Title:Development and Validation of a Fluorescence-Based HTS Assay for the Identification of P/Q-Type Calcium Channel Blockers
Volume: 15
Issue: 5
Author(s): Mario Mezler, David Hermann, Andrew M. Swensen, Andreas Draguhn, Georg C. Terstappen, Gerhard Gross, Hans Schoemaker, Gail Freiberg, Steve Pratt, Sujatha M. Gopalakrishnan and Volker Nimmrich
Affiliation:
Keywords:
Automated patch-clamp, electrophysiology, FLIPR, high throughput screening, P/Q-type voltage-gated calcium
channel, tetracycline-dependent expression
Abstract: Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is
evidence that migraine, Alzheimer’s disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal
presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of
disease symptoms. Although the medical need is high, there are no such compounds on the market.
Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by
automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type
calcium channel under control of a tetracycline (Tet) promoter. The accessory β1.1 and α2δ1 subunits were co-expressed
constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed
functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium
channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the
identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 μM. Hit-to-lead (HTL) efforts
identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized.
An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the
HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp
analysis at inactivated channel state.
The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological
diseases.
