State-Dependent Compound Inhibition of Na v 1.2 Sodium Channels Using the FLIPR V m Dye: On-Target and Off-Target Effects of Diverse Pharmacological Agents

Abstract

Voltage-gated sodiumchannels (NaChs) are relevant targets for pain, 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 conjunctionwith 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 _v1.2NaCh. Channels were opened by addition of veratridine, and Vm 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 Ki but not K _r.FLIPRIC50 values fellwithin 0.1-to 1.5-fold of EPKi 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.