Inhibition of DNA modifying enzymes of category A-C agents

对于生物防御中的应用，期望小分子抑制剂靶向多个类别A-C 因为即使开发一种小分子抑制剂也是困难和昂贵的。相对较少 临床上有用的药物对多种不同的病原体具有活性。在为数不多的几个人中， 部分靶向DMA修饰酶。例子包括用作抗菌剂的拓扑异构酶抑制剂 抗许多细菌，以及抗HIV和HBV的核苷酸逆转录酶抑制剂。在我们 以前的MARCE资助的项目，我们已经开始开发治疗两个DMA修改 在多种A-C类药物中发现的酶。一种是1B型拓扑异构酶，第二种是 霍利迪连接消退酶。最初的研究集中在痘病毒中发现的这些酶的例子。我们 已经建立了体外试验，并使用它们筛选约225，000种小分子的抑制活性， 东北RCE筛选设施和默克研究实验室。我们还开展了 背景技术为我们的抑制剂设计工作提供信息的机理研究。结果包括：（1）合作， 解决与DMA结合的天花拓扑异构酶的结构，以及2）修改我们对 有利于牛痘消退酶的底物。我们已经鉴定出了对每种病毒都有活性的分子 纯化的蛋白质在体外和在细胞培养测定中抗痘病毒复制。我们建议使用这些作为 寻找对痘病毒和球孢子菌有效的抑制剂的起点， Valley Fever最近被添加到选择代理列表中，并编码类似于 痘病毒酶展望未来，我们将开发铅抑制剂，并进行机理研究，以帮助 抑制剂开发。该项目旨在研究计划I，因为它涉及使用小型 分子抑制剂来调节新兴病毒与宿主细胞途径的相互作用，但我们也 预计将与计划IV和VI进行广泛的互动。

For applications in biodefense, it is desirable for small molecule inhibitors to target multiple category A-C agents, because it is difficult and expensive to develop even one small molecule inhibitor. Relatively few clinically useful drugs are active against multiple different pathogens. Of the few that are, a substantial fraction target DMA modifying enzymes. Examples include topoisomerase inhibitors used as antimicrobials against many bacteria, and nucleotide reverse transcriptase inhibitors used against HIV and HBV. In our previous MARCE-funded projects, we have initiated development of therapeutics for two DMA modifying enzymes that are found in multiple category A-C agents. One is a type 1B topoisomerase, the second a Holliday junction resolvase. Initial studies focused on examples of these enzymes found in poxviruses. We have established assays in vitro and used them to screen -225,000 small molecules for inhibitory activity at the Northeast RCE Screening Facility and at Merck Research Laboratories. We have also carried out background mechanistic studies to inform our efforts at inhibitor design. Results include 1) collaborating to solve the structure of the variola topoisomerase enzyme bound to DMA and 2) revising our understanding of favored substrates for the vaccinia resolvase. We have identified molecules with activity against each purified protein in vitro and against poxvirus replication in cell culture assays. We propose to use these as starting points to find inhibitors active against both poxviruses and Coccidioides, the fugus responsible for Valley Fever, which was recently added to the select agent list and encodes a resolvase resembling the poxvirus enzyme. Going forward, we will develop our lead inhibitors and carry out mechanistic studies to aid inhibitor development. This project is intended for Research Program I, since it concerns the use of small molecule inhibitors to modulate interactions of Emerging Viruses with Host Cell Pathways, but we also expect to have extensive interactions with programs IV and VI.