Development of multiplexed microarray binding assays for high-throughput drug discovery

The ability to combine primary hit identification assays with target profiling would significantly streamline the current drug discovery process. Working towards this end, we report here the development of a microarray-based ligand binding assay that supports multiplexed analysis of G protein-coupled receptor systems in a 96-well microplate format that is compatible with the equipment and infrastructure typical of high-throughput screening laboratories. A prototype microarray was generated by pin-printing seven different receptors within the wells of a specially coated glass-bottom microplate and assaying with a cocktail of fluorescent ligands. Development of the multiplexed system included optimization of methods for depositing receptor membrane proteins and establishing a generic set of assay conditions that simultaneously satisfied the pharmacology requirements of all of the receptor systems included on the array. The multiplexed system is shown to produce valid pharmacological results as evidenced by its ability to report K(i) values for receptor-specific fluorescent ligands and rank ordered potencies for diagnostic displacing compounds comparable to values generated by conventional simplexed assays. Moreover, the results of a 40-compound mini-screen confirmed that the assay accurately identifies valid hits. The results suggest the assay may be immediately suitable for routine profiling tasks and demonstrate the potential of the format for high-throughput multiplexed drug discovery.