Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
Department of Pharmacy, University of Washington, Seattle.
Epilepsia. 2020 Oct;61(10):2106-2118. doi: 10.1111/epi.16644. Epub 2020 Aug 14.
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 D rerio. 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 C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABA ) 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 GABA 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.
目前约有三分之一的癫痫患者对现有药物治疗无效。因此，急需开发新的抗癫痫药物来解决这一耐药问题。然而，传统的啮齿动物癫痫发作和癫痫模型不适合高通量化合物筛选。此外，在单一物种中进行测试增加了治疗化合物作用于可能在人类中未保守的分子靶点的可能性。为了解决这些问题，我们使用了四种不同的生物体开发了一种流水线方法。
我们依次在斑马鱼（Danio rerio）中进行化合物文库筛选、在秀丽隐杆线虫（Caenorhabditis elegans）中进行化学遗传学研究、在小鼠和人脑切片中进行电生理分析，以及在小鼠癫痫模型中进行临床前验证，以确定新型抗癫痫药物及其作用机制。
最初，在急性戊四氮癫痫发作模型中使用斑马鱼筛选了 1690 种化合物库。从该筛选中，鉴定出化合物氯苯乙酮不仅在鱼类中，而且在蠕虫中也是一种有效的抗惊厥药物。随后在秀丽隐杆线虫中的遗传筛选揭示了氯苯乙酮的分子靶标为 LGC-37，这是一种蠕虫γ-氨基丁酸 A 型（GABA）受体亚基。这一 GABA 能作用通过来自小鼠和人类的体外脑片制备得到证实，因为氯苯乙酮被证明增强了紧张性和相性抑制，并且这种作用被 GABA 受体拮抗剂荷包牡丹碱逆转。最后，氯苯乙酮在几种小鼠癫痫发作模型中表现出体内抗惊厥作用，包括耐药性癫痫发作的 6-Hz 44-mA 模型。
这些发现建立了一种多器官方法，可以鉴定出具有进化保守的分子靶点和转化潜力的化合物，因此可能有助于癫痫和其他可能疾病的药物发现。
