Multiplex GPCR Assay in Reverse Transfection Cell Microarrays

Abstract

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor:ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists. (Journal of Biomolecular

Cell microarrays and RNA interference chip away at gene function

“…Since this work was done, a handful of overexpression cell microarray studies have been published that offer technological improvements on the basic reverse transfection principle 6,[23][24][25][26] . These studies have so far been limited to assay development or smaller screens.…”

“…Although several groups have demonstrated this capability with overexpression cell microarrays 6,24 , these experiments used arrays printed in the bottom of 12-well plates or in chamber slides. Larger microscope slides fitted with cell-culture chambers will be needed to maximize the printable area on the slides in order to array large sets of RNAi reagents that can be analyzed in real time.…”

“…In this method, nucleic acids (expression plasmid or RNA) are printed on a standard glass microarray slide and used to transfect cells with the help of a transfection reagent 5,6 . The addition of cultured cells in medium to the array forms a living cell microarray of locally transfected cells in a lawn of nontransfected cells.…”

Drug Screening Using Cell Lines: Cell Supply, High‐Throughput and High‐Content Assays

“…An alternative is to infect the cells with Semliki Forest virus (SFV) vectors [24] or adenoviruses (QBiogen) with a broad host range. The transient transfection of every single well of an assay plate requires a long development time, is work-intensive, and tends to be less reproducible, though it can be improved by using automated cell cultivation and plating systems [25].…”

A platform for the advanced spatial and temporal control of biomolecules

“…Cells that attach and grow over this DNA array take up and express the DNA creating regions of localised transfection within a lawn of non-transfected cells 16 . Using such an approach a number of studies have been conducted including the screening of siRNAs and shRNAs for effective silencing of target genes by RNAi, the assignment of the position of subcellular localised proteins, screening for agonists and antagonists of G-protein coupled receptors and screening for gene products that are involved with kinase signalling pathways, stimulation of apoptosis or cell-cell adhesion [16][17][18][19] . Despite the successful application of TCMs a number of inherent limitations remain unsolved.…”