Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima, Saga, Japan.
Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki, Japan.
PLoS Negl Trop Dis. 2019 Aug 19;13(8):e0007633. doi: 10.1371/journal.pntd.0007633. eCollection 2019 Aug.
Amoebiasis, 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 FINDINGS: Here, 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/SIGNIFICANCE: Hence, 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.
由溶组织内阿米巴感染引起的阿米巴病是一个全球性的公共卫生问题。然而，目前可用于治疗阿米巴病的药物有限，并且还没有有效的疫苗。因此，迫切需要开发新的预防阿米巴病的措施。
方法/主要发现：在这里，为了开发针对阿米巴病的新药，我们专注于溶组织内阿米巴腺苷 5'-磷酸硫酸激酶（EhAPSK），这是一种参与 Entamoeba 磺脂代谢的必需酶。脂肪酸二硫酸盐和胆甾醇硫酸盐，在 Entamoeba 中合成的磺脂，在滋养体增殖和囊形成中发挥重要作用。这些过程分别与阿米巴病的临床表现和严重发病机制以及疾病传播密切相关。我们验证了一种组合方法，即基于计算机的分子对接分析和体外酶活性测定的大规模筛选。对接模拟对 EhAPSK 的同源建模结构与 400 种化合物之间的结合自由能进行了排序。400 种化合物也通过 96 孔板基础上的体外 APSK 活性测定进行了筛选。在通过体外系统鉴定为 EhAPSK 抑制剂的 15 种化合物中，有 6 种化合物通过计算机分析被评为对 EhAPSK 具有高亲和力。此外，通过计算机和体外分析均鉴定为 EhAPSK 抑制剂的 2-(3-氟苯氧基）-N-[4-(2-吡啶基）噻唑-2-基]乙酰胺、3-苯基-N-[4-(2-吡啶基）噻唑-2-基]-咪唑-4-甲酰胺和金诺芬不仅阻止了 Entamoeba 滋养体的增殖，还阻止了囊的形成。这三种化合物还剂量依赖性地抑制了溶组织内阿米巴中磺脂的合成。
结论/意义：因此，基于计算机和体外的 EhAPSK 分析的组合方法鉴定了可以评估其对 Entamoeba 影响的化合物。这可以为开发新的抗阿米巴病和阻断阿米巴病传播的药物提供依据。这种策略还可以应用于鉴定特定的 APSK 抑制剂，这将有利于研究硫代谢和普遍存在的途径，最终合成必需的含硫生物分子。
