Inhibition of DNA modifying enzymes of category A-C agents

为了在生物防御中的应用，希望小分子抑制剂靶向多个A-C类 药物，因为即使开发一个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是有效的。在为数不多的几家公司中，有相当一部分 组分靶向DMA修饰酶。例如用作抗菌剂的拓扑异构酶抑制剂 针对许多细菌，以及用于对抗艾滋病毒和乙肝病毒的核苷酸逆转录酶抑制剂。在我们的 之前MARCE资助的项目，我们已经启动了两个DMA修饰的治疗方法的开发 在多种A-C类制剂中发现的酶。一种是1B型拓扑异构酶，第二种是A型 霍利迪交界处解析器。最初的研究集中在痘病毒中发现的这些酶的例子上。我们 已经建立了体外检测方法，并用它们来筛选22.5万个小分子的抑制活性 东北RCE筛查设施和默克研究实验室。我们还开展了 背景机理研究，为我们在抑制剂设计方面的努力提供信息。结果包括1)协作以 解决与DMA结合的天花拓扑异构酶的结构和2)修正我们对 牛痘病毒裂解酶的最佳底物。我们已经确定了对每个分子都有活性的分子 体外纯化蛋白和细胞培养中抗痘病毒复制试验。我们建议将这些用作 从寻找对痘病毒和球孢子虫都有活性的抑制剂开始，这种真菌负责 它最近被添加到选择代理列表中，它编码了一个类似于 痘病毒酶。展望未来，我们将开发我们的铅抑制剂，并进行机理研究，以帮助 抑制剂开发。这个项目是为研究计划I而设计的，因为它涉及到使用小的 调节新出现的病毒与宿主细胞途径相互作用的分子抑制剂，但我们也 预计将与计划IV和计划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.