A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ-aminobutyric acidergic anticonvulsant.

Alistair Jones,Jeremy J Lambert,Celia J Holdsworth,Anthony G Marson,John‐Paul Ashton,Melissa Barker‐Haliski,Sarah Baxendale,H Steve White,Bodiabaduge A P Jayasekera,Andrew J Trevelyan,Murray B Herd,Andrei S Ilie,Marysia Placzek,Vincent T Cunliffe,Graeme J Sills,Alan Morgan,Christopher J A Cowie

doi:10.1111/epi.16644

Abstract

Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using Drerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in Celegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA ) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures. These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

Highlights

Approved antiseizure drugs (ASDs) can provide effective seizure control in around two-thirds of patients
The proportion of medically refractory cases has remained fairly constant for almost 30 years,[1] suggesting that epilepsy drug discovery might benefit from using different animal models for frontline ASD screening
Developing new drugs typically takes >20 years, and much of this time is spent on early drug discovery and translational science rather than on later clinical studies.[46]

Summary

| INTRODUCTION

Approved antiseizure drugs (ASDs) can provide effective seizure control in around two-thirds of patients. Using a chemical-genetic screen, we identify chlorothymol's molecular target in C elegans as the γ-aminobutyric acid type A (GABAA) receptor subunit, LGC-37, and confirm this GABAergic mechanism of action using electrophysiological recording from mouse and human brain slices We validate this novel drug screening approach through the evaluation of chlorothymol in a battery of well-established preclinical acute and chronic seizure models in mice, which have been instrumental to the identification of all clinically available ASDs to date.[3] Of note, chlorothymol demonstrated marked efficacy in the 6-Hz 32-mA and 44-mA pharmacoresistant seizure tests, further confirming the utility of a multispecies drug screening approach for novel ASD discovery. The Johns Hopkins Clinical Compound Library (JHCCL)[24] was supplied by Professor David Sullivan (Baltimore, MD)

| Zebrafish methods

Findings

| DISCUSSION