University of Utah Research Foundation

The Salk Institute for Biological Studies

Cognetix, Inc.

Doju Yoshikami

Baldomero M Olivera

J Michael McIntosh

G Edward Cartier

Siqin Luo

Jennifer M Kulak

David R Hillyard

Ki-Joon Shon

Jean E Rivier

Lourdes J Cruz

Maren Watkins

William R Gray

John Dykert

Maren Marsh

The present invention relates to the use of &#x3b1;-conotoxin peptides having the general formula Xaa-Xaa-Cys-Cys-Xaa-Xaa-Pro-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys (SEQ ID NO:1) for treating disorders regulated at neuronal nicotinic acetylcholine receptors. Such disorders include, but are not limited to, cardiovascular disorders, gastric motility disorders, urinary incontinence, nicotine addiction, mood disorders (such as bipolar disorder, unipolar depression, dysthymia and seasonal effective disorder) and small cell lung carcinoma, as well as the localization of small cell lung carcinoma. In this formula, Xaais des-Xaa, Tyr, mono-iodo-Tyr or di-iodo-Tyr, Xaais any amino acid, Xaais any amino acid, Xaais any amino acid, Xaais any amino acid; Xaais any amino acid, Xaais any amino acid, Xaais any amino acid, Xaais des-Xaaor any amino acid, Xaais des-Xaaor any amino acid, Xaais des-Xaaor any amino acid and Xaais des-Xaaor any amino acid. Disulfide linkages exist between the first and third cysteines and the second and fourth cysteines. Pro may be replaced with hydroxy-Pro. The C-terminus may contain a hydroxyl or an amide group, preferably an amide group.

Uses of &#x3b1;-conotoxin peptides

Uses of &agr;-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.

Conotoxin peptides

Neuronal nicotinic acetylcholine receptors (nAChRs) are believed to mediate nicotine addiction. In addition, stimulation of nAChRs modulates release of neurotransmitters including dopamine, norepinephrine and serotonin. Thus, pharmacological manipulation of nicotinic receptors has implications for a wide variety of disorders including psychotic, mood, movement and cognitive. For most nAChRs, there are no subtype selective ligands. However, .alpha.-conotoxin MII, a small peptide from the carnivorous marine snail Conus magus, was recently isolated. This peptide has been shown to be a specific antagonist for .alpha.3.beta.2 nicotinic receptors. The peptide potently blocks part, but not all, of nicotine-stimulated dopamine release from rat brain striatal synaptosomes. In contrast it has no effect on potassium stimulated dopamine release. Other .alpha.-conotoxins specifically target distinct neuronal nAChR subtypes. .alpha.-Conotoxins thus represent new lead compounds for CNS disorders.

Use of .alpha.-conotoxin MII to treat disorders resulting from

This invention relates to relatively short peptides about 14-17 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogs to the naturally available peptides, and which include two cyclizing disulfide linkages. More specifically, the present invention is directed to conopeptides having the general formula: Gly-Cys-Cys-Ser-Tyr-Xaa.sub.1 -Xaa.sub.1 -Cys-Phe-Ala-Thr-Asn-Xaa.sub.2 -Xaa.sub.3 -Xaa.sub.4 -Cys (SEQ ID NO: 1), wherein Xaa.sub.1 is Pro or Hyp (trans-4-hydroxy-Pro), Xaa.sub.2 is Ser, Pro or Hyp, Xaa.sub.3 is Gly or Asp and Xaa.sub.4 is a Tyr or des- Xaa.sub.4. The disulfide bridges are between the first and third between the second fourth cysteine residues. The C-terminal end is preferably amidated. The invention further relates to the specific peptides AuIA: Gly-Cys-Cys-Ser-Tyr-Pro-Pro-Cys-Phe-Ala-Thr-Asn-Ser-Asp-Tyr-Cys (SEQ ID NO:2); AuIB: Gly-Cys-Cys-Ser-Tyr-Pro-Pro-Cys-Phe-Ala-Thr-Asn-Pro-Asp-Cys (SEQ ID NO:3); and AuIC: Gly-Cys-Cys-Ser-Tyr-Pro-Pro-Cys-Phe-Ala-Thr-Asn-Ser-Gly-Tyr-Cys (SEQ ID NO:4). The invention also includes pharmaceutically acceptable salts of the conopeptides.

Conopeptides AuIA, AuIB and AuIC

Use of .alpha.-conotoxin MII to treat disorders resulting from nicotine

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.

Conotoxin peptide PIIIA

Apparatus for studying the interaction of first and second molecules in a test solution containing fluorescently labeled molecules in addition to the first and second molecules, with the first molecules and the fluorescently labeled molecules being capable of binding with the second molecules. The apparatus comprises a flow channel having opposite, spaced apart walls, with at least one of the walls or a portion thereof being translucent or transparent. A porous matrix is retained in a fixed position between the walls of the flow channel and in direct contact with a translucent or transparent portion of the walls of the flow channel. A test solution flows through the flow channel and around the porous matrix so as to be in contact with the porous matrix. The porous matrix comprises a material that is translucent or transparent and has pores or openings therein which are of a microscopic size such that the unbound fluorescently labeled molecules can permeate the porous matrix, while the second molecules and fluorescently labeled molecules bound to the second molecules cannot permeate the porous matrix. The porous matrix is substantially inert and does not react chemically with any of the molecules contained in the solution. Fluorescence emitted by the fluorescently labeled molecules permeating the porous matrix is detected through the translucent or transparent portion of the walls of the flow channel.

Apparatus for using fluorescently labeled ligands in studying

A method disclosed for studying the interaction in solution of two molecules of the type such as a ligand and a receptor that are capable of reacting or binding with each other. The method comprises preparing an aliquot of a solution containing the first of the molecules. The second of the molecules is then added to the aliquot. A fluorescently labeled molecule is added to the aliquot, wherein the fluorescently labeled molecule is also capable of reacting or binding with the second of the molecules. A porous matrix that is optically transparent is immersed into the aliquot containing the two molecules being studied and the fluorescently labeled molecule, wherein the second molecule and any fluorescently labeled molecule bound thereto is sterically hindered from permeating the porous, optically transparent matrix, while any unbound fluorescently labeled molecule permeates the matrix. The matrix that has been immersed into the aliquot is then viewed with means such as a fluorescent microscope for determining the amount of fluorescence emitted from the matrix.

Growing community of inventors

Salt Lake City, UT, United States of America

Doju Yoshikami