Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chem/Biosensing and Chemometrics, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, and Department of Chemistry, Hunan University, Changsha, 410082, China.
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Biochem Biophys Res Commun. 2019 May 7;512(3):517-523. doi: 10.1016/j.bbrc.2019.03.070. Epub 2019 Mar 21.
Invasive aspergillosis (IA) is a life-threatening disease impacting immunocompromised individuals. Standard treatments of IA, including polyenes and azoles, suffer from high toxicity and emerging resistance, leading to the need to develop new antifungal agents with novel mechanisms of action. Ergosterol biosynthesis is a classic target for antifungals, and squalene synthase (SQS) catalyzes the first committed step in ergosterol biosynthesis in Aspergillus spp. making SQS of interest in the context of antifungal development. Here, we cloned, expressed, purified and characterized SQS from the pathogen Aspergillus flavus (AfSQS), confirming that it produced squalene. To identify potential leads targeting AfSQS, we tested known squalene synthase inhibitors, zaragozic acid and the phosphonosulfonate BPH-652, finding that they were potent inhibitors. We then screened a library of 744 compounds from the National Cancer Institute (NCI) Diversity Set V for inhibition activity. 20 hits were identified and IC values were determined using dose-response curves. 14 compounds that interfered with the assay were excluded and the remaining 6 compounds were analyzed for drug-likeness, resulting in one compound, celastrol, which had an AfSQS IC value of 830 nM. Enzyme inhibition kinetics revealed that celastrol binds to AfSQS in a noncompetitive manner, but did not bind covalently. Since celastrol is also known to inhibit growth of the highly virulent Aspergillus fumigatus by inhibiting flavin-dependent monooxygenase siderophore A (SidA, under iron starvation conditions), it may be a promising multi-target lead for antifungal development.
侵袭性曲霉病（IA）是一种危及生命的疾病，影响免疫功能低下的个体。IA 的标准治疗方法，包括多烯类和唑类药物，存在高毒性和耐药性问题，因此需要开发具有新型作用机制的新型抗真菌药物。麦角固醇生物合成是抗真菌药物的经典靶标，鲨烯合酶（SQS）催化麦角固醇生物合成的第一步，在抗真菌药物开发中具有重要意义。在这里，我们从病原体黄曲霉（AfSQS）中克隆、表达、纯化和表征了 SQS，证实它产生了鲨烯。为了鉴定针对 AfSQS 的潜在先导化合物，我们测试了已知的鲨烯合酶抑制剂扎那米韦酸和膦酸酯 BPH-652，发现它们是有效的抑制剂。然后，我们筛选了来自国立癌症研究所（NCI）多样性集合 V 的 744 种化合物库，以寻找抑制活性。确定了 20 个命中化合物，并通过剂量反应曲线确定了 IC 值。排除了 14 种干扰测定的化合物，对其余 6 种化合物进行了药物相似性分析，结果发现一种化合物 celastrol 对 AfSQS 的 IC 值为 830 nM。酶抑制动力学研究表明，celastrol 以非竞争性方式与 AfSQS 结合，但不与 AfSQS 发生共价结合。由于 celastrol 还已知在缺铁条件下通过抑制黄素依赖性单加氧酶铁载体 A（SidA）抑制高毒力烟曲霉的生长，因此它可能是一种有前途的抗真菌药物多靶标先导化合物。
