High-Throughput Screening for N-Type Calcium Channel Blockers Using a Scintillation Proximity Assay

Abstract

N-type calcium channels located on presynaptic nerve terminals regulate neurotransmitter release, including that from the spinal terminations of primary afferent nociceptors. Accordingly, N-type calcium channel blockers may have clinical utility as analgesic drugs. A selective N-type calcium channel inhibitor, ziconotide (Prialt), is a neuroactive peptide recently marketed as a novel nonopioid treatment for severe chronic pain. To develop a small-molecule N-type calcium channel blocker, the authors developed a 96-well plate high-throughput screening scintillation proximity assay (SPA) for N-type calcium channel blockers using [¹²⁵I]-labeled ω-conotoxin GVIA as a channel-specific ligand. Assay reagents were handled using Caliper’s Allegro automation system, and bound ligands were detected using a PerkinElmer TopCount. Using this assay, more than 150,000 compounds were screened at 10 μM and approximately 340 compounds were identified as hits, exhibiting at least 40% inhibition of [¹²⁵I]GVIA binding. This is the 1st demonstration of the use of [¹²⁵I]-labeled peptides with SPA beads to provide a binding assay for the evaluation of ligand binding to calcium channels. This assay could be a useful tool for drug discovery.