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 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 (Vm) 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 Vm assay of rat Nav1.2 NaCh. 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 (Kr)and inactivated state binding constant (Ki)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not Kr. FLIPRIC50 values fell within 0.1-to 1.5-fold of EP Ki 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.
Benjamin
Elfrida R
ER
Purdue Pharma L.P., 6 Cedarbrook Drive, Cranbury, NJ 08512, USA. Elfrida.Benjamin@pharma.com
Pruthi
Farhana
F
Olanrewaju
Shakira
S
Ilyin
Victor I
VI
Crumley
Gregg
G
Kutlina
Elena
E
Valenzano
Kenneth J
KJ
Woodward
Richard M
RM
D001681:Biological Assay; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D015640:Ion Channel Gating / Q000187:drug effects*; D008564:Membrane Potentials / Q000187:drug effects; D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels; D014701:Veratridine / Q000494:pharmacology
D005456:Fluorescent Dyes; D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C568251:SCN2A protein, human; C568253:Scn2A protein, rat; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D014701:Veratridine