Assay solution compositions and methods for GPCR arrays

Abstract

Buffered assay solutions for performing 1) binding or 2) functional assays on GPCR arrays, along with methods for their use are described. The buffered assay solution has an underlying composition having: a buffer reagent with a pH in the range of about 6.5 to about 7.9; an inorganic salt of either a monovalent or divalent species, at a concentration from about 1 mM to about 500 mM; and optionally a combination of: c) a blocker reagent at a concentration of about 0.01 wt. % to about 2 wt. % of the composition, or d) protease-inhibitor at a concentration of about 0.001 mM to about 100 mM. In an embodiment for functional assay uses, the composition is modified to also include a GTP-analogue, a guanosine 5′-diphosphate (GDP) salt, and / or an anti-oxidant reagent.

Description

FIELD OF INVENTION [0001] The present invention relates to biological assays. In particular, the invention includes compositions for buffer solutions used with binding and functional assays for GPCR arrays. A method is also included which utilizes negatively-charged polymers and / or water-soluble proteins to reduce the background of such assays and improve signal to background ratios. BACKGROUND [0002] G-protein-coupled receptors (GPCRs) are one of the most successful target proteins for drug discovery research to date. Approximately 50% of current drugs target GPCRs; about 20% of the top 50 best selling drugs target GPCRs; more than $23.5 billion in pharmaceutical sales annually are ascribed to medications that address this target class. (Drews, J., “Drug Discovery: A Historical Perspective”Science 2000, 287, 1960-1963; Ma, P., and Zemmel, R., “Value of Novelty”Nat. Rev. Drug Discov. 2002, v. 1, 571-572.) Forming a super-family of seven trans-membrane-spanning proteins that are expr...

AI Technical Summary

Benefits of technology

The present invention provides two embodiments of a buffered solution that can be used for multiplexed binding assays or functional assays. The buffered solution optimizes the binding profiles of target compounds and labeled ligands with GPCRs in a microarray. The solution includes a buffer reagent, an inorganic salt, a blocker reagent, a GTP-analogue, a GDP salt, an anti-oxidant reagent, or a combination of these components. The solution also includes a method for reducing background signal due to non-specific binding. The technical effects of the invention include improved accuracy and reliability of the binding assays and functional assays, as well as reduced background noise.

Problems solved by technology

Effective engineering of these drugs is, however, critical as aberrant binding to such a physiologically significant target class can lead to serious side effects.

Structural data on GPCRs is limited and rational drug design is a significant challenge.

Designing drugs that do not bind to non-targeted GPCRs is almost impossible.

Currently, selectivity studies are conducted downstream in the drug discovery process—discarding compounds because of adverse binding at this stage makes the drug discovery process both expensive and time consuming.

Despite the interest and the overwhelming number of current and future GPCR targets, few methods have been described for simultaneously studying multiple GPCRs.

Although, protein microarrays are naturally suited for testing compounds against multiple proteins simultaneously, some of the fundamental aspects of multiplexed bioassays using protein chips are yet to be fully demonstrated.

Problems due to non-specific binding of labeled ligands to the receptor microspots and the background surface, non-optimal interaction of target compounds and labeled ligands with the receptors in the arrays under the assay conditions, and the lack of general guidelines for assay buffer design and selection have deterred scientists from testing the feasibility of multiplexed binding assays for compound profiling and screening.

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