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.

The positive allosteric modulator BPAM344 and L‐glutamate introduce an active‐like structure of the ligand‐binding domain of GluK2

“…3A,B). However, for the characterization of PAMs, the previous use of the Ca 2+ influx assay utilized stably receptor‐expressing cell lines [28–33]. Therefore, to validate the use of transiently transfected GT‐HEK293 cells in the assay, we determined the potency of BPAM344 at transiently expressed GluA2( Q ) i in parallel with stably expressed GluA2( Q ) i and observed similar EC 50 values (transient expression: 1.28 ± 0.36 μ m , stable expression: 0.86 ± 0.19 μ m ; P = 0.292) that furthermore are similar to the previously determined EC 50 value (0.90 ± 0.10 μ m ) determined on rat primary brain cultures [7] (Fig.…”

Active-like structure of the ligand-binding domain of GluK2 with L-glutamate and the positive allosteric modulator BPAM344

“…However, for characterization of PAMs, the previous use of the Ca 2+ influx assay utilized stably receptor expressing cell lines [28][29][30][31][32]. Therefore, to validate the use of transiently transfected HEK293 cells in the assay, we determined the potency of BPAM344 at transiently expressed GluA2 in parallel with stably expressed GluA2(Q)i, and observed similar EC50 values (transient expression: 1.28 ± 0.36 µM, stable expression: 0.86 ± 0.19 µM; P = 0.292) that furthermore is similar to the previously determined EC50 value (0.90 ± 0.10 µM) determined on rat primary brain cultures [7] (Fig.…”

Novel screening techniques for ion channel targeting drugs

“…For highthroughput screening efforts, indirect readout technologies such as fluorescence assays are still used, and combined with electrophysiological patch clamp compound characterization at later stages of compound development. 29,30 The demand for increasing throughput in electrophysiological screening, while maintaining high resolution data acquisition, however, calls for the introduction of new patch clamp devices with higher throughput. The SyncroPatch 384PE is an example of such a device.…”