Inhibitors of calcium-activated chloride channels

Abstract

Provided herein are methods for identifying compounds that are inhibitors of a calcium-activated chloride channel. Aminothiophene and aminothiazole compounds, and compositions comprising these compounds, described herein that inhibit efflux of chloride through a calcium-activated chloride channel are useful for treating diseases, disorders, and sequelae of diseases, disorders, and conditions that are associated with aberrantly increased chloride and fluid secretion, for example, secretory diarrhea.

1 . A composition comprising a physiologically acceptable excipient and a compound having the following structure (I): 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,
 wherein 
 R 1  is hydrogen or optionally substituted alkyl; 
 R 2  is hydroxy, optionally substituted alkoxy, or optionally substituted phenylamino; 
 R 3  is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted phenyl, or optionally substituted heterocyclyl; and 
 n is 0, 1, or 2, and 
 
       wherein the compound of structure I comprises at least one —COON. 
     
     
         2 . The composition of  claim 1  wherein the compound has the following structure I(A): 
       
         
           
           
               
               
           
         
       
       wherein n is 1 or 2. 
     
     
         3 . The composition of  claim 1  wherein n is 1 and R 1  is hydrogen, tert-butyl, or tert-pentyl, and the compound has the following structure (Ia), (Ib), or (Ic): 
       
         
           
           
               
               
           
         
       
     
     
         4 - 25 . (canceled) 
     
     
         26 . A composition comprising a physiologically acceptable excipient and a compound having the following structure (II): 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,
 wherein R 7  is optionally substituted C 1-6  alkyl, optionally substituted phenyl, or optionally substituted phenylacyl; 
 R 8  is hydrogen, optionally substituted C 1-6  alkyl, or optionally substituted phenyl; 
 R 9  and R 10  are the same or different and independently hydrogen, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted phenoxy. 
 
     
     
         27 . The composition of  claim 26  wherein at least one of R 9  and R 10  is not hydrogen. 
     
     
         28 . The composition of  claim 26 , wherein R 7  is optionally substituted phenyl and the compound has the following structure (IIa): 
       
         
           
           
               
               
           
         
       
       wherein R 11  and R 12  are the same or different and independently hydrogen, hydroxy, carboxy, halo, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted cycloalkyl. 
     
     
         29 .- 112 . (canceled) 
     
     
         113 . An isolated epithelial cell comprising (i) a calcium-activated chloride channel and (ii) a recombinant cytoplasmic indicator protein that binds halide. 
     
     
         114 . The epithelial cell of  claim 113  wherein the epithelial cell is an intestinal epithelial cell or a pulmonary epithelial cell. 
     
     
         115 . The epithelial cell of  claim 113  wherein the intestinal epithelial cell is an HT-29 cell. 
     
     
         116 . The epithelial cell of  claim 113  wherein the cytoplasmic indicator protein is a yellow fluorescent protein (YFP) mutant. 
     
     
         117 . The epithelial cell of  claim 116 , wherein the YFP mutant is YFP-H148Q/I152L. 
     
     
         118 . The epithelial cell of  claim 113  wherein the calcium-activated chloride channel is TMEM16A. 
     
     
         119 . The epithelial cell of  claim 118  wherein TMEM16A is human TMEM16A. 
     
     
         120 . The epithelial cell of  claim 113  wherein the recombinant cytoplasmic indicator protein is introduced into the cell by a recombinant expression vector that is a viral vector. 
     
     
         121 . The method of  claim 120  wherein the viral vector is a retroviral vector. 
     
     
         122 . The method of  claim 121  wherein the retroviral vector is a lentiviral vector. 
     
     
         123 . A method of identifying an agent that is an inhibitor of a calcium-activated chloride channel comprising:
 (a) contacting the isolated epithelial cell of  claim 113  and a candidate agent in a test sample to permit interaction between the candidate agent and the cell;   (b) adding to the test sample (i) at least one calcium-elevating agonist and (ii) iodide, wherein binding of the calcium-elevating agonist to the cell increases the level of calcium ion (Ca 2 ) in the cell; and   (c) determining the level of iodide influx in the presence of the candidate agent and comparing the level of iodide influx in the presence of the candidate agent with the level of iodide influx in the absence of the candidate agent, wherein a decrease in the level of iodide influx in the presence of the candidate agent compared with the level of iodide influx in the absence of the candidate agent, indicates that the candidate agent is an inhibitor of the calcium-activated chloride channel.   
     
     
         124 . The method of  claim 123  wherein the steps of the method are performed in each of a plurality of reaction vessels in a high throughput screening array. 
     
     
         125 . A method of determining influx of an anion in the epithelial cell of  claim 113 , wherein the anion is halide or NO 3   − , said method comprising:
 (a) contacting the epithelial cell with the anion in the presence of a calcium-elevating agonist and in the absence of the calcium-elevating agonist, wherein the cytoplasmic indicator protein binds the anion, and wherein binding of the calcium-elevating agonist to the epithelial cell increase the level of calcium ion (Ca 2+ ) in the epithelial cell; and   (b) determining the level of anion influx in the presence of the calcium-elevating agonist and determining the level of anion influx in the absence of the calcium-elevating agonist, and comparing the level of anion influx in the presence of the calcium-elevating agonist to the level of anion influx in the absence of the calcium-elevating agonist, thereby determining influx of the anion in the epithelial cell.