DEVELOPMENT OF INHIBITORS OF MYCOBACTERIUM TUBERCULOSIS DNA REPLICATION

描述（由申请人提供）：基于针对新细菌靶标的新型化学支架的新型抗生素对于减少全球结核病（TB）感染潮至关重要。由于治疗要求时间长，难以实现治疗依从性，耐多药结核病的发展速度越来越快。此外，在艾滋病流行的地区，结核病和艾滋病毒的合并感染导致三分之一的艾滋病患者死亡。该研究项目的目标是发现和开发有效对抗结核分枝杆菌（Mtb）的新型抗生素。我们的策略是筛选DNA复制途径的新型抑制剂，该途径由多个必需的、药物验证的靶点组成。在第一阶段，我们将设计一种非致病性的M。以构建透性细胞DNA复制途径筛选。我们将验证这个屏幕，优化它的高通量，并将其应用于100 K离散化学和天然产物结构的多样化库。将在重复测定中确认抑制DNA复制的化合物，并在谱分析测定中进行评价，在谱分析测定中，我们测试经验证的命中物的纯度和质量、对完整Mtb细胞的效力、抑制DNA复制的特异性、对培养物中哺乳动物细胞的选择性以及其他药物样性质。最后，我们将尝试确定化合物抑制的酶靶点。我们将开始与2个基本的DNA复制靶DNA聚合酶III（pol III）和DNA促旋酶。Microbiotix在低G：C革兰氏阳性菌中这些酶的制备和测定方面具有相当丰富的经验。本研究的具体目标是：（1）建立一种利用M. smegalloy; Mtb的近亲;（2）用高通量DNA复制测定筛选不同化合物文库并确认所得命中;（3）概况确认对细菌细胞的活性、DNA合成抑制特异性和哺乳动物细胞毒性的命中;和（4）构建来自Mtb的pol III和促旋酶的生化二级测定。在第二阶段，我们将优化最有前途的化合物，以开发新的先导化合物和候选药物，用于在感染动物模型中进行临床前试验。

DESCRIPTION (provided by applicant): New antibiotics based on novel chemical scaffolds against new bacterial targets are vital to reducing the tide of tuberculosis (TB) infection worldwide. Due to the lengthy treatment requirements and the difficulty in achieving treatment compliance, there is an increasing rate of development of multi-drug resistant TB. In addition, the coinfection of TB with HIV, in areas where AIDS is epidemic, is killing 1 in 3 people with AIDS. The goal of this research project is to discover and develop novel antibiotics effective against Mycobacterium tuberculosis (Mtb). Our strategy is to screen for novel inhibitors of the DNA replication pathway, which consists of multiple essential, drug-validated targets. In Phase I, we will engineer a non-pathogenic M. smegmatis strain to build a permeable-cell DNA replication pathway screen. We will validate this screen, optimize it for high throughput, and apply it to a diverse library of 100K discrete chemical and natural product structures. Compounds that inhibit DNA replication will be confirmed in replicate assays and evaluated in profiling assays where we test the validated hits for purity and mass, potency on intact Mtb cells, specificity for inhibition of DNA replication, selectivity with respect to mammalian cells in culture, and other drug-like properties. Finally, we will attempt to identify enzyme targets against which compounds are inhibitory. We will begin with 2 essential DNA replication targets-DNA polymerase III (pol III) and DNA gyrase. Microbiotix has considerable experience with the preparation and assay of these enzymes in low G:C Gram+ bacteria. The specific aims are to (1) develop a permeable-cell, high-throughput DNA replication pathway assay using M. smegmatis; a close relative of Mtb; (2) screen a diverse compound library with the high-throughput DNA replication assay and confirm resulting hits; (3) profile confirmed hits for activity on bacterial cells, specificity for DNA synthesis inhibition, and mammalian cell cytotoxicity; and (4) construct biochemical secondary assays for pol III and gyrase from Mtb. In Phase II, we will optimize the most promising compounds to develop novel leads and candidate drugs for pre-clinical testing in animal models of infection.