DEVELOPMENT OF INHIBITORS OF MYCOBACTERIUM TUBERCULOSIS DNA REPLICATION

描述（由申请人提供）：基于新型化学支架对抗新细菌靶点的新型抗生素对减少全球结核病（TB）感染趋势至关重要。由于治疗需要时间长，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，结核病与艾滋病毒的合并感染导致三分之一的艾滋病患者死亡。本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，该途径由多个基本的药物验证靶点组成。在第一阶段，我们将设计一种非致病性耻垢分枝杆菌菌株来构建可渗透细胞DNA复制途径筛选。我们将验证该筛选，优化其高通量，并将其应用于100K分立化学和天然产物结构的各种文库。抑制DNA复制的化合物将在重复试验中得到确认，并在分析分析中进行评估，我们将测试经过验证的命中物的纯度和质量、对完整结核分枝杆菌细胞的效力、抑制DNA复制的特异性、对培养的哺乳动物细胞的选择性以及其他类似药物的特性。最后，我们将尝试确定对哪些化合物具有抑制作用的酶目标。我们将从2个基本的DNA复制靶点开始-DNA聚合酶III （pol III）和DNA回转酶。Microbiotix在低G:C革兰氏+细菌中这些酶的制备和分析方面有着丰富的经验。具体目标是：(1)利用耻垢分枝杆菌开发一种可渗透细胞、高通量的DNA复制途径试验；Mtb的近亲；(2)利用高通量DNA复制试验筛选多种化合物文库，并确认结果命中；(3)对细菌细胞的活性、DNA合成抑制的特异性和哺乳动物细胞毒性的谱确认命中；(4)构建Mtb中pol III和gyrase的生化二级分析方法。在第二阶段，我们将优化最有希望的化合物，以开发新的先导物和候选药物，用于动物感染模型的临床前测试。

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.