Validation of an ASIC1a Ligand-Gated Assay on an Automated Patch Clamp Platform and its Use for Novel Ligand Screening

Abstract

There is growing interest in automated patch clamp assays for ligand-gated targets, many of which are expressed throughout the nervous system and underlie complex neurological diseases. The Acid-Sensing Ion Channel (ASIC) family comprises combinations of ASIC1-4 proteins that form cation-selective channels in the central and peripheral nervous system. ASIC1a proteins are involved in synaptic plasticity, and implicated in stroke, ischemia and pain. Small molecules and toxins modulate ASIC1a channels, and fragments can bind to ASIC3 channels (Wolkenberg et al., 2010), so we screened a diverse low molecular weight library for novel ASIC1a modulators. All data was generated on the gigaseal QPatch48 system (Sophion) using coated metal pipetting tips and a CHO cell line stably expressing human ASIC1a. Currents were elicited from a holding potential of −60 mV by rapid application of saline or compound-containing solutions of varied pH. EC50 of pH activation and IC50 of test compound inhibition were determined by cumulative applications to the same cell. A proprietary low MW library was tested at 200 μM (N=2) and active hits were confirmed by cumulative IC50 testing (N=3). We validated the pharmacology of the hASIC1a cell line using toxins (e.g. IC50 of 120 nM for Mambalgin-3), small molecules (Benzamil IC50 of 4.0 μM), and clinical development compounds, which paved the way to screen a proprietary library of low molecular weight compounds in an effort to find chemical building blocks for the design of novel and efficacious ASIC1a modulators. A hit rate of 3.0% was achieved which included several novel scaffolds with low μM potency, which can be used in biophysical binding assays and structure-based drug design to develop ligand-efficient modulators of the human ASIC1a channel.