Senomyx, Inc.

Sibia Neurosciences, Inc.

The Salk Institute Biotechnology Industrial Associates, Inc.

Other

Merck + Co., Inc.

Merck Sharp + Dohme Corp.

Paul Brust

Michael Miller Harpold

Guy Servant

Gonul Velicelebi

Alexey Pronin

Michael Anthony Akong

Jon Elliot Adler

Shawn O'Connell

Hong Xu

Huixian Tang

Walter Keung

Judy Connor

Qing Li

Wen Zhao

Xiaodong Li

Donald Karanewsky

Catherine Tachdjian

Vincent Darmohusodo

Andrew P Patron

Lan Zhang

Chad Priest

Victor Selchau

Thomas P Brady

Melissa Arellano

Jing Ling

Brett Weylan Ching

Steven B Ellis

Sumita Ray

Mark Ozeck

Ning Hung

Donald S Karanewsky

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

The present invention relates to the discovery that specific human taste receptors in the T2R taste receptor family respond to particular bitter compounds present in, e.g., coffee. Also, the invention relates to the discovery of specific compounds and compositions containing that function as bitter taste blockers and the use thereof as bitter taste blockers or flavor modulators in, e.g., coffee and coffee flavored foods, beverages and medicaments. Also, the present invention relates to the discovery of a compound that antagonizes numerous different human T2Rs and the use thereof in assays and as a bitter taste blocker in compositions for ingestion by humans and animals.

Compounds that inhibit (block) bitter taste in composition and methods of making same

Assays for identifying compounds that modulate, preferably inhibit bitter taste associated with the activation of hT2R4, hT2R44 and/or hT2R61 are provided. The compounds identified according to these assays should modulate, e.g., inhibit bitter taste associated with bitter tasting compounds including quinine, 6-nitrosaccharin, saccharin and/or denatonium. These compounds are useful additives for foods, beverages or medicinal preparations having a bitter taste.

Use of specific T2R taste receptors to identify compounds that block bitter taste

Compounds that inhibit (block) bitter taste in composition and use thereof

Assays for identifying compounds that modulate, preferably inhibit bitter taste associated with the activation of hT2R4, hT2R44 and/or hT2R61 are provided. The compounds identified according to these assays should modulate, e.g., inhibit bitter taste associated with bitter tasting compounds including quinine, 6-nitrosaccharin, saccharin and/or denatonium. These compounds are useful additives for foods, beverages or medicinal preparationshaving a bitter taste.

Use of specific T2R receptors to identify compounds that block bitter taste

The invention resides in part in the discovery that G proteins other than G&#x3b1;couples to T1R and T2R taste receptors, particularly Gproteins such as G. Related to this discovery, the invention provides cell-based assay methods for identifying compounds that modulate the activity of specific T1R or T2R taste receptors or which modulate the effect of other T1R or T2R modulators on T1R or T2R activity. These assay methods preferably detect the effect of a putative T1R or T2R modulator compound on MAPK activation, cAMP accumulation, or adenylyl cyclase activity or another signaling pathway regulated by Gproteins. The level of MAPK activation, cAMP accumulation or adenylyl cyclase is preferably determined by immunoassay methods that use ligands (monoclonal or polyclonal antibodies) that specifically bind an activated (phosphorylated) MAPK, cAMP, or adenylyl cyclase.

Functional coupling of T1Rs and T2Rs by Gi proteins, and cells-based assays for the identification of T1R and T2R modulators

Automated measurement apparatus and methods for automated drug screening procedures are provided. The apparatus is capable of initiating and measuring rapid or transient events, such as cell receptor and/or ion channel activity. In particular, the apparatus is capable of aligning with predetermined positions, one or more samples contained in a multi-well container, initiating the reaction with reagent addition and measuring a resultant attribute for a period of time. The apparatus can substantially continuously measure and record data corresponding to the measured attribute before, during and after initiation of the reaction so that a time course of the rapid or transient event may be determined. After the reaction(s) are complete in one or more wells of the multi-well container, the apparatus can align one or more different wells to be assayed with the predetermined position and repeat the cycle until a predetermined number of wells are assayed. Automated drug screening methods employing the apparatus are also provided for screening for compounds which activate, inhibit or potentiate cellular ion channel or receptor activity. The assays include fluorescent indicator-based assays that use viable cells containing effective levels of a fluorescent indicator which is responsive to changes in ion concentration. Activation of the ion channels or receptors is initiated in the automated measurement apparatus by injection into one or more predetermined cell-containing wells of a reagent which is a known or putative activator of the ion channels or receptors of interest. The resulting activity of the ion channels or receptors, which causes changes in ion concentration in the cytoplasm, is determined by measurement of fluorescence intensity changes of the indicator in response to an excitation wavelength.

Automated analysis equipment and assay method for detecting cell surface protein and/or cytoplasmic receptor function using same

Automated drug screening method and a method of studying receptor and ion-channel activity are provided.

Automated analysis equipment and assay method for detecting cell surface

Automated assays for detecting and measuring ion channel and cell surface receptor activity and for identifying compounds that modulate or potentiate such activity are provided. Among the assays are fluorescent indicator-based assays that use cells containing effective levels of a fluorescent indicator that is responsive to changes in ion concentration. Activation of the ion channels or receptors is initiated in the automated measurement apparatus by injection into one or more predetermined cell-containing wells of a reagent which is a known or possible activator or inhibitor of the ion channels or receptors of interest. The resulting activity of the ion channels or receptors, which causes changes in ion concentration in the cytoplasm, is determined by measurement of fluorescence intensity changes of the indicator in response to an excitation wavelength.

Automated assays and methods for detecting and modulating cell surface

Human neuronal nicotinic acetylcholine receptor compositions and methods

Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.

Assay methods and compositions for detecting and evaluating the

Recombinant cells are provided which are useful for assaying compounds for their agonist or antagonist activity with respect to ion channels and/or cell surface-localized receptors. In addition, assay methods employing the invention recombinant cells are provided.

Assay methods and compositions useful for measuring the transduction of

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San Diego, CA, United States of America

Paul Brust