Computer-based model for identification and characterization for non-competitive inhibitors of nicotinic acetylcholine receptors and related ligand-gated ion channel receptors

Abstract

A computer readable medium holding data of a molecular model of a ligand-gated ion channel receptor and/or a computer system for modeling said receptor are provided by the instant invention. The molecular model can be used to design novel compounds having activity as non-competitive inhibitors of the ion channel. A preferred embodiment of the invention relates to nicotinic acetylcholine receptors. Compounds having activity as non-competitive inhibitors of ligand-gated ion channel receptors and methods for inhibiting the receptor and treating diseases or disorders mediated by function of the receptor are also disclosed.

What is claimed is:  
     
         1 . A computer system comprising: 
 i) a memory storing positional data of the atomic coordinates of the transmembrane portion of at least one subunit of a ligand-gated neurotransmitter receptor protein; and    ii) a processor generating a molecular model having a three dimensional shape representative of the pore portion of the ligand-gated neurotransmitter receptor based the positional data.    
     
     
         2 . The computer system of  claim 1 , wherein the memory stores data of the atomic coordinates of at least an α 3  chain of a nicotinic acetylcholine receptor and a β 4  chain of a nicotinic acetylcholine receptor.  
     
     
         3 . The computer system of  claim 1 , wherein the memory stores data of the atomic coordinates of at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 through 15.  
     
     
         4 . The computer system of  claim 1 , wherein the processor generates a molecular model of the pore portion of a ligand-gated neurotransmitter receptor having a subunit stoichiometry ranging from (α) 5 (β) 0  to (α) 2 (β) 3 , or (α) 2 βδγ.  
     
     
         5 . The computer system of  claim 4 , in which the stoichiometry is (α) 2 (β) 3 .  
     
     
         6 . The computer system of  claim 1 , wherein the data comprises the atomic coordinates of a portion of the transmembrane portion of the subunit consisting of the amino acid sequence of residues 8 to 19 of at least one of SEQ ID NOS: 1-15.  
     
     
         7 . The computer system of  claim 1 , further comprising a removable, computer readable medium in which the data are stored.  
     
     
         8 . A method for screening a compound for activity as a non-competitive inhibitor of a ligand-gated neurotransmitter receptor comprising; identifying as a compound having activity as a non-competitive inhibitor as one having a ΔG less than −6 kcal/mol by docking a model of the compound in a model of the three-dimensional shape of the pore portion of the ligand-gated neurotransmitter receptor.  
     
     
         9 . The method of  claim 8 , in which the docking step is performed using a computer program implementing a genetic algorithm.  
     
     
         10 . The method of  claim 8 , further comprising assaying the binding of the compound to the receptor and confirming as a non-competitive inhibitor one having a k′ of greater than 8, the assaying step being performed either before or after the docking step.  
     
     
         11 . The method of  claim 10 , further comprising modifying the structure of the compound confirmed as a non-competitive inhibitor and repeating the method using the compound having the modified structure.  
     
     
         12 . A method for making a non-competitive inhibitor of a ligand-gated neurotransmitter receptor comprising: 
 A method for screening a compound for activity as a non-competitive inhibitor of a ligand-gated neurotransmitter receptor comprising:    i) identifying as a compound having activity as a non-competitive inhibitor as one having a ΔG less than −6 kcal/mol by docking a model of the compound in a model of the three-dimensional shape of the pore portion of the ligand-gated neurotransmitter receptor;    ii) testing the compound for binding to the ligand-gated neurotransmitter receptor and/or inhibiting the ion-channel activity thereof;    iii) obtaining as a non-competitive inhibitor of the ligand-gated neurotransmitter receptor the compound that binds to the ligand-gated neurotransmitter receptor with a k′ value greater than 8 and being displaced in chromatographic experiments by mecamylamine and/or inhibits the ion channel activity of the ligand-gated neurotransmitter receptor in nicotine stimulated  86 Rb +  efflux with IC 50  lower than 100 μM.    
     
     
         13 . The method of  claim 10 , in which the ligand-gated neurotransmitter receptor is a neuronal receptor.  
     
     
         14 . The method of  claim 12 , in which the ligand-gated neurotransmitter receptor is a muscle receptor.  
     
