Structure-guided approach to assay development for inhibiting Dengue virus replication

登革热病毒在非洲、亚洲和中美洲通过蚊子传播，每年感染约3.9亿人，导致约2万人死亡，其中包括儿童，主要生活在热带气候。由于没有获得许可的药物，只有在之前感染登革热后才向人们推荐疫苗，因此需要新的抗病毒治疗策略。一种结构导向的方法加速了药物发现，但需要有关蛋白质靶标的结构和组织的信息。登革病毒RNA复制复合体的结构仍然未知，尽管它的7个蛋白质亚基中有5个的结构是可用的。同等的复合体可用于其他抗病毒治疗，如丙型肝炎(登革热的近亲)、艾滋病毒、SARS-CoV-2和单纯疱疹病毒1型。然而，登革热病毒复制复合体的组装仍然缺乏特征。在这里，对登革热病毒复制复合体核心的两种酶--非结构蛋白NS3和NS5进行严格的结构和生物物理研究将使开发用于体外筛选的新的高通量分析成为可能，同时也为未来的硅药物发现工作提供支持。该项目将涉及收集电子显微镜和小角X射线散射(SAXS)数据，以利用个别蛋白质先前的X射线结构建立登革热复制复合体的模型。随后，将开发新的高通量分析方法来筛选抑制该复合体的组装和酶活性的小分子。

Dengue virus, transmitted by mosquitos in Africa, Asia and central America, infects ~390 million people and kills ~20,00 people, including children, annually, mostly in tropical climates. With no licensed drugs and a vaccine recommended to people only after a previous Dengue infection, new anti-viral therapeutic strategies are needed. A structure-guided approach accelerates drug discovery but requires information about the structure and organization of protein targets. The architecture of the Dengue virus RNA replication complex remains unknown, despite structures of 5 out of its 7 protein subunits being available. Equivalent complexes are targeted in other anti-viral treatments, such as against Hepatitis C (a close relative of Dengue), HIV, SARS-CoV-2 and the Herpes Simplex type 1 virus. However, the assembly of the Dengue virus replication complex remains poorly characterised. Here, a rigorous structural and biophysical study of the two enzymes, non-structural proteins NS3 and NS5, at the heart of the Dengue viral replication complex will enable the development of new high-throughput assays for in vitro screening, while also supporting future in silico drug discovery efforts. This project will involve collecting electron microscopy and small angle X-ray scattering (SAXS) data to build a model of the Dengue replication complex using previous X-ray structures of the individual proteins. Subsequently, new high-throughput assays will be developed to screen small molecules that inhibit the assembly and enzyme activities of this complex.