Novel Targets for Treatment of Smallpox

描述（由申请人提供）：本项目的目标是确定用于治疗和预防天花的新靶点和药物。抗痘病毒药物靶点是一个紧迫的问题，因为人们担心天花可能被用作针对未接种疫苗人群的生物恐怖主义武器。我们建议发现新的痘病毒复制抑制剂，靶向病毒基因表达和DNA代谢所需的病毒编码的酶。痘病毒mRNA加帽装置，由RNA三磷酸酶组成。RNA鸟苷酰转移酶和RNA（鸟嘌呤-7）-甲基转移酶是一种有前途的药物靶标，因为三个催化位点的组织不同于人类宿主细胞加帽系统的组织。痘病毒18型DNA拓扑异构酶是一个有吸引力的目标，鉴于其独特的DNA识别特异性，紧凑的结构，和独特的药理学敏感性相比，人类拓扑异构酶I。 本申请的具体目的是：（1）通过体外筛选固定在固体珠支持物（每个珠一种化合物）上的编码的分裂-合成组合文库来鉴定与靶病毒酶结合的小分子。(2)检测初步筛选中鉴定的单个化合物抑制靶向三磷酸酶、鸟苷酰转移酶、甲基转移酶和拓扑异构酶催化活性的能力。(3)通过对封端和拓扑异构酶反应的组分步骤进行动力学分析，剖析二级筛选中鉴定的化合物抑制催化活性的机制。(4)采用空斑减少法和一步生长测定法测定酶抑制剂对细胞培养中牛痘病毒复制的影响。(5)通过评估先导药物化合物对痘病毒复制周期主要标志的影响来评价抗病毒作用机制：病毒mRNA和蛋白质合成、DNA复制、端粒分解和病毒体形态发生。

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.