Novel Targets for Treatment of Smallpox

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。考虑到天花可能被用作针对未接种疫苗的人口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。我们建议发现新的针对病毒编码酶的痘病毒复制抑制剂，这些酶是病毒基因表达和DNA代谢所必需的。痘病毒信使核糖核酸封端装置，由三磷酸核糖核酸组成。RNA鸟苷酸转移酶和RNA(鸟嘌呤-7)甲基转移酶是一种很有前途的药物靶点，因为这三个催化位点的组织结构不同于人类宿主细胞的封顶系统。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、致密的结构和独特的药理敏感性而成为一个有吸引力的靶点。 本应用的具体目的是：(1)通过体外筛选固定在固体小球载体上的编码裂解合成组合文库(每个小球一种化合物)，鉴定与靶病毒酶结合的小分子。(2)测试初步筛选中确定的单个化合物对目标三磷酸酶、鸟苷酸转移酶、甲基转移酶和拓扑异构酶的催化活性。(3)通过对封端反应和拓扑异构酶反应组成步骤的动力学分析，剖析二次筛选中确定的化合物抑制催化活性的机理。(4)采用空斑减少法和一步生长法检测酶抑制剂对细胞培养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物：病毒的mRNA和蛋白质合成、DNA复制、端粒分解和病毒粒子形态发生的影响，来评价其抗病毒作用的机制。

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.