Discovery of Inhibitors of DNA Replication in Gram-Positive Bacteria

描述(申请人提供)：使用模式革兰氏(+)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，以使发现小分子抑制剂，有可能开发为抗菌药物和DNA复制途径的步骤特异性扰动。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DNA复制这一关键过程的抗菌药。细菌DNA复制是由一种特定来源的结合蛋白启动的，该结合蛋白招募解旋酶组装蛋白和复制的解旋酶。解旋酶一旦组装到DNA上，就为底物酶提供了一个相互作用的部位，底物酶是一种为DNA合成产生RNA引物的酶。解旋酶还在招募细胞复制酶DNA聚合酶III全酶方面发挥作用，DNA聚合酶III全酶具有不解离地合成整个染色体的过程。尽管存在这种可能性，大多数复制都会遭到破坏，导致复制分叉崩溃。这可以通过一种特殊的与原点无关的复制重启设备来抵消，该设备可以重新组装复制叉子。这些过程总共使用了至少20种不同的基本蛋白质。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。与公共卫生相关的细菌病原体越来越多地对社区和医院环境中常用的抗生素产生抗药性，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药的需要，这些抗菌药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 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.