University of Utah Research Foundation

Yissum Research Development Company of the Hebrew University of Jerusalem Ltd.

The Salk Institute for Biological Studies

Univ. of Utah Research Foundation

Ki-Joon Shon

Baldomero M Olivera

J Michael McIntosh

David R Hillyard

Doju Yoshikami

Jean E Rivier

Lourdes J Cruz

Maren Watkins

William R Gray

John Dykert

Micha E Spira

Michelle Grilley

Heinrich Terlau

Maren Marsh

Michelle M Grilley

Arik Hasson

The present invention is directed to conotoxin peptides having 25-35 amino acids, six cysteines which form three disulfide bonds between the first and fourth, second and fifth, and third and sixth cysteines, respectively. The invention is directed to .delta.-conotoxin GmVIA having the formula Val-Lys-Pro-Cys-Arg-Lys-Glu-Gly-Gln-Leu-Cys-Asp-Pro-Ile-Phe-Gln-Asn-Cys-Cy s-Arg-Gly-Trp-Asn-Cys-Val-Leu-Phe-Cys-Val (SEQ ID NO:1). This peptide activates sodium channels. The invention is further directed to .mu.O-conotoxin peptides of the generic formula Ala-Cys-Xaa.sub.1 -Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Xaa.sub.2 -Gly-Phe-Xaa.sub.3 -Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val, wherein Xaa.sub.1 is Arg or Ser, Xaa.sub.2 is Ile or Leu and Xaa.sub.3 is Ile or Val (SEQ ID NO:2). These peptides block sodium channels. Examples of .mu.O-conotoxin peptides of the present invention are MrVIA, having the formula Ala-Cys-Arg-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Ile-Gly-Phe-Ile-Tyr-Cy s-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val (SEQ ID NO:3), and MrVIB, having the formula Ala-Cys-Ser-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Leu-Gly-Phe-Val-Tyr-Cy s-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val (SEQ ID NO:4).

Conotoxin peptides

Substantially pure conotoxin peptides are provided which inhibit synaptic transmissions at the neuromuscular junctions and which are useful both in vivo and in assays because they specifically target particular skeletal nAChRs to the exclusion of neuronal nAChRs. The peptides are of such length that they can be made by chemical synthesis, and the preferred peptides have formula: H-His-4Hyp-4Hyp-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-4Hyp-Gly-Cys-Ser-S er-Ala-Ser-Cys-Cys-Gln-Xaa.sub.24 -NH.sub.2 wherein Xaa.sub.24 is Arg or Gly.

The present invention is directed to .delta.-conotoxin PVIA having the formula Glu-Ala-Cys-Tyr-Ala-Xaa.sub.1 -Gly-Thr-Phe-Cys-Gly-Ile-Lys-Xaa.sub.2 -Gly-Leu-Cys-Cys-Ser-Glu-Phe-Cys-Leu-Pro-Gly-Val-Cys-Pro-Gly (Xaa.sub.1 or Xaa.sub.2 is Pro or 4-trans-hydroxyproline) (SEQ ID NO:7). The C-terminus may be free or amidated. This latter conotoxin is vertebrate-specific which targets voltage-sensitive Na channels.

The present invention is directed to conotoxin peptides having 25-35 amino acids, six cysteines which form three disulfide bonds between the first and fourth, second and fifth, and third and sixth cysteines, respectively. The invention is directed to .delta.-conotoxin GmVIA having the formula Val-Lys-Pro-Cys-Arg-Lys-Glu-Gly-Gln-Leu-Cys-Asp-Pro-Ile-Phe-Gln-Asn-Cys-Cy s-Arg-Gly-Trp-Asn-Cys-Val-Leu-Phe-Cys-Val (SEQ ID NO:1). This peptide activates sodium channels. The invention is further directed to .mu.O-conotoxin peptides of the generic formula Ala-Cys-Xaa.sub.1 -Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Xaa.sub.2 -Gly-Phe-Xaa.sub.3 -Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val, wherein Xaa.sub.1 is Arg or Ser, Xaa.sub.2 is Ile or Leu and Xaa.sub.3 is Ile or Val (SEQ ID NO:2). These peptides block sodium charmels. Examples of .mu.O-conotoxin peptides of the present invention are MrVIA, having the formula Ala-Cys-Arg-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Ile-Gly-Phe-Ile-Tyr-Cy s-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val (SEQ ID NO:3), and MrVIB, having the formula Ala-Cys-Ser-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Leu-Gly-Phe-Val-Tyr-Cy s-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val (SEQ ID NO:4).

A new peptide, .kappa.-conotoxin PVIIA, is disclosed. This peptide is found naturally in the cone snail Conus purpurascens and has the amino acid sequence Cys-Arg-Ile-Xaa-Asn-Gln-Lys-Cys-Phe-Gln-His-Leu-Asp-Asp-Cys-Cys-Ser-Arg-Ly s-Cys-Asn-Arg-Phe-Asn-Lys-Cys-Val (SEQ ID NO:1) where Xaa represents 4-trans-hydroxyproline hydroxyproline or proline. This peptide together with a previously disclosed peptide, .delta.-conotoxin PVIA, act synergistically to rapidly immobilize fish which are injected with the two peptides. Injection of .kappa.-conotoxin PVIIA alone results in different symptoms with an injected fish becoming hyperactive and then contracting and suddenly extending all major fins. This 'fin-popping' occurs repeatedly resulting in a series of jerky movements, but injection of only .kappa.-conotoxin PVIIA does not immobilize or kill the fish.

Conotoxin peptide PVIIA

The invention is directed to a new .mu.-conotoxin named GIIIA. .mu.-Conotoxin PIIIA consists of 22 amino residues and is found in the Eastern Pacific fish-hunting species Conus purpurascens. This conotoxin is a new Na.sup.+ channel blocker and can be used to resolve tetrodotoxin-sensitive sodium channels into three categories: 1) sensitive to .mu.-PIIIA and .mu.-conotoxin GIIIA; 2) sensitive to .mu.-PIIIA but not to .mu.-GIIIA; and 3) sensitive to neither of these two .mu.-conotoxins. In rat brain, binding competition studies between the two .mu.-conotoxins and saxitoxin suggest at least three pharmacologically distinguishable binding sites. Thus, .mu.-conotoxin PIIIA should be a key tool for distinguishing among different sodium channel subtypes.

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Salt Lake City, UT, United States of America

Ki-Joon Shon