Screening for Inhibitors of DNA Replication in Gram-Positive Bacteria

描述（由申请人提供）：使用模型革兰氏菌（+）生物，枯草芽孢杆菌，我们建议转移到MLPCN，这是一种针对DNA聚合酶III全酶的强大的高通量筛选试验。通过筛选该靶点发现的小分子将优先使用适当的生化特异性和微生物活性测定来选择抑制剂，这些抑制剂有可能发展成为抗菌和步骤特异性DNA复制途径的干扰。这种模式生物与大多数常见的革兰氏（+）人类病原体密切相关，如金黄色葡萄球菌、化脓性葡萄球菌和生物防御A类生物，炭疽芽孢杆菌。在过去的三十年里，FDA只批准了20种新的化学抗生素，人们普遍认为细菌对现有抗生素的耐药性正在增加。目前还没有针对细菌DNA复制这一重要过程的抗菌药物。细菌的DNA复制是由细胞复制酶进行的，DNA聚合酶III全酶具有复制整个染色体而不解离的能力。这个过程使用至少5种不同的必需蛋白质和酶活性。这些蛋白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，也将作为开发化学遗传方法来干扰各种相互作用和反应阶段的理想系统。

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.