Discovery of 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，我们建议开发和运行强大的高通量筛选分析以及适当的特异性分析和反筛选，以发现有潜力开发成抗菌药和DNA复制途径的步骤特异性干扰物的小分子抑制剂。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DNA的复制是由一个特定的起始结合蛋白发起的，该蛋白招募解旋酶组装蛋白和复制解旋酶。解旋酶一旦组装在DNA上，就为引物酶提供了一个相互作用位点，引物酶产生用于DNA合成的RNA引物。解旋酶还在募集细胞复制酶、DNA聚合酶III全酶中发挥作用，该全酶具有合成整个染色体而不解离的能力。尽管存在这种可能性，但大多数复制酶都会受到损害，从而导致复制分叉崩溃。这可以通过一个特殊的独立于起点的复制重启装置来抵消，该装置可以重新组装复制分叉。这些过程总共使用了至少20种不同的必需蛋白质。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。在社区和医院环境中，细菌病原体对常用抗生素的耐药性日益增强，这是一个日益严重的公共卫生问题。这促使人们需要研究发现新的抗菌药，这些抗菌药可以影响没有耐药性的未开发靶点。这项工作将探索各种此类未开发的靶点，这些靶点对于与许多常见人类致病菌密切相关的模式生物中的细菌DNA复制至关重要，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 develop and run robust high throughput screening assays as well as appropriate specificity assays and counterscreens to enable the discovery of small molecule inhibitors that have the potential to be developed into antibacterials 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 exisiting classes of antibiotics is increasing. Presently there are no antibacterials targeting the essential process of DNA replication in bacteria. Bacterial DNA replication is initiated by a specific origin binding protein that recruits helicase assembly proteins and the replicative helicase. The helicase, once assembled on DNA, provides an interaction site for primase, the enzyme that generates RNA primers for DNA synthesis. The helicase also plays a role in recruiting the cellular replicase, DNA polymerase III holoenzyme, which has the processivity to synthesize the entire chromosome without dissociation. In spite of this potential, most replicases encounter damage, resulting in replication fork collapse. This can be counteracted by a special origin-independent replication restart apparatus that can reassemble replication forks. Altogether, these processes employ at least 20 different essential proteins. These protein targets and the essential interactions that occur between them provide attractive targets for the development of antibacterials, 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 antibacterials 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.