title: High-throughput screening for Kv1.3 channel blockers using an improved FLIPR-based membrane-potential assay.
abstract: Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates.
authors.affiliation: Department of Screening Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA. liuk2@wyeth.com
authors.forename: Kun
authors.initials: K
authors.lastname: Liu
authors.forename: Manoj
authors.initials: M
authors.lastname: Samuel
authors.forename: Jeff
authors.initials: J
authors.lastname: Tillett
authors.forename: James K
authors.initials: JK
authors.lastname: Hennan
authors.forename: Belew
authors.initials: B
authors.lastname: Mekonnen
authors.forename: Veronica
authors.initials: V
authors.lastname: Soloveva
authors.forename: Richard K
authors.initials: RK
authors.lastname: Harrison
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Paslay
authors.forename: James
authors.initials: J
authors.lastname: Larocque
mesh_terms: D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004594:Electrophysiology; D005470:Fluorometry; D057166:High-Throughput Screening Assays; D020128:Inhibitory Concentration 50; D051664:Kv1.3 Potassium Channel / Q000037:antagonists & inhibitors*; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D019810:Sodium Azide / Q000494:pharmacology
chemical_list: D051664:Kv1.3 Potassium Channel; D026902:Potassium Channel Blockers; D019810:Sodium Azide