Identification of Novel Kv1.3 Blockers Using a Fluorescent Cell-Based Ion Channel Assay

Abstract

A functional cell-based assay was developed using a generic proprietary assay protocol, based on a membrane-potential sensitive dye, for the identification of small-molecule antagonists against the Kv1.3 potassium ion channel. A high-throughput screen (HTS) was subsequently performed with 20,000 compounds from the Evotec library, preselected using known smallmolecule antagonists for both sodium and potassium ion channels. Following data analysis, the hit rate was measured at 1.72%, and subsequent dose-response analysis of selected hits showed a high hit confirmation rate yielding approximately 50 compounds with an apparent IC 50 value lower than 10 µM. Subsequent electrophysiological characterization of selected hits confirmed the initial activity and potency of the identified hits on the Kv1.3 target and also selectivity toward Kv1.3 through measurements on HERG as well as Kv1.3-expressing cell lines. Follow-up structure-activity relationship analysis revealed a variety of different clusters distributed throughout the library as well as several singlicates. In comparison to known Kv1.3 blockers, new chemical entities and scaffolds showing potency and selectivity against the Kv1.3 ion channel were detected. In addition, a screening strategy for ion channel drug discovery HTS, medicinal chemistry, and electrophysiology is presented. (Journal of Biomolecular Screening 2006:57-64)

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine

“…Membrane potential (⌬⌿) was evaluated using 3,3-dipentyloxacarbocyanine (DiOC 5 ), a charged lipophilic dye that emits a fluorescent signal proportional to ⌬⌿. The validity of this method to assess channel activity approaches the reliability of classical patch clamp methods, as borne out by a compelling body of evidence (8)(9)(10). Overall, hBD-2 and crotamine altered ⌬⌿ in several target organisms versus untreated controls (Fig.…”

Novel screening techniques for ion channel targeting drugs

“…For highthroughput screening efforts, indirect readout technologies such as fluorescence assays are still used, and combined with electrophysiological patch clamp compound characterization at later stages of compound development. 29,30 The demand for increasing throughput in electrophysiological screening, while maintaining high resolution data acquisition, however, calls for the introduction of new patch clamp devices with higher throughput. The SyncroPatch 384PE is an example of such a device.…”

Recombinant Kv Channels at the Membrane of Escherichia coli Bind Specifically Agitoxin2

“…Advanced drug screening techniques employed today for ion channels utilize intact cells bearing functionally active target protein (Slack et al 2006;Falconer et al 2002;Gill et al 2007;Murphy et al 2006;Baxter et al 2002). To achieve physiologically relevant results, cell-based assays are mostly used with eukaryotic cells.…”