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

[0013] According to another aspect of the present invention, a method for reducing background signal due to non-specific binding of either a labeled-ligand or GTP-analogue to a substrate surface is described. In one embodiment the method comprises: a) providing a buffered solution containing a blocker reagent; b) applying the solution to an array of GPCRs; c) applying a second solution containing a labeled ligand or GTP-analogue, in either the absence or presence of a target compound; and d) monitoring or determining the binding of the labeled ligand to a receptor, or the GTP-analogue to a G-protein coupled with the receptor in the array. The method may further involve a washing and dry step before data acquisition. Alternatively, the method may comprise: a) providing a solution containing a blocker reagent and a labeled ligand or GTP-analogue, in either the absence or presence of a target compound; b) applying the solution to a microarray of GPCRs; and c) monitoring or determining the binding of the labeled ligand to a receptor, or the GTP-analogue to a G-protein coupled with the receptor in the microarray.

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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