Azole Resistance Reduces Susceptibility to the Tetrazole Antifungal VT-1161
Azole Resistance Reduces Susceptibility to the Tetrazole Antifungal VT-1161
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DOI：
10.1128/aac.02114-18
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发表时间：
2019-01-01
影响因子：
4.9
通讯作者：
Tyndall, Joel D. A.
中科院分区：
文献类型：
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作者：
Monk, Brian C.;Keniya, Mikhail V.;Tyndall, Joel D. A.
针对真菌羊毛甾醇14-14α-脱甲基酶(LDM)设计的四氮唑抗真菌药物似乎对一系列真菌病原体有效。此外，还得到了白念珠菌LDM与四唑VT-1161络合物的催化域的晶体结构。我们已经解决了人们对VT-1161与截短的重组CYP51s结晶可能产生的伪影的担忧，并测量了已知的赋予唑类耐药性的基因对VT-1161敏感性的影响。利用酵母系统超量表达带有C端六组氨酸标签(ScLDM6 X His)的重组酿酒酵母LDM，并用VT-1161或广泛使用的三氮唑类药物泊沙康唑(PCZ)进行表型分析和结晶学研究。我们确定了LDM中特征性突变对VT-1161活性的影响，并确定了来自真菌的药物外排泵，包括关键的真菌病原体，外排VT-1161。使用白色念珠菌和光滑念珠菌的临床分离株验证了这些基于酵母的观察结果与药物外排的相关性。当泊沙康唑结合时，VT-1161结合引起全长和截短酶之间的显著构象差异。三磷酸腺苷结合盒(ABC)和主要促进剂超家族(MFS)药物外排泵、LDM的过度表达以及LDM药物结合口袋内影响药物与叔醇相互作用的突变会降低对VT-1161的敏感性。
Tetrazole antifungals designed to target fungal lanosterol 14 14 alpha-demethylase (LDM) appear to be effective against a range of fungal pathogens. In addition, a crystal structure of the catalytic domain of Candida albicans LDM in complex with the tetrazole VT-1161 has been obtained. We have addressed concern about artifacts that might arise from crystallizing VT-1161 with truncated recombinant CYP51s and measured the impact on VT-1161 susceptibility of genotypes known to confer azole resistance. A yeast system was used to overexpress recombinant full-length Saccharomyces cerevisiae LDM with a C-terminal hexahistidine tag (ScLDM6 x His) for phenotypic analysis and crystallographic studies with VT-1161 or with the widely used triazole drug posaconazole (PCZ). We determined the effect of characterized mutations in LDM on VT-1161 activity and identified drug efflux pumps from fungi, including key fungal pathogens, that efflux VT-1161. The relevance of these yeast-based observations on drug efflux was verified using clinical isolates of C. albicans and Candida glabrata. VT-1161 binding elicits a significant conformational difference between the full-length and truncated enzymes not found when posaconazole is bound. Susceptibility to VT-1161 is reduced by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) drug efflux pumps, the overexpression of LDM, and mutations within the drug binding pocket of LDM that affect interaction with the tertiary alcohol of the drug.