High Throughput Screening Assay for the TRPM5 Ion Channel

Abstract

There exists a need in the art for high throughput screening assays that can identify compounds that specifically modulate the activity of fast-acting ion channels, such as TRPM5. Current methods suffer from a lack of sensitivity, low throughput, and are labor intensive. The claimed methods provide fluorescent assays with an optical readout that gives rapid readout of the results, has a high signal to noise background ratio, are easy to use, can be modified for automation and miniaturization, and provide verification that a compound specifically modulates TRPM5.

1 - 14 . (canceled) 
   
   
       15 . A high throughput screening assay for determining whether a test compound is a TRPM5 ion channel-specific modulator comprising:
 (a) contacting a cell that expresses TRPM5 and has been preloaded with a membrane potential fluorescent dye, with a test compound in the presence of potassium chloride;   (b) using an optical detector, measuring the fluorescent intensity of said cell in the presence of said potential modulating compound;   (c) comparing the measured fluorescent intensity determined in step (b) to the fluorescent intensity of a different cell that expresses TRPM5 and has been preloaded with a membrane potential fluorescent dye, in the presence of potassium chloride and the absence of the test compound; and   (d) evaluating whether the test compound may be a TRPM5-specific modulator by determining if the ratio of the fluorescent intensity with potassium chloride and the test compound to the intensity with potassium chloride in the absence of the test compound is less than or greater than 1.   
   
   
       16 . The assay of  claim 15 , further comprising selecting a test compound that enhances TRPM5 activity. 
   
   
       17 . The assay of  claim 15 , further comprising selecting a test compound that inhibits TRPM5 activity. 
   
   
       18 . The assay of  claim 15 , wherein said cells are located in a multi-well vessel. 
   
   
       19 . The assay of  claim 18 , wherein said multi-well vessel comprises up to 96 wells. 
   
   
       20 . The assay of  claim 18 , wherein said multi-well vessel comprises greater than 96 wells. 
   
   
       21 . The assay of  claim 18 , wherein said multi-well vessel comprises 384 wells. 
   
   
       22 . The assay of  claim 18 , wherein said multi-well vessel comprises 1536 wells. 
   
   
       23 - 26 . (canceled) 
   
   
       27 . The assay of  claim 15 , wherein said membrane potential fluorescent dye is a Fluorescent Imaging Plate Reader Membrane Potential (FMP) dye. 
   
   
       28 . The assay of  claim 15 , wherein said optical detector is selected from the group consisting of: Fluorescent Imaging Plate Reader (FLIPR®), FLEXStation, Voltage/Ion Probe Reader (VIPR), fluorescent microscope and charge-coupled device (CCD) camera, and Fathway HT. 
   
   
       29 . The assay of  claim 28 , wherein said optical detector is a FLIPR®. 
   
   
       30 - 63 . (canceled) 
   
   
       64 . The assay of  claim 15 , wherein said cell of step (a) is a cell selected from the group consisting of: CHO, COS-7, HeLa, HEK 293, PC-12, and BAF. 
   
   
       65 . The assay of  claim 64 , wherein said cell is a CHO cell. 
   
   
       66 . The assay of  claim 64 , wherein said cell is a HEK 293 cell. 
   
   
       67 . The assay of  claim 15 , wherein the amount of said test compound that contacts the cell in step (a) is between 0.1 and 1000 μM. 
   
   
       68 . The assay of  claim 15 , wherein the test compound is a small organic compound having a molecular weight of more than 100 and less than about 10,000 Daltons. 
   
   
       69 . The assay of  claim 15 , wherein the test compound is selected from the group consisting of: synthetic organic compounds, chemical compounds, naturally occurring products, polypeptides, peptides, and nucleic acids. 
   
   
       70 . The assay of  claim 15 , wherein said membrane potential fluorescent dye is a voltage-sensitive dye selected from the group consisting of: modified bisoxonol dyes, sodium dyes, potassium dyes, and thorium dyes. 
   
   
       71 . The assay of  claim 15 , wherein said membrane potential fluorescent dye is a dual wavelength FRET-based dye selected from the group consisting of: DiSBAC2, DiSBAC3, and CC-2-DMPE. 
   
   
       72 . The assay of  claim 15 , wherein said optical detector is a FLIPR-Tetra™.