Mechanistic understanding and inhibition of Zika NS5 protein

寨卡病毒NS5蛋白的机制理解和抑制 摘要 寨卡病毒（ZIKV）属于单链RNA黄病毒家族。它最近的爆发和 对人类疾病（如神经系统疾病）的影响已经引起了全球健康警报， 开发针对ZIKV感染的治疗策略。然而，目前还没有批准的抗病毒药物 针对ZIKV的攻击本申请寻求开发一种针对非结构性的抗病毒策略， ZIKV的蛋白5（NS5），其负责病毒特异性基因组复制。一方面，目前 将评估鉴定的黄病毒抑制剂对ZIKV抑制的效率。另一方面，在一项研究中， 将研究ZIKV NS5介导的RNA复制的机制细节，从而为ZIKV NS5介导的RNA复制提供基础。 开发靶向ZIKV NS 5的各种酶促步骤的协同抑制策略。在目标1中， 将采用结构、生物化学和细胞方法来评估ZIKV NS5介导的ZIKV NS5的抑制。 通过噻吩基炔丙醇（TPA）化合物的从头RNA合成，非核苷抑制剂 在体外和离体研究中，已经鉴定为登革病毒（DENV）NS5的抑制剂的NNI。我们最近 ZIKV NS 5的结构研究揭示DENV NS 5的TPA结合位点在ZIKV NS 5中是保守的。 通过评估TPA化合物对ZIKV NS 5的抑制作用，本申请将解决以下问题： TPA化合物是否可以作为ZIKV NS5的抑制剂，更重要的是，为ZIKV NS5的抑制提供基础。 ZIKV NS5的基于结构的药物优化。在目的2中，ZIKV NS5介导的RNA的机制基础是： 复制将通过复制起始和延伸的结构解析来确定 ZIKV NS5的复合物，结合突变和酶分析。结构上的知识 然后，ZIKV NS5从复制起始到延伸的构象转变将为ZIKV NS5从复制起始到延伸的构象转变提供框架。 用于全面抑制ZIKV NS5活性基于结构的药物设计。总的来说，拟议的研究 将为NS5介导的基因组复制提供关键的机制见解，并为 开发针对ZIKV的有效抑制剂。

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.