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Journal of Biomolecular Screening
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Article

State-Dependent Compound Inhibition of Nav 1.2 Sodium Channels Using the FLIPR Vm Dye: On-Target and Off-Target Effects of Diverse Pharmacological Agents

Elfrida R. Benjamin*, Farhana Pruthi, Shakira Olanrewaju, Victor I. Ilyin, Gregg Crumley, Elena Kutlina*, Kenneth J. Valenzano*, Richard M. Woodward*

Purdue Pharma Discovery Research, Cranbury, NJ.

* To whom correspondence should be addressed. E-mail: Elfrida.Benjamin{at}pharma.com.


  Abstract

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 Vmassay of rat Nav1.2 NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50values 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.

Key Words: sodium, channel, membrane potential, inactivation, state dependent, fluorescent imaging plate reader

First published on October 18, 2005, doi:10.1177/1087057105280918

Journal of Biomolecular Screening 2006;11:29.

A more recent version of this article appeared on February 1, 2006

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