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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.
Keywords: Automated patch-clamp, electrophysiology, FLIPR, high throughput screening, P/Q-type voltage-gated calcium channel, tetracycline-dependent expression
Combinatorial Chemistry & High Throughput Screening
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
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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.
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Cite this article as:
Mezler Mario, Hermann David, M. Swensen Andrew, Draguhn Andreas, C. Terstappen Georg, Gross Gerhard, Schoemaker Hans, Freiberg Gail, Pratt Steve, M. Gopalakrishnan Sujatha and Nimmrich Volker, Development and Validation of a Fluorescence-Based HTS Assay for the Identification of P/Q-Type Calcium Channel Blockers, Combinatorial Chemistry & High Throughput Screening 2012; 15 (5) . https://dx.doi.org/10.2174/138620712800194503
DOI https://dx.doi.org/10.2174/138620712800194503 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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