Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California, United States of America.
PLoS Negl Trop Dis. 2012;6(7):e1736. doi: 10.1371/journal.pntd.0001736. Epub 2012 Jul 31.
Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.
METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM.
CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.
恰加斯病是一种世界卫生组织和美国国立卫生研究院指定的被忽视的热带病，在拉丁美洲流行，由于人口流动，已成为北美和欧洲的一种新出现的传染病。尽管它是心力衰竭导致发病率和死亡率的主要原因，并且在受影响地区造成了沉重的经济负担，但由于经济激励措施不利，制药行业对恰加斯病的关注甚少。发现和开发治疗恰加斯病的新化疗途径是当务之急。
方法/主要发现：致病真菌的膜甾醇需求与寄生原生动物克氏锥虫（引起恰加斯人心肌病的病原体）的膜甾醇需求相似，这导致了抗真菌唑抑制剂 14α-去甲基酶（CYP51）的重新利用，用于治疗恰加斯病。为了使抗恰加斯病候选药物的治疗管道多样化，我们利用了一种方法，该方法包括直接用合成小分子文库探测 T. cruzi CYP51 活性位点。基于靶标的高通量筛选将约 104,000 种小分子的文库减少到 185 个具有估计纳摩尔 K(D)值的命中，而针对 T. cruzi 感染的骨骼肌母细胞的交叉验证产生了 57 个具有 EC(50) <10 µM 的活性命中。两个命中池部分重叠。排名最高的命中以 17 nM 的 EC(50)抑制 T. cruzi，以 40 nM 的 EC(50)具有杀变形虫作用。
结论/意义：这些命中具有结构多样性，表明 CYP51 是小分子的相当宽松的酶靶标。命中的化学生信分析表明，CYP51 的药理学与其他细胞色素 P450 治疗靶标相似，包括血栓烷合酶（CYP5）、脂肪酸 ω-羟化酶（CYP4）、17α-羟化酶/17,20-裂解酶（CYP17）和芳香酶（CYP19）。令人惊讶的是，强烈的相似性表明谷氨酰胺肽环转移酶与 CYP51 在序列或结构上没有关系。制药公司针对这些靶标开发的先导化合物也可用于探索其对 T. cruzi 的疗效。
