Multiplexing Label-Free and Fluorescence-Based Methods for Pharmacological Characterization of GPCR Ligands
Cell-based assays are essential to drug discovery and biomedical research. Most cell-based assays have been targeting specific components of signaling pathways with considerable mechanistic significance. G protein-coupled receptors (GPCRs) represent a major class of drug targets. In addition to G protein-dependent pathways, G protein-independent signaling mechanisms such as β-arrestin pathways, allosteric modulation of receptor function, and receptor oligomerization are among currently active research areas. High-throughput calcium- and membrane potential-based assays have been widely used as efficient screening platforms. The Hamamatsu FDSS7000 instrument is capable of simultaneous compound addition and fluorescence monitoring in 96- or 384-wells for fluorescence-based calcium or membrane potential assays. However, phenotypic or holistic cellular measurements of combined effects contributed by multiple signaling pathways may also be required to complement label-based assays that target specific signaling events. For this purpose, the effects mediated by GPCRs may be measured by dynamic mass redistribution (DMR) through Resonant Waveguide Grating (RWG) biosensors embedded in 384-well plates using a Corning EPIC BT label-free assay system. Herein, we describe experimental protocol for profiling ADX88178, a potent and selective positive allosteric modulator (PAM) of the metabotropic glutamate 4 receptor (mGluR4) using both Ca2+ and DMR phenotypic readouts, and discuss the complementary features of each assay type. Further, we demonstrate, for the first time, a unique integrated procedure multiplexing FDSS calcium mobilization and EPIC label-free assays using a single set of cell/compound plates.
Key wordsDynamic mass redistribution Functional drug screening system G protein-coupled receptor Label-free Resonant waveguide grating
The authors thank Drs. Shouming Du, Yiwen Wang, Alex Sanchez, and Al McGrath of Hamamatsu Corporation, and Drs. Hung Cuong Louie Tran and Ye Fang of Corning Incorprated for technical assistance. We are also grateful to Mr. Michael Collins and Noel J. Boyle for technical support.
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