IonWorks Barracuda Assay for Assessment of State-Dependent Sodium Channel Modulators

Abstract

Voltage-gated sodium channels represent important drug targets. The implementation of higher throughput electrophysiology assays is necessary to characterize the interaction of test compounds with several conformational states of the channel, but has presented significant challenges. We describe a novel high throughput approach to assess the effects of test agents on voltage-gated sodium currents. The multiple protocol mode of the automated electrophysiology instrument IonWorks Barracuda was used to control the level of inactivation and monitor current stability. Good temporal stability of currents and spatial uniformity of inactivation were obtained by optimizing the experimental conditions. The resulting assay allowed for robust assessment of state-dependent effects of test agents and enabled direct comparison of compound potency across several sodium channel subtypes at equivalent levels of inactivation.

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors

“…Fifty compounds, or just under one-sixth, were not confirmed by either platform. Since compound potency on IWB correlated well with manual patch clamping for Nav inhibitors, 21 we conclude that the Optopatch platform has lower sensitivity but retains high specificity. Sensitivity is here defined as the proportion of active hits that are correctly identified as such in the assay, while specificity is defined as the proportion of negative compounds that are correctly identified as such in the assay.…”

“…The IWB assay for Nav1.7 had an average plate Z′ of 0.6, with 99.2% of all wells passing criteria of a minimum 25 MΩ seal resistance and ≥0.5 nA peak current size cutoff. Reported Nav1.7 automated electrophysiology assays on the IWB, 21 IWQ, 11 Qube, 12 and SynchroPatch 22 utilized variations of a two-pulse protocol to measure compound activity at closed and/or inactivated states. Another Nav1.7 automated electrophysiology assay on the IW-HT platform used a use-dependent voltage protocol where cells were repeated depolarized to 20 mV.…”

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE

“…IonWorks Barracuda was launched in 2010 and has been reported for compound screening on hERG, Ca V 2.2 and Nav channels [16–18]. Different from the other two systems, Barracuda uses perforated patch configuration, and the averaged seal resistance was ~120 MΩ for single-hole mode and ~35 MΩ for population patch mode (64 holes).…”

Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors

“…To identify subtype-selective chemical scaffolds, large libraries of compounds, sometimes exceeding millions of individual compounds, are screened against the targets of interest (e.g., Nav1.7), followed by selectivity tests on other isoforms (e.g., Nav1.5). The effects of compounds are determined using in vitro assays such as electrophysiology ( 17 – 19 ) or fluorescence-based membrane potential assays ( 20 – 22 ). The major considerations for assay design have been signal robustness, throughput, and cost, whereas mechanisms of action, such as drug binding sites and molecular basis for selectivity, are often not taken into consideration.…”