Analogs of shk toxin and their uses in selective inhibition of kv1. 3 potassium channels

Abstract

Analogs of ShK toxin and methods for using such ShK analogs. The ShK analogs generally comprise ShK toxin attached to a chemical entity (e.g. an atom, molecule, group, residue, compound, moiety, etc.) that has an anionic charge. In some embodiments, the chemical entity attached to the ShK toxin may comprise an amino acid residue. The ShK analogs may be administered to human or non-human animal subjects to cause inhibition of potassium channels or to otherwise treat diseases or disorders. In some embodiments, the chemical entity to which the ShK toxin is attached may be chosen to provide selective inhibition of certain potassium channels (e.g., Kv1.3 channels) over other potassium channels (e.g., Kv1.1 channels). In come embodiments, the chemical entity to which the ShK toxin is attached may include a fluorophore, thereby providing a fluorophore tagged ShK analog. Such fluorophore tagged ShK analogs may be used in flow cytometry alone, or in conjunction with class II tetramers that can detect autoreactive cells.

1 . A composition of matter comprising ShK attached to an organic or inorganic chemical entity that has an anionic charge. 
     
     
         2 . A composition according to  claim 1  wherein the chemical entity is selected from the group consisting of: amino acids; polypeptides; amino acid residues; unnatural amino acid residues; threonine; threonine derivatives; phospho-threonine; serine; serine derivatives; phospho-serine; glutamic acid; glutamic acid derivatives; gammacarboxy-glutamic acid; aspartic acid; aspartic acid derivatives; inorganic compounds or groups; organic compounds or groups; succinic anhydride; and phthalic anhydride. 
     
     
         3 . A composition according to  claim 1  wherein the ShK toxin is obtained from a natural source. 
     
     
         4 . A composition according to  claim 1  wherein the ShK toxin is synthetic. 
     
     
         5 . A composition according to  claim 1  wherein the chemical entity is attached to an N terminus of the ShK polypeptide. 
     
     
         6 . A composition according to  claim 5  wherein the chemical entity is attached to an N-terminus of ShK through a linking molecule or linking group. 
     
     
         7 . A composition according to  claim 5  wherein the chemical entity is attached to an N-terminus of ShK by an aminoethyloxyethyloxy-acetyl linker. 
     
     
         8 . A composition according to  claim 1  wherein the chemical entity includes a fluorophore tag. 
     
     
         9 . A composition according to  claim 1  wherein the chemical entity is selected from the group consisting of: AEEAc-L-Pmp(OH 2 ), AEEAc-D-Pmp(OH 2 ), AEEAc-D-Pmp(OH, Et), AEEAc-L-Pmp(Et 2 ), AEEAc-D-Pmp(Et 2 ), AEEAc-L-Tyr, AEEAc-L-Tyr(PO 3 H 2 ), AEEAc-L-Phe(p-NH 2 ), AEEAc-L-Phe(p-CO 2 H), AEEAc-L-Aspartate, AEEAc-D-Aspartate, AEEAc-L-Glutamate, and AEEAc-D-Glutamate. 
     
     
         10 - 21 . (canceled) 
     
     
         22 . A method for causing inhibition of Kv1.3 potassium channels in a human or animal subject, said method comprising the step of:
 (A) administering to the subject a composition that comprises ShK toxin attached to an organic or inorganic chemical entity that has an anionic charge, in a form and amount that is effective to inhibit of Kv1.3 potassium channels.   
     
     
         23 . A method according to  claim 22  wherein the method is carried out to prevent or treat an autoimmune disorder. 
     
     
         24 . A method according to  claim 23  wherein the autoimmune disorder is selected from the group consisting of: Multiple sclerosis, Myasthenia gravis, Autoimmune neuropathies such as Guillain-Barré, Autoimmune uveitis, Crohn's Disease, Ulcerative colitis, Primary biliary cirrhosis, Autoimmune hepatitis, Autoimmune thrombocytopenia, Type 1 diabetes mellitus, Addison's Disease, Grave's Disease, Hashimoto's thyroiditis, Autoimmune oophoritis and orchitis, Behcet's disease, Rheumatoid arthritis, bone resorption associated with periodontal disease, Systemic lupus erythematosus, Scleroderma Polymyositis, dermatomyositis, Pemphigus vulgaris, Spondyloarthropathies such as ankylosing spondylitis, and Sjogren's syndrome. 
     
     
         25 . A method according to  claim 22  wherein the method is carried out to prevent or treat graft vs. host disease; resection of a transplanted tissue or organ; metabolic syndrome; Type 2 diabetes; obesity; or bone resorption associated with periodontal disease. 
     
     
         26 .- 30 . (canceled) 
     
     
         31 . A method according to  claim 22  wherein the chemical entity is selected from the group consisting of: amino acids; polypeptides; amino acid residues; unnatural amino acid residues; threonine; threonine derivatives; phospho-threonine; serine; serine derivatives; phospho-serine; glutamic acid; glutamic acid derivatives; gammacarboxy-glutamic acid; aspartic acid; aspartic acid derivatives; inorganic compounds or groups; organic compounds or groups; succinic anhydride; and phthalic anhydride. 
     
     
         32 . A method according to  claim 22  wherein the chemical entity is selected from the group consisting of: AEEAc-L-Pmp(OH 2 ), AEEAc-D-Pmp(OH 2 ); AEEAc-D-Pmp(OH, Et), AEEAc-L-Pmp(Et 2 ), AEEAc-D-Pmp(Et 2 ), AEEAc-L-Tyr, AEEAc-L-Tyr(PO 3 H 2 ), AEEAc-L-Phe(p-NH 2 ), AEEAc-L-Phe(p-CO 2 H), AEEAc-L-Aspartate, AEEAc-D-Aspartate, AEEAc-L-Glutamate, and AEEAc-D-Glutamate. 
     
     
         33 .- 44 . (canceled) 
     
     
         45 . A method for performing flow cytometry, said method comprising the steps of:
 (B) providing a composition that comprises ShK attached to an organic or inorganic chemical entity that has an anionic charge and a fluorophore tag;   (C) combining the composition provided in Step A with cells; and   (D) using a flow cytometer to count, isolate, or distinguish cells that have affinity for the composition provided in Step A.   
     
     
         46 . A method according to  claim 45  wherein Step C comprises using the flow cytometer to count, isolate or distinguish T lymphocytes. 
     
     
         47 . A composition according to  claim 1  wherein the ShK is modified by substitution of the Met residue at position 21. 
     
     
         48 . A composition according to  claim 47  wherein the substitution at Met residue 21 deters oxidation. 
     
     
         49 . A composition according to  claim 1  wherein the ShK is modified by substitution of the of the C-terminal acid function with an amide. 
     
     
         50 .- 54 . (canceled)