Ruma Yasmeen N, Keniya Mikhail V, Tyndall Joel D A, Monk Brian C
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin 9016, New Zealand.
School of Pharmacy, University of Otago, Dunedin 9016, New Zealand.
J Fungi (Basel). 2022 Jan 10;8(1):69. doi: 10.3390/jof8010069.
The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole-enzyme interactions and functional studies of recombinant CYP51 from have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.
真菌细胞色素P450羊毛甾醇14α-脱甲基酶（CYP51）是真菌特异性麦角甾醇生物合成所必需的，并且是唑类抗真菌药物的作用靶点。尽管作为唑类抗真菌药的临床靶点已被证明是成功的，但迫切需要开发靶向CYP51的新一代抗真菌药，以克服致病真菌对现有唑类药物的耐药性、毒性不良反应以及由于人类药物代谢细胞色素P450引起的药物相互作用。白色念珠菌是一种易于传播的机会性真菌病原体，可在医疗环境中引起念珠菌病。在本研究中，我们通过在酿酒酵母宿主系统中表达这些酶，对野生型白色念珠菌CYP51及其具有临床意义的耐药性引起点突变Y132F进行了表征。在某些情况下，这些酶与它们同源的NADPH-细胞色素P450还原酶（CPR）共表达。组成型表达的白色念珠菌CYP51 Y132F对氟康唑和伏立康唑产生了10至12倍的耐药性，对四氮唑类VT-1161和VT-1129的耐药性降低至约6倍，但对长尾三唑类没有耐药性。在共表达白色念珠菌CPR的情况下，敏感性没有变化。II型结合光谱显示亲和纯化的重组白色念珠菌CYP51与三唑类和四氮唑类紧密结合。我们报告了酿酒酵母CYP51与VT-1129复合物的X射线晶体结构，分辨率为2.1 Å。对唑类与酶相互作用的结构分析以及来自白色念珠菌的重组CYP51的功能研究，提高了我们对它们对唑类药物敏感性的理解，并将有助于推进基于结构的抗真菌药物发现。
