Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC₅₀ 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

中文翻译：

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棘阿米巴强效选择性抑制剂的鉴定：对甾醇 14α-脱甲基酶作为关键药物靶点的结构见解

棘阿米巴是自由生活的致病原虫，可引起致盲性角膜炎、播散性感染和肉芽肿性阿米巴脑炎，通常是致命的。开发高效、安全的药物是一个未满足的关键需求。棘阿米巴甾醇 14α-去甲基酶 (CYP51) 是甾醇生物合成途径的必需酶。已有报道将抗真菌唑类药物重新用于阿米巴感染，但其对棘阿米巴CYP51 酶活性的抑制作用尚未研究。在这里，我们报告了卡氏棘阿米巴CYP51 的催化特性、抑制作用和结构特征。该酶对钝叶醇的底物偏好是羊毛甾醇的 100 倍，支持病原体中类似植物的环木菠萝烯醇途径。伏立康唑 (1 h IC ₅₀ 0.45 μM)、VT1598 (0.25 μM) 和 VT1161 (0.20 μM)观察到最强的抑制作用。本文介绍了结合了 VT1161 (2.24 Å) 且不含抑制剂 (1.95 Å) 的A.castellanii CYP51的晶体结构，可用于开发基于唑的支架，以实现最佳的杀阿米巴效果。