Department of Applied Biological Chemistry, Faculty of Agriculture (W.K., S.O., S.Fur., K.N., K.T., S.Fuj., T.M., R.K., M.I., K.M.) and Agricultural Technology and Innovation Research Institute (K.M.), Kindai University, Nara, Japan; Graduate School of Agriculture (K.K., H.H.) and Graduate School of Human Life and Ecology (M.S.A., E.K-N.), Osaka Metropolitan University, Osaka, Japan; Faculty of Food Science and Technology, Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh (M.S.A.); and Centre for Respiratory Biology, UCL Respiratory, Division of Medicine, University College London, London, UK (D.B.S.).
Department of Applied Biological Chemistry, Faculty of Agriculture (W.K., S.O., S.Fur., K.N., K.T., S.Fuj., T.M., R.K., M.I., K.M.) and Agricultural Technology and Innovation Research Institute (K.M.), Kindai University, Nara, Japan; Graduate School of Agriculture (K.K., H.H.) and Graduate School of Human Life and Ecology (M.S.A., E.K-N.), Osaka Metropolitan University, Osaka, Japan; Faculty of Food Science and Technology, Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh (M.S.A.); and Centre for Respiratory Biology, UCL Respiratory, Division of Medicine, University College London, London, UK (D.B.S.)
Mol Pharmacol. 2023 Jun;103(6):299-310. doi: 10.1124/molpharm.122.000601. Epub 2023 Mar 22.
The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant nematodes and functionally expressed nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the -AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.
驱虫药帕拉喹胺 A 选择性作用于线虫 L 型烟碱型乙酰胆碱受体 (nAChR)，但其选择性机制尚不清楚。本研究通过确定与化合物结合的乙酰胆碱结合蛋白 (AChBP) 的 X 射线晶体结构，即 nAChR 配体结合结构域的替代物，以及测量其对野生型和突变线虫和功能表达 nAChR 的作用，确定了帕拉喹胺 A 选择性的基础。帕拉喹胺 A 对氯硝柳胺敏感的 [L 型 (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR 的疗效高于尼古丁敏感的 [N 型 (ACR-16)] nAChR，这一结果与野生型线虫和两种受体亚基突变线虫的体内研究一致。-AChBP-帕拉喹胺 A 复合物的 X 射线晶体结构和定点氨基酸突变研究首次表明，变构受体结合位点的环 C、环 E 和环 F 在观察到的帕拉喹胺 A 对 L 型 nAChR 的选择性作用中起关键作用。 意义声明：氧吲哚生物碱帕拉喹胺 A 已被证明选择性作用于线虫中的 L 型而非 N 型 nAChR，但选择性机制尚不清楚。我们已经将帕拉喹胺 A 与乙酰胆碱结合蛋白共结晶，乙酰胆碱结合蛋白是 nAChR 的替代物，我们发现环 C、环 E 和环 F 的结构特征有助于该生物碱对 L 型 nAChR 的选择性。这些结果为设计针对寄生线虫胆碱能神经传递的驱虫药物提供了新的平台。
