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Using Ligand-Induced Conformational Change to Screen for Compounds Targeting G-Protein-Coupled Receptors

Departments of Pharmacology, University of Toronto, Toronto, Ontario, Canada; Centre for Addiction and Mental Health, Toronto, Ontario, Canada

Mohammad Alijaniaram

Xiaodong Ji

Departments of Pharmacology, University of Toronto, Toronto, Ontario, Canada

Tuan Nguyen

Centre for Addiction and Mental Health, Toronto, Ontario, Canada

Richard M. Eglen

DiscoveRx, Freemont, CA

Susan R. George

Departments of Pharmacology, University of Toronto, Toronto, Ontario, Canada; Centre for Addiction and Mental Health, Toronto, Ontario, Canada

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.

Key Words: G-protein-coupled receptor • green fluorescent protein • nuclear localization sequence • confocal microscopy • enzyme fragment complementation

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