeptides of the present
invention may be delivered in the form of drug cocktails. A
cocktail is a mixture of any one of the compounds useful with this
invention with another drug or agent. In this embodiment, a common
administration vehicle (e.g., pill, tablet, implant, pump,
injectable solution, etc.) would contain both the instant
composition in combination supplementary potentiating agent. The
individual drugs of the cocktail are each administered in
therapeutically effective amounts. A therapeutically effective
amount will be determined by the parameters described above; but,
in any event, is that amount which establishes a level of the drugs
in the area of body where the drugs are required for a period of
time which is effective in attaining the desired effects.
[0105] The present invention also relates to rational drug design
for the indentification of additional drugs which can be used for
the pursposes described herein. The goal of rational drug design is
to produce structural analogs of biologically active polypeptides
of interest or of small molecules with which they interact (e.g.,
agonists, antagonists, inhibitors) in order to fashion drugs which
are, for example, more active or stable forms of the polypeptide,
or which, e.g., enhance or interfere with the function of a
polypeptide in vivo. Several approaches for use in rational drug
design include analysis of three-dimensional structure, alanine
scans, molecular modeling and use of anti-id antibodies. These
techniques are well known to those skilled in the art. Such
techniques may include providing atomic coordinates defining a
three-dimensional structure of a protein complex formed by said
first polypeptide and said second polypeptide, and designing or
selecting compounds capable of interfering with the interaction
between a first polypeptide and a second polypeptide based on said
atomic coordinates.
[0106] Following identification of a substance which modulates or
affects polypeptide activity, the substance may be further
investigated. Furthermore, it may be manufactured and/or used in
preparation, i.e., manufacture or formulation, or a composition
such as a medicament, pharmaceutical composition or drug. These may
be administered to individuals.
[0107] A substance identified as a modulator of polypeptide
function may be peptide or non-peptide in nature. Non-peptide
"small molecules" are often preferred for many in vivo
pharmaceutical uses. Accordingly, a mimetic or mimic of the
substance (particularly if a peptide) may be designed for
pharmaceutical use.
[0108] The designing of mimetics to a known pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound. This approach might be desirable where
the active compound is difficult or expensive to synthesize or
where it is unsuitable for a particular method of administration,
e.g., pure peptides are unsuitable active agents for oral
compositions as they tend to be quickly degraded by proteases in
the alimentary canal. Mimetic design, synthesis and testing is
generally used to avoid randomly screening large numbers of
molecules for a target property.
[0109] Once the pharmacophore has been found, its structure is
modeled according to its physical properties, e.g.,
stereochemistry, bonding, size and/or charge, using data from a
range of sources, e.g., spectroscopic techniques, x-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can be used in this
modeling process.
[0110] A template molecule is then selected, onto which chemical
groups that mimic the pharmacophore can be grafted. The template
molecule and the chemical groups grafted thereon can be
conveniently selected so that the mimetic is easy to synthesize, is
likely to be pharmacologically acceptable, and does not degrade in
vivo, while retaining the biological activity of the lead compound.
Alternatively, where the mimetic is peptide-based, further
stability can be achieved by cyclizing the peptide, increasing its
rigidity. The mimetic or mimetics found by this approach can then
be screened to see whether they have the target property, or to
what extent it is exhibited. Further optimization or modification
can then be carried out to arrive at one or more final mimetics for
in vivo or clinical testing.
[0111] The present invention further relates to the use of a
labeled (e.g., radiolabel, fluorophore, chromophore or the like) of
the .beta.-conotoxins described herein as a molecular tool both in
vitro and in vivo, for discovery of small molecules that exert
their action at or partially at the same functional site as the
native toxin and capable of elucidation similar functional
responses as the native toxin. In one embodiment, the displacement
of a labeled .beta.-conotoxin from its receptor or other complex by
a candidate drug agent is used to identify suitable candidate
drugs. In a second embodiment, a biological assay on a test
compound to determine the therapeutic activity is conducted and
compared to the results obtained from the biological assay of a
.beta.-conotoxin. In a third embodiment, the binding affinity of a
small molecule to the receptor of a .beta.-conotoxin is measured
and compared to the binding affinity of a .beta.-conotoxin to its
receptor.
[0112] In view of the targets of the .beta.-conotoxins, they may be
used for treating the following conditions: cancer (neoplasm, solid
tumor, diabetic nephropathy, fibrosis, hypophysis tumor, GI
disease, IBS, restinosis, angiogenesis disorder, diabetes mellitus,
endocrine tumor, diarrhea, pancreatic disease, prostate tumor,
bleeding, apoptosis), inflammation, pain, diabetes, obesity, sexual
dysfunction, acromegaly, glaucoma, cardiovascular, diabetic,
retinopathy, depression, myocardial infarction, stroke, epilepsy,
anorexia, wasting diseases, seborrheic dermatitis, schizophrenia,
mood disorders, chemotherapeutic induced emesis, disorders
associated with changes in blood pressure, immune disorders, nerve
damage, acne, GI infections, myocardial infarction, angina,
thromboembolism and cardiovascular disease.
[0113] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA, genetics,
immunology, cell biology, cell culture and transgenic biology,
which are within the skill of the art. See, e.g., Maniatis et al.,
1982; Sambrook et al., 1989; Ausubel et al., 1992; Glover, 1985;
Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988; Jakoby
and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S.
J. Higgins eds. 1984); Transcription And Translation (B. D. Hames
& S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.
Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes
(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular
Cloning (1984); the treatise, Methods In Enzymology (Academic
Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.
H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition,
Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986).
EXAMPLES
[0114] 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
Isolation of DNA Encoding .beta.-Superfamily Conopeptides
[0115] DNA coding for .beta.-superfamily conotoxin peptides was
isolated and cloned in accordance with conventional techniques
using general procedures well known in the art, such as described
in Olivera et al. (1996), including using primers based on the DNA
sequence of known conotoxin peptides. Alternatively, cDNA libraries
was prepared from Conus 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. Clones
having a size of approximately 300-500 nucleotides were sequenced
and screened for similarity in sequence to known conotoxins. The
DNA sequences and encoded propeptide sequences are set forth in
Table 1. DNA sequences coding for the mature toxin can also be
prepared on the basis of the DNA sequences set forth in Table 1. An
alignment of the conopeptides of the present invention is set forth
in Table 2. Sequences of truncated and analog peptides are set
forth in Table 3.
1TABLE 1 Sequences of .beta.-Superfami1y Conotoxins Name: Fd14.1
Species: flavidus Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGT- GATGATGATGGTGTGGATTACAGC (SEQ ID
NO:1) CCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCAGATGA
CTTAACCCCACAGCTTATTTTGCAAAGTCTGGATTCCCGTCGTCATGATCACGGCAT
TCGTCCGAAGAGAGTCGACATATGTAACTGGAGGATATGTGCACCAAACCCATTGA
GACGACATGATCTTAAGAAAGGAAACAATTGACGTCAGACAACCGCCACAACTTGA
GTACGACATCGTTAATACGACTTCAGCAAATATGAAATTTTCAGCATCACTGTGGTT
GTGAAGAAATCAGTTGCTTTAAAAGGTTGGATTTGTCCTTGTTTAAGCCGTTGTACT
GATGACATCTCTGCACTATGAAATAAAGCTGATGTGACAAACTAAAAAAAAAAAAA AAAA
Translation: MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGL-
VPDDLTPQLILQSLDSRRLHDHGIRP (SEQ ID NO:2) KRVDICNWRICAPNPLRRHDLKKGNN
Toxin Sequence: His-Asp-His-Gly-Ile-Arg-Xaa3-Lys-A-
rg-Val-Asp-Ile-Cys-Asn-Xaa4-Arg-Ile-Cy5-Ala-Xaa3- (SEQ ID NO:3)
Asn-Xaa3-Leu-Arg-Arg-His-Asp-Leu-Lys-Lys-Gly-Asn-Asn-{circumflex
over ( )} Name: Mi14.1 Species: miles Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGGTGGGGCTCAC (SEQ ID
NO:4) CGTCGGGAGTCACGTCCATCGGTCTCACAGTCCTACATCGCGCAGCCATGGTGATGA
CTCCATTCATGACAAGACGATTCATCAACATCTGTTTGCCCGTCTTCCTCTGGAGAA
CAACGACGACCATCGTTCTGTGGATCTTCCTGCAGGGAATGGTGCAGGCAACACCA
AGCAACAAGACCAAAGTCCTCATCATGTGTGTTGTGCTATTGGTCCGGTTCTTCCAT
TCTGTTGTGTCAGTTGGCTGCACAAACTCCATTGAACTGGCCAATGAAAATAACTCA
GGAATAGACAGAAAGGCAAAAAAAAAAAAAAAAA Translation:
MQTAYWVMVMMMVVGLTVGSHVTTRSHSPTSRSHGDDSIHDKTIHLFARLPLE- NND (SEQ ID
NO:5) DHRSVDLPAGNGAGNTKQQDQSPHHVCCAIGPVLPFCCVSWLHKLH Toxin
Sequence: Xaa2-Gln-Asp-Gln-Ser-Xaa3-His-His-Val-
-Cys-Cys-Ala-Ile-Gly-Xaa3-Val-Leu-Xaa3-Phe-Cys- (SEQ ID NO:6)
Cys-Val-Ser-Xaa4-Leu-His-Lys-Leu-His-{circumflex over ( )} Name:
Mil4.2 Species: miles Isolated: No Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGGTGGGGTTCAC (SEQ ID
NO:) CGTCGGGGGTCACGTCCATCGGTCTCACAGTCCTACATCGCGCAGCCATGGTGATGA
CTCCATTCATGACAAGACGATTCATCAACATCTGTTTGCCCGTCTTCCTCAGGAGAA
CAACGACGACCATCGTTCTGTGGATCTTCCTGCAGGGACTAGCGCAGGCGACATGA
AACCACAACGCCAAAGACGTCTCTGCTGCATCTTTGCCCCGATTCTTTGGTTCTGTT
GTCACGGTTAACAGCTCAAATTACACTGCACTGGCCGATTGAAAGAACTGCAATAA
ACGGAAAAAAAAAAAAAAAA Translation:
MQTAYWVMVMMMVVGFTVGGHVHRSHSPTSRSHGDDSIHDKTIHQHLFARLPQENN (SEQ ID
NO:8) DDHRSVDLPAGTSAGDMKPQRQRRLCCIFAPILWFCCHG Toxin Sequence:
Leu-Cys-Cys-ILe-Phe-Ala-Xaa3-ILe-LeU-Xaa4-Phe-Cys-Cys-Hi- s-# (SEQ
ID NO:9) Name: Cp14.1 Species: capitaneus Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGGTGGGGTTCAC (SEQ ID
NO:10) CGTCGGGGGTCACGTCCATCGGTCTCACAGTCCTACATCGCGCAGCCATGGTGATGA
CTCCATTCATGACGAGACGATTCATCAACATCTGTTTGCCCGTCTTCCTCAGGAGAA
CAACGACGACCATCGTTCTGTGGATCTTCCTGCAGGGACTAGCGCAGGCGACATGA
AACCACAACGCCAAAGAGGTTTCTGCTGCGACTTTCCCCCGATTTTTTGGTTCTGTT
GTATCGGTTAACAGCACAAATTACACTGCACTGGCCGATTGAAAGAACTGCAATAA
ACGGAAAAAAAA Translation:
MQTAYWVMVMMMVVGFTVGGHVHRSHSPTSRSHGDDSIHDETIHQHLFARLPQENN (SEQ ID
NO:11) DDHRSVDLPAGTSAGDMKPQRQRGFCCDFPPIFWFCCIG Toxin Sequence:
Gly-Phe-Cys-Cys-Asp-Phe-Xaa3-Xaa3-Ile-Phe-Xaa4-Phe-Cys-C- ys-Ile-#
(SEQ ID NO:12) Name: Ge14.1 Species: generalis Cloned: Yes DNA
Sequence: GGATCCATGCAGACGGCCTACTGGGTAATGGTGATGATGATGGTGTGGATTAAAGG
(SEQ ID NO:13)
CCCTGTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCAGACG
ACTTGACCCCAGTGTTTGCCTTGCATCATCCGGTTTCCCATCGTCGGTCTCACAGCA
GTAGTTTGTGGTGTGTATGTCCATTCAGGGTGTGTCCACCATGCCATGGAAGATGAC
CTGGTCCCAAACCAACAAAATAACGTCAGACAACCGCCACAACTTTAGTACGACAT
CCCTTAATACGACTTCAGCAAGTATTTTAACATCACTATGGTGTGATGAAATCAGTT GCTTTAAAA
Translation:
MQTAYWVMVMMMVWIKGPVSEGGKLNDVIRGLVPDDLTPVFALHHPVSHRRSHSSSL (SEQ ID
NO:14) WCVCPFRVCPPCHGR Toxin Sequence:
Ser-His-Ser-Ser-Ser-Leu-Xaa4-Cys-Val-Cys-Xaa3-Phe-Arg-Val-Cys-Xaa3-Xaa3-C-
ys-His-# (SEQ ID NO:15) Name: Wi14.1 Species: wittigi Cloned: Yes
DNA Sequence:
ATGATGTTGGTGTGGATTACAGCCCCTCTGCCTGAAGGTGGTAAACTGAAGCACGT (SEQ ID
NO:16) AATTCGGGGTTTGGTGCCAGACGACTTAACCCCACAGCTTATCTTGCGAAGTCTGAT
TTCCCGTCGTAGTTCTGACGGCAGTGATCCGAAGGCAAAAAAACAGTGTATGTGGA
AGAGATGTATACCAGACCAATCGAGACTAGAAGAAGATGAATGATGTCAGACAAC
CGCCATCACTGTAGTATGACATCGTTAATACGACTTAAGCAAATATTTTAACATCAC
TGTGGTTCTGAAGACATCAGTTGCTTTAAAAGATTGGATTCTTCCTTGTTTAAGAGTT
GTACTGANATCATTCCTGCCCTGTGAAATAAAGCTGATGTTGACANNCAAACAAAA AAAAAAAAAA
Translation:
MMLVWITAPLPEGGKLKHVIRGLVPDDLTPQLIILRSLISRRSSDGSDPKAKKQCMWKRC (SEQ
ID NO:17) IPDQSRLEEDE Toxin Sequence:
Ser-Ser-Asp-Gly-Ser-Asp-Xaa3-Lys-Ala-Lys-Lys-Gln-Cys-Met-Xaa4-Lys-Arg-Cys-
-Ile-Xaa3- (SEQ ID NO:18)
Asp-Gln-Ser-Arg-Leu-Xaa1-Xaa1-Asp-Xaa1-{c- ircumflex over ( )}
Name: Cn14.1 Species: consors Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGC (SEQ ID
NO:19) CCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGTCACACAT
CTTAATCCCACAGCATACCTTGCGAAGTCTGACTTCCCGTGATCGTTCTGACAACGG
TGGTTCGAGTGGAGCACAAATATGCATCTGGAAGGTATGTCCACCATCCCCATAGA
GACGACCACGAGGAAAAAGATGAACGGCGTCAGACAACCGCCACAACTGTAGTAC
GACATCGTTGATACGACTTCAGCAACTATTTTAACATCACTGTGGTTGTGAAGAAAT
CAGTCGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCT
GCACTATGAAATAAAGCTGATGTGACATAAAAAAAAAAAAAAAAAGTACTCTGCGT
TGTTACTCGAG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVSHILIPQHTLRSLTSRDRSDNGGSS (SEQ ID
NO:20) GAQICIWKVCPPSP Toxin Sequence:
Asp-Arg-Ser-Asp-Asn-Gly-Gly-Ser-Ser-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val--
Cys-Xaa3- (SEQ ID NO:21) Xaa3-Ser-Xaa3-{circumflex over ( )} Name:
Cn14.2 Species: consors Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGT- GATGATGATGGTGTGGATTACAGC (SEQ ID
NO:22) CCCTCTGTCTGAAGGTGGTAAATTGAACGACGCAATTCGGGGTTTGGTGTCACACAT
