In vitro screening strategies for nicotinic receptor ligands

Abstract

A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR™) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat α7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC 50 values of 5.5 and 1.6 μM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known α7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration–response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC 50 values for acetylcholine-evoked currents in GH4C1/α7 cells were 55 and 576 μM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of α7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC 50 values are highly comparable to those obtained using conventional manual patch clamp.