Novel Targets for Treatment of Smallpox

天花治疗的新靶点

• 批准号: 6561451
• 负责人: Stewart H Shuman
• 金额: $ 19.33万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561451
• 关键词: DNA replication; DNA topoisomerases; Poxviridae disease; RNA biosynthesis; acid anhydride hydrolase; antiviral agents; bioterrorism /chemical warfare; combinatorial chemistry; drug discovery /isolation; enzyme inhibitors; methyltransferase; nucleotidyltransferase; protein biosynthesis; smallpox virus; telomere; tissue /cell culture; vaccinia virus; virus replication

DESCRIPTION (provided by applicant): The goal of this project is to identify novel targets and drugs for the treatment and prophylaxis of smallpox. Anti-poxvirus drug targets are a pressing issue, given the concern that smallpox may be used as a bioterrorism weapon against an unvaccinated population. We propose to discover new inhibitors of poxvirus replication targeted to essential virus-encoded enzymes that are required for viral gene expression and DNA metabolism. The poxvirus mRNA capping apparatus, consisting of RNA triphosphatase. RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase enzymes, is a promising drug target because the organization of the three catalytic sites is distinct from that of human host cell capping system. The poxvirus type 18 DNA topoisomerase is an attractive target in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared to human topoisomerase I. The specific aims of this application are: (1) To identify small molecules that bind to the target viral enzymes by in vitro screening of an encoded split-synthesis combinatorial library immobilized on a solid bead support (one compound per bead). (2) To test the individual compounds identified in the primary screen for their ability to inhibit the catalytic activities of the target triphosphatase, guanylyltransferase, methyltransferase, and topoisomerase enzymes. (3) To dissect the mechanisms of inhibition of catalytic activity by the compounds identified in the secondary screen, via kinetic analysis of the component steps of the capping and topoisomerase reactions. (4) To assay the enzyme inhibitors for their effects on vaccinia virus replication in cell culture, using plaque reduction and one-step growth assay methods. (5) To evaluate the mechanism of antiviral action by assessing the effects of the lead drug compounds on the major landmarks of the poxvirus replication cycle: viral mRNA and protein synthesis, DNA replication, telomere resolution, and virion morphogenesis.

描述（由申请人提供）：该项目的目标是确定治疗和预防天花的新靶点和药物。鉴于人们担心天花可能被用作针对未接种疫苗的人群的生物恐怖主义武器，抗痘病毒药物目标是一个紧迫的问题。我们建议寻找新的痘病毒复制抑制剂，针对病毒基因表达和 DNA 代谢所需的病毒编码酶。痘病毒 mRNA 加帽装置，由 RNA 三磷酸酶组成。 RNA 鸟苷基转移酶和 RNA (鸟嘌呤-7)-甲基转移酶是一个有前途的药物靶点，因为这三个催化位点的组织与人类宿主细胞加帽系统的组织不同。与人类拓扑异构酶 I 相比，痘病毒 18 型 DNA 拓扑异构酶因其独特的 DNA 识别特异性、紧凑的结构和独特的药理敏感性而成为一个有吸引力的靶标。 该应用的具体目标是：（1）通过体外筛选固定在固体珠支持物（每个珠一个化合物）上的编码分割合成组合文库来鉴定与目标病毒酶结合的小分子。 (2) 测试在初级筛选中鉴定的各个化合物抑制目标三磷酸酶、鸟苷基转移酶、甲基转移酶和拓扑异构酶的催化活性的能力。 (3) 通过对封端和拓扑异构酶反应的组成步骤进行动力学分析，剖析二次筛选中鉴定的化合物抑制催化活性的机制。 (4)使用噬菌斑减少和一步生长测定方法来测定酶抑制剂对细胞培养物中痘苗病毒复制的影响。 (5) 通过评估先导药物化合物对痘病毒复制周期的主要标志的影响来评估抗病毒作用的机制：病毒mRNA和蛋白质合成、DNA复制、端粒解析和病毒颗粒形态发生。