Inhibition of DNA modifying enzymes of category A-C agents

A-C 类药物 DNA 修饰酶的抑制

• 批准号: 8233378
• 负责人: Frederic D Bushman
• 金额: $ 30.52万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233378
• 关键词: Advanced Development; Bacteria; Binding; Biological Assay; Categories; Cell Culture Techniques; Cells; Coccidioides; Complex; DNA; DNA Topoisomerases; Development; Enzymes; Fever; Funding; Goals; HIV; Hepatitis B Virus; Holliday Junction Resolvases; In Vitro; Infection; Laboratory Research; Lead; Microbe; Molecular Target; New England; Nucleotides; Pathway interactions; Pharmaceutical Preparations; Poxviridae; Proteins; Research; Resolvase; Reverse Transcriptase Inhibitors; Screening procedure; Smallpox; Structure; Therapeutic; Topoisomerase; Topoisomerase Inhibitors; Vaccinia; Vaccinium; Virus; antimicrobial; biodefense; design; drug structure; fungus; improved; in vitro Assay; inhibitor/antagonist; pathogen; programs; small molecule; therapeutic development

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.

对于生物防御应用，小分子抑制剂需要靶向多类 A-C 药物，因为即使开发一种小分子抑制剂也是困难且昂贵的。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。在少数几个中，有相当一部分是 分数目标 DMA 修饰酶。例子包括用作抗菌剂的拓扑异构酶抑制剂 对抗许多细菌，以及用于对抗 HIV 和 HBV 的核苷酸逆转录酶抑制剂。在我们的 在之前的 MARCE 资助项目中，我们已经启动了两种 DMA 修饰疗法的开发 多类 A-C 制剂中存在的酶。一种是 1B 型拓扑异构酶，第二种是 霍利迪连接酶。最初的研究集中在痘病毒中发现的这些酶的例子。我们 已经建立了体外测定并用它们筛选-225,000个小分子的抑制活性 东北 RCE 筛查机构和默克研究实验室。我们还开展了 背景机制研究为我们的抑制剂设计工作提供信息。结果包括 1) 合作 解决与 DMA 结合的天花拓扑异构酶的结构，2) 修改我们的理解 牛痘分解酶的首选底物。我们已经鉴定出对每种药物都具有活性的分子 体外纯化蛋白质并在细胞培养测定中对抗痘病毒复制。我们建议将这些用作 寻找对痘病毒和球孢子菌均具有活性的抑制剂的起点，球孢子菌是负责 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，我们将开发先导抑制剂并进行机制研究以帮助 抑制剂的开发。该项目旨在用于研究计划 I，因为它涉及小型 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 期望与计划 IV 和 VI 进行广泛的互动。