GPCRs are a tempting target for cell-based assays. Clay Scott, Ph.D., associate director for lead generation at AstraZeneca, discussed his company’s pursuit of assays that will identify the ligands that bind to this family of transmembrane receptors. GPCRs play a critical role in the detection of molecular signals from outside the cell that activate signal-transduction pathways and cellular responses.
Dr. Scott and his colleagues compared optical- and impedance-based biosensors for their ability to detect ligand binding to the GPCRs. According to Dr. Scott, impedance-based assays measure changes in electrical impedance (roughly equivalent to resistance) relative to a voltage applied to a cell monolayer.
Optically based methods, on the other hand, quantify the shift in wavelength of reflected light that occurs due to the refractive properties of the biomass. As the morphology and mass redistribution of the cell changes in response to GPCR-ligand driven dynamics, these events can be detected by the two technologies. However, the AstraZeneca team investigates questions of the suitability these approaches for drug and pharma discovery.
In comparisons of GPCR agonists and antagonists, Dr. Scott noted that, in general, similar values were obtained with optical- or impedance-based assays. Both platforms provide sensitive, label-free precise measurement of these agents’ effects on cellular-response. However, using the impedance-based CellKey assay system (MDS Analytical Technologies), novel data was generated, including temporal responses that distinguish Gi, Gq, and Gs signaling.
Johannes Pschorr, Ph.D., European application scientist for MDS Analytical Technologies, also discussed the CellKey platform. “The system measures impedance changes occurring in response to stimulation or activation of signaling pathways within the cell. A monolayer of cells is seeded into a custom microplate that contains electrodes patterned at the bottom of each well. The CellKey system applies small voltages across a range of frequencies, measuring changes due to transcellular and extracellular current, reported kinetically for each well.”
Dr. Pschorr’s team is confident that these physiological changes occur as a direct result of signaling-pathway activation. The platform offers the sensitivity to measure functional activity of endogenous targets in live cells, as opposed to being limited to detecting overexpressed recombinant expression systems, he said. Additionally, he reported that the platform has the ability to measure any cell type—adherent and nonadherent cell lines and primary cells.
Analyzing receptors that are expressed with the appropriate accessory proteins gives researchers a picture of receptor function in a physiologically relevant environment. This flexibility allows researchers to conveniently screen a wide variety of targets with a single instrument platform, which helps standardize data to support lead compound selection.
One of the significant challenges Dr. Pschorr faces is the complexity of GPCR pathways and their interdependencies. “When screening for receptor modulators, we need to use methods that are as close to the actual cellular response as possible. Trying to interpret results from cells that are not physiologically relevant can lead to less-than-optimal lines of investigation to pursue.”
According to Dr. Pschorr, combining the ability to measure a whole live-cell assay with the ability to measure multiple signal pathways within the cell provides a more informative data result. He predicted that the measurement of integrated response of whole cells rather than measurement of one specific point in one specific pathway will lead to a more thorough understanding of complex receptor activity and compound mechanisms of action.
“The ability to do so in a label-free manner also provides an advantage, both in enabling the universality of the measurement and in allowing researchers to have a robust system to easily weed out nonspecific effects due to labels of the currently employed technologies.”