CTTAATCCCACAGCATACCTTGCGAAGTCTGACTTCCCGTGCTCGTTCTGACAACGG
TGGTTCGAGTGGAGCACAAATATGCATCTGGAAGGTATGTCCACCATCCCCATGGA
GACGACCACAAGGAAAAAGATGAATGACGTCAGACAACCGCCACAACTGTAGTAC
GACATCGTTGATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAAT
CAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCT
GCACTATGAAATAAAGCTGATGTGACAAACAATAAAAAAGAAAAAAAAAAAAGTA
CTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDAIRGLVSHILIPQHTLRSLTSRARSDNGGSS (SEQ ID
NO:23) GAQICIWKVCPPSPWRRPQGKR Toxin Sequence:
Ala-Arg-Ser-Asp-Asn-Gly-Gly-Ser-Ser-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val--
Cys-Xaa3- (SEQ ID NO:24) Xaa3-Ser-Xaa3-Xaa4-Arg-Arg-Xaa3-Gln-#
Name: Cn14.3 Species: consors Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGGT- GATGATGATGGTGTGGATTACAGC (SEQ ID
NO:25) CCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCACACTT
CTTAACCCCACAGCATATCTTGCAAAGTCTGACTTCCCGTAATGGTTCTGGCAGCAG
TAATCAGAAAGAAGCACAACTATGCATCTGGAAGGTATGTCCACCATCCCCATGGA
GATGACCACAAGGAAAAAGATGAACGGCGTCAGACAACCGCCACAACTGTAGTGG
GACATCGTTGATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAAT
CAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAATTGTACTGATATCAGCTCT
GCACTATGAAATAAAGCTGATGTGACAACCCAAAAAAAAAAAAAAAAAAAAGTAC
TCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMYWITAPLSEGGKLNDVIRGLVPHFLTPQHILQSLTSRNGSGSSNQ (SEQ ID
NO:26) KEAQLCIWKVCPPSPWR Toxin Sequence:
Asn-Gly-Ser-Gly-Ser-Ser-Asn-Gln-Lys-Xaa1-Ala-Gln-Leu-Cys-Ile-Xaa4-Lys-Val-
-Cys-Xaa3- (SEQ ID NO:27) Xaa3-Ser-Xaa3-Xaa4-Arg-{circumflex over (
)} Name: T14.1 Species: tulipa Cloned: Yes DNA Sequence:
GGATCCATGCAGACGGCCTACTGGGTGATGCTGATGATGATGGTGTGGATTACAGC (SEQ ID
NO:28) CCCTCTGTCTGAAGGTGGTAAACTGAACGACGTAATTCGGGGTTTGGTGCCACACGT
CTTAACCCCACAGCATATCTTGCAAAGTCTGGTTTCCCGTCGTCATTTTAACAGCGTT
GTTCCGACGGTATACATATGCATGTGGAAGGTATGTCCACCATCGCCATAGAGACG
ACCATAAGGAAAAAGATGAATGACGTCAGACAACCGCCACAACTGTAGTACGACAT
CGTTAATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATCAGTT
GCTTTAAAAGATTGGATTTTTCCTTGTTTCAGAGTTGTACTGATATCAGCTCTGCAC- T
ATCAAATAAAGCTGAAGTGACAAACCNNAAAAAAAAAAAAAAAAAAAAAAAAGTA
CTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMLMMMVWJTAIPLSEGGKLNDVIIRGLVPHVLTPQHILQSLVSRRHFNSVVP (SEQ ID
NO:29) TVYICMWKVCPPSP Toxin Sequence:
His-Phe-Asn-Ser-Val-Val-Xaa3-Thr-Val-Xaa5-Ile-Cys-Met-Xaa4-Lys-Val-Cys-Xa-
a3-Xaa3-Ser- (SEQ ID NO:30) Xaa3-{circumflex over ( )} Name: T14.2
Species: tulipa Isolated: No Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGCTGTTGATGATGGTGGGCATTACAGCCCCTCTG (SEQ ID
NO:31) CCTGAAGGTGGTAAACCGAACAGCGTAATTCGGGGTTTGGTGCCAAACGACTTAAC
TCCACAGCATACCTTGCGAAGTCTGATTTCCCGTCGTCAAACTGACGTTCTTCTGGA
GGCTACCCTTTTGACAACACCAGCCCCCGAGCAGAGATTGTTCTGCTTCTGGAAGTC
ATGTTGGCCAAGGCCCTACCCTTGGAGACGACGTGATCTTAATGGAAAACGATGAA
TGACGTCAGACAACCGCCACAACTGTAGTACGACATCATTAATACGACTTCAGCAA
ATATTTTAACATTACTGTGGTTGTGAAGAAATCACTTGCTTTAAAAGATTGGTTTTT- T
CCTTGTTTCAGAGTTGTACTGATATCAGCTCTGCCCTATGAAATAAAGCTGATG Translation:
MQTAYVVMLLMMVGITAPLPEGGKPNSVIIRGLVPNDLT- PQHTLRSLISRRQTDVLLEA (SEQ
ID NO:32) TLLTTPAPEQRLFCFWKSCWPRPYPWRRRDL- NGKR Toxin Sequence:
Xaa2-Thr-Asp-Val-Leu-Leu-Xaa1--
Ala-Thr-Leu-Leu-Thr-Thr-Xaa3-Ala-Xaa3-Xaa1-Gln-Arg (SEQ ID NO:33)
Leu-Phe-Cys-Phe-Xaa4-Lys-Ser-Cys-Xaa4-Xaa3-Arg-Xaa3-Xaa5-Xaa3-Xaa4-Arg-Ar-
g-Arg- Asp-Leu-Asn-# Name: S114.2 Species: sulcatus Cloned: Yes DNA
Sequence: ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTCT
(SEQ ID NO:34) GTCTGAAGGTGGTAAACCGAACGACGTAATTCGGGGTTTGGTGCCAGACG-
ACTTAA CCCCACAGCGTGTCTTGCGAAGTCTGATTTCCCGTCGTCAATCTGGCTGCA- GAGTCC
CGTTTGAATTGAAATGCATCTGGAAGTTCTGTACAATATACCCATCGAGAC- CATTTG
CTTCTCTGGAAGAAAAAGACGAATGTCAGACAGTCACCATAACTGTAACAT- GGGAT
TTTTAATACGTCTCCAGCAAGTATTTTAACATCACTGTGGTTGTGAAGAAAT- CAGTT
GCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTC- TGCCCT
GTGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSEGGKPNDVIRGLVPDDLTPQRVLRSLISRRQSGCRVP (SEQ ID
NO:35) FELKCIWKFCTIYPSRPFASLEEKDECQTVTITVTWDF Toxin Sequence:
Xaa2-Ser-Gly-Cys-Arg-Val-Xaa3-Phe-Xaa1-Leu-Lys-Cys-
-Ile-Xaa4-Lys-Phe-Cys-Thr-Ile-Xaa5- (SEQ ID NO:36)
Xaa3-Ser-Arg-Xaa3-Phe-Ala-Ser-Leu-Xaa1-Xaa1-Lys-Asp-Xaa1-Cys-Gln-Thr-Val--
Thr-Ile-Thr- Val-Thr-Xaa4-Asp-Phe-{circumflex over ( )} Name:
S114.1 Species: sulcatus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTG- TGGATTACAGCCTCTCTG (SEQ ID
NO:37) TCTGAAGGTGGTAAACCGAACGAC- GTCATTCGGGGTTTTGTGCCAGACGACTTAAC
CCCACAGCTTATCTTGCGAAGTCTG- ATTTCCCGTCGTCGTTCTGACAAGGATGTTGG
GAAGAGAATGGAATGTTACTGGAAG- GCATGTAGACCCACGCTATCGAGACGACATG
ATCTTGGGTAAAAGATGAATGACGTC- AGACAACAGCCACAACTATAGTATGACATC
GTTAATACGACTTCAGCAAATATTTTA- ACATCACTGTGGTTGTGAAGAAATCAGTTG
CTTTAAAAGATTGGATTTTTCCGTGTT- TAAGAGTTGTACTGATATCAGCTCTGCCCTG
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITASLSEGGKPNDVIRGFVPDDLTPQLILRSL- ISRRRSDKDVGK (SEQ
ID NO:38) RMECYWKACRPTLSRRHDLG Toxin Sequence:
Arg-Ser-Asp-Lys-Asp-Val-Gly-Lys-Arg-Met-Xaa1-Cys-X-
aa5-Xaa4-Lys-Ala-Cys-Arg-Xaa3- (SEQ ID NO:39)
Thr-Leu-Ser-Arg-Arg-His-Asp-Leu-# Name: M14.1 Species: magus
Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGCTGATGATGATGGTGTGCATCACAGCCCCTCTG (SEQ ID
NO:40) CCTGAAGGTGGTAAACCGAACAGCGGAATTCGGGGTTTGGTGCCAAACGA- CTTAAC
TCCACAGCATACCTTGCGAAGTCTGATTTCCCGTCGTCAAACTGACGTTCT- TCTGGA
TGCTACCCTTTTGACAACACCAGCCCCCGAGCAGAGATTGTTCTGCTTCTG- GAAGTC
ATGTTGGCCAAGGCCCTACCCTTGGAGACGACGTAATCTTAATGGAAAACG- ATGAA
TGACGTCAGACAACCGCCACAACTGTAGTACGACATCGTTAATACGACTTCA- GCAA
ATATTTTAACATAACTGTGGTTGTGAAGAAATCGGTTGCTTTAAAAGATTGGA- TTTT
TCCTTGTTTCAGAGTTGTACTGATATGAGCTCTGCCCTGTGAAATAAAGCTGA- TG
Translation: MQTAYWVMLMMMVCITAPLPEGGKPNSGIRGLVPN-
DLTPQHTLRSLISPRRQTDVLLDA (SEQ ID NO:41)
TLLTTPAPEQRLFCFWKSCWPRPYPWR- RRNLNGKR Toxin Sequence:
Xaa2-Thr-Asp-Val-Leu-Leu-A-
sp-Ala-Thr-Leu-Leu-Thr-Thr-Xaa3-Ala-Xaa3-Xaa1-Gln-Arg- (SEQ ID
NO:42)
Leu-Phe-Cys-Phe-Xaa4-Lys-Ser-Cys-Xaa4-Xaa3-Arg-Xaa3-Xaa5-Xaa3-Xaa4-Arg--
Arg-Arg- Asn-Leu-Asn-# Name: Em14.1 Species: emaciatus Cloned: Yes
DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGCGATGATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:43) GTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCAGATG- ACTTAA
CCCCACAGCTTGTTTTGCAAAGTCTGGATTCCCGTCGTCATACTCACGGCA- TTCGTC
CGAAGGGAGACGGCATATGTATCTGGAAGGTATGTCCACCAGACCCATGGA- GACGA
CATCGTCTTAAGAAAAGAAACAATTGACGTCAGACAACCGCCACAACTTGAG- TACG
ACATCGTTAATACGACTTCAGCAAATATGAAATTTTCAGCATCACTGTGGTTG- TCAA
GAAATCAGTTGCTTTAAAAGATTGGATTTGTCCTTGTTTAAGAGTTGTACTGA- TGTC
AGCTCTGCCCTGTGAAATAAAGCTGATG Translation:
MQTAYWVMAMMMVWITAPLSEGGKLNDVIRGLVPDDLTPQLVLQSLDSRRHTH- GIRP (SEQ ID
NO:44) KGDGICIWKVCPPDPWRRHRLKKRNN Toxin Sequence:
His-Thr-His-Gly-Ile-Arg-Xaa3-Lys-Gly-Asp-Gly-Ile-C-
ys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3- (SEQ ID NO:45)
Asp-Xaa3-Xaa4-Arg-Arg-His-Arg-Leu-Lys-Lys-Arg-Asn-Asn-{circumflex
over ( )} Name: Cr14.1 Species: circumeisus Cloned: Yes DNA
Sequence: ATGCAGACGGCCTACTGGGTGATGGTGATGATGGTGGTGTGGATTACAGCCCCTCT
(SEQ ID NO:46)
GTCTGAAGGTGGTAAATCGAACGACGTAATTCGGGGTTTGGTGCCACACATCTTAA
CCCCACAGCATATCTTGCAAAGTCTGACTTCCCGTCTTCGTTCTGACAGCAGTGGTC
AGAAAGGAGCACAAATATGCATCTGGAAGGTATGTCCACTATCCCCATGGAGACGA
CCACAAGGAAAAAGATGAATGACGTCAGACAACCGCTACAACTGTAGTACGACATC
GTTGATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATCAGTTG
CTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCTGCCCTG
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMVVWITAPLSEGGKSNDVIRGLVPHILTPQHILQSLTSRLRSDSSGQK (SEQ ID
NO:47) GAQICIWKVCPLSPWRRPQGKR Toxin Sequence:
Leu-Arg-Ser-Asp-Ser-Ser-Gly-Gln-Lys-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val--
Cys-Xaa3-Leu (SEQ ID NO:48) Ser-Xaa3-Xaa4-Arg-Arg-Xaa3-Gln-# Name:
Bt14.1 Species: betulinus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGAT- GATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:49) GTCCGAAGGTGGTAAACTGAACGATGTAATTCGGGCTTTGGCGCCAGACGACGTAA
CCCCACAGTTTATCTTGCGAAGTCTGATTTCCCGTCGTCGTTCTGACAGCGATGTTCG
GGAGGTACCCGTATGTTCCTGGAAGATATGTCCACCATAGCCATAGAGACGACATG
ATCTTAAGGAAAAAGAGAAATGACGTCAGACAACCGCCACAACTGTAGTACGGCAT
CGTTAATACGACTTCAGCAAATGTTTTAACATCACTGTGGTTGTGAAGAAATCAGCT
GCTTTAAAAGATTGGATTTTTCCTTAAGAGTTGCACTGATGTCAGTTCTGCCCTGTG
AAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNPVIRALAPDDVTPQFILRSLISRRRSDSDVRE (SEQ ID
NO:50) VPVCSWKICPP Toxin Sequence:
Arg-Ser-Asp-Ser-Asp-Val-Arg-Xaa1-Val-Xaa3-Val-Cys-Ser-Xaa4-Lys-Ile-Cys-Xa-
a3-Xaa3-{circumflex over ( )} (SEQ ID NO:51) Name: A14.1 Species:
aurisiadus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGCGATGATGATGGTGTGGATTACAGCCC- CTCT (SEQ ID
NO:52) GTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTT- TGGTGCCAGATGACTTAA
CCCCACAGCTTGTTTTGCAAAGTCTGGATTCCCGTCGTC- ATACTCACGGCATTCGTC
CGAAGGGAGACGGCATATGTATCTGGAAGGTATGTCCAC- CAGACCCATGGAGACGA
CATCATCTTAAGAAAAGAAACAATTGACGTCAGACAACCG- CCACAACTTGAGTACG
ACATCGTTAATACGACTTCAGCAAATATGAAATTTTCAGCA- TCACTGTGGTTGTCAA
GAAATCAGTTGCTTTAAAAGATTGGATTTGTCCTTGTTTAA- GAGTTGTACTGATGTC
AGCTCTGCCCTATGAAATAAAGCTGATG
Translation: MQTAYWVMAMMMVWITAPLSEGGKLNDVIRGLVPDDLTPQLVLQSLDSRR-
HTHGIRP (SEQ ID NO:53) KGDGICIWKVCPPDPWRRHHLKKRNN Toxin Sequence:
His-Thr-His-Gly-Ile-Arg-Xaa3-Lys-Gly-Asp-Gly-Ile-C-
ys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-(SEQ ID NO: 54)
Asp-Xaa3-Xaa4-Arg-Arg-His-His-Leu-Lys-Lys-Arg-Asn-Asn-{circumflex
over ( )} Name: A14.2 Species: aurisiacus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:55) GTCTGAAGGTGGTAAATTGAACGACGTAATTTGGGGTTTGGTGCCACACATCTTAAC
CCCACAGCATATCTTGCAAAGCCTGACTTCCCGTCTTCATTCTGACAGCAGTGATCA
GAAAGGAGGCATGAACGCATGGACAGGAGCAGGAGCACAAATATGCATCTGGAAG
GTATGTCCACCACCCCCATGGAGATGAACACAAGGAAAAAGATGAATGACGTCAGA
CAACCGCCACAACTGTAGTACGACATCGTTGATACGACTTCAGCAAATATTTTAACA
TCACTGTGGTTGTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAG
AGTTGTACTGATATCAGCTCTGCCCTGTGAAGTAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIWGLVPHILTPQHILQSLTSRLHSD- SSDQ (SEQ ID
NO:56) KGGMNAWTGAGAQICIWKVCPPPPWR Toxin Sequence:
Leu-His-Ser-Asp-Ser-Ser-Asp-Gln-Lys-Gly-Gly-Met-As-
n-Ala-Xaa4-Thr-Gly-Ala-Gly-Ala-Gln- (SEQ ID NO:57)
Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Xaa3-Xaa3-Xaa4-Arg-{circumflex
over ( )} Name: A14.3 Species: aurisiacus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:58) GTCTGAAGGTGGTAAATTGAACGACGTAATTTGGGGTTTGGTGCCACACATCTTAAC
CCCACAGCATATCTTGCAAAGCCTGACTTCCCGTCTTCATTCTGACAGCAGTGATCA
GAAAGGAGCACAAATATGCATCTGGAAGGTATGTCCACCACCCCCATGGAGATGAA
CACAAGGAAAAAGATGAATGACGTCAGACAACCGCCACAACTGTAGTACGACATC
GTTGATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATCAGTTG
CTTTAAAAGATTGGATTTTTCCTTGTTTAGGAGTTGTATTGATATCAGCTCTGCCCTG
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIWGLVPHILTPQHILQSLTSRLHSDSSDQ (SEQ ID
NO:59) KGAQICIWKVCPPPPWR Toxin Sequence:
Leu-His-Ser-Asp-Ser-Ser-Asp-Gln-Lys-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val--
Cys-Xaa3-(SEQ lID NO:60) Xaa3-Xaa3-Xaa3-Xaa4-Arg-{circumflex over (
)} Name: A14.4 Species: aurisiacus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:61) GTTTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCACACATCTTAAC
CCCACAGCATATCTTGCAAAGCCTGACTTCCCGTCTTCGTTCTGACAGCAGTGATCA
GAAAGGAGGCATGAACGCATCGACAGGAGCAGGAGCACAAATATGCATCTGGAAG
GTATGTCCACCATCCCCATGGAGACGAACACAAGGAAAAAGATGAATGACGTCAGA
CAACCGCCACAACTGTAGTACGACATCGTTGATACGACTTCAGCAAATATTTTAACA
TCACTGTGGTTGTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAG
AGTTGTACTGATATCAGCTCTGCACTGTGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLFEGGKLNDVIRGLVPHILTPQHILQSLTSRLRSD- SSDQK (SEQ
ID NO:62) GGMNASTGAGAQICIWKVCPPSPWRRTQGKR Toxin Sequence:
Leu-Arg-Ser-Asp-Ser-Ser-Asp-Gln-Lys-Gly-Gly-Met-A-
sn-Ala-Ser-Thr-Gly-Ala-Gly-Ala-Gln-(SEQ ID NO:63)
Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Ser-Xaa3-Xaa4-Arg-Arg-Thr-Gln-#
Name: Ac14.1 Species: achatinus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGAT- GATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:64) GTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCACACATCTTAAC
CCCACAGCATATCTTGCAAAGTCTGACTTCCCGTCTTCGTTCTGACAACGGTGGTTC
GAGTGGAGCACAAATATGCATCTGGAAGGTGTGTCCACCATCCCCATGGAGACGAC
CACAAGGAAAAAGATGAACGGCGTCAGACAACCGCCACAACTGTAGTGGGACATC
GTTGATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATCAGTTG
CTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCTGCCCTA
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVPHILTPQHILQSLTSRLRSDNGGSS (SEQ ID
NO:65) GAQICIWKVCPPSPWRRPQGKR Toxin Sequence:
Leu-Arg-Ser-Asp-Asn-Gly-Gly-Ser-Ser-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val--
Cys-Xaa3- (SEQ ID NO:66) Xaa3-Ser-Xaa3-Xaa4-Arg-Arg-Xaa3-Gln-#
Name: P14.2 Species: purpurascens Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGAT- GACGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:67) GTCTGAAGGTGGAAAACTGAACGATGTAATTCGGGGTTTGGTGCCAGACGACTTAG
CCCTACAGCTTATCTTGCAAAGTCCGGTTTTCCGTCGTCAATCTGAAGAGGAAAAAA
TATGCCTCTGGAAGATATGTCCACCACCCCCATGGAGACGATCATAAGGAAAAAAA
AATGAATGACGTCAGACAACCACCACAACTGTAATACGACATCGTTAATACGACTT
CAGCAAACATTTTAACATCACTGTGGTTGTGAAGAAATCAGTTGCTTTAGAAGCTTG
GATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCTGCCCTATGAAATAAAGCT GATG
Translation: MQTAYWVMVMTMVWITAPLSEGGKLNDVIRGL-
VPDDLALQLILQSPVFRRQSEEEKIC (SEQ ID NO:68) LWKICPPPPWRRS Toxin
Sequence: Xaa2-Ser-Xaa1-Xaa1-Xaa1-Lys-Ile-Cys-Leu-Xaa4-L-
ys-Ile-Cys-Xaa3-Xaa3-Xaa3-Xaa3-Xaa4- (SEQ ID NO:69)
Arg-Arg-Ser-{circumflex over ( )} Name: P14.1 Species: purpurascens
Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:70) GTCTGAGGGTAGAAAACCGAACGATGTAATTCGGGGTTTGGT- GCCAGATGACTTAG
CCCTACAGCTTATCTTGCAAAGTCAGGTTTCCCGTCGTGAATC- TAATGGGGTGGAAA
