Senomyx, Inc.

Sumita Ray

Guy Servant

Bryan Moyer

Hong Chang

Imran Clark

Cyril Redcrow

Paul Brust

Ning Hung

In one aspect, the present invention relates to a mammalian cell-based high-throughput assay for the profiling and screening of human epithelial sodium channel (hENaC) cloned from a human kidney c-DNA library and is also expressed in other tissues including human taste tissue. The present invention further relates to amphibian oocyte-based medium-throughput electrophysiological assays for identifying human ENaC modulators, preferably ENaC enhancers. Compounds that modulate ENaC function in a cell-based ENaC assay are expected to affect salty taste in humans. The assays described herein have advantages over existing cellular expression systems. In the case of mammalian cells, such assays can be run in standard 96 or 384 well culture plates in high-throughput mode with enhanced assay results being achieved by the use of a compound that inhibits ENaC function, preferably an amiloride derivative such as Phenamil. In the case of the inventive oocyte electrophysiological assays (two-electrode voltage-clamp technique), these assays facilitate the identification of compounds which specifically modulate human ENaC. The assays of the invention provide a robust screen useful to detect compounds that facilitate (enhance) or inhibit hENaC function. Compounds that enhance or block human ENaC channel activity should thereby modulate salty taste in humans.

Electrophysiological assays using oocytes that express human ENaC and the use of phenamil to improve the effect of ENaC enhancers in assays using membrane potential reporting dyes

In one aspect, the present invention relates to a mammalian cell-based high-throughput assay for the profiling and screening of human epithelial sodium channel (hENaC) cloned from a human kidney c-DNA library and is also expressed in other tissues including human taste tissue. The present invention further relates to amphibian oocyte-based medium-throughput electrophysiological assays for identifying human ENaC modulators, preferably ENaC enhancers. Compounds that modulate ENaC function in a cell-based ENaC assay are expected to affect salty taste in humans.

Electrophysiological assays using oocytes that express human enac and the use of phenamil to improve the effect of enac enhancers in assays using membrane potential reporting dyes

This invention relates to improved electrophysiological assays that measure sodium conductance activity of a delta or alpha human epithelial sodium channel (ENaC) expressed in a test cell in the presence and absence of delta hENaC enhancers. The improvement comprises contacting the test cells with an amount of sodium ion, typically from at least 15 mM to 140 mM, for a time sufficient, e.g. for at least 5 minutes to an hour, prior to the test cells being screened against at least one putative enhancer. It has been surprisingly discovered that this sodium pretreatment enhances assay sensitivity, especially assays that use membrane potential or ion sensitive dyes that detect changes in conductance fluorimetrically. These enhancer compounds have potential application in modulating (enhancing) salty taste perception and for treating disorders involving aberrant hENaC function.

Cell-based fluorescent assays for identifying alpha and delta ENaC modulators

Method of modulating human ENaC sodium channel

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San Diego, CA, United States of America

Sumita Ray