title: Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers.
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.
authors.affiliation: Abbott GmbH & Co. KG, Germany. mario.mezler@abbott.com
authors.forename: Mario
authors.initials: M
authors.lastname: Mezler
authors.forename: David
authors.initials: D
authors.lastname: Hermann
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Swensen
authors.forename: Andreas
authors.initials: A
authors.lastname: Draguhn
authors.forename: Georg C
authors.initials: GC
authors.lastname: Terstappen
authors.forename: Gerhard
authors.initials: G
authors.lastname: Gross
authors.forename: Hans
authors.initials: H
authors.lastname: Schoemaker
authors.forename: Gail
authors.initials: G
authors.lastname: Freiberg
authors.forename: Steve
authors.initials: S
authors.lastname: Pratt
authors.forename: Sujatha M
authors.initials: SM
authors.lastname: Gopalakrishnan
authors.forename: Volker
authors.initials: V
authors.lastname: Nimmrich
mesh_terms: D002121:Calcium Channel Blockers / Q000032:analysis* / Q000494:pharmacology; D020865:Calcium Channels, P-Type / Q000378:metabolism*; D020907:Calcium Channels, Q-Type / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D018408:Patch-Clamp Techniques / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods; D014162:Transfection
chemical_list: D002121:Calcium Channel Blockers; D020865:Calcium Channels, P-Type; D020907:Calcium Channels, Q-Type