A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential-Sensitive Dye and FLIPR

Abstract

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR3 relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µ ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r2 = 0.56) between the respective IC50s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128). © 2009, Society for Biomolecular Sciences. All rights reserved.