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人群的生物恐怖主义武器，抗痘病毒药物靶点是一个紧迫的问题。我们建议发现针对病毒基因表达和DNA代谢所必需的病毒编码酶的新的痘病毒复制抑制剂。痘病毒mRNA盖帽装置，由RNA三磷酸酶组成。RNA（鸟嘌呤-7）-甲基转移酶和RNA（鸟嘌呤-7）-甲基转移酶是一个很有前景的药物靶点，因为这三个催化位点的组织与人类宿主细胞盖层系统的组织不同。与人类拓扑异构酶I相比，痘病毒18型DNA拓扑异构酶具有独特的DNA识别特异性、紧凑的结构和独特的药理学敏感性，是一个有吸引力的靶标。

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.