Pharmacological chaperones for treating obesity

Abstract

The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation which affects protein folding and/or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, for example in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance the activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance the activity of the MC4R at the cell surface.

1 . A method for enhancing activity of an MC4R polypeptide, which method comprises contacting an MC4R-expressing cell with a pharmacological chaperone that binds to the MC4R polypeptide in an amount effective to increase MC4R activity.  
     
     
         2 . The method of  claim 1 , wherein the MC4R polypeptide is a wild-type MC4R polypeptide.  
     
     
         3 . The method of  claim 2 , wherein the MC4R polypeptide has a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.  
     
     
         4 . The method of  claim 1 , wherein the MC4R polypeptide is a mutant MC4R polypeptide.  
     
     
         5 . The method of  claim 4 , wherein the mutant MC4R polypeptide comprises a mutation associated with misfolding of the MC4R polypeptide.  
     
     
         6 . The method of  claim 4 , wherein the mutant MC4R polypeptide comprises a mutation selected from the group consisting of P78L, R165Q, R165W, I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R, N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X, and P299H.  
     
     
         7 . The method of  claim 4 , wherein the mutant MC4R polypeptide is R165Q, R165W, S58C, N62S, or P299H.  
     
     
         8 . The method of  claim 1 , wherein the pharmacological chaperone is an MC4R antagonist.  
     
     
         9 . The method of  claim 8 , wherein the MC4R antagonist has a structure as set forth in  FIG. 5  or  FIG. 6 .  
     
     
         10 . The method of  claim 1 , wherein the pharmacological chaperone binds to the MC4R polypeptide as it is being folded into a functional conformation.  
     
     
         11 . The method of  claim 1 , wherein the activity enhanced by the pharmacological chaperone is adenylyl cyclase activation.  
     
     
         12 . A method for enhancing cell surface expression of an MC4R polypeptide, which method comprises contacting a MC4R-expressing cell with a pharmacological chaperone that binds to the MC4R polypeptide in an amount effective to increase MC4R cell surface expression.  
     
     
         13 . The method of  claim 12 , wherein the pharmacological chaperone dissociates from the MC4R polypeptide when it is folded into a functional conformation.  
     
     
         14 . The method of  claim 12 , wherein the MC4R polypeptide is a wild-type MC4R polypeptide.  
     
     
         15 . The method of  claim 12 , wherein the MC4R polypeptide has a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.  
     
     
         16 . The method of  claim 12 , wherein the MC4R polypeptide is a mutant MC4R polypeptide.  
     
     
         17 . The method of  claim 16 , wherein the mutant MC4R polypeptide comprises a mutation associated with misfolding of the MC4R polypeptide.  
     
     
         18 . The method of  claim 16 , wherein the mutant MC4R polypeptide comprises a mutation selected from the group consisting of P78L, R165Q, R165W, I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R, N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X, and P299H.  
     
     
         19 . The method of  claim 16 , wherein the mutant MC4R polypeptide is. R165Q, RI 65W, S58C, N62S, or P299H.  
     
     
         20 . The method of  claim 12 , wherein the pharmacological chaperone is an MC4R antagonist.  
     
     
         21 . The method of  claim 20 , wherein the MC4R antagonist has a structure as set forth in  FIG. 5  or  FIG. 6 .  
     
     
         22 . A method of treating an individual in need of increased stability of an MC4R polypeptide, which method comprises administering to the individual a pharmacological chaperone that binds to the MC4R polypeptide in an amount effective to increase stability.  
     
     
         23 . The method of  claim 22 , wherein the pharmacological chaperone is an MC4R antagonist having a structure as set forth in  FIG. 5  or  FIG. 6 .  
     
     
         24 . The method of  claim 22 , wherein the pharmacological chaperone increases trafficking of the MC4R polypeptide to the cell membrane.  
     
     
         25 . The method of  claim 22 , wherein the individual is obese or is at risk for becoming obese.  
     
     
         26 . The method of  claim 22 , wherein the individual comprises a mutation in an MC4R gene which has been associated with obesity.  
     
     
         27 . The method of  claim 22 , wherein the pharmacological chaperone is administered in-a pharmaceutically-acceptable carrier.  
     
     
         28 . The method of  claim 22 , wherein the MC4R polypeptide is a wild-type MC4R polypeptide.  
     
     
         29 . The method of  claim 22 , wherein the MC4R polypeptide is a mutant MC4R polypeptide.  
     
     
         30 . A method for identifying a pharmacological chaperone for an MC4R, which method comprises: 
 (a) contacting a test compound to a reaction mixture that comprises a cell or cell extract expressing an MC4R polypeptide, wherein the reaction mixture conditions permit binding of the test compound to the MC4R polypeptide;    (b) detecting stability, activity, or cell surface localization of the MC4R polypeptide in the reaction mixture in the presence of the test compound; and    (c) comparing stability, activity, or cell surface localization of the MC4R polypeptide in the presence of the test compound to stability, activity, or cell surface localization of the MC4R polypeptide in the absence of the test compound,    wherein detection of enhanced stability, activity, or cell surface localization in the presence of the test compound relative to the absence of the test compound indicates that the test compound is a pharmacological chaperone for the MC4R.    
     
     
         31 . The method of  claim 30 , wherein the MC4R polypeptide is a wild-type MC4R polypeptide.  
     
     
         32 . The method of  claim 31 , wherein the MC4R polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.  
     
     
         33 . The method of  claim 30 , wherein the MC4R polypeptide is a mutant MC4R polypeptide.  
     
     
         34 . The method of  claim 33 , wherein the MC4R polypeptide comprises a mutation associated with misfolding of the MC4R polypeptide.  
     
     
         35 . The method of  claim 34 , wherein the MC4R misfolding mutation is selected from the group consisting of P78L, R165Q, R165W, I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R, N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X, P299H, and S58C, relative to the wild-type MC4R polypeptide sequence as set forth in SEQ ID NO: 2.  
     
     
         36 . The method of  claim 30 , wherein the reaction mixture is cell-based.  
     
     
         37 . The method of  claim 30 , wherein the reaction mixture is cell-free.  
     
     
         38 . The method of  claim 30 , further comprising detecting activity of an MC4R polypeptide located at the cell surface.  
     
     
         39 . The method of  claim 38 , wherein the activity detected is cAMP activation.  
     
     
         40 . The method of  claim 1 , wherein the pharmacological chaperone has a structure as depicted in  FIG. 13 .  
     
     
         41 . The method of  claim 1 , wherein the pharmacological chaperone has a structure as depicted in  FIG. 14 .  
     
     
         42 . The method of  claim 1 , wherein the pharmacological chaperone has a structure as depicted in  FIG. 15 .  
     
     
         43 . The method of  claim 1 , wherein the pharmacological chaperone has a structure as depicted in  FIG. 16 .