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

¹ Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada.; Centre for Addiction and Mental Health, Toronto, Ontario, Canada.
² Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada.
³ Centre for Addiction and Mental Health, Toronto, Ontario, Canada.
⁴ DiscoveRx, Freemont, CA.
⁵ Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada.; Department of Medicine, University of Toronto, Toronto, Ontario, Canada.; Centre for Addiction and Mental Health, Toronto, Ontario, Canada.

^* To whom correspondence should be addressed. E-mail: brian.odowd{at}utoronto.ca.

	Abstract

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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