State-dependent compound inhibition of Nav1.2 sodium channels using the FLIPR Vm dye:: On-target and off-target effects of diverse pharmacological agents

Voltage-gated sodium channels (NaChs)are relevant targets forpain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (V-m) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus. the authors have developed a FLIPR V-m assay of rat Na(v)1.2 NaCh. Channels were opened by addition of veratridine, and V-m dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (K-r) and inactivated state binding constant (K-i) obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with K-i but not K-r. FLIPR IC50 values fell within 0.1 - to 1.5-fold of EP K-i values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.