GPCR Activation Response
John Gatfield, Ph.D., senior lab head of molecular biology at Actelion Pharmaceuticals, described the use of his company’s technology to characterize coupling pathways of a prototypic GPCR activation response in recombinant and nonrecombinant cells. GPCRs comprise one of the largest classes of drug targets; about 50% of all marketed drugs act directly or indirectly on this receptor family. These receptors are the focus of multiple drug discovery efforts currently under way in pharmaceutical companies.
Dr. Gatfield described cell-based assays that use impedance measurements to analyze GPCR activation response patterns in recombinant and nonrecombinant cells. Cellular parameters such as attachment, spreading growth, death, and morphology cause impedance change in response to ligand binding and receptor pathway activation.
Acea Biosciences’ xCELLigence System, developed with Roche, consisting of a microelectronic biosensor array incorporated into each well of 16 and 96 well microplates, enables cell activity measurements in cell monolayers without labels or reporters. The system measures activity in real time, providing instantaneous readings of experimental results and improving the ability to identify cellular changes that reflect response of the cells to certain stimuli (added drugs, hormones, etc.).
Measurements analyze the interaction of living cells with the microelectronic sensor array. The system detects and quantitates changes in electrical impedance as the living cells interact with and electrically insulate the biocompatible microelectrode surface in the microplate well. The degree of electrical insulation is then converted by an algorithm to a specific parameter, the Cell Index, which can be used to characterize cellular activities including cell proliferation, cytotoxicity, adhesion, receptor tyrosine kinase activity, and G-coupled protein pathway responses. The cell index is directly proportional to the cell layer induced electrical impedance.
The system can be applied to functional monitoring of GPCR linked to different activation pathways; the sensitivity of the assay parallels those of standard assays such as calcium and cAMP measurements. As a label-free technology it theoretically allows for the simultaneous analysis of multiple pathways in one experimental well.
Indeed, one especially interesting use of the technology is the dissection of GPCR signaling in recombinant and nonrecombinant cells in response to natural ligands or synthetic compounds. As an illustration, Dr. Gatfield presented the response pattern of a Gq- and Gs-coupled GPCR to its natural ligand as being biphasic. By using specific pathway inhibitors of the Ca2+ pathway or the cAMP pathway he could attribute the two response phases to the two different coupling pathways.
“As one potential application in the pharmaceutical industry, this multiplexing of several GPCR signaling pathways in one assay format would allow a rapid characterization of full agonists versus biased agonists after a screening campaign,” Dr. Gatfield added.