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

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey, vasylyd{at}wyeth.com

Qin J. Shan

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey

Yan T. Lee

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey

Veronica Soloveva

Chemical and Screening Sciences, Wyeth Research, Princeton, New Jersey

Stanley P. Nawoschik

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey

Edward J. Kaftan

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey

John Dunlop

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey

Scott C. Mayer

Chemical and Screening Sciences, Wyeth Research, Princeton, New Jersey

Mark R. Bowlby

'Discovery Neuroscience, Wyeth Research, Princeton, New Jersey

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 µM 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)

Key Words: HCN1 channel • membrane potential-sensitive dye • high-throughput screening • FLIPR • Ion Works

This version was published on October 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 9, 1119-1128 (2009)
DOI: 10.1177/1087057109345526


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