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Unveiling drug selectivity via functional profiling and multi-prong approach to hit validation using ChemiScreen™ GPCR cell lines |
| FLIPR® and aequorin technologies have become the systems of choice for measuring the changes in intracellular calcium in a high throughput manner. They both provide rapid and sensitive read-out for many GPCR drug targets. Not all GPCRs, however, couple to Gq leading to calcium mobilization. We have developed a novel cell- based assay to make all GPCRs signal through calcium mobilization using cell lines with endogenous promiscuous G proteins and CRAC channel. It eliminates the need and problem associated with co- transfecting either promiscuous or chimeric G proteins. The functional readout of each GPCR expressed in this system has been validated for over 150 targets of different G protein coupling status, and compared to the binding assay and native readouts using small molecule agonists, antagonists, and allosteric regulators. Case study and SAR cascade are presented using chemokine receptor CXCR2 as an example. To further validate the screening hits from a FLIPR assay we compared the result to a conventional radioligand filtration binding assay and many other different functional assays using ChemiScreen transfected cell line and native cell line expressing CXCR2. Here we evaluated the activities for known compounds to be selective for the CXCR2 receptor, SB 225002 and SB 265610. Also, we evaluated the activity of non-selective compounds for CXCR2 but are selective for other chemokine receptors, RS 102895 (CCR2b), RS 504393 (CCR2), and UCB 35625 (CCR1 and CCR3). Sample activity across different assay platforms and preserving sample rank order is presented to illustrate the versatility and multi-purpose applications for Millipore’s GPCR product line. Click the PDF above to view the entire poster. |