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Multiplex GPCR Assay in Reverse Transfection Cell Microarrays

Akceli, Inc., Medford, MA

Christopher J. Wilson

Akceli, Inc., Medford, MA

Linda Bruett

Akceli, Inc., Medford, MA

Jesse J. Smith

Akceli, Inc., Medford, MA

Chatanika Stoop-Myer

Akceli, Inc., Medford, MA

Sena Jong

Akceli, Inc., Medford, MA

Lizabeth P. Amaral

Akceli, Inc., Medford, MA

Robin Pedersen

Akceli, Inc., Medford, MA

Susan K. Lyman

Akceli, Inc., Medford, MA

Vic E. Myer

Akceli, Inc., Medford, MA

Brent L. Kreider

Akceli, Inc., Medford, MA

Craig M. Thompson

Akceli, Inc., Medford, MA

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.

Key Words: reverse transfection • cell microarray • multiplexed assay • GPCR drug discovery • GPCR de-orphaning

Journal of Biomolecular Screening, Vol. 9, No. 3, 196-207 (2004)
DOI: 10.1177/1087057103261880


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