Screening Assay for Inhibitors of TRPA1 Activation by a Lower Alkyl Phenol

Abstract

The invention relates an assay useful for screening and identifying compounds as modulators of lower alkyl phenol activation of TRPA1. Thymol, a lower alkyl phenol anti-infective and the active ingredient in, e.g., mouthwashes, is stringent and has an objectionable burning taste sensation. Thymol activates the transient receptor potential like ion channel TRPA1. The assay described and claimed herein involves measurement of activation of TRPA1 and enables the screening of compounds that inhibit lower alkyl phenol, or thymol activation of TRPA1. Inhibitors of thymol activation of TRPA1 can be used to prevent the objectionable taste of thymol in medical uses where taste limits acceptance.

1 . A method for screening a candidate compound to identify a compound that modulates activation of TRPA1 by Compound I, wherein Compound I is represented by Formula I: 
     
       
         
         
             
             
         
       
       wherein each instance of R 1  is independently hydrogen or a C 1-6  alkyl, and n is an integer from 1-5, said method comprising: 
       (i) measuring the level of activation of Compound I-activated-TRPA1 in the presence of a candidate compound to obtain a measured value; and 
       (ii) comparing said measured value to a standard value, wherein a difference between the measured value and the standard value indicates that the candidate compound modulates Compound I activation of TRPA1. 
     
   
   
       2 . The method of  claim 1 , wherein a difference in the level of activation of the Compound I-activated TRPA1 in the presence of the candidate compound, compared to the standard value, provides a quantitative measure of modulation of TRPA1 activation. 
   
   
       3 . The method of  claim 1 , wherein the standard value is the value determined by measurement, in the absence of the candidate compound, of the level of activation of Compound I-activated TRPA1. 
   
   
       4 . The method of  claim 1 , wherein the standard value is the value determined by measurement, in the presence of the candidate compound, of the level of activation of TRPA1 that has not been activated by Compound I. 
   
   
       5 . The method of  claim 1 , wherein the standard value is the value determined by measurement, in the presence of Compound I and the candidate compound, of the level of activation of Compound I-activated-TRPA1 in cells that do not express TRPA1. 
   
   
       6 . The method of  claim 1 , wherein the standard value is the value determined by measurement, in the presence of Compound I and the candidate compound, of the level of activation of Compound I-activated-TRPA1 in cells transfected with both TRPA1 and an siRNA molecule. 
   
   
       7 . The method of  claim 1 , wherein the level of activation of Compound I-activated TRPA1 is determined by measuring the flux of ions into or out of a TRPA1 expressing cell in the presence of Compound I. 
   
   
       8 . The method of  claim 7 , wherein the flux of ions is measured using an indicator capable of producing a signal that signifies the movement of ions into or out of a cell. 
   
   
       9 . The method of  claim 8 , wherein the indicator is selected from the group consisting of calcium sensitive fluorescent dyes, sodium-sensitive fluorescent dyes, membrane potential-sensitive fluorescent dyes and mixtures thereof. 
   
   
       10 . The method of  claim 9 , wherein the indicator is in acetoxymethyl ester form. 
   
   
       11 . The method of  claim 7 , wherein the cells are selected from a group comprising HEK 293, CHO, COS-7, HeLa, PC-12, and BA, or mixtures thereof. 
   
   
       12 . The method of  claim 11 , wherein the cells are HEK 293 or CHO cells. 
   
   
       13 . The method of  claim 1 , wherein to measure the level of activation of TRPA1, one or more techniques selected from the following group is used: fluorimetry, flow cytometry, fluorescent microscopy, fluorescent imaging plate reading or patch clamp technique. 
   
   
       14 . The method of  claim 1 , wherein to measure the level of activation of TRPA1 a fluorescent imaging plate reader is used. 
   
   
       15 . The method of  claim 1 , wherein the TRPA1 is expressed in cells maintained in a culture vessel having a single compartment. 
   
   
       16 . The method of  claim 1 , wherein the TRPA1 is expressed in cells maintained in a divided culture vessel having an array of individual compartments. 
   
   
       17 . The method of  claim 16 , wherein a candidate compound is added to more than one compartment at a time. 
   
   
       18 . The method of  claim 16 , wherein said measuring is effected in more than one compartment at a time. 
   
   
       19 . A method for identifying a compound that modulates the activation of TRPA1 by Compound I, wherein Compound I is represented by Formula I: 
     
       
         
         
             
             
         
       
       wherein each instance of R 1  is independently hydrogen or a C 1-6  alkyl, and n is an integer from 1-5, said method comprising: 
       (a) incubating cells expressing TRPA1 with an indicator for a time sufficient to allow incorporation of the indicator into the cells, wherein the indicator is capable of signaling activation of TRPA1; 
       (b) incubating said cells with a candidate compound while maintaining said cells in a solution that contains Compound I and is suitable to allow a flux of ions through TRPA1; 
       (c) after completing steps (a) and (b), measuring a signal from the indicator in the presence of the candidate modulator compound; and 
       (d) comparing the signal of step (c) to a standard value, wherein the indicator indicates the flux of ions via Compound I-activated TRPA1 and a difference between the signal of step (c) and the standard value indicates that the candidate compound modulates TRPA1 activation. 
     
   
   
       20 - 54 . (canceled) 
   
   
       55 . A method for identifying a compound that modulates the activation of TRPA1 by thymol, said method comprising:
 (a) incubating cells expressing TRPA1 with an indicator for a time sufficient to allow incorporation of the indicator into the cells, wherein the indicator is capable of signaling activation of TRPA1;   (b) incubating said cells with a candidate compound while maintaining said cells in a solution that contains thymol and is suitable to allow a flux of ions through TRPA1;   (c) after completing steps (a) and (b), measuring a signal from the indicator in the presence of the candidate modulator compound; and   (d) comparing the signal of step (c) to a standard value, wherein the indicator indicates the flux of ions via thymol-activated TRPA1 and a difference between the signal of step (c) and the standard value indicates that the candidate compound modulates TRPA1 activation.   
   
   
       56 - 72 . (canceled)