G-proteins are important binding proteins from physiological and a drug discovery standpoint. They mobilize a variety of GPCR-mediated signal transduction pathways. cAMP and Ca2+ mobilization assays were the classical assays to measure GPCR binding and activation. Kinase mobilization, gene transcription, and regulation of signal transduction are some recently developed assays that are being increasingly used in a cellular context to determine not just GPCR binding but also help assess receptor function.
Richard Eglen, Ph.D.,vp and general manager of discovery and research reagents for PerkinElmer’s Life & Analytical Sciences (www.perkinelmer.com), and Beth Rayl, Ph.D., scientific and exeution marketing manager at Perkin Elmer, discussed various cellular screening technologies for G protein-coupled receptors.
“Cellular models provide a rich data set. The availability of advanced detection systems, improved reagents, and automated robotic workstations has eased the running of cellular GPCR assays in an HTS mode. It has also enabled several companies to perform GPCR screening at the primary screening stage of their drug discovery program,” commented Dr. Eglen.
PerkinElmer offers a diverse portfolio of cellular screening technologies for GPCR assays that address receptor-ligand, phospho-ERK quantification, cAMP measurements, reporter gene assays, IP3 quantification, GTP binding, and novel Ca2+ mobilization detection assays. Dr. Rayl’s discussion profiled a Flash luminescence aequorin technology coupled to the company’s LumiLux Cellular screening platform and described the Janus™ Cellular Workstation for use in multiple cellular GPCR assays.
The Flash luminescence aequorin assay, developed at Euroscreen (www.euroscreen.com), is a cellular assay that measures changes in intracellular Ca2+ . It is based on the principle that the apo-enzyme ApoAequorin is converted to active enzyme Aequorin in presence of coelenterazine. Upon Ca2+ binding, Aeqourin oxidizes coelenterazine into coelenteramide resulting in flash luminescent light emission, which is measured using LumiLux.
Calcium mobilization data generated using a CHO cell line expressing recombinant aequorin and the G-protein coupled receptor was presented. Results of 384-well format correlated well with the 1,536 cellular assay format run with 1,500 cells per well.
The data also has excellent correspondence to data generated with traditional fluorescent Ca2+ assays. The assay can be run with suspension or adherent cells and can be performed in one day.
“A key differentiator of the flash luminescence technology compared to the glow luminescence technology is that the signal is intense as it is expressed over a short span of time. This high signal intensity allows one to look at partial activators and allosteric inhibitors, thus providing a wider range of potential drug compounds. When compared to traditional fluorescent-based Ca2+ assays, the flash luminescence signal is 20 to 30 times higher, which is a large window to assay for novel compounds that modulate a signaling pathway,’’ explained Dr. Eglen.
Dr. Eglen also described in detail PerkinElmer’s Janus Cellular Workstation that enables complete assay automation. The workstation comes with prevalidated templates and data for Lance™ cAMP, britelite™, steadylite™ HTS, ATPlite™ and the AlphaScreen™ SureFire cellular kinase cellular assays performed on CHO cells engineered with appropriate receptors.