TATGCATGTGGAAGGTATGTCCACCATCCCCATGGAGACGATC- ATAAGGAAAAAAA
ATGAATGACGTCAGACAACCACCACAACTGTAATACGACATCGT- TAATACGACTTC
AGCAAACATTTTAACATCACTGTGGTTGTGAAGAAATCAGTTGCT- TTAAAAGATTGG
ATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCTGCCCTAT- GAAATAAAGCTG ATG
Translation:
MQTAYWVMVMMMVWITAPLSEGRKPNDVIRGLVPDDLALQLILQSQVSRRESNGVEI (SEQ ID
NO:71) CMWKVCPPSPWRRS Toxin Sequence:
Xaa1-Ser-Asn-Gly-Val-Xaa1-Ile-Cys-Met-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Ser-Xaa3-
-Xaa4-Arg- (SEQ ID NO:72) Arg-Ser-{circumflex over ( )} Name:
Sm14.1 Species: stercusmuscarum Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGAT- GATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:73) GTCTGAAGGTGGTAAATTGACCGACGTAATTCGGGGTTTGGTGCCACACATCTTAAC
CCCACAGCATATCTTGCAAAGTATGACTTCCCGTCTTGGTATTGGCAGCAGTGATCA
AAATGCACAAATATGCATCTGGAAGGTATGTCCACCATCCCCATAGAGACGACCAT
AAGGAAAAAGATGAATGACGTCAGACAACCGCCACAACTGTAGTACGACATCGTTG
ATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATCAGTTGCTTT
AAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCTGCCCTGTGA
AATAAAGCTGATG Translation: MQTAYWVMVMMMVWITAPLSEGG-
KLTDVIRGLVPHILTPQHILQSMTSRLGIGSSDQN (SEQ ID NO:74) AQICIWKVCPPSP
Toxin Sequence: Leu-Gly-Ile-Gly-Ser-Ser-Asp-Gln-Asn-A-
la-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Ser- (SEQ ID NO:75)
Xaa3-{circumflex over ( )} Name: Ba14.1 Species: baileyi Cloned:
Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATAATGGTGTGGATTACAGTCCCTCTG (SEQ ID
NO:76) TCTGAAGGTGGTAAATTGAACGACATAATTCGGGGTTTGTTGCCAGACAA- CTTCCCC
CCACAGCTTACCTTGCATCGTCTGGTTTCCCGTCGTCATTCTGACAGCAT- TATTCTGA
GGGGCTTATGTATCTGGAAGGTGTGTGAACCTCCGCCACAAAGATGATC- TGGTCCA
AAGCCAAAAAACGAATGATGTCAGACAACCGCCACAGCTTTAGTACGACA- TGGTTA
ATACGACTTCAGCAAATATTTCAACATCACTGTGGTTGTGAAGAAATCAGT- TACTTT
AAAAGATTGGAATGATGTCAGCTGTGCACTATCAAATAAAGTTGATGTGAC- AAAAA
AAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMIMVWITVPLSEGGKLNDIIRGLLPDNFPPQLTLHRLVSRRHSD- SIILRG (SEQ
ID NO:77) LCIWKVCEPPPQR Toxin Sequence:
His-Ser-Asp-Ser-Ile-Ile-Leu-Arg-Gly-Leu-Cys-Ile-Xaa4-Lys-Val-Cys-X-
aa1-Xaa3-Xaa3-Xaa3- (SEQ ID NO:78) Gln-Arg-{circumflex over ( )}
Name: Bk14.1 Species: bocki Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGAT- GATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:79) GTCTGAAAGTGATAAACTGAACGACGTAATTCGGGGTTTGGTGCCAGACAACTTAA
CCCCACAGCTTATCTTGCGAAGTCTGATTTCCCGTCGTCGTTCTGACAAGGATGATC
CGGGAGGACAAGAATGTTACTGGAACGTATGTGCACCAAACCAGGGAGACCACAT
GATCTTAAGAAAAAAGATGAATGACGACAGACAACCGCCACAACTGTAATACGAC
ATCGTTAATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATCAG
TTGCTTTAAAAGATTGGATTTTTCCGTGTTTAAGAGCTGTACTGATATCTGCTCTGCC
CTGTGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSESDKLNDVIRGLVPDNLTPQLILRSLISRRRSDKDDPG (SEQ ID
NO:80) GQECYWNVCAPNQGDHMILRKKMNDDRQPPQL Toxin Sequence:
Arg-Ser-Asp-Lys-Asp-Asp-Xaa3-Gly-Gly-Gln-Xaa1-Cys-Xaa5-Xaa4-Asn-Val--
Cys-Ala-Xaa3- (SEQ ID NO:81)
Asn-Gln-Gly-Asp-His-Met-Ile-Leu-Arg-Ly-
s-Lys-Met-Asn-Asp-Asp-Arg-Gln-Xaa3-Xaa3 -Gln- Leu-{circumflex over
( )} Name: Cd14.1 Species: chaldaeus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGATGGGGATGATG (SEQ ID
NO:82) ATGGTGTGGATTACAGCCCCTCTGTCTGGAGGTGGTAAACTGAACGACGTAATTCG
GGGTTTGGTGCCAGACGACTTAACCCTACAGCGTATGTTCGAAACTCCGGTTTCCCA
TCGTCTTTCTGAGGGCAGAAATTCGACGGTACACATATGTACGTGGAAGGTATGTCC
ACCTCCCCCATGGAGACGACCACATGGACAAAGATGAATGACGTCAGACAACCTCC
ACAACTGTAGTACGACATCGTTAACACGACGTCAGCTAATCTTTTAACATCACTGTG
GCTGTGAAGAACTCGGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTG
CTGATATGAACTCTGCACTACGAAATAAAGCTGATGTGACAAACAAAAAAAAGAAA
AAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMMGMMMVWITAPLSGGGRLNDVIRGLVPDDLTLQRMIFETPVSHRL- SEGR (SEQ ID
NO:83) NSTVHICTWKVCPPPPWRRPHGQR Toxin Sequence:
Leu-Ser-Xaa1-Gly-Arg-Asn-Ser-Thr-Val-His-Ile-Cys-Thr-Xaa-
4-Lys-Val-Cys-Xaa3-Xaa3-Xaa3- (SEQ ID NO:84)
Xaa3-Xaa4-Arg-Arg-Xaa3-His-Gly-Gln-Arg-{circumflex over ( )} Name:
Cd14.2 Species: chaldaeus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGG- TGATGATGGGGATGATG (SEQ ID
NO:85) ATGGTGTGGATTACAGCCCCTCTGT- CTGGAGGTGGTAAACTGAACGACGTAATTCG
GGGTTTGGTGCCAGACGACTTAACCC- TACAGCGTATGTTCGAAACTCCGGTTTCCCA
TCGTCTTTCTGAGGGCAGAAATTCGA- CGGTACACATATGTATGTGGAAGGTATGTCC
ACCTCCCCCATGGAGACGACCACATG- GACAAAGATGAATGACGTCAGACAACCTCC
ACAACTGTAGTACGACATCGTTAACAC- GACGTCAGCTAATCTTTTAACATCACTGTG
GTTGTGAAGAAATCGGTTGCTTTAAAA- GATTGGATTTTTCCTTGTTTAAGAGTTGTG
CTGATATGAACTCTGCACTACGAAATA- AAGCTGATGTGACAAACGGAAAAAAAAAA
AAAAAAAAAAGTACTCTGCGTTGTTACT- CGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMMGMMMVWITAPLSGGGKLNDVIRGLVPDDLTLQRMFETPVSHRLSEGR (SEQ ID
NO:86) NSTVHICMWKVCPPPPWRRPHGQR Toxin Sequence:
Leu-Ser-Xaa1-Gly-Arg-Asn-Ser-Thr-Val-His-Ile-Cys-Met-Xaa4-Lys-Val-Cys-Xaa-
3-Xaa3- (SEQ ID NO:87)
Xaa3-Xaa3-Xaa4-Arg-Arg-Xaa3-His-Gly-Gln-Arg-- {circumflex over ( )}
Name: Ci14.1 Species: cinereus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGTTGG (SEQ ID
NO:88) TGTGGATTACAGCCCCTCTGCCTGAGGGTGGTAAACCGAAGCACGTAATTCGGGGT
TTGGTACCAGACGACTTAACCCCACAGCATATCTTGCGAAGTTTGATTTCCCGTCGT
TCATCTGGCTGCAGTGTTTCGTTGGGCTTCAAATGCTTCTGGAAGAGCTGTACAGTA
ATCCCAGTGAGACCATTTGTATCTCTGGAAGAAGAAAATGAATGCCAGAAAGTCCA
AATAAGTGCAGTATGGGGTCCTTGATACGACTTCAGCAAGGATCACTGTGGTTGTG
AAGAAATCAGTTGCTTTAAAAGATTTGATTTTTCCTTGTTTAAGAGTTGTACTGATA- T
CAGCTCTGTACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAAAAAAAAAAA
AGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMLVWITAPLPEGGKPKHVIRGLVPDDLTPQHILRSLRSRRSSG- CSVSLG (SEQ
ID NO:89) FKCFWKSCTVIPVRPFVSLEEENECQKVQISAVWGP Toxin Sequence:
Ser-Ser-Gly-Cys-Ser-Val-Ser-Leu-Gly-Phe-Lys-
-Cys-Phe-Xaa4-Lys-Ser-Cys-Thr-Val-Ile-Xaa3-(SEQ ID NO:90)
Val-Arg-Xaa3-Phe-Val-Ser-Leu-Xaa1-Xaa1-Xaa1-Asn-Xaa1-Cys-Gln-Lys-Val-Gln--
Ile-Ser- Ala-Val-Xaa4-Gly-Xaa3-{circumflex over ( )} Name: Ci14.2
Species: cinereus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGA- TGGTGATGATGGTG (SEQ ID
NO:91) GTGGTGTGGATTACAGCCCCTCTGCCTG- AAGGTGGTAAACCGGAGCACGTAATTCG
GGGTTTGGTGCCAGACGACTTAACCCCAC- AGCTTATCTTGCGAAGTCTGATTTCCCG
TCGTAGTTCTGACGGCAAGGCAAAAAGAA- ATTGTTTCTGGAAGGCATGTGTACCAG
AACAATGGAGACAACGTGATCTTAAGGAAA- AAGATGAATGATGTCAGACAACCGC
CATCACTGTAGTATGACATCGTTAATACGACT- TAAGCAAATATTTTAACATCACTGT
GGATCTGAAGAAATCAGTTGCTTTAAAAGATT- GGATTTTTCCTCGTTTAAGAGTTGT
ACTGATGTCAGCTCTGCACTGTGAAATAAAGC- TGATGTGACAAACGAAAAAAAAAA
AAAAAAAAAAGTACTCTGCGTTGTTACTCGAGC- TTAAGGGCGAATTC Translation:
MQTAYWVMVMMVVVWITAPLPEG- GKPEHVIRGLVPDDLTPQLILRSLISRRSSDGKAK (SEQ
ID NO:92) RNCFWKACVPEQWRQRDLKEKDE Toxin Sequence:
Ser-Ser-Asp-Gly-Lys-Ala-Lys-Arg-Asn-CyS-Phe-Xaa4-Lys-Ala-Cys-Val-Xaa3-Xaa-
1-Gln- (SEQ ID NO:93)
Xaa4-Arg-Gln-Arg-Asp-Leu-Lys-Xaa1-Lys-Asp-Xaa- 1-{circumflex over (
)} Name: Ci14.3 Species: cinereus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG (SEQ ID
NO:94) GTGGTGTGGATTACAGCCCCTCTGCCTGAAGGTGGTAAACCGAAGCACGTAATTCG
GGGTTTGGTGCCAGACGACTTAACCCCACAGCTTATCTTGCGAAGTCTGATTTCCCG
TCGTAGTTCTGACGGCAAGGCAAAAAGAAATTGTTTCTGGAAGGCATGTGTACCAG
AACAATGGAGACAACGTGATCCTAAGGAAAAAGATGAATGATGTCAGACAACCGC
CATCACTGTAGTATGACATCGTTAATACGACTTAAGCAAATATTTTAACATCACTGT
GGATCTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTCGTTTAAGAGTTGT
ACTGATGTCAGCTCTGCACTGTGAAATAAAGCTGACGTGACAAGCAAAAAAAAAAA
AAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVVWITAPLPEGGKPKHVIRGLVPDDLTPQLILRSLISRRSS- DGKAK (SEQ
ID NO:95) RNCFWKACVPEQWRQRDPKEKDE Toxin Sequence:
Ser-Ser-Asp-Gly-Lys-Ala-Lys-Arg-Asn-Cys-Phe-Xaa4-Lys-Ala-
-Cys-Val-Xaa3-Xaa1-Gln-(SEQ ID NO:96)
Xaa4-Arg-Gln-Arg-Asp-Xaa3-Lys- -Xaa1-Lys-Asp-Xaa1-{circumflex over
( )} Name: Ci14.4 Species: cinereus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATAATGATG (SEQ ID
NO:97) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAACCGAAG- CACGTAATTCGGGG
TTTGGTGCCAGTCGACTTAACCCCACAGCTTATCTTGCGAAGT- CTGATTTCCCGTCGT
AGTTCTGACGGCAAGGCAAAAAAACAATGTGCCTGGAAGACA- TGTGTACCAACCCA
ATGGAGACGACGTGATCTTAAGGAAAAAGATGAATGATGTCAG- ACAACCGCCATCA
CTGTAGTATGACATCGTTAATACGACTTAAGCAAATATTTTAAC- ATCACTGTGGTTC
TGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTA- AGAGTTGTACTGA
TATCAGCTCTGCACTGTGAAATAAAGCTGATGTGACAAACAAAA- AAAAAAAAAAAA
AAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVIMMVWITAPLSEGGKPKHVIRGLVPVDLTPQL- ILRSLISRRSSDGKAKK (SEQ
ID NO:98) QCAWKTCVPTQWRRRDLKEKDE Toxin Sequence:
Ser-Ser-Asp-Gly-Lys-Ala-Lys-Lys-Gln-Cys-Ala-Xa-
a4-Lys-Thr-Cys-Val-Xaa3-Thr-Gln-Xaa4 (SEQ ID NO:99)
Arg-Arg-Arg-Asp-Leu-Lys-Xaa1-Lys-Asp-Xaa1-{circumflex over ( )}
Name: Cr14.2 Species: circumcisus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCC- TACTGGGTGATGGTGATGATGATG (SEQ ID
NO:100) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGT
TTGGTGCCACACATCTTAACCCCACAGCATATCTTGCAAGGTCTGACTTCCCGTCTT
CGTTCTGACAGCAGTGGTCAGAAAGGAGCACAAATATGCATCTGGAAGGTATGTCC
ACTATCCCCATGGAGACGACCACAAGGAAAAGATGAATGACGTCAGACAACCGCTA
CAACTGTAGTACGACATCGTTGATACGACTTCAGCAAATATTTTAACATCACTGTGG
TTGTGAAGAAATCAGCTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACT
GATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAAAAAAA
AAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVPHILTPQHILQGLTSRLRSDSSGQK (SEQ ID
NO:101) GAQICIWKIVCPLSPWRRPQGKDE Toxin Sequence:
Leu-Arg-Ser-Asp-Ser-Ser-Gly-Gln-Lys-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-
-Val-Cys-Xaa3-Leu-(SEQ ID NO:102)
Ser-Xaa3-Xaa4-Arg-Arg-Xaa3-Gln-Gl- y-Lys-Asp-Xaa1-{circumflex over
( )} Name: Cn14.4 Species: consors Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG (SEQ ID
NO:103) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAA- TTCGGGGT
TTGGTGCCACACTTCTTAACCCCACAGCATATCTTGCAAAGTCTGACTT- CCCGTAAT
GGTTCTGGCAGCAGTAATCAGAAAGAAGCACAACTATGCATCTGGAAGG- TATGTCC
ACCAACCCCATGGAGATGACCACAAGGAAAAAGATGAACGGCGTCAGACA- ACCGC
CACAACTGTAGTGGGACATCGTTGATACGACTTCAGCAAATATTTTAACATC- ACTGT
GGTTGTGAAGAAATCAGTTGTTTTAAAAGATTGGATTTTTCCTTGTTTAAGA- GTTGT
ACTGATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACAAGCAAAAAAA-
AAAA AAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVPHFLTPQHILQS- LTSRNGSGSSNQ (SEQ ID
NO:104) KEAQLCIWKVCPPTPWR Toxin Sequence:
Asn-Gly-Ser-Gly-Ser-Ser-Asn-Gln-Lys-Xaa1-Ala-Gln-L-
eu-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3- (SEQ ID NO:105)
Xaa3-Thr-Xaa3-Xaa4-Arg-{circumflex over ( )} Name: Cn14.5 Species:
consors Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGAT- GATG (SEQ ID
NO:106) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAAC- TGAACGGCGTAATTCGGGG
TTTGGTGTCACACATCTTAATCCCACAGCATACCTTGC- GAAGTCTGACTTCCCGTGA
TCGTTCTGACAACGGTGGTTCGAGTGGAGCACAAATAT- GCATCTGGAAGGTATGTC
CACCATCCCCATGGAAATGACCACAAGGAAAAAGATGAA- CGGCGTCAGACAACCA
CCACAACTGTAGTGGGACATCGTTGATACGACTTCAGCAAA- TATTTTAACATCACTG
TGGTCGTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTT- CCTTGTTTAAGAGTTG
TACTGATATCAGCTCTGCACTATGAA~ATAAAGCTGATGTG- ACAAACAAAAAAAAAA
AAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCG- AATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNGVIRGL- VSHILIPQHTLRSLTSRDRSDNGGSS (SEQ
ID NO:107) GAQICIWKVCPPSPWK Toxin Sequence:
Asp-Arg-Ser-Asp-Asn-Gly-Gly-Ser-Ser-Gly-Al-
a-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3- (SEQ ID NO:108)
Xaa3-Ser-Xaa3-Xaa4-Lys-{circumflex over ( )} Name: Ct14.1 Species:
coronatus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGATGATGATGATGATGGTGTGGATTACAG- CCCC (SEQ ID
NO:109) TCTGTCTGAAGGTGGTAAACTGAACGACGTAATTCGG- GGTTTGGTGCCAGACGACTT
AACCCTACAGCGTATGTTCAAAGCTCTGGTTTCCCAT- CGTCTTTCTGACGGCAGAGA
TTGGACGGGATACATATGTATCTGGAAGGCATGTCCA- CGTCCCCCATGGATCCCACC
AAAGGGAAAAAGATGAATGACGTCAGACAACCGCCAC- AACTGTAGTACGACATCG
TTAACACAACTTCAGCTAATATTTTAACATCACTGTGGT- TGTGAAGAAATCGGTTGC
TTTAAAAGATTGAATTTTTCGTTTAAGAGTTGTGCTGAT- ACGAGCTCTGCACTATGA
AATAAAGCTGATGTGACAAACAAAAAAAAAAAAAAAAAA- AAGTACTCTGCGTTGTT ACTCGAG
Translation:
MQTAYWVMMMMMMVWITAPLSEGGKLNDVIRGLVPDDLTLQRMFKALVSHRLSDG (SEQ ID
NO:110) RDWTGYICIWKACPRPPWIPPKGKR Toxin Sequence:
Leu-Ser-Asp-Gly-Arg-Asp-Xaa4-Thr-Gly-Xaa5-Ile-Cys-Ile-Xaa4-Lys-Ala-
-Cys-Xaa3-Arg-(SEQ ID NO:111) Xaa3-Xaa3-Xaa4-Ile-Xaa3-Xaa3-Lys-#
Name: Eb14.1 Species: ebraeus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGC- CTACTGGGTGATGATGATGATGATG (SEQ ID
NO:112) ATGGTGTGGATTACAGCCCCTCTGTCTGAAGGCGGTAAACTGAACGACGTAATTCG
GGGTTTGGTGCCAGACGACTTAACCCTACAGCGTATGTTCAAAAGTCTGTTTTCCCA
TCGTCTTTCTGGCGGCACATATTCGAGGGTAGACACATGCATCTGGAAGGTATGTCC
ACAATCTCCATAGGGACGATCATATGGAAAAAGATGAGTGACATCAGACAACTGCC
ACAACTGTAGTACGACATCGTTAACACGACTTCAGCTAATATTTTAACATCACTGTG
GTTGTGAAGAAATCGGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTG
CTGATATGAGCTCTGCACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAAAAA
AAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMMMMMMVWITAPLSEGGKLNDVIRGLVPDDLTLQRMFKSLFSHRLSGGT (SEQ ID
NO:113) YSRVDTCIWKVCPQSP Toxin Sequence:
Leu-Ser-Gly-Gly-Thr-Xaa5-Ser-Arg-Val-Asp-Thr-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa-
3-Gln-Ser- (SEQ ID NO:114) Xaa3-{circumflex over ( )} Name: G14.2
Species: geographus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGT- GATGCTGATGATGATGG (SEQ ID
NO:115) TGTGCATCACAGCCCCTCTGCCTG- AAGGTGGTAAACCGAACAGCGGAATTCGGGGT
TTGGTGCCAAACGACTTAACTCCAC- AGCATACCTTGCGAAGTCTGATTTCCCGTCGT
CAAACTGACGTTCTTCTGGAGGCTA- CCCTTTTGACAACACCAGCCCCCGAGCAGAG
ATTGTTCTGCTTCTGGAAGTCATGTA- CGTGGAGGCCCTACCCTTGGAGACGACGTGA
TCTTAATGGAAAACGATGAATGACGC- CAGACAACCGCCACAACTGTAGTACGACAT
CGTTAATACGACTTCAGCAAACATTTT- AACATAACTGTGGTTGTGAAGAAATCAGTT
GCTTTAAAAGATTGGATTTTTCCTTGT- TTCAGAGTTGTACTGATATGAGCTCTGCACC
ATGAAATAAAGCTGAAGTGACGAACA- AAAAAAAAAAAAAAAAAAAAGTACTCTGC
GTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMLMMMVCITAPLPEGGKPNSGIRGLVPNDL- TPQHTLRSLISRRQTDVLLEA (SEQ
ID NO:116) TLLTTPAPEQRLFCFWKSCTWRPYPWRRR- DLNGKR Toxin Sequence:
Xaa2-Thr-Asp-Val-Leu-Leu-Xaa-
1-Ala-Thr-Leu-Leu-THr-Thr-Xaa3-Ala-Xaa3-Xaa1-Gln-Arg- (SEQ ID
NO:117)
Leu-Phe-Cys-Phe-Xaa4-Lys-Ser-Cys-Thr-Xaa4-Arg-Xaa3-Xaa5-Xaa3-Xaa4-Arg-A-
rg-Arg-Asp- Leu-Asn-# Name: Gd14.1 Species: gladiator Cloned: Yes
DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGGTTACAGTCCCTCG (SEQ ID
NO:118) ATCTGAAGGTGGCACGTGGAACTACTTAATTCGGGGTTTGGTGCCAGAC- GACCTAA
CCCCACAGCTTACCTTGCATCGTCTGGTTACCCGTCGTCATCCTGCCAAC- GTTAGAC
AGCAGGGGAAAATATGTGTATGGAAGGTGTGTCCACCATGGCCAGTAAGA- TCACCT
GGTCCACAGCCAAAAAACAAATGACGTCAGACAACCGCCACAACTTTAGTA- CGACA
TCGTTGATACAACTTCAGCAAGTATTTTAACATCACTGTGGCTCTGAAGAAA- TCAGT
TGCTTTAAAAGATTGGATTTTTCCTTGTTTTAGAGTTTTACTGATATCAGCT- CTGCAC
TATGAAATAAAGATGTGACGAAAAAAAAAAAAAAAAAAAGTACTCTGCGTT- GTTAC TCGAG
Translation:
MQTAYWVMVMMMVWVTVPRSEGGTWNYLIRGLVPDDLTPQLTLHRLVTRRHPANV (SEQ ID
NO:119) RQQGKICVWKVCPPWPVRSPGPQPKNK Toxin Sequence:
His-Xaa3-Ala-Asn-Val-Arg-Gln-Gln-Gly-Lys-Ile-Cys-Val-Xaa4-Lys-Val-Cys-Xaa-
3-Xaa3 (SEQ ID NO:120)
Xaa4-Xaa3-Val-Arg-Ser-Xaa3-Gly-Xaa3-Gln-Xaa3-
-Lys-Asn-Lys-{circumflex over ( )} Name: Gd14.2 Species: gladiator
Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGGTTACAGTCCCTCG (SEQ ID
NO:121) ATCTGAAGGTGGCACGTGGAACTACTTAATTCGGGGTTTGGTGCCAGAC- GACCTAA
CCCCACAGCTTACCTTGCATCGTCTGGTTACCCGTCGTCATCCTGCCAAC- GTTAGAC
AGCAGGGGAAAATATGTGTATGGAAGGTGTGTCCACCATCGCCAGTAAGA- TCACCT
GGTCCACTGCCAAAAAACAAATGACGTCAGACAACCGCCACAACTTTAGTA- CGACA
TCGTTGATACAACTTCAGCAAGTATTTTAACATCACTGTGGCTCTGAAGAAA- TCAGT
TGCTTTAAAAGATTGGATTTTTCCTTGTTTTAGAGTTTTACTGATATCAGCT- CTGCAC
TATGAAATAAAGATGTGACGGACAAAAAAAAAAAAAAAAAAGTACTCTGCG- TTGTT ACTCGAG
Translation:
MQTAYWVMVMMMVWVTVPRSEGGTWNYLIRGLVPDDLTPQLTLHRLVTRRHPANV (SEQ ID
NO:122) RQQGKICVWKVCPPSPVRSPGPLPKNK Toxin Sequence:
His-Xaa3-Ala-Asn-Val-Arg-Gln-Gln-Gly-Lys-Ile-Cys-Val-Xaa4-Lys-Val-Cys-Xaa-
3-Xaa3-Ser- (SEQ ID NO:123)
Xaa3-Val-Arg-Ser-Xaa3-Gly-Xaa3-Leu-Xaa3- -Lys-Asn-Lys-{circumflex
over ( )} Name: Ly14.1 Species: litoglyphus Cloned: Yes DNA
Sequence: GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG
(SEQ ID NO:124) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGATAAATTGAA-
CGACGTAATTCGGGGT TTGGTGCCAGATAACTTAGCCCCACAGCTTGTTTTGCAAAG-
TCTGGATTCCCGTCGT CATCCTCACGGCATTCGTCAGGATGGAGCCCAAATATGTAT-
CTGGAAGATATGTCCA CCATCCCCATGGAGACGACTTGGATCTTAAGAAAAGAAACA-
ATTGACGTCAGACAA CCGCCACATCTTGAGTACGACATCGTTAATACGACTTCAGCA-
AATATGAAATTTTCA GCATCACTGTGGTTGTGAAGAAATCAGTTGCTTTAAAAGATT-
GGATTTGTCCTTGTT TAAGAGTTGTACTGATGTCATCTCTGCACTATGAAATAAAGC-
TGATGTGAAAAAAA AAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAA- TTC
Translation: MQTAYWVMVMMMVWITAPLSEGDKLNDVIRGLVP-
DNLAPQLVLQSLDSRRHPHGIRQ (SEQ ID NO:125) DGAQICIWKICPPSPWRRLGS Toxin
Sequence: His-Xaa3-His-Gly-Ile-Arg-Gln-Asp-Gly-Ala-
-Gln-Ile-Cys-Ile-Xaa4-Lys-Ile-Cys-Xaa3-Xaa3- (SEQ ID NO:126)
Ser-Xaa3-Xaa4-Arg-Arg-Leu-Gly-Ser-{circumflex over ( )} Name:
Ly14.2 Species: litoglyphus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGT- GATGGTGATGATGATG (SEQ ID
NO:127) GTGTGGATTACAGCCCCTCTGTCTG- AAGGTGATAAATTGAACGACGTAATTCGGGGT
TTGGTGCCAGATAACTTAGCCCCAC- AGCTTGTTTTGCAAAGTCTGGATTCCCGTCGT
CATCCTCACGGCATTCGTCAGGATG- GAGCCCAAATATGTATCTGGAAGATATGTCCA
CCATCCCCATGGAAACGACTTGGAT- CTTAAGAAAAGAAACAATTGACGTCAGACAA
CCGCCACAACTTGAGTACGACATCGT- TAATACAACTTCAGCAAATATGAAATTTTCA
GCATCACTGTGGTTGTGAAGAAATCA- GTTGCTTTAAAGGATTGGATTTGTCCTTGTT
TAAGAGTTGTACTGATGTCATCTCTG- CACTATGAAATAAAGCTGATGTGACAAGCA
AAAAAAAAAAAAAAAAAGTACTCTGCG- TTGTTACTCGAGCTTAAGGGCGAATTC
Translation:
MQTAYWVMVMMMVWITAPLSEGDKLNDVIRGLVPDNLAPQLVLQSLDSRRHPHGIRQ (SEQ ID
NO:128) DGAQICIWKICPPSPWKRLGS Toxin Sequence:
His-Xaa3-His-Gly-Ile-Arg-Gln-Asp-Gly-ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Ile-Cys-
-Xaa3-Xaa3- (SEQ ID NO: 129)
Ser-Xaa3-Xaa4-Lys-Arg-Leu-Gly-Ser-{cir- cumflex over ( )} Name:
Lt14.1 Species: litteratus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG (SEQ ID
NO:130) GTGGGGATTACAGCCCCTCTGTCTGAAGGTCGTAAATTGAACGACGCAATTCGGGG
TTTGGTGCCAGATGACTTAACCCCACAGCTTTTGCGAAGTCCGGTTTCGACTCCTTA
TCCTGAGTTTCATCTTGATGAACCTTATCTGAAGATACCCGTATGTATCTGGAAGAT
ATGTCCACCAAACCTATTGAGACGACGTGATCTTAAGAAAAGAAACAAAGTACGTC
AGACAACCGCCACAACTTGAGTACGACATCGTTCATACAACTTGAGCAAATATTTC
AGCATCACTATGGTTGTGAAGAAATCAGTTGCTTTAAAAGATTGGATCTTTCCTTGT
TTAAGAGTTGTATTGATGTCAGCTCTGCACTCTGAAATAAAGCTGATGTGACAAACA
AAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC
Translation: MQTAYWVMVMMMVGITAPLSEGRKLNDAIRGLVPDDLTP-
QLLRSPVSTPYPEFHLDEP YLKIPVCIWKICPPNLLRRRDLKKRNKVRQTTATT Toxin
Sequence: Ser-Xaa3-Val-Ser-Thr-Xaa3-Xaa5-Xaa3-Xaa1-Phe-H-
is-Leu-Asp-Xaa1-Xaa3-Xaa5-Leu-Lys-Ile- (SEQ ID NO:132)
Xaa3-Val-Cys-Ile-Xaa4-Lys-Ile-Cys-Xaa3-Xaa3-Asn-Leu-Leu-Arg-Arg-Arg-Asp-L-
eu-Lys-Lys- Arg-Asn-Lys-Val-Arg-Gln-Thr-Thr-Ala-Thr-Thr-{circumflex
over ( )} Name: Lt14.2 Species: litteratus Cloned: Yes DNA
Sequence: GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG
(SEQ ID NO:133)
GTGGGGATTACAGCCCCTCTGTCTGAAGGTCGTAAATTGAACGACGCAATTCGGGG
TTTGGTGCCAAATGACTTAACCCCACAGCTTTTGCAAAGTCTGGTTTCCCGTCGTCA
TCGTGTGTTTCATCTTGACAACACTTATCTCAAGATACCCATATGTGCCTGGAAGGT
ATGTCCACCAACCCCATGGAGACGACGTGATCTTAAGAAAAGAAACAAATGACGTC
AGACAACCGCCACAACTTGAGTACGACATTGTTAATGCGACTTGAGCAAATTTTTCA
GCATCACTATGGTTGTAAAGAAATCAGCTGCTTTAAACGATTGGATCTTTCCTTATT
TAAGAGTTGTATTGATGTCAGCTCTGCACTCTGAAATAAAGCTGATGTGACAAACA
AAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC
Translation: MQTAYWVMVMMMVGITAPLSEGRKLNDAIRGLVPNDL-
TPQLLQSLVSRRHRVFHLDN (SEQ ID NO:134) TYLKIPICAWKVCPPTPWRRRDLKKRNK
Toxin Sequence: His-Arg-Val-Phe-His-Leu-Asp-Asn-Thr--
Xaa5-Leu-Lys-Ile-Xaa3-Ile-Cys-Ala-Xaa4-Lys-Val-(SEQ ID NO:135)
Cys-Xaa3-Xaa3-Thr-Xaa3-Xaa4-Arg-Arg-Arg-Asp-Leu-Lys-Lys-Arg-Asn-Lys-{circ-
umflex over ( )} Name: Ls14.1 Species: loroisii Cloned: Yes DNA
Sequence: GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG
(SEQ ID NO:136)
GTGTGGATTAAAGGCCCTGTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGG
TTTGGTGCCAGACGACTTAACCCCACAGCTTATCTTGCAAAGTCTGATGTCCCGTCG
TCGTTCTGACAGCGATGTTCGGGAGGTGTACATATTATGCATCTGGAAGATATGTCC
ACCATTGCCATGAAGACGACATGATCTTAAGGAAAAGGATAAACGACGTCAGACAA
CCGCTACAACTGTAGTACGACATCGTTAATACGACTTCAGCAAATATTTGAACATCA
CTGTGGTTGTGAAGAAATCAGTTGCTTTAAACGATTGGATTTTTCCTTAAGAGTTGC
ACTGATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACTACCAAAAAAAAAAA
AAAAAAAAAGTACTNTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWIKGPVSEGGKLNDVIRGLVPDDLTPQLILQSLMS- RRRSDSDVR (SEQ ID
NO:137) EVYILCIWKICPPLP Toxin Sequence:
Arg-Ser-Asp-Ser-Asp-Val-Arg-Xaa1-Val-Xaa5-Ile-Leu-Cys-Il-
e-Xaa4-Lys-Ile-Cys-Xaa3-Xaa3 - (SEQ ID NO:138) Leu-Xaa3-{circumflex
over ( )} Name: M14.2 Species: magus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG (SEQ ID
NO:139) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGT
TTGGTGCCACACTCCTTAACCCCACAGCATATCTTGCAAAGTCTGACTTCCCGTAAT
GGTTCTGGCAGCAGCAATCAGAAAGAAGCACAACTATGCATCTGGAAGGTATGTCC
ACCATCCCCATGGAGATGACCACAAGGAAAAAGATGAACGGCGTCAGACAACCGC
CACAACTGTAGTGGGACATCGTTGATACGACTTCAACAAATATTTTAACATCACTGT
GGTTGTAAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGT
ACTGATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAAAA
AAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVPHSLTPQHILQSLTSRNGSG- SSNQ (SEQ ID
NO:140) KEAQLCIWKVCPPSPWR Toxin Sequence:
Asn-Gly-Ser-Gly-Ser-Ser-Asn-Gln-Lys-Xaa1-Ala-Gln-Leu-Cys-
-Ile-Xaa4-Lys-Val-Cys-Xaa3- (SEQ ID NO:141)
Xaa3-Ser-Xaa3-Xaa4-Arg-- {circumflex over ( )} Name: Mi14.3
Species: miles Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGATGATGGTGGTG (SEQ ID
NO:142) ATGATGGTGGGGGTTACTGTCGCTGGCTCCCTGCCTGTGTTTGATGACGACAACGAC
TCTGACCCCGCTGTCAAGCGCGCTATCACGTGGTCCCGCATCCTGGGCGTGTCTCCA
GCCTTCCTGGCACAGCAGCGAGCGCTGGTTCCCTTCGCCAACCGATTCATCAGTGAG
CAGAAACGTTTCCGACCCGCCATGCAGAGCCGATCAGGAGGAATGTCGCTGTGCCT
ATGGAAAGTGTGTCCTGCAGCCCCCTGGCTGGTCGCCAAACGTAAACAGGAAACCA
GCGACTACTGACGTCATACCTCTAAAGACCCACTCATGACGTCAACGCTGAACTGA
CGTCACCGACAGCTCCAACGTCACAGCAGGAGCGAGAGAGAGGCTGGAGCATTTCT
CTTTCTTTTGGTTTTTCGAGTTGAAGTGTGATCAGCTGGGCTGGTGAAAAAATTGTT- G
AGTAAAGTTGAATGAAAATCAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGGTA
CTCGAGGCTTAAAGGCGNAATTC Translation:
MQTAYWVMMMVVMMVGVTVAGSLPVFDDDNPSDPAVKRAITWSRILGVSPAFLAQQ (SEQ ID
NO:143) RALVPFANRFISEQKRFRPAMQSRSGGMSLCLWKVCPAAPWLVAKRKQETSDY Toxin
Sequence: Phe-Arg-Xaa3-Ala-Met-Gln-Ser-Arg-Ser-Gl-
y-Gly-Met-Ser-Leu-Cys-Leu-Xaa4-Lys-Val-Cys- (SEQ ID NO:144)
Xaa3-Ala-Ala-Xaa3-Xaa4-Leu-Val-Ala-Lys-Arg-Lys-Gln-Xaa1-Thr-Ser-Asp-Xaa5--
{circumflex over ( )} Name: Mi14.4 Species: miles Cloned: Yes DNA
Sequence: GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG
(SEQ ID NO:145)
GTGGTGGGTTCACCGTCGGGAGTCACGTCCATCGGTCTCACAGTCCTACGTCGCGCA
ACCATGGTGATGACTCCATTCATGACAAGACGATTCATCAACATCTGTTTGCCCGTC
TTCCTCTGGAGAACAACGACGACCATCGTTCTGTGGATCTTCCTGCAGTGTATGCGC
CGGGCCAGGCACGTGTGCGTTCTACTTTTGTTCTTGACCTCATTGCAGATAGGGGTT
GGTGCAGACGACATGAAACTACAGCGCCAAAGACGTCAAGGTTTCTGTTGCGTCGT
TATCCCGATTCTTTGGTTCTGTTGTGGGGGTTACCGCACAAATGGCACTGCACTGGC
CGATTGAAAGAACTGCAATAAACGGAATGGCAAGAAGGAATAAAAAAAAAAAAAA
AAAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVVGSPSGVTSIGLTVLRRATMVMTPFMTRRFINICLP- VFLWRTTT (SEQ ID
NO:146) TIVLWIFLQCMRRARHVCVLLLFLTSLQIGVGADDMKLQRQR-
RQGFCCVVIPILWFCCG GYRTNGTALAD Toxin Sequence:
Xaa2-Gly-Phe-Cys-Cys-Val-Val-Ile-Xaa3-Ile-Leu-Xaa4-Phe-Cys-Cys-Gly-Gly-X-
aa5-Arg-Thr- (SEQ ID NO:147)
Asn-Gly-Thr-Ala-Leu-Ala-Asp-{circumfle- x over ( )} Name: Mu14.1
Species: muriculatus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTTTG (SEQ ID
NO:148) TCTGAAGGTGGTAAACTGAACGATGTAATTCGGGGTTTCGCGCTAGATGACTTAGCC
CAAAGCCGTATTATGCAAAGTCTGGTTTTCAGTCATCAGCCTCTTCCAACGGCATCC
ATATGTATCTGGAAGATATGTCCACCAGACCCATGGAGACGACATGATCTTCAGAA
AAGTAACAAATGACGTCAGACAACCGCCACAACTTGAATACAACATCATTAATACG
ACTTCAGCAAATATTTTAACATCACTGTGATTGTTCGGAAGTCAGTTGCTTTAAAGG
ATTGGATTTGTCCCTGTTGTATTGATGTCAACTCTGCACTATGAAATAAAGCTGATG
TGACAAACAAGAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNPVIRGFALDDLAQSR- IMQSLVFSHQPLPTAS (SEQ ID
NO:149) ICIWKICPPDPWRRHDLQKSNK Toxin Sequence:
Ile-Met-Gln-Ser-Leu-Val-Phe-Ser-His-Gln-Xaa3-L-
eu-Xaa3-Thr-Ala-Ser-Ile-Cys-Ile-Xaa4-Lys- (SEQ ID NO:150)
Ile-Cys-Xaa3-Xaa3-Asp-Xaa3-Xaa4-Arg-Arg-His-Asp-Leu-Gln-Lys-Ser-Asn-Lys-{-
circumflex over ( )} Name: Ms14.1 Species: musicus Cloned: Yes DNA
Sequence: ATGCAGACGGCCTACTGGGTGATGATGATGACGATGATGGTGTGGATGACAGCCCC
(SEQ ID NO:151)
TCTGTCTGAAGGTCGTCCACTGAGCGACGAAGTTCGGGGTATGGTGCCAGGCGACT
TGGTCCTACAGTATCTGTTCCCAAGTCTGGCTTTCAGTCCTCCGGACATATGTACGT
GGAAGGTATGTCCACCACCCCCATGGAGACGACCAAAAAAAATAACAGACGTCAG
ACAGCCGCCACAACTGTAGTACGACATCGTTGATACGGCTTCAGCAAATATTTTCAA
CATCACTGCGGTTGTGAAGAAATCAGTTGCTTTAAAATGTTGGATTTTTCCTTGTTTA
AAAGAGCTGTACTGATGTCAGCCCTGCATTACGAAATAAAGCTGATGTGACAAACA
AAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMMMTMMVWMTAPLSEGRIPLSDEVRGMVPGDLVLQYLFPSLAFSP- PDICT (SEQ ID
NO:152) WKVCPPPPWRRPKIKITDVRQPPQL
Toxin Sequence: Gly-Met-Val-Xaa3-Gly-Asp-Leu-Val-Leu-Gln-Xaa5-Leu--
Phe-Xaa3-Ser-Leu-Ala-Phe-Ser- (SEQ ID NO:153)
Xaa3-Xaa3-Asp-Ile-Cys-Thr-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Xaa3-Xaa3-Xaa4-Arg-A-
rg-Xaa3-
Lys-Lys-Ile-Thr-Asp-Val-Arg-Gln-Xaa3-Xaa3-Gln-Leu-{circumf- lex
over ( )} Name: Ms14.2 Species: musicus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGATGATGATGATGATGGTGTGGATGACAGCCCC (SEQ ID
NO:154) TCTGTCTGAAGGTCGTAAACTGATCGACAAAGTTCGGGGTATGGGGCCAGGCGACT
TATCCCTACAGAAAATGTTCCCAAGTCTGGCTTTAGGTCCTGGGGGAGACGTAATAT
GTAGGTGGAAGGTATGTCCACCAACCCCATGGAAACGACTAATAAAATAACTGACG
TCAGACAGCCGCCACAACTGTAGTACGACATCGTTGATACGACTTCAGCAAATATTT
CAACATCACTGCGGTTGTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTG
TTTAAAGAGTTGTACTGATATCAGCTCTGCATTACGAAATAAAGCTGATGTGACAAA
CAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMMMMMMVWMTAPLSEGRKLIDKVRGMGPGDLSLQKMFPSLALGPGG- D (SEQ ID
NO:155) VICRWKVCPPTPWKRLTK Toxin Sequence:
Gly-Met-Gly-Xaa3-Gly-Asp-Leu-Ser-Leu-Gln-Lys-Met-Phe-Xaa-
3-Ser-Leu-Ala-Leu-Gly-Xaa3- (SEQ ID NO:156)
Gly-Gly-Asp-Val-Ile-Cys-
-Arg-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Thr-Xaa3-Xaa4-Lys-Arg-Leu-
Ile-Lys-{circumflex over ( )} Name: Ms14.3 Species: musicus Cloned:
Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGATGATGATGACGATGATGGTGTGGATGACAGC (SEQ ID
NO:157) CCCTCTGTCTGAAGGTCGTCCACTGAGCGACAAAGTTCGGGGTATGGTG- CCAGGCG
ACTTAGCCCTGCAGTATCTGTTCCCAAGTCTGGCTTTCAATCCCCCGGAC- ATATGTA
CGTGGAAGGTATGTCCACCACCCCCATGGAGACGACCAAAAAAAATAACT- GACGTC
GGACAGCCGCCACAACTGTAGTACGACATCGTTGATACGACTTCAGCAAAT- ATTTTC
AACATCACTGCGGTTGTGAAGAAATCAGTTGTTTTAAAAGGTTGGATTTTT- CCTTGT
TTAAAAGAGCTGTACTGATGTCAGCTCTGCATTACGAAATAAAGCTGATGT- GACAA
ACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCG- AG
Translation: MQTAYWVMMMMTMMVWMTAPLSEGRPLSDKVRGMV-
PGDLALQYLFPSLAFNPPDI (SEQ ID NO:158) CTWKVCPPPPWRRPKKITDVGQPPQL
Toxin Sequence: Gly-Met-Val-Xaa3-Gly-Asp-Leu-Ala-Leu-G-
ln-Xaa5-Leu-Phe-Xaa3-Ser-Leu-Ala-Phe-Asn-(SEQ ID NO: 159)
Xaa3-Xaa3-Asp-Ile-Cys-Thr-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Xaa3-Xaa3-Xaa4-Arg-A-
rg-Xaa3-
Lys-Lys-Ile-Thr-Asp-Val-Gly-Gln-Xaa3-Xaa3-Gln-Leu-{circumf- lex
over ( )} Name: Ms14.4 Species: musicus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGATGATGACGATGATGGTGTGGATGACAGCCCC (SEQ ID
NO:160) TCTGTCTGAAGGTCGTCCACTGAGCGACAAAGTTCGGGGTATGGTGCCAGGCGACT
TAGTCCTGCAGTATCTGTTCCCAAGTCTGGCTTTCAATCCTCCGGACATATGTACGT
GGAAGGTATGTCCACCACCCCCATGGAGACGACCAAAAAAAATAACTGACGTCAGA
CAGCCGCCACAACTGTAGTACGACATCGTTGATACGACTTCAGCAAATATTTTCAAC
