Applera Corporation

Tropix, Inc.

Michelle A J Palmer

Melissa Gee

Xiao-jia Chang

Bonnie Tillotson

Irena Y Bronstein

John C Voyta

Anthony C Chiulli

Neil Carlson

Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR/ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.

Systems for sensitive detection of G-protein coupled receptor and orphan receptor function using reporter enzyme mutant complementation

Methods for detecting G-protein coupled receptor (GPCR) activity; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described.

Receptor function assay for G-protein coupled receptors and orphan receptors by reporter enzyme mutant complementation

A method and apparatus for displaying data presents results from a single assay across multiple wells and plates on a single screen in a format that preserves the spatial relationship between the wells on a plate and the sequential relationship between plates. Also provided to the user is the ability to display these results from multiple wells in a color coded format corresponding to a binary (e.g. good/bad) or quadripartite (e.g. good/marginally good/marginally bad/bad) division. Also displayed are plate-wide statistics, such as signal-to-noise ratio and variability, for multiple plates on a single screen so that problem plates can be readily identified. The ability for a user to view a summary of performance on a well-by-well basis across all compounds/plates is also provided to aid the user in detecting general trends, including the possible existence of mechanical errors. Finally, the user is also provided with the ability to select or deselect plates and assays for review in the above formats.

Data display software for displaying assay results

A competitive assay for the concentration of a cyclic nucleotide such as cyclic adenosine monophosphate combines, in a reaction chamber provided with a capture antibody, an antibody for the cAMP or other cyclic nucleotide, the sample to be assayed and a conjugate of cAMP and alkaline phosphatase. The mixture is incubated and washed, and after washing, a 1,2-dioxetane which is caused to decompose when contacted by the alkaline phosphatase of said conjugate is added. The light emission caused by decomposition is measured, with the strength of the signal being inversely related to the concentration of the cyclic nucleotide. Optionally, an enhancement agent in the form of a polymeric onium salt may be added, to further enhance the light emission.

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Arlington, MA, United States of America

Michelle A J Palmer