title: High-Throughput Screen of GluK1 Receptor Identifies Selective Inhibitors with a Variety of Kinetic Profiles Using Fluorescence and Electrophysiology Assays.
abstract: GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC50 values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.
authors.affiliation: Screening and Protein Sciences, Merck Research Laboratories, North Wales, PA, USA.
authors.forename: Kelli
authors.initials: K
authors.lastname: Solly
authors.affiliation: Pain & Migraine Pharmacology, Merck Research Laboratories, West Point, PA, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klein
authors.affiliation: Exploratory Chemistry, Merck Research Laboratories, West Point, PA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Rudd
authors.affiliation: Structural Chemistry, Merck Research Laboratories, West Point, PA, USA.
authors.forename: M Katharine
authors.initials: MK
authors.lastname: Holloway
authors.affiliation: Screening and Protein Sciences, Merck Research Laboratories, North Wales, PA, USA Wuxi AppTec, Plainsboro, NJ, USA.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Johnson
authors.affiliation: Pain & Migraine Pharmacology, Merck Research Laboratories, West Point, PA, USA.
authors.forename: Darrell
authors.initials: D
authors.lastname: Henze
authors.affiliation: Screening and Protein Sciences, Merck Research Laboratories, North Wales, PA, USA Michael_Finley@merck.com.
authors.forename: Michael F A
authors.initials: MF
authors.lastname: Finley
mesh_terms: D057205:Automation, Laboratory; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D044463:Receptor, PAR-1 / Q000378:metabolism; D018092:Kainic Acid Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D010975:Platelet Aggregation Inhibitors
chemical_list: D044463:Receptor, PAR-1; D018092:Kainic Acid Receptors; D016194:Receptors, N-Methyl-D-Aspartate; D054852:Small Molecule Libraries; D010975:Platelet Aggregation Inhibitors; C121001:NR2B NMDA receptor
keywords: GluK1; electrophysiology; high-throughput screening