ATCACTGCGGTTGTGAAGAAATCAGTTGTTTTAAAAGGTTGGATTTTTCCTTGTTTA- A
AAGAGCTGTACTGATGTCAGCTCTGCATTACGAAATAAAGCTGATGTGACAAGCAA
AAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVAMTMMVWMTAPLSEGRPLSDKVRGMVPGDLVLQYLFPSLAFNPPDI- CT (SEQ ID
NO:161) WKVCPPPPWRRPKKITDVRQPPQL Toxin Sequence:
Gly-Met-Val-Xaa3-Gly-Asp-Leu-Val-Leu-Gln-Xaa5-Leu-Phe-Xa-
a3-Ser-Leu-Ala-Phe-Asn-(SEQ ID NO:162)
Xaa3-Xaa3-Asp-Ile-Cys-Thr-Xa-
a4-Lys-Val-Cys-Xaa3-Xaa3-Xaa3-Xaa3-Xaa4-Arg-Arg-Xaa3-
Lys-Lys-Ile-Thr-Asp-Val-Arg-Gln-Xaa3-Xaa3-Gln-Leu-{circumflex over
( )} Name: Mt14.2 Species: mustelinus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGCGTGGTATACAACCCCTGT (SEQ ID
NO:163) GTCTGAATGTGGGAAATTGAACAACGTAATTCGGGGTTTTGTGCCAAAGGACTGGA
CCCCAATGCTTCCCTGGCGTCGTCTAGTTTCCCATACCAGCAGCAAGTATCCAGGTG
TGACTTTTTGTCCATGGAAGGTGTGTCCGCCAGCGCCATGGAGAATACTTGGGGTCT
AACGCAAAAAAATACATGACGTCAGACAACCGCCACCGCTTTAGTACGACATCGTT
CATACGTCTCCAGCAAGTATTTTAACATCACTGTGGTTGTGAAGAAGTCAGTAGCTT
TAAAAGATTGGATTTTTTCCTTGTTTAAGAGTTGTACTGACATGAGTTCTGCACTAT- G
AAATAAAGTTGATGTGACGAACGAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTT GTTACTCGAG
Translation:
MQTAYWVMVMMMAWYTTPVSECGKLNNVIRGFVPKDWTPMLPWRRLVSHTSSKYP (SEQ ID
NO:164) GVTFCPWKVCPPAPWRILGV Toxin Sequence:
Leu-VaL-Ser-His-Thr-Ser-Ser-Lys-Xaa5-Xaa3-Gly-Val-Thr-Phe-Cys-Xaa3-Xaa4-L-
ys-Val-Cys- (SEQ ID NO:165)
Xaa3-Xaa3-Ala-Xaa3-Xaa4-Arg-Ile-Leu-Gly- -Val-{circumflex over ( )}
Name: Nb14.1 Species: nobilis Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGATGATGGTGGTG (SEQ ID
NO:166) GTGATGATGGTGGGGGTTACTGTCGCTGGCTCACTGTCTGTGTTTGATG- ATGACAAC
GACTCTGACCCAGCTGTCAAGCGCGCCATCACGTGGTCTCGATTCCTGG- GCGCGTCT
CCAGCCTTCCTGGCACAGCAGCGAGCGTGGCTCCCTTCGCCAACCGACC- CATCAAT
GAGCAGAAACGTTTCCGACCTGCCGTGAAGAGCCGATCACGACGAGCGCC- GCCGTG
CGTGTGGAAGGTGTGTCCCGCTCCCCCCTGGCTGGTCACCAAACGTAAACA- GGAAA
CCAGCGACTACTGACGTCATACCTCAATAGACCGACTCATGACTTCAACGCT- GAATT
GACGTCACCGAGAGCTCCAACGTCACAGCAGGAGCGAGAGAGAGAGAGAGAG- AGA
GAGAAAGAGAGAGAGAAAGGCTGGAGTATTTCTCTTTCTTTTGGTTTTTCGTGT- TGA
AGTGTGATCAGCTGGGCTGGTTCAAAATTGTTGAATAAAGTTGAATGAAAATCA- AA
AAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC
Translation: MQTAYWVMMMVVVMMVGVTVAGSLSVFDDDNDSDPAV-
KRAITWSRFLGASPAFLA (SEQ ID NO:167)
QQRALAPFANRPEQKRFRPAVKSRSRRAPPCV- WKVCPAPPWLVTKRKQETSDY Toxin
Sequence:
Phe-Arg-Xaa3-Ala-Val-Lys-Ser-Arg-Ser-Arg-Arg-Ala-Xaa3-Xaa3-Cys-Val-Xaa4-L-
ys-Val-Cys- (SEQ ID NO:168)
Xaa3-Ala-Xaa3-Xaa3-Xaa4-Leu-Val-Thr-Lys-
-Arg-Lys-Gln-Xaa1-Thr-Ser-Asp-Xaa5-{circumflex over ( )} Name:
Nb14.2 Species: nobilis Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATG- GTGATGATGATG (SEQ ID
NO:169) GTGTGGATTACAGCCCCTCTGTCTGAAGG- TGGTAAATTGAACGACGTAATTCGGGGT
TTGGTGCCACACTTCTTAACCCCACAGCA- TATCTTGCAAAGTCTGACTTCCCGTAAT
GGTTCTGGCAGCAGTAATCAGAAAGAAGC- GCAACTATGCATCTGGAAGGTATGTCC
ACCAACCCCATGGAGATGATCACAAGGAAA- AAGATGAACGGCGTCAGACAACCGC
CACAACTGTAGTGGGACATCGTTGATACGACT- TCAGCAAATATTTTAACATCACTGT
GGTTGTGAAGAAATCAGTTGTTTTAAAAGATT- GGATTTTTCCTTGTTTAAGAGTTGT
ACTGATATCAGCTCTGCACTATGAAATAAAGC- TGATGTGACAAGCAAAAAAAAAAA
AAAAAAGTACTCTGCGTTGTTACTCGAGCTTAA- GGGCGAATTC Translation:
MQTAYWVMVMMMVWITMLSEGGKNDVR- GLVPHLFLTPQHILQSLTSRNGSGSSNQ (SEQ ID
NO:170) KEAQLCIWKVCPPTPWR Toxin Sequence:
Asn-Gly-Ser-Gly-Ser-Ser-Asn-Gln-Lys-Xaa-
1-Ala-Gln-Leu-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3- (SEQ ID NO:171)
Xaa3-Thr-Xaa3-Xaa4-Arg-{circumflex over ( )} Name: Nb14.3 Species:
nobilis Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGATGATGGT- GGTG (SEQ ID
NO:172) GTGATGATGGTGGGGGTTACTGTCGCTGGCTCACTGT- CTGTGTTTGATGACGACAAT
GACTCTGACCCAGCTGTCAAGCGCGCCATCACGTGGT- CTCGATTCCTGGGCGCGTCT
CCAGCCTTCCTGGCACAGCAGCGAGCGCTGGCTCCCT- TCGCCAACCGACCCATCT
GAGCAGAAACGTTTCCGACCTGCCGTGAAGAGCCGATCA- CGACGAGCGCCGCCGTG
CGTATGGAAGGTGTGTCCCGCTCCCCCCTGGCTGGTCACC- AAACGTAAACAGGAAA
CCAGCGACTACTGACGTCATACCTCAATAGACCGACTCATG- ACTTCAACGCTGAATT
GACCTCACCGAGAGCTCCAACGTCACAGCAGGAGCGAGAGA- GAGAGAGAGAGAGA
GAGAGAGAGAAAGGCTGGAGTATTTCTCTTTCTTTCGGTTTTT- CGTGTTGAAGTGTG
ATCAGCTGGGCTGGTTCAAAATTGTTGAATAAAGTTGAATAAA- AAAAAAAAAAAAA
AAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVM1VVVMMVGVTVAGSLSVFDDDNDSDPAVKRAIT- WSRFLGASPAFLA (SEQ ID
NO:173) QQRALAPFANRPINEQKRFRPAVKSRSRRAPPCVWKV- CPAPPWLVTKRKQETSDY
Toxin Sequence:
Phe-Arg-Xaa3-ALa-VaL-Lys-Ser-Arg-Ser-Arg-Arg-ALa-Xaa3-Xaa3-Cys-Val-Xaa4-L-
ys-Val-Cys- (SEQ ID NO:174)
Xaa3-Ala-Xaa3-Xaa3-Xaa4-Leu-VaL-Thr-Lys-
-Arg-Lys-Gln-Xaa1-Thr-Ser-Asp-Xaa5-{circumflex over ( )} Name:
Pr14.1 Species: parius Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGG- TGATGATGATG (SEQ ID
NO:175) GTGGTGTGGATTACAGCCCCTTTGTCTGAA- GGTGGTAAACCGAAGCACGCAATTCG
GGGTTTGGTGCCAGACGACTTAACCCCACAG- CTTATCTTGCGAAGTCTGATTTCCCG
TCGTAGTTCTTTCGGCAAGGATGCGAAACCC- CCCTTTAGTTGTTCAGGCCTCCGAGG
GGGTTGCGTCCTACCTCCCAATCTCAGGCCA- AAGTTCAACAAAGGTGGATAACAAA
CCCAAGCGTTCCTAGTTATACGAATGCCAGCA- AATAAAAGCAGTTTGATTGTGAAA
AAAAAAAAAAAAAAAAGTACTCTGCGTTGTTAC- TCGAGCTTAAGGGCGAATTC
Translation:
MQTAYWVMVMMMVVWITAPLSEGGKPKHMRGLVPDDLTPQLILRSLISRRSSFGKDA (SEQ ID
NO:176) KPPFSCSGLRGGCVLPPNLRPKENKGG Toxin Sequence:
Xaa5-Xaa3-Phe-Ser-Cys-Ser-Gly-Leu-Arg-Gly-Gly-Cys-Val-Leu-Xaa3-Xaa3-Asn-L-
eu-Arg-(SEQ ID NO:177) Xaa3-Lys-Phe-Asn-Lys-Gly-# Name: Pr14.2
Species: parius Cloned: Yes DNA Sequence:
GAATTCGCCCTTGGATCCATGCAGACGGCCTACTGGGT- GATGGTGATGATGATGGT (SEQ ID
NO:178) GATGTGGATTACAGCCCCTCTGT- CTGAAGGTGGTAAACCGAAGCTCATAATTCGGG
GTTTGGTGCCAAACGACTTAACCC- CACAGCGTATCTTGCGAAGTCTGATTTCCGGGC
GTACTTATGGCATCTATGATGCGA- AACCCCCCTTTAGTTGTGCAGGCCTCCGAGGGG
GTTGCGTCCTACCTCCCAATCTCA- GGCCAAAGTTCAAGGAAGGTCGATAAAAAACC
CAAGCGTTCCTAGTTATACGAATGC- CAGCAAATAAAAGCAGTTTGATTGCGAAAAA
AAAAAAAAAAAAAAAGTACTCTGCGT- TGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVMWLTAPLSEGGKPRLURGLVPNDLTPQRILRSLISGRTYGIYDA (SEQ ID
NO:179) KPPFSCAGLRGGCVLPPNLRPKFKEGR Toxin Sequence:
Xaa3-Xaa3-Phe-Ser-Cys-Ala-Gly-Leu-Arg-Gly-Gly-Cys-Val-Leu-Xaa3-Xaa3-Asn-L-
eu-Arg- (SEQ ID NO:180) Xaa3-Lys-Phe-Lys-Xaa1-# Name: P114.1
Species: planorbis Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGA- TGATGATGATGATG (SEQ ID
NO:181) GTGTGGATTACAGGCCATCTGTCTGAA- GGTGGCAAATTGAAGGATGCAATTAGGGG
TTTGGTGCCAGACGACTTGACCTCAATG- TTTGCGTTGCATCTTCCGGTTTCCCATTCT
CGGTCTAGCAGCAATGGTCTGAAGAGA- GCTGACCTATGTATCCACAAGATTTGTCC
ACCACGGTATCACCAAAGCCAACAATAA- AAGACGTCAGACAACCACCACAACTTTA
GTATGACATCGTTAATAGGACTTCAGCAA- GTATTTTAACATCACTGTGGTTGTGATG
AAATCAGTCGCCTTAAAAGATTGGCTTTT- TCCTTGTTTAAGAGTTGTACTTGTATCAG
CTTTGCACTTCGAAATAAAGTTGATGTG- ATGAACCAAAAAAAAAAAAAAAAAAAAG
TACTCTGCGTTGTTACTCGAGCTTAAGGG- CGAATTC Translation:
MQTAYWVMMMMMVWITGHLSEGGKLKDAIR- GLVPDDLTSMFALHLPVSHSRSSSNG (SEQ ID
NO:182) LKRADLCIHKICPPRYHQSQQ Toxin Sequence:
Ser-Ser-Ser-Asn-Gly-Leu-Lys-Arg-Ala-A-
sp-Leu-Cys-Ile-His-Lys-Ile-Cys-Xaa3-Xaa3-Arg- (SEQ ID NO:183)
Xaa5-His-Gln-Ser-Gln-Gln-{circumflex over ( )} Name: Pu14.1
Species: pulicarius Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGATGGTGTGGGTTACAG- CGCC (SEQ ID
NO:184) TGTGTCTGAAGGTGGTAAATTGAGCGACGTAATTCGG- GGTTTGGTGCCAGACGACA
TAACCCCACAGATTATTTTGCAAAGTCTGAATGCCAGT- CGTCATGCTTACAGACGTG
TTCGTCTGAGAGGACAGATATGTATCTGGAAGGTATGT- CCACCACTACTACAATGG
ATACATCCATTAGTAAAAAGATGAATGACATCAGACAAC- CGCCACAACTGTAGTAC
GACATCGTTAACACGACTTCAGCAAATATTCTAACATCAC- AGTGGGTTGTGAAGAN
ATCGGGTTGGCTTTAAAAAAAANAATGGGGGNTTTTCCCCN- TGGGTTTAAAAAAAN
NTNGGNNCCGGGNAANNNCCCNNNNTNNNCCCCCCCCNNTNG- GGAGAAAAAAAAA
ANNCCNNTNNNGGGGGGNNNNCNAAAAAAAAAAAAAAAAAAAAA- AAAAAAAAAN
CCCCNGGGGGGNTGNTTTNNCCCCCCNCCCCNGGGGGGGGGGGNGNT- TTNNCCCCC
CCCCCGNGGGGGGGGGGGNTTTTNNTTTNNGGGGGNGCCCCCCCCCCC- CCCNNNCN
NNNNAANAANNNNNGGGGGGGGGGAANAAAAANANNNNNNNNNNNNNNN- NNNTT
TTNTCNNTCNNCCGNGNNGNNAAAAAAAAAANTTNATTTNTNNANNNCNNCN- NNCC
NNCNNCNNACCCNNCCCCNNCCNCNNCNNCNNAGANNANGAGGGGGGGGNGNN
NNGGNGNANNNANNNNNNNGAANNNGAGGNGNGNNNCNCGNCNNCGCNCNNG NC
Translation: MQTAYWVMVMMMMVWVTAPVSEG-
GKLSDVIRGLVPDDITPQIILQSLNASRHAYRRV (SEQ ID NO:185)
RLRGQICIWKVCPPLLQWIHPLVKR Toxin Sequence:
Val-Arg-Leu-Arg-Gly-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Leu-Gln-Xa-
a4-Ile- (SEQ ID NO:186) His-Xaa3-Leu-Val-Lys-Arg-{circumflex over (
)} Name: Pu14.2 Species: pulicarius Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGATGGTGTGGGTTACAGCGCC (SEQ ID
NO:187) TGTGTCTGAAGGTGGTAAATTGAGCGACGTAATTCGGGGTTTGGTGCCAGACGACTT
AACCCCACAGATTATCTTGCAAAGTCTGAATGCCAGTCGTCATGCTTACAGACGTGT
TCGTCCGAGAGGACAGATATGTATCTGGAAGGTATGTCCACCACTACTACAATGGA
TACATCCATTAGTAAAAAGATGAATGACATCAGACAACCGCCACAACTGTAGTACG
GCATCGTTAACACGACTTCAGCAAATATTTTAACATCACAGTGGTTGTGAAGAAATC
GGTTGCTTTAAAAAAAGATTGGGTTTTTCCTTGTTTAAGAGTTGTACTGATATCAGT- T
CTGCACTATGAAATAAAGCTGATGTGACGAACAAAAAAAAAAAAAAAAAAAAGTA
CTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMMVWVTAPVSEGGKLSDVRGLVPDDLTPQIILQSLNASRHAYRR (SEQ ID
NO:188) VRPRGQICIWKVCPPLLQWIHPLVKR Toxin Sequence:
Val-Arg-Xaa3-Arg-Gly-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Lcu-Lcu-G-
ln-Xaa4-Ile- (SEQ ID NO:189) His-Xaa3-Leu-Val-Lys-Arg-{circumflex
over ( )} Name: Pu14.3 Species: pulicarius Cloned: Yes DNA
Sequence: ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGATGGTGTGGGTTACAGCGCC
(SEQ ID NO:190)
TGTGTCTGAAGGTGGTAAATTGAGCGACGTAATTCGGGGTTTGGTGCCAGACGACA
TAACCCCACAGATTATCTTGCAAAGTCTGAATGCCAGTCGTCATGCTTACAGACCTG
TTCGTCTGAGAGGACAGATATGTATCTGGAAGGTATGTCCACCACTACTACAATGG
ATACATCCATTAGTAAAAAGATGAATGACATCAGACAACCGCCACAACTGTAGTAC
GACATCGTTAACACGACTTCAGCAAATATTTTAACATCACAGTGGTTGTGAAGAAAT
CGGTTGCTTTAAAAAAAGATTGGGTTTTTCCTTGTTTAAGAGTTGTACTGATATCAG- T
TCTGCACTATGAAATAAAGCTGATGTGACGAACAAAAAAAAAAAAAAAAAAAAGT
ACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMMVWVTAPVSEGGKLSDVIRGLVPDDITPQIILQSLNASRHAYRPV
RLRGQICIWKVCPPLLQWIHPLVKR Toxin Sequence:
Xaa3-Val-Arg-Leu-Arg-Gly-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Leu-L-
eu-Gln- (SEQ ID NO:192)
Xaa4-Ile-His-Xaa3-Leu-Val-Lys-Arg-{circumfl- ex over ( )} Name:
Ra14.1 Species: rattus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGGTGGTGGTGGGGTTCACCGTCGG (SEQ ID
NO:193) GGGTCACGTCCATCAATCTCACAGTCCTACATCGCGCAGCCATGGTGATGACTCCAT
TCATGACAAGACGATTCATCAACATCTGTTTGCCCGTCTTCCTCTGGAGAACAACGA
CGACCATCGTTCTGTGGATCTTCCTGCAGGGACCAGCGCAGGCGACATGAAACCAC
AACGCCAAAGACGTCTCTGCTGCATCTTTGCCATTCTTTGGTTCTGTTGTCTCGGTT- A
ACAGTACAAATTGCAATGCACTGGCCGATTGAAAGAACTGCAATAAACGGAAAAA
ANAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMVVVGFTVGGHVHQSHSPTSRSHGDDSIHDKTIHQHLFARLPL- ENND (SEQ ID
NO:194) DHRSVDLPAGTSAGDMKPQRQRRLCCIFAILWFCCLG Toxin Sequence:
Leu-Cys-Cys-Ile-Phe-Ala-Ile-Leu-Xaa4-Phe-Cy- s-Cys-Leu-# (SEQ ID
NO:195) Name: S14.2 Species: striatus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG (SEQ ID
NO:196) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAA- TTCGGGGT
TTGGTGCCACACATCTTAACCCCACAGCATATCTTGCAAAGTCTGATTT- CCCCTCTTC
GTTCTAACAACGGTCGTTCGAGTGGAGCACAAATATGCATCTGGAAGG- TATGTCCA
CCATCCCCATGGAGACAACCACAAGAAATGATGAATGACATCAGACAAC- CGCCACA
ACTGTAGTACGACATCGTTGATACGACTTTAGCAAATATTTTAACATCAC- TGTGGTT
GTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGT- TGTACTG
ATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAA- AAAAAA
AAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVRGLVPHILTPQHILQSLI- SPLRSNNGRSS (SEQ ID
NO:197) GAQICIWKVCPPSPWRQPQEMMNDIRQPPQL Toxin Sequence:
Ser-Asn-Asn-Gly-Arg-Ser-Ser-Gly-Ala-Gln-Il-
e-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Ser- (SEQ ID NO:198)
Xaa3-Xaa4-Arg-Gln-Xaa3-Gln-Xaa1-Met-Met-Asn-Asp-Ile-Arg-Gln-Xaa3-Xaa3-Gln-
-Leu-{circumflex over ( )} Name: Sx14.1 Species: striolatus Cloned:
Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGACCCTCT (SEQ ID
NO:199) GTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGTTTGGTGCCACGC- ATCTTAAC
CCCACAGCATACCTTGCGAAGTCCGACTTCCCTTCTTCGTTCTAACACC- GGTGGTTC
GAGTGGAGCACAAATATGCATCTGGAAGGTATGTCCACCATCCCCATGG- AGACGAT
CACAAGGAAAAAGATGAATGACGTCAGACAAGCGCCACAACTGTAGTACG- ACATC
GTTGATACGACTTCAGCAAGTATTTTAACATCACTGTGGTTGTGAAGAAATC- AGTTG
CTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTGATATCAGCTCT- GCCCTG
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWLTDPLSEGGKLNDVIRGLVPRILTPQHTLRSPTSLLRSNTGGSS
GAQICIWKVCPPSPWRRSQGKR Toxin Sequence:
Ser-Asn-Thr-Gly-Gly-Ser-Ser-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys-Xaa3-
-Ser- (SEQ ID NO:201) Xaa3-Xaa4-Arg-Arg-Ser-Gln-# Name: Sx14.2
Species: striolatus Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTG- GATTACAGCCCCTCT (SEQ ID
NO:202) GTCTGAAGGTGGTAAATTGAACGACG- TAATTCGGGGTTTGGTGCCACACATCTTAAC
CCCACAGCATATCTTGCAAAGTCTGA- TTTCCCCTCTTCGTTCTAACAACGGTCGTTC
GAGTGGAGCACAAATATGCATCTGGA- AGGTATGTCCACCATCCCCATGGAGACGAT
CACAAGGAAAAAGATGAATGACGTCAG- ACAAGCGCCACAACTGTAGTACGACATC
GTTGATACGACTTCAGCAAGTATTTTAAC- ATCACTGTGGTTGTGAAGAAATCAGTTG
CTTTAAAAGATTGGATTTTTCCTTGTTTA- AGAGTTGTACTGATATCAGCTCTGCACTG
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVPHILTPQHILQSLISP- LRSNNGRSS
GAQICIWKVCPPSPWRRSQGKR Toxin Sequence:
Ser-Asn-Asn-Gly-Arg-Ser-Ser-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Val-Cys--
Xaa3-Xaa3-Ser- (SEQ ID NO:204) Xaa3-Xaa4-Arg-Arg-Ser-Gln-# Name:
Sx14.3 Species: striolatus Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCC- TACTGGGTGATGGTGATGATGATG (SEQ ID
NO:205) GTGTGGATTAAAGACCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGG
TTTGGTGCCACACATCTTAACCCCACAGCATATCTTGCAAAGTCTGATTTCCCCTCTT
CGTTCTAACAACGGTCGTTCGAGTGGAGCACAAATATGCAACTGGAAGGTATGTCC
ACCATCCCCATGGAGACGACCACGAGGAAAATGATGAATGACATCAGACAACCGCC
ACAACTGTAGTACGACTTCGTTGATACGACTTTAGCAAATATTTTAACATCACTGTG
GTTGTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTA
CTGATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAAAAA
AAAAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWIKDPLSEGGKLNDVIRGLVPHILTPQHILQSLISPLRSN- NGRSS (SEQ
ID NO:206) GAQICNWKVCPPSPWRRPRGK Toxin Sequence:
Ser-Asn-Asn-Gly-Arg-Ser-Ser-Gly-Ala-Gln-Ile-Cys-Asn-Xaa4-
-Lys-Val-Cys-Xaa3-Xaa3-Ser- (SEQ ID NO:207)
Xaa3-Xaa4-Arg-Arg-Xaa3-- Arg-# Name: Sx14.4 Species: striolatus
Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGATGATG (SEQ ID
NO:208) GTGTGGATTACAGCCCCTCTGTCTGAAGGTGGTAAATTGAACGACGTAATTCGGGGT
TTGGTGCCACACATCTTAACCCCACAGCATATCTTGCAAAGTCTGATTTCCCCTCTT- C
GTTCTAACAACGGTCGTTCGAGTGGAGCACAAATATGCATCTGGAAGGTATGTCCA
CCATCCCCATGGAGACAACCACAAGAAATGATGAATGACATCAGACAACCGCCACA
ACTGTAGTACGACATCGTTGATACGACTTTAGCAAATATTTTAACATCACTGTGGTT
GTGAAGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGTACTG
ATATCAGCTCTGCACTATGAAATAAAGCTGATGTGACAAACGAAAAAAAAAAAAAA
AAAAAAGTACTCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNDVIRGLVPHILTPQHILQSLISPLR- SNNGRSS (SEQ
ID NO:209) GAQICIWKVCPPSPWRQPQEMMNDIRQPPQL Toxin Sequence:
Ser-Asn-Asn-Gly-Arg-Ser-Ser-Gly-Ala-Gln-Ile-Cy-
s-Ile-Xaa4-Lys-Val-Cys-Xaa3-Xaa3-Ser- (SEQ ID NO:210)
Xaa3-Xaa4-Arg-Gln-Xaa3-Gln-Xaa1-Met-Met-Asn-Asp-Ile-Arg-Gln-Xaa3-Xaa3-Gln-
-Leu-{circumflex over ( )} Name: S114.1 Species: sulcatus Cloned:
Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCTCTCTG (SEQ ID
NO:211) TCTGAAGGTGGTAAACCGAACGACGTCATTCGGGGTTTTGTGCCAGACG- ACTTAAC
CCCACAGCTTATCTTGCGAAGTCTGATTTCCCGTCGTCGTTCTGACAAGG- ATGTTGG
GAAGAGAATGGAATGTTACTGGAAGGCATGTAGACCCACGCTATCGAGAC- GACATG
ATCTTGGGTAAAAGATGAATGACGTCAGACAACAGCCACAACTATAGTATG- ACATC
GTTAATACGACTTCAGCAAATATTTTAACATCACTGTGGTTGTGAAGAAATC- AGTTG
CTTTAAAAGATTGGATTTTTCCGTGTTTAAGAGTTGTACTGATATCAGCTCT- GCCCTG
TGAAATAAAGCTGATG Translation:
MQTAYWVMVMMMVWITASLSEGGKPNDVIRGFVPDDLTPQLILRSLISRRRSDKDVGK (SEQ ID
NO:212) RMECYWKACRLPTLSRRHDLG Toxin Sequence:
Arg-Ser-Asp-Lys-Asp-Val-Gly-Lys-Arg-Met-Xaa1-Cys-Xaa5-Xaa4-Lys-Ala-Cys--
Arg-Xaa3- (SEQ ID NO:213) Thr-Leu-Ser-Arg-Arg-His-Asp-Leu-# Name:
Tr14.1 Species: terebra Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATG- GTGTGGATTACAGCCCCTCT (SEQ ID
NO:214) GTCTGAAGGTGATAAATTGAACGACGTAATTCGGGGTTTGGTGCCAGATAACTTAG
CCCCACAGCTTGTTTTGCAAAGTCTGGATTCCCGTCGTCATCCTCACGGCATTCGTC
AGGATGGAGCCCAAATATGTATCTGGAAGATATGTCCACCATCCCCATGGAAACGA
CTTGGATCTTAAGAAAAGAAACAATTGACGTCAGACAACCGCCACAACTTGAGTAC
GACATCGTTAATACAACTTCAGCAAATATGAAATTTTCAGCATCACTGTGGTTGTGA
AGAAATCAGTTGCTTTAAAAGATTGGATTTGTCCTTGTTTAAGAGTTGTACTGATGT
CATCTCTGCACTGTGAAATAAAGCTGATGTGACAAACAAAAAAAAAAAAAAAAAA
GTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMVWITAPLSEGDKLNDVIRGLVPDNLAPQLVLQSLDSRRHPHGIRQ (SEQ ID
NO:215) DGAQICIWKICPPSPWKRLGS Toxin Sequence:
His-Xaa3-His-Gly-Ile-Arg-Gln-Asp-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Ile-Cys-
-Xaa3-Xaa3- (SEQ ID NO:216)
Ser-Xaa3-Xaa4-Lys-Arg-Leu-Gly-Ser-{circ- umflex over ( )} Name:
Tr14.2 Species: terebra Cloned: Yes DNA Sequence:
ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGTGGATTACAGCCCCTCT (SEQ ID
NO:217) GTCTGAAGGTGATAAATTGAACGACGTAATTCGGGGTTTGGTGCCAGATAACTTAG
CCCCACAGCTTGTTTTGCATAGTCTGGATTCCCGTCGTCATCCTCACGGCATTCGTC- A
GGATGGAGCCCAAATATGTATCTGGAAGATATGTCCACCATCCCCATGGAGACGAC
TTGGATCTTAAGAAAAGAAACAATTGACGTCAGACAACCGCCACATCTTGAGTACG
ACATCGTTAATACGACTTCAGCAAATATGAAATTTTCAGCATCACTGTGGTTGTGAA
GAAATCAGTTGCCTTAAAAGATTGGATTTGTCCTTGTTTAAGAGTTGTACTGATGTC
ATCTCTGCACTATGAAATAAAGCTGATGTGACAAACAAAAAAAAAAAAAAAAAAA
AGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMVWITAPLSEGDKLNDVIRGLVPDNLAPQLVLHSLDSRRHPHGIRQ (SEQ ID
NO:218) DGAQICIWKICPPSPWRRLGS Toxin Sequence:
His-Xaa3-His-Gly-Ile-Arg-Gln-Asp-Gly-Ala-Gln-Ile-Cys-Ile-Xaa4-Lys-Ile-Cys-
-Xaa3-Xaa3- (SEQ ID NO:219)
Ser-Xaa3-Xaa4-Arg-Arg-Leu-Gly-Ser-{circ- umflex over ( )} Name:
Vx14.1 Species: vexillum Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGATGGCCTACTGGGTGATGGTGATGATGATGG (SEQ ID
NO:220) TGTGGATTAAAGGCCCTGTGTCCGAAGGTGGTAAATTGAACGACGTAATTCGGGGT
TTGGTGCCAGACGACTTGACCCCAGTGTCTGCCTTGCATCATCCGGTTTCCCATCGT
CGGTCTCACAGCAGTAGTTTGTGGTGTGTATGTCCATTCAGGGTGTGTCCACCATGC
CATGGAAGATGACCTGGTCCCAAACCAACAAAATAACGTCAGACAACCGCCACAAC
TTTAGTACGACATCCCTTAATACGACTTCAGCAAGTATTTTAACATCACTATGGTGT
GATGAAATCAGTTGCTTTAAAAGATTGGATTTTTCCTTGTTTAAGAGTTGCACTGAT
AACAGCCCAGCAGTATGAAATAAAGTTGATGTGGCAAAAAAAAAAAAAAAAGTAC
TCTGCGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQMAYWVMVMMMVWIKGPVSEGGKLNDVIRGLVPDDLTPVSALHHPVSHRRSH- SSS (SEQ ID
NO:221) LWCVCPFRVCPPCHGR Toxin Sequence:
Ser-His-Ser-Ser-Ser-Leu-Xaa4-Cys-Val-Cys-Xaa3-Phe-Arg-Va-
l-Cys-Xaa3-Xaa3-Cys-His-# (SEQ ID NO:222) Name: Vx14.2 Species:
vexillum Cloned: Yes DNA Sequence:
GAATTCGCCCTTATGGATCCATGCAGACGGCCTACTGGGTGATGGTGATGAT- GATG (SEQ ID
NO:223) GTGTGGATTACAGCCCCTTTGTCTGAAGGTGGTAAAC- TGAACGATGTAATTCGGGGT
TTCGCGCTAGATGACTTAGCCCAAAGCCGTATTATGC- AAAGTCTGGTTTTCAGTCAT
CAGCCTCTTCCAACGGCATCCATATGTATCTGGAAGA- TATGTCCACCAGACCCATGG
AGACGACATGATCTTCAGAAAGTAACAAATGACGTCA- GACAACCGCCACAACTTG
AATACAACATCATTAATACGACTTCAGCAAATATTTTAG- CATCACTGTGATTGTTCG
GAAGTCAGTTGCTTTAAAAGATTGGATTTGTCCCTGTTG- TATTGATGTCACTCTGC
ACTATGAAATAAAGCTGATGTGACAAGCAAAAAAAAAAAA- AAAAAAAGTACTCTG
CGTTGTTACTCGAGCTTAAGGGCGAATTC Translation:
MQTAYWVMVMMMVWITAPLSEGGKLNPVIRGFALDDLAQSRIMQSLVFSH- QPLPTAS (SEQ ID
NO:224) ICIWKICPPDPWRRHDLQKSNK Toxin Sequence:
Ile-Met-Gln-Ser-Leu-Val-Phe-Ser-His-Gln-Xaa3-Leu-X-
aa3-Thr-Ala-Ser-Ile-Cys-Ile-Xaa4-Lys- (SEQ ID NO:225)
Ile-Cys-Xaa3-Xaa3-Asp-Xaa3-Xaa4-Arg-Arg-His-Asp-Leu-Gln-Lys-Ser-Asn-Lys-{-
circumflex over ( )} Name: Vx14.3 Species: vexillum Cloned: Yes DNA
Sequence: ATGCAGACGGCCTACTGGGTGATGGTGATGATGATGGTGGTGGGGTTCACCGTCGA
(SEQ ID NO:226)
GAGTCACGTCCATCAGTCTCACAGTCCTACATCGCGCAGCCATGGTGATGACTCCAT
TCATGACAAGACGATTCATCAACATCTGTTTGCCCGTCTTCCTCTGGAGAACAACGA
CGACCATCGTTCTGTGGATCTTCCTGCAGGGACTAGCGCAGGCGACATGAAACCAC
AACGCCAGAAACGTTTCTGCTGCATCTTTGCCCCGATTCTTTTGTTCTGTTGTTTCG- G
TTAACAGCACAAATTACACTGCACTGGCCGATTGAAAGAACTGCAATAAACGGTAA
AGCAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACTCGAG Translation:
MQTAYWVMVMMMVVGFTVESHVHQSHSPTSRSHGDDSIHDKTHIHQHLFA- RLPLENND (SEQ
ID NO:227) DHRSVDLPAGTSAGDMKPQRQKRFCCIFAPILLFCCFG Toxin Sequence:
Phe-Cys-Cys-Ile-Phe-Ala-Xaa3-Ile-Leu-L- eu-Phe-Cys-Cys-Phe-# (SEQ
ID NO:228) Xaa1 is Glu or .gamma.-carboxy-Glu Xaa2 is Gln or
pyro-Glu Xaa3 is Pro or hydroxy-Pro Xaa4 is Trp, D-Trp or
6-bromo-[D or L] Trp Xaa5 is Tyr, .sup.125I-Tyr, mono-iodo-Tyr,
di-iodo-Tyr, O-sulpho-Tyr or O-phospho-Tyr {circumflex over ( )} is
free carboxyl or amidated C-terminus, preferably free carboxyl # is
free carboxyl or amidated C-terminus, preferably amidated ? is free
carboxyl or amidated C-terminus
[0116]
2TABLE 2 Alignment of .beta.-Superfamily Conotoxins (SEQ ID NO:)*
Type 2: T14.2 tulipa ----ZTDVLLEATLLTTPAPEQRLFCFWKSCWPRPYPWRRRDLN#
(229) M14.1 magus ----ZTDVLLDATLLTTPAPEQRLFCFWKSCWPRPYPWRRRNLN#
(230) G14.2 geographus
----ZTDVLLEATLLTTPAPEQRLFCFWKSCTWRPYPWRRRDLN# (231) T14.2 tulipa
-----------------------LFCFWKSCWPRPYPWRRRDLN# (232) M14.1 magus
-----------------------LFCFWKSCWPRPYPWR- RRNLN# (233) G14.2
geographus -----------------------LFCFW- KSCTWRPYPWRRRDLN# (234)
Type 3: Ge14.1 generalis
-----------------------SHSSSLWCVCPFRVCPPCH# (235) Vx14.1 vexillum
-----------------------SHSSSLWCVCPFRVCPPCH# (236) Type 4: Fd14.1
flavidus ---HDHGIRPKR-----------------VDICNWRICAPN-
PLRRHDLKKGNN{circumflex over ( )} (237) Em14.1 emaciatus
---HTHGIRPKG-----------------DGICIWKVCPPDPWRRHRLKKRNN{circumflex
over ( )} (238) A14.1 aurisiacus ---HTHGIRPKG-----------------DG-
ICIWKVCPPDPWRRHHLKKRNN{circumflex over ( )} (239) Tr14.1 terebra
---HPHGIRQDG-----------------AQICIWKICPPSPWKRLGS{circumflex over (
)} (240) Tr14.2 terebra ---HPHGIRQDG-----------------AQ-
ICIWKICPPSPWRRLGS{circumflex over ( )} (241) Ly14.2 litoglyphus
---HPHGIRQDG-----------------AQICIWKICPPSPWRRLGS{circumflex over (
)} (242) Ly14.1 litoglyphus
---HPHGIRQDG-----------------AQICIWKICPPSPWRRLGS{circumflex over (
)} (243) Type 5: Cn14.1 consors
---DRSDNGGSSG----------------AQICIWKVCPPSP{circumflex over ( )}
(244) Cn14.5 consors
---DRSDNGGSSG----------------AQICIWKVCPPSPWK{c- ircumflex over ( )}
(245) Cn14.2 consors
---ARSDNGGSSG----------------AQICIWKVCPPSPWRRPQ# (246) Sx14.1
striolatus -----SNTGGSSG----------------AQICIWKVCPPSPWRRSQ# (247)
Sx14.3 striolatus -----SNNGRSSG----------------AQICNWKVCP-
PSPWRRPR# (248) Sx14.2 striolatus -----SNNGRSSG------------
-----AQICIWKVCPPSPWRRSQ# (249) Sx14.4 striolatus
-----SNNGRSSG----------------AQICIWKVCPPSPWRQPQEMMNDIRQPPQL{circumflex
over ( )} (250) S14.2 striatus -----SNNGRSSG--------------
---AQICIWKVCPPSPWRQPQEMMNDIRQPPQL{circumflex over ( )} (251) A14.3
aurisiacus ---LHSDSSDQKG----------------AQICIWKVCPPPPWR{circumfle-
x over ( )} (252) A14.2 aurisiacus ---LHSDSSDQKGGMNAWTGAGA-
QICIWKVCPPPPWR{circumflex over ( )} (253) A14.4 aurisiacus
---LRSDSSDQKGGMNASTGAGAQICIWKVCPPSPWRRTQ# (254) Cr14.1 circumcisus
---LRSDSSGQKG---------AQICIWKVCPLSPWRRPQ# (255) Cr14.2 circumcisus
---LRSDSSGQKG---------AQICIWKVCPLSPWRRPQGKDE{circumf- lex over ( )}
(256) Ac14.1 achatinus ---LRSDNGGSSG---------AQICIWKVCPPSPPWRRPQ#
(257) Sm14.1 stercus-
---LGIGSSDQN----------AQICIWKVCPPSP{circumflex over ( )} (258)
muscarum Cn14.3 consors ---NGSGSSNQKE---------AQLCI-
WKVCPPSPWR{circumflex over ( )} (259) Cn14.4 consors
---NGSGSSNQKE---------AQLCIWKVCPPTPWR{circumflex over ( )} (260)
M14.2 magus ---NGSGSSNQKE---------AQLCIWKVCPPSPWR{circumflex over (
)} (261) Nb14.2 nobilis ---NGSGSSNQKE---------AQLCIWKVC-
PPTPWR{circumflex over ( )} (262) Type 6: S114.1 sulcatus
-------------RSDKDVGKRME-CYWKACRPTLSRRHDL# (263) Bk14.1 bocki
-------------RSDKDDPGGQE-CYWNVCAPNQGDHMILRKKMNDDRQPPQL{circu- mflex
over ( )} (264) Bt14.1 betulinus
-------------RSDSDVREV-PVCSWKICPP{circumflex over ( )} (265) Ls14.1
loroisii -------------RSDSDVREVYILCIWKICPPLP{circumflex over ( )}
(266) Type 7: Gd14.1 gladiator
-------------HPANVRQQGKICVWKVCPPWPVRSPGPQPKNK{circumflex over ( )}
(267) Gd14.2 gladiator -------------HPANVRQQGKICVWKVCPPSPVRSPGP-
LPKNK{circumflex over ( )} (268) Type 8: Ms14.2 musicus
GMGPGDLSLQKMFPSLALGPGGDVICRWKVCPPTPWKRLIK{circumflex over ( )}
(269) Ms14.3 musicus GMVPGDLALQYLFPSLAFNP-PD-ICTWKVCPPPPW-
RRPKKITDVGQPPQL{circumflex over ( )} (270) Ms14.1 musicus
GMVPGDLVLQYLFPSLAFSP-PD-ICTWKVCPPPPWRRPKKITDVRQPPQL{circumflex over
( )} (271) Ms14.4 musicus GMVPGDLVLQYLFPSLAFNP-PD-ICTWKVCPPPPW-
RRPKKITDVRQPPQL{circumflex over ( )} (272) Type 9: Mi14.1 miles
ZQDQSPHHVCCAIGPVLPFCCVSWLHKLH{circumflex over ( )} (273) Mi14.2
miles --------LCCIFAPILWFCCH# (274) Ral4.1 rattus
--------LCCIFA-ILWFCCL# (275) Cp14.1 capitaneus
-------GFCCDFPPIFWFCCI# (276) Mi14.4 miles
------ZGFCCVVIPILWFCCGGYRTNGTALAD{circumflex over ( )} (277) Vx14.3
vexillum --------FCCIFAPTLLFCCF# (278) Type 10: S114.2 sulcatus
--------------ZSGCRVPFELKCIWKFCTIYPSRPFASLEEKDECQT-
VTITVTWDF{circumflex over ( )} (279) Ci14.1 cinereus
--------------SSGCSVSLGFKCFWKSCTVIPVRPFVSLEEENECQKVQISAVWGP{circumflex
over ( )} (280) Type 11: Pr14.1 parius
---------------------PPFSCSGLRGGCVLPPNLRPKFNKG# (281) Pr14.2 parius
---------------------PPFSCAGLRGGCVLPPNLRPKFKE# (282) Type 12:
Wi14.1 wittigi -------------SSDGSDPKAKKQCMWKRCIP-
DQSR---L-EEDE{circumflex over ( )} (283) Ci14.4 cinereus
-------------SSDG---KAKKQCAWKTCVPTQWRRRDLKEKDE{circumflex over ( )}
(284) Ci14.3 cinereus -------------SSDG---KAKRNCFWKACVPEQWRQRDL-
KEKDE{circumflex over ( )} (285) Ci14.2 cinereus
-------------SSDG---KAKRNCFWKACVPEQWRQRDLKEKDE{circumflex over ( )}
(286) Type 13: Nb14.1 nobilis -------------FRPAVKSRSRRAP-
PCVWKVCPAPPWLVTKRKQETSDY{circumflex over ( )} (287) victor Nb14.3
nobilis -------------FRPAVKSRSRRAPPCVWKVCPAPPWLVTKRKQETSDY-
{circumflex over ( )} (288) skinneri Mi14.3 miles
-------------FRPAMQSRSGGMSLCLWKVCPAAPWLVAKRKQETSDY{circumflex over
( )} (289) Miscellaneous: T14.1 tulipa
-------------HFNSVVPTVYICMWKVCPPSP{circumflex over ( )} (290) P14.2
purpurascens -----------------ZSEEEKICLWKICPPPPWRRS{circumflex over
( )} (291) P14.1 purpurascens
-----------------ESNGVEICMWKVCPPSPWRRS{circumflex over ( )} (292)
Vx14.2 vexillum -------IMQSLVFSHQPLPTASICIWKICPPDPWRRHDLQKSNK{cir-
cumflex over ( )} (293) Mu14.1 muriculatus
-------IMQSLVFSHQPLPTASICIWKICPPDPWRRHDLQKSNK{circumflex over ( )}
(294) Pu14.1 pulicarius -----------------VRLRGQICIWKVCPPLLQWIHP-
LVKR{circumflex over ( )} (295) Pu14.2 pulicarius
-----------------VRPRGQICIWKVCPPLLQWIHPLVKR{circumflex over ( )}
(296) Pu14.3 pulicarius ----------------PVRLRGQICIWKVCPPLLQWIHPLVK-
R{circumflex over ( )} (297) Mt14.2 mustelinus
----------LVSHTSSKYPGVTFCPWKVCPPAPWRILGV{circumflex over ( )} (298)
Ba14.1 baileyi --------------HSDSIILRGLCIWKVCEPPPQR{circumflex over
( )} (299) P114.1 planorbis -------------SSSNGLKRADL-
CIHKICPPRYHQSQQ{circumflex over ( )} (300) Lt14.2 litteratus
----------HRVFHLDNTYLKIPICAWKVCPPTPWRRRDLKKRNK{circumflex over ( )}
(301) Lt14.1 litteratus ----SPVSTPYPEFHLDEPYLKIPVCIW-
KICPPNLLRRRDLKKRNKVRQTTATT{circumflex over ( )} (302) Ct14.1
coronatus --------------LSDGRDWTGYICIWKACPRPPWIPPK# (303) Cd14.2
chaldaeus --------------LSEGRNSTVHICMWKVCPPPPWRRPHGQR{circu- mflex
over ( )} (304) Cd14.1 chaldaeus
--------------LSEGRNSTVHICTWKVCPPPPWRRPHGQR{circumflex over ( )}
(305) Eb14.1 ebraeus --------------LSGGTYSRVDTCIWKVCPQSP{circumfle-
x over ( )} (340) *The W or F in the .beta.-turn motif may be in
the D or L configuration. Additionally, the K or F in the backbone
may also be in the D or L configuration.
