Mechanistic understanding and inhibition of Zika NS5 protein

寨卡病毒NS5蛋白的作用机制及其抑制 摘要 寨卡病毒(Zika Virus，ZIKV)属于单链RNA黄病毒家族。它最近的爆发和 对人类疾病(例如神经性疾病)的影响已经引起了全球健康警报，并迫切需要 制定针对寨卡病毒感染的治疗策略。然而，目前还没有批准的抗病毒药物 对阵ZIKV的比赛还没开始。此应用程序寻求开发一种抗病毒策略，以对抗非结构性 ZIKV的蛋白5(NS5)，负责病毒特异性基因组复制。一方面，目前 将评估已确定的黄病毒抑制剂对ZIKV的抑制效果。另一方面， 将研究ZIKV NS5介导的RNA复制的机制细节，从而为 针对ZIKV NS5各种酶步骤的协同抑制策略的开发。在目标1中， 将采用结构、生化和细胞方法评价ZIKV NS5介导的抑制作用 非核苷抑制剂--苯丙醇(TPA)类化合物从头合成RNA (NNIS)已被确定为登革病毒(DENV)NS5的体外和体外抑制物。我们最近 对ZIKV NS5的结构研究表明，DENV NS5的TPA结合位点在ZIKV NS5中是保守的。 通过评估TPA化合物对ZIKV NS5的抑制作用，本申请将解决 TPA化合物能否作为ZIKV NS5的抑制剂，更重要的是为 基于结构的ZKV NS5药物优化。在目标2中，ZIKV NS5介导的RNA的机制基础 复制将通过复制起始和延伸的结构说明来确定 ZIKV NS5的复合体，结合突变和酶分析。关于结构的知识 ZIKV NS5从复制起始到延伸的构象转换将提供一个框架 基于结构的药物设计用于综合抑制ZIKV NS5活性。总之，拟议的研究 将为NS5介导的基因组复制提供关键的机械性见解，并为 ZIKV有效抑制剂的研究进展。

Mechanistic understanding and inhibition of Zika NS5 protein ABSTRACT Zika virus (ZIKV) belongs to the single-stranded RNA-containing flavivirus family. Its recent outbreak and implication in human diseases (e.g. neurological disorders) have raised a global health alarm, and urgency to develop a therapeutic strategy against ZIKV infection. However, there are no currently approved antivirals against ZIKV available yet. This application seeks to develop an antiviral strategy against the non-structural protein 5 (NS5) of ZIKV, which is responsible for virus-specific genomic replication. On one hand, the currently identified flavivirus inhibitors will be evaluated for their efficiency on ZIKV inhibition. On the other hand, mechanistic details of ZIKV NS5-mediated RNA replication will be investigated, thereby providing a basis for development of synergistic inhibition strategies targeting various enzymatic steps of ZIKV NS5. In Aim 1, structural, biochemical and cellular approaches will be taken to evaluate the inhibition of ZIKV NS5-mediated de novo RNA synthesis by the thiophenyl propargyl alcohol (TPA) compounds, the non-nucleoside inhibitors (NNIs) that have been identified as inhibitors for Dengue virus (DENV) NS5, in vitro and ex vivo. Our recent structural study of ZIKV NS5 revealed that the TPA-binding site of DENV NS5 is conserved in ZIKV NS5. Through evaluation of the inhibitory effects of the TPA compounds on ZIKV NS5, this application will address whether the TPA compounds can serve as inhibitors to ZIKV NS5, and more importantly, to provide a basis for structure-based drug optimization for ZIKV NS5. In Aim 2, the mechanistic basis of ZIKV NS5-mediated RNA replication will be determined through structure elucidation of the replication initiation and elongation complexes of ZIKV NS5, combined with mutational and enzymatic analyses. The structural knowledge on the conformational transition of ZIKV NS5 from replication initiation to elongation will then provide a framework for structure-based drug design for comprehensive inhibition of ZIKV NS5 activity. Together, the proposed studies will provide key mechanistic insights into the NS5-mediated genome replication and establish a foundation for development of effective inhibitors against ZIKV.