An Overview of High Throughput Screening at G Protein Coupled Receptors

Abstract

Technologies used for high throughput screening (HTS) at G protein coupled receptors (GPCRs) comprise two major approaches; those generally conducted measuring signal intensity changes using a microtiter plate format, and those measuring cellular protein redistribution via imaging-based analysis systems. Several homogeneous assays, i.e. those without wash and fluid phase separation steps, measure changes of second messenger signaling molecules including cAMP, Ins P 3 and calcium. Imaging based assays determined the translocation of GPCR associated proteins such as β arrestin, or internalization of the receptor labeled with fusion tags. Generally, the former assays are used in a primary screening campaign, whilst the latter are used in secondary screening and lead optimization. However, increasing use of automated confocal imaging systems and prevalence of modified cell lines has expanded use of protein redistribution assays. Finally, radiometric techniques are widely used, frequently to measure GPCR ligand binding, using a scintillation proximity assay format. In this paper, the various assay methods used for HTS at G protein coupled receptors are compared and contrasted. G PROTEIN COUPLED RECEPTORS AND HIGH THROUGHPUT SCREENING G protein coupled receptors (GPCRs) are a proven class of targets for drug discovery and are frequent targets entering high throughput screening (HTS) laboratories. Modern HTS is a highly automated approach to compound identification using robotic, fluid dispensing systems and sensitive signal detection instruments [1]. Several assay techniques for GPCR screening employ non-radiometric assay platforms. Radiometric techniques dominate in assays in which ligand binding to the GPCR per se is measured, [2]. There is increased adoption of functional assays in which the effects of the activated GPCR on function are determined, with concomitant development and implementation of cell based assay technologies, and automated cell culture and dispensing [3]. Compounds active at GPCRs have therapeutic benefit in many diseases ranging from central nervous system disorders, including pain, schizophrenia and depression, and metabolic disorders, such as cancer, obesity or diabetes [5]. GPCRs are considered a highly 'druggable' class of proteins, with over 40% of marketed drugs (such as Zyprexa, Clarinex, Zantac and Zelnorm) acting to modulate their function. Interestingly, approximately 9% of global pharmaceutical sales are realized from drugs targeted

Discovery and Lead Optimization of Atropisomer D1 Agonists with Reduced Desensitization

“…Available literature suggested that potent and even modestly selective activators of D1 that lack the catecholamine motif are rare, although recent reports by Bristol-Myers Squibb and Astellas Pharma have disclosed the discovery of non-catecholamine D1-selective positive allosteric modulators (PAMs). In our effort to identify novel activators of this recalcitrant target, we opted to run a high-throughput screen (HTS) . The HTS was run using cAMP homogeneous time-resolved fluorescence (HTRF) kits from Cisbio.…”