Inhibition and mechanism of flavivirus methyltransferase

描述（由申请人提供）：许多黄病毒如登革热病毒、西尼罗河病毒和黄热病病毒引起重大的人类疾病。然而，没有临床批准的抗病毒治疗可用于治疗黄病毒感染。因此，开发预防和治疗黄病毒感染的疫苗和抗病毒药物显然是公共卫生的优先事项。在此之前，我们发现了一个普遍的机制，即黄病毒NS5依次甲基化帽上的鸟嘌呤N7和第一个转录核苷酸上的核糖2'-OH。我们还发现N7甲基转移酶（MTase）对黄病毒复制至关重要，因为N7 MTase活性的缺陷会导致病毒复制的缺陷。我们还在甲基供体sam结合袋附近发现了一个黄病毒保守袋。此外，我们鉴定了一种MTase先导抑制剂，并发现抑制WNV MTase N7活性的抑制剂可以抑制病毒生长。这些结果清楚地表明，黄病毒MTase是开发抗病毒治疗的新靶点。本应用程序的目的是研究黄病毒必需MTase的分子抑制作用。我们将通过追求三个具体目标来实现这个应用程序的总体目标。目的1。黄病毒甲基转移酶的鉴定。我们将使用诱变和反向遗传方法进一步表征辅助因子sam结合位点附近的黄病毒保守口袋。目标2。基于结构的MTase抑制剂的设计与开发。我们将进行基于结构的设计和合成针对黄病毒MTases的特异性抑制剂。目标3。进行深入的结构-活性研究，并确定选定抑制剂的x射线结构，以获得分子洞察力。我们将利用体外MTase实验、细胞复制子抑制实验和病毒还原实验来评估合成的MTase抑制剂的抑制能力。高级化合物将通过体内动物研究、深入的耐药研究和mtase抑制剂复合物的共晶结构测定来进一步表征。本研究具有创新意义。从这项研究中获得的知识将大大提高我们对黄病毒帽甲基化的理解，并导致开发用于抗病毒治疗的黄病毒MTase抑制剂。

DESCRIPTION (provided by applicant): Many flaviviruses such as Dengue virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. Previously, we found a general mechanism that the flavivirus NS5 sequentially methylates the guanine N7 on the cap and ribose 2'-OH on the first transcribed nucleotide. We also found that the N7 methyltransferase (MTase) is essential for flavivirus replication, since defects in N7 MTase activity lead to defects in viral replication. We also identified a flavivirus-conserved pocket near the methyl- donor SAM-binding pocket. In addition, we identified a lead MTase inhibitor, and found that inhibitors against the N7 activity of the WNV MTase can inhibit viral growth. These results clearly demonstrate that flavivirus MTase is a novel target for development of antiviral therapy. The goal of this application is to study the molecular inhibition of the essential flavivirus MTase. We will accomplish the overall objective of this application by pursuing three specific aims. Aim 1. Characterization of the flavivirus methyltransferase. We will further characterize the flavivirus- conserved pocket near the co-factor SAM-binding site using mutagenesis and reverse genetic approaches. Aim 2. Structure-based design and development of MTase inhibitors. We will perform structure-based design and synthesis of specific inhibitors against flavivirus MTases. Aim 3. Performance of in-depth structure-activity studies and determination of X-ray structures of selected inhibitors to gain molecular insight. We will evaluate the inhibition ability of synthesized MTase inhibitors using in vitro MTase assay, cell-based replicon inhibition assay, and viral reduction assay. Advanced compounds will be further characterized by in vivo animal studies, in-depth drug resistance studies, and co-crystal structure determination of the MTase-inhibitor complexes. The proposed research is innovative and significant. The knowledge acquired from this study will significantly advance our understanding of flavivirus cap methylation and lead to the development of inhibitors of the flavivirus MTase for antiviral therapy.