Characterization of Entamoeba histolytica adenosine 5′-phosphosulfate (APS) kinase; validation as a target and provision of leads for the development of new drugs against amoebiasis

BackgroundAmoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed.Methodology/Principal findingsHere, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5′-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica.Conclusions/SignificanceHence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules.

**背景**
溶组织内阿米巴（Entamoeba histolytica）感染引发的阿米巴病是全球性公共卫生难题。当前临床可用的抗阿米巴病药物十分有限，且尚无有效疫苗，因此亟需开发新型阿米巴病防治手段。

**方法与主要发现**
本研究为开发新型抗阿米巴病药物，聚焦于溶组织内阿米巴腺苷5'-磷酸硫酸激酶（EhAPSK）——该酶是阿米巴硫脂代谢通路中的关键必需酶。阿米巴自身合成的脂肪醇二硫酸盐与硫酸胆固醇均为硫脂，二者分别在滋养体增殖与包囊形成过程中发挥重要作用：前者与阿米巴病的临床症状及重症发病机制密切相关，后者则直接关联疾病传播。本研究验证了一套结合计算机虚拟分子对接分析（in silico molecular docking analysis）与体外酶活性测定的大规模筛选方案：首先通过同源建模获得EhAPSK的三维结构，利用分子对接模拟计算该结构与400种化合物的结合自由能并完成排序；同时采用基于96孔板的体外APSK活性测定法对这400种化合物进行筛选。经体外实验体系鉴定出的15种EhAPSK抑制剂中，有6种经计算机虚拟分析被评定为对EhAPSK具有高亲和力。进一步研究显示，经两种分析方法共同验证的2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide、3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide以及金诺芬（auranofin），不仅能够抑制溶组织内阿米巴滋养体的增殖，还可阻断包囊形成；且这三种化合物均能以剂量依赖性方式损害溶组织内阿米巴的硫脂合成过程。

**结论与意义**
综上，本研究通过结合计算机虚拟与体外实验的EhAPSK分析策略，筛选出可用于评估抗阿米巴活性的候选化合物，为开发新型抗阿米巴病药物以及阻断阿米巴病传播的治疗手段提供了先导化合物。该筛选策略还可用于鉴定特异性APSK抑制剂，将有助于硫代谢以及终末合成必需含硫生物分子的普遍保守通路相关研究的推进。