Unattended Drug Discovery Screening Workflow in a 384-Well Automated Patch Clamp System

Abstract

Automated patch clamp (APC) electrophysiology is an established and accepted technique in most safety testing laboratories. Recently published cross-site studies [1] highlight the need for standardized protocols for reliable results, for example, for Nav1.5 recordings. A standardization of protocols should be backed up by a robust screening assay before a screening workflow is implemented in a lab. Challenging state-of-the-art APC instrumentation with full-day unattended runs and probing them for reliable results was the goal of this study. We used a previously validated assay methodology [2] and challenged it with significant HTS deliverables (success rate, number of data points, Z′). Experimental runs were executed and 4-point dose-response curves of 7 reference compounds, amongst them bepridil and verapamil, were recorded on Nav1.5 in CHO cells. Positive and negative controls were also performed to determine false positive and negative rates and Z’ values were calculated to determine reliability of the assay. Rundown of 0.3% DMSO as vehicle control solution was found to be 5.5 ± 0.3% (n = 364 wells) and success rates for completed experiments were > 93% for every plate. In a full working day 224 IC50s derived from 112 full concentration response curves (4 point) with n = 6 replicates were generated. Z’ values of 0.91 ± 0.004 for a 1st peak Nav amplitude and 0.89 ± 0.006 for the 2nd peak identifying state-dependent inhibition prove low variability of the data. Our statistical measures of assay performance over a typical full working day in a HT ion channel screen showed that the 384-well APC system used in our study (SyncroPatch 384i) delivers reproducible and reliable results for the human Nav1.5 channel. [1] Kramer et al. Sci Rep 10 (2020). https://doi.org/10.1038/s41598-020-62344-w [2] Brinkwirth et al., JPTM (2020), https://doi.org/10.1016/j.vascn.2020.106884