Institute of Parasitology, McGill University, 21111 Lakeshore Road Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada.
Institute of Parasitology, McGill University, 21111 Lakeshore Road Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada; School of Biological Sciences, Queen's University - Belfast, 19 Chlorine Gardens, Belfast BT9 5DL, Northern Ireland, United Kingdom.
Biomed Pharmacother. 2022 Jan;145:112380. doi: 10.1016/j.biopha.2021.112380. Epub 2021 Nov 5.
Nematode glutamate-gated chloride channels (GluCls) are targets of ivermectin (IVM) and moxidectin (MOX), structurally dissimilar macrocyclic lactone (ML) anthelmintics. IVM and MOX possess different pharmacokinetics and efficacy profiles but are thought to have the same binding site, through which they allosterically activate GluCls, apart from the GLC-2 receptor, which is antagonized by IVM. Our goal was to determine GLC-2 sensitivity to MOX, investigate residues involved in antagonism of GLC-2, and to identify differences in receptor-level pharmacology between IVM and MOX.
Two-electrode voltage clamp electrophysiology was used to study the pharmacology of Caenorhabditis elegans GLC-2 receptors heterologously expressed in Xenopus laevis oocytes. In silico homology modeling identified Cel-GLC-2 residues Met291 and Gln292 at the IVM binding site that differ from other GluCls; we mutated these residues to those found in ML-sensitive GluCls, and those of filarial nematode GLC-2.
We discovered that MOX inhibits wild-type C. elegans GLC-2 receptors roughly 10-fold more potently than IVM, and with greater maximal inhibition of glutamate activation (MOX = 86.9 ± 2.5%; IVM = 57.8 ± 5.9%). IVM was converted into an agonist in the Met291Gln mutant, but MOX remained an antagonist. Glutamate responses were abrogated in a Met291Leu Gln292Thr double mutant (mimicking filarial nematode GLC-2), but MOX and IVM were converted into positive allosteric modulators of glutamate at this construct.
Our data provides new insights into differences in receptor-level pharmacology between IVM and MOX and identify residues responsible for ML antagonism of GLC-2.
线虫谷氨酸门控氯离子通道（GluCls）是伊维菌素（IVM）和莫昔克丁（MOX）的靶标，这两种药物是结构不同的大环内酯类驱虫药。IVM 和 MOX 具有不同的药代动力学和疗效特征，但据认为它们具有相同的结合位点，通过该位点它们变构激活 GluCls，除了 GLC-2 受体，该受体被 IVM 拮抗。我们的目标是确定 GLC-2 对 MOX 的敏感性，研究参与 GLC-2 拮抗的残基，并确定 IVM 和 MOX 之间在受体水平药理学上的差异。
使用双电极电压钳电生理学研究异源表达于非洲爪蟾卵母细胞中的秀丽隐杆线虫 GLC-2 受体的药理学。计算机同源建模确定了伊维菌素结合位点处的 Cel-GLC-2 残基 Met291 和 Gln292，它们与其他 GluCls 不同；我们将这些残基突变为 ML 敏感型 GluCls 以及丝虫 GLC-2 的残基。
我们发现 MOX 对野生型秀丽隐杆线虫 GLC-2 受体的抑制作用比 IVM 强约 10 倍，对谷氨酸激活的最大抑制作用也更强（MOX = 86.9 ± 2.5%；IVM = 57.8 ± 5.9%）。在 Met291Gln 突变体中，IVM 转变为激动剂，但 MOX 仍然是拮抗剂。在 Met291Leu Gln292Thr 双突变体（模拟丝虫 GLC-2）中，谷氨酸反应被阻断，但 MOX 和 IVM 在该构建体中转变为谷氨酸的正变构调节剂。
我们的数据为 IVM 和 MOX 之间在受体水平药理学上的差异提供了新的见解，并确定了负责 ML 拮抗 GLC-2 的残基。
