Screening for Inhibitors of DNA Replication in Gram-Positive Bacteria

描述(申请人提供)：使用模式革兰氏(+)生物体，枯草芽孢杆菌，我们建议转移到MLPCN，一个强大的高通量筛选试验，针对DNA聚合酶III全酶。通过筛选这个靶点发现的小分子将使用适当的生化特异性和微生物学活性分析来优先选择有可能发展成抗菌和步骤特异性DNA复制途径扰动的抑制剂。这种模式生物与最常见的革兰氏阳性人类病原体如金黄色葡萄球菌、化脓性葡萄球菌和生物防御A类生物炭疽杆菌密切相关。在过去的三十年里，FDA只批准了两类新的化学类抗生素，而且人们普遍认为，细菌对现有类抗生素的耐药性正在增加。目前还没有针对细菌中DNA复制的关键过程的抗菌药物。细菌DNA复制是由细胞复制酶DNA聚合酶III全酶执行的，它具有复制整个染色体的过程而不解离。这一过程使用了至少5种不同的必需蛋白质和酶活性。这些蛋白质靶点和它们之间发生的基本相互作用为抗菌药物的开发提供了有吸引力的靶点，也将成为开发扰乱各种相互作用和反应阶段的化学遗传方法的理想系统。 与公共卫生相关：在社区和医院环境中，细菌病原体对常用抗生素的抗药性越来越强，这是一个日益严重的公共卫生问题。这推动了研究发现新的抗菌素的需要，这些抗菌素影响到没有耐药性的未开发的靶标。这项工作将探索各种这样的未开发的靶点，对于细菌DNA在与许多常见的人类病原体密切相关的模式生物中复制是必不可少的，这将有助于发现此类新的抗菌化合物。

DESCRIPTION (provided by applicant): Using a model Gram (+) organism, Bacillus subtilis, we propose to transfer to the MLPCN, a robust high throughput screening assay targeting the DNA Polymerase III holoenzyme. Small molecules discovered via screening of this target will be prioritized using appropriate biochemical specificity and microbiological activity assays to select inhibitors that have the potential to be developed into antibacterial and step-specific perturbants of DNA replication pathways. This model organism is closely related to most common Gram (+) human pathogens such as S. aureus, S. pyogenes and the biodefense category A organism, Bacillus anthracis. Over the last three decades only 2 new chemical classes of antibiotics have been approved by the FDA and it is widely recognized that bacterial resistance to existing classes of antibiotics is increasing. Presently there are no antibacterial targeting the essential process of DNA replication in bacteria. Bacterial DNA replication is performed by the cellular replicase, DNA polymerase III holoenzyme that has the processivity to replicate the entire chromosome without dissociation. This process employs at least 5 different essential proteins and enzymatic activities. These protein targets and the essential interactions that occur between them provide attractive targets for the development of antibacterial, and will also serve as an ideal system for developing chemical genetic approaches to perturb the various interactions and reaction stages. PUBLIC HEALTH RELEVANCE: Bacterial pathogens are increasingly becoming resistant to commonly used antibiotics in both community and hospital settings, representing a growing public health problem. This has driven the need for research to discover new antibacterial that affect unexploited targets for which resistance is absent. This work will explore a variety of such unexploited targets, essential for bacterial DNA replication in a model organism closely related to many common human pathogenic bacteria, which will aid the discovery of such new antibacterial compounds.