[0117]
3TABLE 3 Analogs and Truncations of .beta.-Superfamily Conoto- xins
(SEQ ID NO:) Q663 ZCMWKRCIPDQSR{circumflex over ( )} (306) F531
VDICNWRICAPNPLR{circumflex over ( )} (307) .beta.G-C1325
LCFX1KSCRPYPWR{circumflex over ( )} (308) .beta.M1
LFCFX1WKSCWPRPYWR{circumflex over ( )} (309) .beta.M2
LFCFX1KSCWPRPYPWRA{circumflex over ( )} (310) .beta.M3
LX2CFWKSCWPRPYWR{circumflex over ( )} (311) .beta.M4
LX2CFX1KSCWPRPYWR{circumflex over ( )} (312) .beta.M5
LX2CFWKSCWPR{circumflex over ( )} (313) .beta.M6
LFCFX1KSCWPR{circumflex over ( )} (314) .beta.M7
LX2CFX1KSCWPR{circumflex over ( )} (315) .beta.M8
LX2CFWKSCW{circumflex over ( )} (316) .beta.M9
LFCFX1KSCW{circumflex over ( )} (317) .beta.M10
LX2CFX1KSCW{circumflex over ( )} (318) .beta.M11
FCFX1KSCWPR{circumflex over ( )} (319) .beta.M12
FCFWX3SCWPR{circumflex over ( )} (320) .beta.M13
FCFX1FSCWPR{circumflex over ( )} (321) .beta.M14
FCFWKSCWPR{circumflex over ( )} (322) .beta.P2
ESNGVEICMX1KVCPPSPWRRS{circumflex over ( )} (323) .beta.S1
MECYX1KACRPTLSR{circumflex over ( )} (324) .beta.S12
FELKCIX1KFCTIYPSR{circumflex over ( )} (325) .beta.S12b
FELKCIX1KFCTIYPSRPF{circumflex over ( )} (326) .beta.T
TVYICMX1KVCPPSP{circumflex over ( )} (327) .beta.A-CTL03
SDSSDQKAQICIX1KVCPPPPWR{circumflex over ( )} (328) .beta.Cn2
GAQICIX1KVCPPSPWR{circumflex over ( )} (329) .beta.Ms14.5
MFPSLALGPGGDVICRX1KVCPPTPWKRLIK{circumflex over ( )} (330)
.beta.Fd-F531 VDICNX1RICAPNPLRRHDLKKGNN{circumflex over ( )} (331)
.beta.F531-dW VDICNX1RICAPNPLR{circumflex over ( )} (332)
.beta.G14.1 RLFCFX1KSCTWRPYPWRRRDLN#(333) .beta.D919 [1-4]
SLWCVCPFRVCPPCHGR{circumflex over ( )} (334) .beta.D919 [2-4]
SLWCVCPFRVCPPCHGR{circumflex over ( )} (335) .beta.Ge [1-4]
SLWCVCPX2RVCPPCH# (336) .beta.Ge [2-4] SLWCVCPX2RVCPPCH# (337) X1
is D-Trp or L-Trp X2 is D-Phe or L-Phe X2 is D-Lys or L-Lys
Example 2
Activity of Type 2 .beta.-Superfamily Conopeptide on Tumor Cell
Lines
[0118] Test Substance and Concentration: A .beta.-M14.1 derivative,
.beta.-M14.1-D1 (LFCFXKSCWPRPYPWR (SEQ ID NO:309, where X is dW)
was used for in vitro anti-tumor studies. The test compound was
dissolved and diluted with sterile distilled water to obtain
initial working solutions of 10000, 1000, 100, 10, and 1 .mu.M. In
testing, 100-fold dilution was made in culture media to get final
assay concentrations of 100, 10, 1, 0.1, and 0.01 .mu.M.
[0119] Cell Culture Media: The culture medium used for the MCF-7
cell line was Minimum Essential Medium, 90%; Fetal Bovine Serum,
10%. The culture medium used for the MIA PaCa-2 cell line was
Dulbecco's Modified Eagle's Medium, 90%; Fetal Bovine Serum, 10%.
All media were supplemented with 1% Antibiotic-Antimycotic.
[0120] Cell Lines: The cell line MCF-7, which is a breast
adenocarcinoma, pleural effusion, human, was obtained from the
American Type Culture Collection (ATCC HTB-22). The cell line MIA
PaCa-2, which is a pancreatic carcinoma, human, was obtained form
the American Type culture Collection (ATCC CRL-1420). The tumor
cells were incubated in an air atmosphere of 5% CO.sub.2 at
3.degree. C.
[0121] Chemicals: The sources of the chemicals were as follows:
AlamarBlue (Biosource, USA), Antibiotic-Antimycotic (GIBCO BRL,
USA), Dulbecco's Modified Eagle's Medium (GIBCO BRL, USA), Fetal
Bovine Serum (HyClone, USA), Minimum Essential medium (GIBCO BRL,
USA) and Mitomycin (Kyowa, Japan).
[0122] Equipment: Centrifuge 5810R (Eppendorf, Germany), CO.sub.2
Incubator (Forma Scientific Inc., USA), Hemacytometer(Hausser
Scientific Horsham, USA), Inverted Microscope CK-40 (Olympus,
Japan), Spectrafluor Plus (Tecan, Austria), System Microscope E-400
(Nikon, Japan) and Vertical Laminar Flow (Tsao-Hsin, Taiwan).
[0123] Reference Methods: Ahmed et al. (1994), Boyd et al. (1989),
Boyd et al. (1992).
[0124] Aliquots of 100 .mu.l of cell suspension (about
2.5.times.10.sup.3/well) were placed in 96-well microtiter plates
in an air atmosphere of 5% CO.sub.2 at 37.degree. C. After 24
hours, 100 .mu.l of growth medium and 2 .mu.l of test solution, or
mitomycin or vehicle (sterile distilled water), were added
respectively per well in duplicate for an additional 72-hour
incubation. The test compound, .beta.-M14.1 derivative, was
evaluated at concentrations of 100, 10, 1, 0.1 and 0.01 .mu.M. At
the end of incubation, the media in microplate were all removed,
and then 200 .mu.l of fresh media and 20 .mu.l of 90% alamarBlue
reagent were added to each well for another 6-hour incubation
before detection of cell viability by fluorescent intensity.
Fluorescent intensity was measured using a Spectraflour Plus plate
reader with excitation at 530 nm and emission at 590 nm.
[0125] The measured results was calculated by the following
formula:
PG(%)=100.times.(Mean F.sub.test-Mean F.sub.time0)/(Mean
F.sub.ctr1-Mean F.sub.time0)
[0126] If (Mean F.sub.test-Mean F.sub.time0)<0, then
PG(%)=100.times.(Mean F.sub.test-Mean F.sub.time0)/(Mean
F.sub.time0-Mean F.sub.blank)
[0127] Where:
[0128] PG: percent growth
[0129] Mean F.sub.time0=The average of 2 measured fluorescent
intensities of reduced alamarBlue at the time just before exposure
of cells to the test substance.
[0130] Mean F.sub.test=The average of 2 measured fluorescent
intensities of alamarBlue after 72-hour exposure of cells to the
test substance.
[0131] Mean F.sub.ctr1=The average of 2 measured fluorescent
intensities of alamarBlue after 72-hour incubation without the test
substance.
[0132] Mean F.sub.blank=The average of 2 measured fluorescent
intensities of alamarBlue in medium without cells after 72-hour
incubation.
[0133] A decrease of 50% or more (.gtoreq.50%) in fluorescent
intensity relative to vehicle-treated control indicated significant
growth inhibition, cytostatic or cytotoxic activity, and a
semi-quantitative IC.sub.50, TGI and LC.sub.50 were then determined
by nonlinear regression using GraphPad Prism (GraphPad Software,
USA).
[0134] The assays were used to detect changes in cell proliferation
based on the ability of viable cells to cause alamarBlue to change
from its oxidized (non-fluorescent, blue) to a reduced
(fluorescent, red) form. With the results obtained from the
alamarBlue reaction, cell proliferation can be quantified and
metabolic activity of viable cells can be examined. The
.beta.-M14.1-D1 was tested for its effect upon the proliferation of
2 different human tumor cell lines, MCF-7 (breast) and MIA PaCa-2
(pancreas), at five final assay concentrations from 0.01 to 100
.mu.M through serial 10-fold dilutions.
[0135] Based on the results obtained, the .beta.-M14.1-D1 exhibited
significant growth inhibition (>50%) relative to the respective
vehicle treated control group at concentrations between 10 .mu.M to
100 .mu.M in the 2 human tumor cells lines (Table 4). Significant
activity was observed for the concurrently tested standard
reference agent Mitomycin at <10 .mu.M (Table 1). Consequently,
semi-quantitative determinations of estimated IC.sub.50 (50%
inhibition concentration), TGI (total growth inhibition) and
LC.sub.50 (50% lethal concentration) by nonlinear regression
analysis were calculated (Table 5).
4TABLE 4 Effect of Test Substance Tumor Cells Percent Growth (Mean
.+-. SEM, n = 2) Concentration (.mu.M) Treatment Assay Name Blank
Time.sub.0 Vehicle 100 10 1 0.1 0.01 0.001 PT# 1018911-ADD 370000
Breast, -100 0 100 -43 .+-. 11 102 .+-. 14 87 .+-. 9 88 .+-. 12 100
.+-. 8 .sup.a-- .beta.-M14.1-D1 MCF-7 Mitomycin 370000 Breast, -100
0 100 -- -96 .+-. 0 -38 .+-. 16 5 .+-. 12 88 .+-. 8 95 .+-. 7 MCF-7
PT# 1018911-ADD 371700 Pancreas, -100 0 100 -14 .+-. 9 109 .+-. 2
100 .+-. 4 102 .+-. 6 102 .+-. 3 -- .beta.-M14.1-D1 MIA PaCa-2
Mitomycin 371700 Pancreas, -100 0 100 -- -93 .+-. 2 -44 .+-. 16 1
.+-. 6 76 .+-. 5 105 .+-. 10 MIA PaCa-2 .sup.a--: Not tested
[0136]
5TABLE 5 Estimated IC.sub.50, TGI and LC.sub.50 Values Treatment
Prot. # Assay Name .sup.aIC.sub.50 .sup.bTGI .sup.cLC.sub.50 PT#
1018911-ADD 370000 Tumor, Breast, MCF-7 62 .mu.M 81 .mu.M >100
.mu.M .beta.-M14.1-D1 Mitomycin 370000 Tumor, Breast, MCF-7 0.035
.mu.M 0.18 .mu.M 0.93 .mu.M PT# 1018911-ADD 371700 Tumor Pancreas,
79 .mu.M 95 .mu.M >100 .mu.M .beta.-M14.1-D1 MIA PaCa-2
Mitomycin 371700 Tumor Pancreas, 0.028 .mu.M 0.15 .mu.M 0.78 .mu.M
MIA PaCa-2 .sup.aIC.sub.50 (50% Inhibition Concentration): Test
compound concentration where the increase from time0 in the number
or mass of treated cells was only 50% as much as the corresponding
increase in the vehicle-control at the end of experiment. .sup.bTGI
(Total Growth Inhibition): Test compound concentration where the
number or mass of treated cells at the end of experiment was equal
to that at time.sub.0. .sup.cLC.sub.50 (50% Lethal Concentration):
Test compound concentration where the number or mass of treated
cells at the end of experiment was half that at time.sub.0.
Example 3
In Vitro Functional Activity of Type 2 .beta.-Superfamily
Conopeptide
[0137] The in vitro functional activity of .beta.-M14.1-D1 with
respect to somatostatin sst.sub.2 and sst.sub.5, was tested using
the following assays.
6 Somatostatin sst.sub.2 (Feniuk et al., 1993) Tissue: Duncan
Hartley Guinea pig 325 .+-. 25 g Vehicle: 0.1 mL Distilled Water
Incubation Time/Temp: 5 minutes @ 32.degree. C. Incubation Buffer:
Krebs, pH 7.4 Administration Volume: 10 .mu.L Bath Volume: 10 mL
Time of Assessment: 5 minutes Quantitation Method: Isometric (gram
changes) Significance Criteria -Ag: .gtoreq.50% Inhibition of
contraction relative to somatostatin.sub.28-responses Significance
Criteria -Ant: .gtoreq.50% Inhibition of somatostatin.sub.28
relaxant response Somatostatin sst.sub.5 (Feniuk et al., 1993)
Tissue: Duncan Hartley Guinea pig 325 .+-. 25 g Vehicle: 0.1 mL
Distilled Water Incubation Time/Temp: 5 minutes @ 32.degree. C.
Incubation Buffer: Krebs, pH 7.4 Administration Volume: 10 .mu.L
Bath Volume: 10 mL Time of Assessment: 5 minutes Quantitation
Method: Isometric (gram changes) Significance Criteria -Ag:
.gtoreq.50% Inhibition of contraction relative to
somatostatin.sub.28-responses Significance Criteria -Ant:
.gtoreq.50% Inhibition of somatostatin.sub.28 relaxant response
[0138] Biochemical assay results are presented as the percent
inhibition of specific binding or activity. All other results are
expressed in terms of that assay's quantitation method. For primary
assays, only the lowest concentration with a significant response
judged by the assays' criteria, is shown. Primary screening in
duplicate with quantitative data are shown where applicable for
individual assays. Significant responses were noted in the primary
assays shown in Table 6.
7TABLE 6 Primary Tests.sup.a Primary Tissue Assay Tissue, gp Conc.
Criteria AG ANT EC.sub.50/IC.sub.50 Somatostatin sst.sub.2 ileum 1
.mu.M .gtoreq.50% 68% ND 0.49 .mu.M Somatostatin sst.sub.5 vas 1
.mu.M .gtoreq.50% 61% ND 0.59 .mu.M deferens .sup.aA standard error
of the mean is presented where results are based on multiple
independent determinations. Gp = guinea pig; AG = Agonist; ANT =
Antagonist; ND = Assay Test Not Done
Example 4
Radioligand Binding Assay of Type 2 .beta.-Superfamily
Conopeptide
[0139] The radioligand binding activity of .beta.-M14.1-D1 with
respect to somatostatin sst.sub.1, sst.sub.2, sst.sub.3 and
sst.sub.4 and sst.sub.5 was tested using the following assays.
8 Somatostatin sst.sub.1 (Liapakis et al., 1996; Patel and Srikant,
1994) Source: Human recombinant CHO-K1 Ligand: 0.1 nM .sup.125I
Somatostatin-14 Vehicle: 0.4% DMSO Incubation Time/Temp: 2 hours @
25.degree. C. Incubation Buffer: 25 mM Hepes, pH 7.4, 5 mM
MgCl.sub.2, 1 mM CaCl.sub.2, 0.5% BSA NonSpecific Ligand: 1 .mu.M
Somatostatin-14 K.sub.d: 1.9 nM (historical value) B.sub.max: 0.5
pmol/mg Protein (historical value) Specific Binding: 60%
(historical value) Quantitation Method: Radioligand Binding
Significance Criteria: .gtoreq.50% of max stimulation or inhibition
Somatostatin sst.sub.2 (Patel and Srikant, 1994) Source: Human
recombinant CHO-K1 Ligand: 0.03 nM .sup.125I Somatostatin-14
Vehicle: 1% H.sub.2O Incubation Time/Temp: 4 hours @ 25.degree. C.
Incubation Buffer: 25 mM Hepes, pH 7.4, 5 mM MgCl.sub.2, 1 mM
CaCl.sub.2, 0.5% BSA NonSpecific Ligand: 1 .mu.M Somatostatin-14
K.sub.d: 0.034 nM (historical value) B.sub.max: 11 pmol/mg Protein
(historical value) Specific Binding: 90% (historical value)
Quantitation Method: Radioligand Binding Significance Criteria:
.gtoreq.50% of max stimulation or inhibition Somatostatin sst.sub.3
(Liapakis et al., 1996; Patel and Srikant, 1994) Source: Human
recombinant CHO-K1 Ligand: 0.1 nM .sup.125I Somatostatin-14
Vehicle: 0.4% DMSO Incubation Time/Temp: 2 hours @ 25.degree. C.
