G-protein-coupled receptor microarrays for multiplexed compound screening.

Conventional assay methods for discovering and profiling drug-target interactions are typically developed on a target-by-target basis and hence can be cumbersome to enable and orchestrate. Herein the authors report a solid-state ligand-binding assay that operates in a multiplexed mode to report compound activity against a micorarray-configured panel of G-protein-coupled receptor (GPCR) targets. The pharmacological fidelity of the system is high, and its miniaturized "plug-and-play" format provides improved efficiency both in terms of execution time and reagent consumption. Taken together, these features make the system ideally suited to explore the structure-activity relationship of compounds across a broad region of target class space.

Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays.

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.

A reporter system for reverse transfection cell arrays.

The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conventional gene discovery approaches in the pharmaceutical industry. High-throughput approaches for studying gene function in vivo are greatly needed. One potential answer to this challenge is reverse transfection, a high-throughput gene expression method for examining the function of hundreds to thousands of genes in parallel. One limitation of reverse transfection technology is the need for posttransfection processing of the arrays to analyze the activity of the expressed proteins. The authors have investigated the use of a reporter construct cotransfected with other genes of interest to monitor and screen gene function on reverse transfection microarrays. They developed a serum response element (SRE) reporter linked to the green fluorescent protein (GFP) that is cotransfected with target genes on reverse transfection arrays for monitoring mitogen-activated protein (MAP) kinase signaling by multiple targets in parallel. The authors show that this reporter system is able to detect inhibition of upstream MAP kinase signaling proteins by the MEK inhibitor U0126. The ability to monitor the activity of multiple signaling proteins in a multiwell format suggests the utility of reverse transfection reporter arrays for high-throughput screening applications.

Functional GPCR microarrays.

This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.