     
         15 . A compound comprising: a bulky hydrophobic moiety and primary, secondary or tertiary amino group located 5 to 10 Å from said hydrophobic moiety, said compound having activity of inhibiting the ion-channel activity of a ligand-gated neurotransmitter receptor in an assay of nicotine stimulated  86 Rb +  efflux with an IC 50  lower than 50 μM.  
     
     
         16 . The compound of  claim 15 , wherein the bulky hydrophobic moiety is selected from the group consisting of a phenyl ring, a napthyl ring, a cyclopentyl ring, a cyclohexyl ring, a fused ring.  
     
     
         17 . The compound of  claim 16 , wherein the hydrophobic moiety is a fused ring selected from the group consisting of bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane, morphinan and dibenzo [1.41] diazepine.  
     
     
         18 . The compound of  claim 15 , that does not compete with the neurotransmitter ligand of a receptor for binding of a neurotransmitter ligand binding site of the receptor located on the external surface at the interface of two subunits in a pocket approximately 30-35 Å from the transmembrane portion of the subunits.  
     
     
         19 . The compound of  claim 15 , wherein said receptor is a neuronal nicotinic acetylcholine receptor.  
     
     
         20 . The compound of  claim 15 , wherein the receptor is a muscular nicotinic acetylcholine receptor.  
     
     
         21 . A compound comprising a bulky hydrophobic moiety and a primary, secondary or tertiary amino group placed 5 to 10 Å from said hydrophobic moiety, said compound having activity of binding to the non-competitive inhibitor site of a ligand-gated neurotransmitter receptor with a ΔG of at least −6 kcal/mol.  
     
     
         22 . The compound of  claim 21 , that does not compete with the neurotransmitter ligand of the receptor for binding to the neurotransmitter ligand binding site of the receptor.  
     
     
         23 . The compound of  claim 21 , wherein said receptor is a neuronal nicotinic acetylcholine receptor.  
     
     
         24 . The compound of  claim 21 , wherein the receptor is a muscular nicotinic acetylcholine receptor.  
     
     
         25 . A kit comprising a computer readable medium having stored therein positional data of the atomic coordinates of the transmembrane portion of at least one subunit of a ligand-gated neurotransmitter receptor protein; and 
 a composition comprising the ligand-gated neurotransmitter receptor protein.    
     
     
         26 . The kit of  claim 25 , in which the composition comprising the receptor protein is an affinity chromatography medium.  
     
     
         27 . The kit of  claim 26 , in which the receptor is a nicotinic acetylcholine receptor.  
     
     
         28 . A computer readable medium having stored therein positional data of the atomic coordinates of the transmembrane portion of at least one subunit of a ligand-gated neurotransmitter receptor protein.  
     
     
         29 . The computer readable medium of  claim 28 , wherein the data comprise the atomic coordinates of at least an α 3  chain of a nicotinic acetylcholine receptor and a β 4  chain of a nicotinic acetylcholine receptor.  
     
     
         30 . The computer readable medium of  claim 28 , wherein the data comprise the atomic coordinates of at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 through 15.  
     
     
         31 . The computer readable medium of  claim 28 , wherein the data comprise a molecular model of the pore portion of a ligand-gated neurotransmitter receptor having a subunit stoichiometry ranging from (α) 5 (β) 0  to (α) 2 (β) 3 , or (α) 2 βδγ.  
     
     
         32 . The computer readable medium of  claim 31 , in which the stoichiometry is (α) 2 (β) 3 .  
     
     
         33 . A method for non-competitively inhibiting a ligand-gated ion channel receptor comprising contacting the ligand-gated ion channel receptor with a compound of  claim 15 , except for bupropion, ketamine, laudanosine, mecamylamine, methadone, MK-801, phenylcylclidine, ethidium, and dextromethorphan.  
     
     
         34 . A method for treating Tourette's syndrome, or a cognitive disorder, pain, anxiety, depression, neurodegeneration or an addiction caused by an overactive ligand-gated ion channel receptor, comprising administering to a subject an amount of a compound of  claim 15  effective to inhibit ion flux through said ligand-gated ion channel.  
     
     
         35 . The method of  claim 34 , that is a method of treating smoking addiction and the receptor is a neuronal nicotinic acetylcholine receptor.  
     
     
         36 . A method for evaluating cardiovascular toxicity or GI spasming or diarrheal side effects of a compound comprising testing a compound for activity in the method of  claim 8 , wherein the receptor is a muscular nicotinic acetylcholine receptor subtype.