Inhibition and mechanism of flavivirus methyltransferase

黄病毒甲基转移酶的抑制及其机制

• 批准号: 8434274
• 负责人: ARUN K GHOSH
• 金额: $ 59.75万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2016-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8434274
• 关键词: Adenine; Animals; Antiviral Agents; Antiviral Therapy; Arthropods; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Cell Culture Techniques; Cells; Clinical Trials; Complex; Defect; Dengue Virus; Development; Disease; Drug resistance; Encephalitis; Ensure; Enzymes; Family; Flaviviridae; Flavivirus; Flavivirus Infections; Future; Genome; Goals; Growth; Guanine; Human; In Vitro; Japanese encephalitis virus; Knowledge; Lead; Mediating; Methylation; Methyltransferase; Modification; Molecular; Mutagenesis; National Institute of Allergy and Infectious Disease; Nucleotides; Performance; Play; Prevention; RNA; Replicon; Research; Ribose; Risk; Roentgen Rays; Role; Structure; Surface; Therapeutic; Tick-Borne Encephalitis Virus; Tick-Borne Encephalitis Viruses; Toxic effect; Vaccines; Viral; Virus; Virus Diseases; West Nile virus; World Health Organization; Yellow fever virus; analog; base; design; human disease; in vivo; inhibitor/antagonist; innovation; insight; intermolecular interaction; novel; pathogen; positional cloning; pre-clinical; public health priorities; public health relevance; sinefungin; vaccine development; viral RNA

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.

描述(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会引起严重的人类疾病。然而，目前还没有临床批准的抗病毒疗法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。以前，我们发现了一个一般的机制，即黄病毒NS5顺序甲基化第一个转录的核苷酸上的鸟嘌呤N7和核糖2‘-OH。我们还发现N7甲基转移酶(MTase)是黄病毒复制所必需的，因为N7甲基转移酶活性的缺陷会导致病毒复制的缺陷。我们还在甲基供体SAM结合口袋附近发现了一个黄病毒保守的口袋。此外，我们鉴定了一种先导MTase抑制剂，并发现针对WNV MTase N7活性的抑制剂可以抑制病毒的生长。这些结果清楚地表明，黄病毒MTase是一个新的抗病毒治疗的靶点。本应用的目的是研究必需的黄病毒MTase的分子抑制作用。我们将通过追求三个具体目标来实现此应用程序的总体目标。目的1.黄病毒甲基转移酶的特性。我们将使用突变和反向遗传方法进一步鉴定辅助因子SAM结合位点附近的黄病毒保守口袋。目的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MTase的特异性抑制剂。目的3.对选定的抑制剂进行深入的构效研究和X-射线结构测定，以获得分子洞察力。我们将使用体外MTase实验、细胞复制子抑制实验和病毒抑制实验来评估合成的MTase抑制剂的抑制能力。先进的化合物将通过体内动物研究、深入的耐药性研究以及MTase-抑制剂复合体的共晶结构测定来进一步表征。本文的研究具有创新性和重要意义。从这项研究中获得的知识将极大地促进我们对黄病毒帽甲基化的理解，并导致用于抗病毒治疗的黄病毒MTase抑制剂的开发。