Incubation Buffer: 25 mM Hepes, pH 7.4, 5 mM MgCl.sub.2, 1 mM
CaCl.sub.2, 0.5% BSA NonSpecific Ligand: 1 .mu.M Somatostatin-14
K.sub.d: 0.79 nM (historical value) B.sub.max: 1.1 pmol/mg Protein
(historical value) Specific Binding: 78% (historical value)
Quantitation Method: Radioligand Binding Significance Criteria:
.gtoreq.50% of max stimulation or inhibition Somatostatin sst.sub.4
(Patel and Srikant, 1994) Source: Human recombinant CHO-K1 Ligand:
0.12 nM .sup.125I Somatostatin-14 Vehicle: 0.4% DMSO Incubation
Time/Temp: 2 hours @ 25.degree. C. Incubation Buffer: 25 mM Hepes,
pH 7.4, 5 mM MgCl.sub.2, 1 mM CaCl.sub.2, 0.5% BSA NonSpecific
Ligand: 1 .mu.M Somatostatin-14 K.sub.d: 0.87 nM (historical value)
B.sub.max: 0.5 pmol/mg Protein (historical value) Specific Binding:
60% (historical value) Quantitation Method: Radioligand Binding
Significance Criteria: .gtoreq.50% of max stimulation or inhibition
Somatostatin sst.sub.5 (Greenwood et al., 1997; Patel and Srikant,
1994) Source: Human recombinant HEK-293 EBNA cells Ligand: 0.1 nM
.sup.125I Somatostatin-14 Vehicle: 1% H.sub.2O Incubation
Time/Temp: 60 minutes @ 37.degree. C. Incubation Buffer: 50 mM
Hepes, pH 7.4, 5 mM MgCl.sub.2, 1 mM CaCl.sub.2, 0.5% BSA
NonSpecific Ligand: 1 .mu.M Somatostatin-14 K.sub.d: 0.5 nM
(historical value) B.sub.max: 1.2 pmol/mg Protein (historical
value) Specific Binding: 94% (historical value) Quantitation
Method: Radioligand Binding Significance Criteria: .gtoreq.50% of
max stimulation or inhibition
[0140] Biochemical assay results are presented as the percent
inhibition of specific binding or activity. All other results are
expressed in terms of that assay's quantitation method. For primary
assays, only the lowest concentration with a significant response
judged by the assays' criteria, is shown. Primary screening in
duplicate with quantitative data (e.g., IC.sub.50.+-.SEM,
K.sub.i.+-.SEM and nH) are shown where applicable for individual
assays. In screening packages, primary screening in duplicate with
semi-quantitative data (e.g., estimated IC.sub.50, K.sub.i and nH)
are shown where applicable (concentration range of 4 log units).
Significant responses were noted in the primary assays shown in
Table 7.
9TABLE 7 Primary Test Primary Biochemical Assay Species Conc. %
Inh. IC.sub.50 K.sub.i n.sub.h Somatostatin sst.sub.1 hum 0.1 .mu.M
61 0.053 .mu.M 0.05 .mu.M 0.736 Somatostatin sst.sub.2 hum 0.1
.mu.M 90 0.018 .mu.M 9.35 nM 1.24 Somatostatin sst.sub.3 hum 10 nM
61 6.14 nM 5.45 nM 0.714 Somatostatin sst.sub.4 hum 10 .mu.M 67
5.63 .mu.M 4.95 .mu.M 1.26 Somatostatin sst.sub.5 hum 0.1 .mu.M 61
0.082 .mu.M 0.068 .mu.M 0.961 hum = human
Example 5
Radioligand Binding Assay of .beta.-Superfamily Conopeptides
[0141] The radioligand binding activity of truncations of
.beta.-M14.1-D1 and other .beta.-superfamily conopeptides with
respect to somatostatin sst.sub.1, sst.sub.2, sst.sub.3 and
sst.sub.4 and sst.sub.5 was tested as described in Example 4. The
peptides which were tested are set forth in Table 8. The
significant responsers (.gtoreq.50% inhibition or stimulation) are
set forth in Table 9.
10 TABLE 8 Conopeptide Type Sequence (SEQ ID NO:).sup.a
.beta.-M14.1-6 2 LFCFX.sub.1KSCWPR (314) .beta.-M14.1-9 2
LFCFX.sub.1KSCW (317) .beta.-M14.1-10 2 LX.sub.2CFX.sub.1KSCW (318)
.beta.-T14.1-D1 Misc TVYICMX.sub.1KVCPPSP (327) .beta.-Sl14.1-D1 6
MECYX.sub.1KACRPTLSR (324) .beta.-Cn14.2-D1 5
GAQICIX.sub.1KVCPPSPWR (329) .sup.aX.sub.1 is dW and X.sub.2 is
dF
[0142]
11TABLE 9 Primary Test Primary Biochemical Assay Species Peptide
Conc. % Inh. Somatostatin sst.sub.1 hum .beta.-Cn14.2-D1 10 .mu.M
61 Somatostatin sst.sub.3 hum .beta.-M14.1-6 0.1 .mu.M 63
Somatostatin sst.sub.3 hum .beta.-M14.1-9 0.1 .mu.M 72 Somatostatin
sst.sub.3 hum .beta.-Sl14.1-D1 10 .mu.M 82 Somatostatin sst.sub.3
hum .beta.-Cn14.2-D1 10 .mu.M 84 Somatostatin sst.sub.4 hum
.beta.-Cn14.2-D1 10 .mu.M 60 Somatostatin sst.sub.5 hum
.beta.-M14.1-6 0.1 .mu.M 57 Somatostatin sst.sub.5 hum
.beta.-M14.1-9 0.1 .mu.M 55 Somatostatin sst.sub.5 hum
.beta.-M14.1-10 0.1 .mu.M 65 Somatostatin sst.sub.5 hum
.beta.-T14.1-D1 10 .mu.M 65 Somatostatin sst.sub.5 hum
.beta.-Sl14.1-D1 10 .mu.M 86 Somatostatin sst.sub.5 hum
.beta.-Cn14.2-D1 10 .mu.M 56 hum = human
Example 6
Radioligand Binding Assay of Type 3 .beta.-Superfamily
Conopeptide
[0143] The radioligand binding activity of .beta.-Ge14.1 D1
(SLWCVCPFRVCPPCH#; SEQ ID NO:335 with 1-3 fold), D919 (with 1-4
fold, SEQ ID NO:334) and D919 (with 2-4 fold, SEQ ID NO:335) with
respect to melanocortin MC.sub.3, MC.sub.4, MC.sub.5 and MCH (h)
was tested using the following assays.
12 Melanocortin MC.sub.3 (Schioth et al., 1995) Source: Human
recombinant HEK-293 cells Ligand: 0.035 nM .sup.125I NDP-.alpha.MSH
Vehicle: 0.4% DMSO Incubation Time/Temp: 60 min @ 37.degree. C.
Incubation Buffer: 25 mM HEPES-KOH, 0.2% BSA, pH 7.0,100 mM NaCl, 1
mM 1,10-phenanthroline, 1.5 mM CaCl2, 1 mM MgSO4, and one complete
protease inhibitor tablet/100 ml NonSpecific Ligand: 3 .mu.M
NDP-.alpha.MSH K.sub.d: 0.53 nM (historical value) B.sub.max: 6
pmol/mg Protein (historical value) Specific Binding: 85%
(historical value) Quantitation Method: Radioligand Binding
Significance Criteria: .gtoreq.50% of max stimulation or inhibition
Melanocortin MC.sub.4 (Schioth et al., 1995) Source: Human
recombinant HEK-293 cells Ligand: 0.02 nM .sup.125I NDP-.alpha.MSH
Vehicle: 01% H.sub.2O Incubation Time/Temp: 2 hours @ 37.degree. C.
Incubation Buffer: 25 mM HEPES-KOH, 0.2% BSA, pH 7.0,100 mM NaCl, 1
mM 1,10-phenanthroline, 1.5 mM CaCl2, 1 mM MgSO4, and one complete
protease inhibitor tablet/100 ml NonSpecific Ligand: 3 .mu.M
NDP-.alpha.MSH K.sub.d: 0.5 nM (historical value) B.sub.max: 3.9
pmol/mg Protein (historical value) Specific Binding: 90%
(historical value) Quantitation Method: Radioligand Binding
Significance Criteria: .gtoreq.50% of max stimulation or inhibition
Melanocortin MC.sub.5 (Schioth et al., 1995) Source: Human
recombinant HEK-293 cells Ligand: 0.035 nM .sup.125I NDP-.alpha.MSH
Vehicle: 1% H.sub.2O Incubation Time/Temp: 2 hours @ 37.degree. C.
Incubation Buffer: 25 mM HEPES-KOH, 0.2% BSA, pH 7.0,100 mM NaCl, 1
mM 1,10-phenanthroline, 1.5 mM CaCl2, 1 mM MgSO4, and one complete
protease inhibitor tablet/100 ml NonSpecific Ligand: 3 .mu.M
NDP-.alpha.MSH K.sub.d: 0.53 nM (historical value) B.sub.max: 6
pmol/mg Protein (historical value) Specific Binding: 85%
(historical value) Quantitation Method: Radioligand Binding
Significance Criteria: .gtoreq.50% of max stimulation or inhibition
MCH (h) (Chambers et al., 1999) Source: Human recombinant CHO cells
Ligand: 0.1 nM .sup.125I [Phe.sup.13, Tyr.sup.19]-MCH Incubation
Time/Temp: 60 min @ 22.degree. C. NonSpecific Ligand: 1 .mu.M
NDP-.alpha.MSH K.sub.d: 0.05 nM (historical value) Quantitation
Method: Radioligand Binding Significance Criteria: .gtoreq.50% of
max stimulation or inhibition Melanocortin MC.sub.1 (Siegrist et
al., 1988) Source: Human recombinant CHO cells Ligand: 0.05 nM
.sup.125I NDP-.alpha.MSH Incubation Time/Temp: 90 min @ 22.degree.
C. NonSpecific Ligand: 0.1 .mu.M MCH K.sub.d: 0.62 nM (historical
value) Quantitation Method: Radioligand Binding Significance
Criteria: .gtoreq.50% of max stimulation or inhibition
[0144] Biochemical assay results are presented as the percent
inhibition of specific binding or activity. All other results are
expressed in terms of that assay's quantitation method. For primary
assays, only the lowest concentration with a significant response
judged by the assays' criteria, is shown. Primary screening in
duplicate with quantitative data (e.g., IC.sub.50.+-.SEM,
K.sub.i.+-.SEM and nH) are shown where applicable for individual
assays. In screening packages, primary screening in duplicate with
semi-quantitative data (e.g., estimated IC.sub.50, K.sub.i and nH)
are shown where applicable (concentration range of 4 log units).
Significant responses were noted in the primary assays shown in
Tables 10 and 11.
13TABLE 10 Primary Test for .beta.-Ge14.1-D1 Primary Biochemical
Assay Species Conc. % Inh. IC.sub.50 K.sub.i n.sub.h Melanocortin
hum 1 .mu.M 71 0.294 .mu.M 0.276 .mu.M 0.762 MC.sub.5 hum =
human
[0145]
14TABLE 11 Primary Test Primary Biochemical Assay Species Peptide
Conc. % Inh. Melanocortin MC.sub.3 hum D919 [2,4] 10 .mu.M 67
Melanocortin MC.sub.4 hum D919 [2,4] 10 .mu.M 67 Melanocortin
MC.sub.4 hum D919 [1,4] 10 .mu.M 58 Melanocortin MC.sub.5 hum D919
[2,4] 10 .mu.M 96 Melanocortin MC.sub.5 hum D919 [1,4] 10 .mu.M 89
Melanocortin MC.sub.1 hum D919 [2,4] 10 .mu.M 60 Melanocortin
MC.sub.1 hum D919 [1,4] 10 .mu.M 66 MCH(h) hum D919 [2,4] 10 .mu.M
83 MCH(h) hum D919 [1,4] 10 .mu.M 65 hum = human
[0146] 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.
BIBLIOGRAPHY
[0147] Abiko, H. et al. (1986). Brain Res. 38:328-335.
[0148] Ahmed, S. A. et al. (1994). J Immunol Methods
170:211-224.
[0149] Aldrete, J. A. et al. (1979). Crit. Care Med. 7:466-470.
[0150] Barnay, G. et al. (2000). J. Med. Chem.
[0151] Beeley, N. R. A. (2000). Drug Disc Today 5:454.
[0152] Bitan, G. et al. (1997). J. Peptide Res. 49:421-426.
[0153] Bodansky et al. (1966). Chem. Ind. 38:1597-98.
[0154] Boyd, M. R. et al. (1989). Principles and Practices of
Oncology Updates 3:1-12
[0155] Boyd, M. R. et al. (1992). Data display and analysis
strategies for the NCI disease-oriented in vitro antitumor drug
screen. In Cytotoxic anti-cancer drugs: models and concepts for
drug discovery and development, Kluwer Academic, Boston, pp.
11-34.
[0156] Bulbring, W. and Wajda, J. (1945). J. Pharmacol. Exp. Ther.
85:78-84.
[0157] Cartier, G. E. et al. (1996). J. Biol. Chem.
271:7522-7528.
[0158] Chambers, J. et al. (1999). Nature 400:261-265.
[0159] Chandler, P. et al. (1993). J. Biol. Chem.
268:17173-17178.
[0160] Chaplan S. R. (1994). J Neuroscience Methods 53:55-63.
[0161] Chaplan S. R. (1997). J Pharmacol. Exp. Ther.
280:829-838.
[0162] Civelli, 0. et. al. (2001). Trends Neurosci. 24:230-7.
[0163] Clark, C. et al. (1981). Toxicon 19:691-699.
[0164] Codere, T. J. (1993). Eur. J. Neurosci. 5:390-393.
[0165] Craig, A. G. et al. (1997). J. Biol. Chem 272:4689-4698.
[0166] Cruz, L. J. at al. (1976). Verliger 18:302-308.
[0167] Cruz, L. J. et al. (1987). J. Biol. Chem.
262:15821-15824.
[0168] Dorr et al. (1994). Cancer Chemotherapy Handbook, 2d Ed.,
pp. 15-34, Appleton & Lange, Connecticut.
[0169] Ettinger, L. J. et al. (1978). Cancer 41:1270-1273.
[0170] Fainzilber, M. et al. (1998). Biochemistry 37:1470-1477.
[0171] Feniuk, W. et al. (1993). Br. J. Pharmacol
110:1156-1164.
[0172] Golebiowski, Q. et al. (2001). Curr Opin Drug Disc Dev
4:428-434.
[0173] Greenwood, M. T. et al. (1997). Pharmacol Exp Ther
52:807-814.
[0174] Goodman and Gilman's The Pharmacological Basis of
Therapeutics, Seventh Ed., Gilman, A. G. et al., eds., Macmillan
Publishing Co., New York (1985).
[0175] Hammerland et al. (1992). Eur. J. Pharmacol.
226:239-244.
[0176] Heading, C. (1999). Curr. Opin. CPNS Invest. Drugs
1:153-166.
[0177] Hopkins, C. et al. (1995). J. Biol. Chem.
270:22361-22367.
[0178] Horiki, K. et al. (1978). Chemistry Letters 165-68.
[0179] Horwell, D. C. (1996). Bioorg Med Chem. 4:1573-1576.
[0180] Horwell, D. C. (2000). Drug Discovery Today, [insert volume
and page numbers]
[0181] Hubry, V. et al. (1994). Reactive Polymers 22:231-241.
[0182] Hylden, J. L. K. and Wilcox, G. (1980). Eur. J. Pharmacol.
67:313-316.
[0183] Jacobsen, R. et al. (1997). J. Biol. Chem.
272:22531-22537.
[0184] Jimenez, E. C. et al. (1996). J. Biol. Chem.
271:28002-28005.
[0185] Kaiser et al. (1970). Anal. Biochem. 34:595.
[0186] Kapoor (1970). J. Pharm. Sci. 59:1-27.
[0187] Kornreich, W. D. et al. (1986). U.S. Pat. No. 4,569,967.
[0188] Kruszynski, M. et al. (1990). Experientia 46:771-773.
[0189] Liapakis, G. et al. (1996). J Biol Chem 271:20331-20339.
[0190] Luer, M. S. & Hatton, J. (1993). Annals Pharmcotherapy
27:912-921.
[0191] Liu, H. et al. (1997). Nature 386:721-724.
[0192] Malmberg, A. B. and Basbaum, A. I. (1998). Pain
76:215-222.
[0193] Maric, M. et al. (1989). Physiol. Pharmacol.
67:1437-1441.
[0194] Martinez, J. S. et al. (1995). Biochem. 34:14519-14526.
[0195] Mayer, E. A. et al. (1994). Gastroenterology
107:271-293.
[0196] McIntosh, J. M. et al. (1998). Methods Enzymol.
294:605-624.
[0197] The Merck Manual of Diagnosis and Therapy, 16 Ed., Berkow,
R. et al., eds., Merck Research Laboratories, Rahway, N.J., pp.
1436-1445 (1992).
[0198] Methoden der Organischen Chemie (Houben-Weyl): Synthese von
Peptiden, E. Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger.
(1974).
[0199] Murphy, A. J. et al. (1998). Curr. Opin. Drug Disc. And
Devel. 1:192-199
[0200] Nehlig, A. et al. (1990). Effects of phenobarbital in the
developing rat brain. In Neonatal Seizures, Wasterlain, C. G. and
Vertt, P. (eds.), Raven Press, New York, pp. 285-194.
[0201] Nishiuchi, Y. et al. (1993). Int. J. Pept. Protein Res.
42:533-538.
[0202] Okarvi, S. M. (2001). Eur. J. Nucl. Med 28:929-938.
[0203] Olivera, B. M. et al. (1984). U.S. Pat. No. 4,447,356.
[0204] Olivera, B. M. et al. (1985). Science 230:1338-1343.
[0205] Olivera, B. M. et al. (1990). Science 249:257-263.
[0206] Olivera, B. M. et al. (1996). U.S. Pat. No. 5,514,774.
[0207] Ornstein, et al. (1993). Biorganic Medicinal Chemistry
Letters 3:43-48.
[0208] Patel, Y. C. and Srikant, C. B. (1994). Endocrinol
135:2814-1817.
[0209] Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa.).
[0210] Rivier, J. R. et al. (1978). Biopolymers 17:1927-38.
[0211] Rivier, J. R. et al. (1987). Biochem. 26:8508-8512.
[0212] Sambrook, J. et at. (1989). Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring
[0213] Harbor Laboratory, Cold Spring Harbor, N.Y.
[0214] Schioth, H. B. et al. (1995). Eur J Pharmacol
288:311-317.
[0215] Shaaban, S. (2001). Cur. Opin Drug Disc Dev 4:535-547
[0216] Shon, K. J. et al. (1994). Biochemistry 33:11420-11425.
[0217] Shon, K.-J. et al. (1997). Biochemistry 36:9581-9587.
[0218] Siegrist, W. et al. (1988). J Recep Res 8:323-343.
[0219] Slooter, G. D. et al. (2001). Br. J. Surg. 88:31-40.
[0220] Stewart and Young, Solid-Phase Peptide Synthesis, Freeman
& Co., San Francisco, Calif. (1969).
[0221] Troupin, A. S. et al. (1986). MK-801. In New Anticonvulsant
Drugs, Current Problems in Epilepsy 4, Meldrum, B. S. and Porter,
R. J. (eds.), John Libbey, London, pp. 191-202.
[0222] Vale et al. (1978). U.S. Pat. No. 4,105,603.
[0223] Van de Steen, P. et al. (1998). Critical Rev. in Biochem.
and Mol. Biol. 33:151-208.
[0224] Virgolini, I. Q. (2001). J Nucl Med 45:153-159.
[0225] White, H. S., et al. (1992). Epilepsy Res. 12:217-226.
[0226] White, H. S., et al. (1995). Experimental Selection,
Quantification, and Evaluation of Antiepileptic Drugs. In
Antiepileptic Drugs, 4th Ed., Levy, R. H., eds., Raven Press, N.Y.,
pp. 99-110.
[0227] Wong, E. H. P. et al. (1986). Proc. Natl. Acad. Sci. USA
83:7104-7108.
[0228] Zhou L. M., et al. (1996). J. Neurochem. 66:620-628.
[0229] Zimm, S. et al. (1984). Cancer Res. 44:1698-1701.
[0230] U.S. Pat. No. 3,842,067.
[0231] U.S. Pat. No. 3,862,925.
[0232] U.S. Pat. No. 3,972,859.
[0233] U.S. Pat. No. 5,514,774.
[0234] U.S. Pat. No. 5,550,050.
[0235] U.S. Pat. No. 5,670,622.
[0236] U.S. Pat. No. 5,719,264.
[0237] U.S. Pat. No. 5,844,077.
[0238] U.S. Pat. No. 5,889,147.
[0239] U.S. Pat. No. 5,969,096.
[0240] U.S. Pat. No. 6,077,934.
[0241] Published PCT Application WO 92/19195.
[0242] Published PCT Application WO 94/25503.
[0243] Published PCT Application WO 95/01203.
[0244] Published PCT Application WO 95/05452.
[0245] Published PCT Application WO 96/02286.
[0246] Published PCT Application WO 96/02646.
[0247] Published PCT Application WO 96/40871.
[0248] Published PCT Application WO 96/40959.
[0249] Published PCT Application WO 97/12635.
[0250] Published PCT Application WO 98/03189.
[0251] Published PCT Application WO 00/23092.
Sequence CWU 0
0
* * * * *
References