Pharmacoperones for correcting disease states involving protein misfolding

Abstract

This application relates to methods of identifying pharmacoperone agents that can restore function to a misfolded protein, such as a misfolded protein that causes disease. Also disclosed are methods of using such pharmacoperone agents to treat a disease or disorder that results from the misfolded protein.

1 . An assay for detecting a pharmacoperone agent that restores protein function, comprising:
 selecting a test agent that is capable of penetrating a cell membrane and specifically binding to the protein, wherein the test agent binds specifically to the protein but does not significantly interfere with protein activity, wherein the protein includes a mutation that affects protein folding; and   determining whether the test agent restores protein function, wherein restoration of protein function indicates that the test agent is a pharmacoperone agent.   
     
     
         2 . The assay of  claim 1 , wherein the protein is a receptor protein, and wherein the mutation that affects protein folding can also affect routing of the receptor to the cell membrane surface. 
     
     
         3 . The assay of  claim 1 , wherein the mutation that affects protein folding can also affect aggregation of the protein. 
     
     
         4 . The assay of  claim 2 , wherein selecting the test agent comprises selecting a test agent that is a non-antagonist of the receptor. 
     
     
         5 . The assay of  claim 2 , wherein selecting the test agent comprises selecting a test agent that binds outside a ligand binding site of the receptor. 
     
     
         6 . The assay of  claim 1 , wherein the test agent comprises selecting a test agent that acts as an agonist at the protein. 
     
     
         7 . The assay of  claim 1 , wherein selecting the test agent that specifically binds to the protein comprises selecting a test agent that binds to the protein with a dissociation constant (Kd) of less than 1 μM. 
     
     
         8 . The assay of  claim 1 , wherein selecting the test agent comprises selecting an agent which is a peptidomimetic of a molecule that binds to the protein. 
     
     
         9 . The assay of  claim 2 , wherein determining whether the agent restores receptor function comprises determining whether the agent restores function of a mutant receptor in which a mutation adversely affects receptor folding and diminishes expression of the receptor at a cell membrane surface. 
     
     
         10 . The assay of  claim 9 , wherein the assay further comprises determining a specific mutation in the receptor that adversely affects receptor function, and selecting the test agent comprises selecting an agent that has an effect against the specific mutation. 
     
     
         11 . The assay of  claim 1 , wherein determining whether the agent restores protein function comprises expressing the protein in a cell, exposing the cell to a protein agonist, and measuring an intracellular marker of protein function in the cell. 
     
     
         12 . The assay of  claim 1 , wherein the protein is a G-coupled protein receptor and the intracellular marker comprises inositol phosphate (IP). 
     
     
         13 . The assay of  claim 2 , wherein selecting a test agent comprises selecting a test agent having activity at a concentration that does not interfere with an ability of a ligand to bind to the receptor. 
     
     
         14 . The assay of  claim 1 , wherein the protein that includes a mutation results in a disease. 
     
     
         15 . The method of  claim 14 , wherein the disease is cystic fibrosis, nephrogenic diabetes insipidus, familial hypercholesterolemia, cataracts, Alzheimer's, retinitis pigmentosa, or hypogonadotropic hypogonadism (HH). 
     
     
         16 . The assay of  claim 15 , wherein the protein that includes a mutation is a mutant cystic fibrosis transmembrane conductance regulator (CFTR), mutant amyloid receptor protein, mutant gonadotropin-releasing hormone receptor (GnRHR), mutant alpha-1 antitrypsin receptor, mutant rhodopsin, mutant lens crystalline protein, mutant low-density lipoprotein (LDL) receptor, mutant arginine-vasopressin receptor protein, or mutant insulin receptor protein. 
     
     
         17 . The assay of  claim 1 , wherein the test agent is an indole, macrolide or quinolone. 
     
     
         18 . The assay of  claim 13 , wherein selecting a test agent having activity at a concentration that does not interfere with an ability of a ligand to bind to the receptor comprises determining if the test agent competes with a ligand, wherein the presence of competition for the ligand binding site indicates that the test agent binds to the ligand binding site, and wherein the absence of detectable competition for the ligand binding site indicates that the test agent does not bind to the ligand binding site. 
     
     
         19 . The assay of  claim 1 , wherein the assay further comprises determining whether the test agent can dissociate from the protein. 
     
     
         20 . A method of treating a subject having a disease resulting from a misfolded mutant protein, comprising:
 determining mutations present in the misfolded mutant protein, wherein the presence of a particular mutation indicates that the subject can be treated with a pharmacoperone specific for the misfolded mutant protein;   administering the pharmacoperone at a therapeutically effective amount to the subject having a particular mutation which indicates that the subject can be treated with the pharmacoperone.   
     
     
         21 . The method of  claim 20 , wherein administering comprises administering the pharmacoperone to the subject, followed by a period when the pharmacoperone is not administered to the subject, and subsequently administering the pharmacoperone at a therapeutically effective amount to the subject. 
     
     
         22 . The method of  claim 20 , wherein the method further comprises administering a therapeutically effective amount of an agonist of the protein to the subject following administering the pharmacoperone. 
     
     
         23 . A method of treating a subject having a disease resulting from a misfolded receptor protein, comprising:
 administering a therapeutically effective amount of a pharmacoperone to the subject, wherein the pharmacoperone is cell membrane permeable, is substantially specific for the receptor but does not bind to a ligand binding site of the receptor.   
     
     
         24 . The method of  claim 23 , wherein the pharmacoperone is an antagonist of the receptor protein, and the method further comprises administration of a therapeutically effective amount of an agonist of the misfolded receptor protein to the subject following administering the pharmacoperone. 
     
     
         25 . A method of determining the severity of hypogonadotropic hypogonadism (HH) in a subject, comprising:
 determining mutations present in a GnRHR protein sequence in the subject, wherein the presence of a E 90 K, S 168 R, S 217 R, or L 266 R mutation indicates that the subject has a severe form of HH.