Lynagh Timothy, Cromer Brett A, Dufour Vanessa, Laube Bodo
Neurophysiology and Neurosensory Systems, Technical University of Darmstadt, 64287 Darmstadt, Germany.
Health Innovations Research Institute and School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.
Int J Parasitol Drugs Drug Resist. 2014 Aug 10;4(3):244-55. doi: 10.1016/j.ijpddr.2014.07.004. eCollection 2014 Dec.
Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.
靶向谷氨酸门控氯离子通道（GluCls）的药理学方法是一种有效的驱虫策略，大环内酯类药物通过激活线虫的GluCls消除多种线虫感染就证明了这一点。鉴于最近已鉴定出扁虫的GluCls，且迫切需要抗血吸虫病的药物，扁虫的GluCls应作为潜在的驱虫靶点进行评估。本研究旨在鉴定一种这样的GluCl，即来自寄生扁虫曼氏血吸虫的SmGluCl-2的激动剂或调节剂。通过电生理学方法测定了九种谷氨酸样化合物和三种单萜类离子通道调节剂对在非洲爪蟾卵母细胞中重组表达的SmGluCl-2的影响。为了与已确定的驱虫靶点进行比较，还对寄生线虫捻转血矛线虫的AVR-14B GluCl进行了实验。L-谷氨酸是两种GluCl的最有效激动剂，但L-2-氨基己二酸、D-谷氨酸和D-2-氨基己二酸激活SmGluCl-2（EC50分别为1.0±0.1 mM、2.4±0.4 mM、3.6±0.7 mM）的效力比AVR-14B更强。quisqualate仅激活SmGluCl-2，而L-天冬氨酸仅激活AVR-14B GluCl。关于单萜类化合物，两种GluCl均被丙泊酚、百里酚和薄荷醇抑制，SmGluCl-2对百里酚的抑制作用最强（IC50为484±85 μM），对薄荷醇的抑制作用最弱（IC50>3 mM）。计算对接表明，激动剂和抑制剂的效力归因于与细胞外或跨膜氨基酸残基的特定相互作用。这些结果表明，扁虫的GluCl在药理学上对多种激动剂和调节剂敏感，并表明谷氨酸γ-羧基或丙泊酚6-异丙基的变化可改变扁虫和线虫GluCl的药理学差异。这应为开发更有效的化合物提供信息，进而带来新型驱虫药。
