Targeting a New Essential Virulence Mechanism in Drug-Resistant Mycobacteria

描述（由申请人提供）：结核分枝杆菌（Mtb）感染至少30%的世界人口，估计每年造成180万人死亡。耐药结核菌株的出现，占先前治疗的结核病病例的20%，加剧了这一本已令人担忧的流行病。目前结核病治疗的不足之处需要发现新的药物来治疗结核分枝杆菌感染。我们已经发现了Mtb蛋白RelMtb在小鼠中急性和慢性Mtb感染中的重要作用。RelMtb合成并水解一种重要的细菌信号分子，称为（p）ppGpp。我们已经表明，Mtb感染的所有阶段都需要RelMtb的（p）ppGpp水解酶活性。这表明活动性和慢性结核病都可以通过以下方法治疗： 用小分子抗生素抑制RelMtb水解酶结构域。重要的是，RelMtb尚未被开发为抗菌靶标，因此，在其他药物靶标中具有突变的耐药Mtb菌株仍然对RelMtb的化学抑制剂敏感。该项目第一阶段的目标是：1）开发筛选RelMtb介导的（p）ppGpp水解抑制剂的测定方法，2）验证RelMtb作为可药用靶标。具体而言，我们将追求以下三个目标：R21-1。开发RelMtb（p）ppGpp水解酶活性的非放射性高通量体外测定。 R21-2开发基于分枝杆菌细胞的体内试验，以筛选RelMtb活性的抑制剂。 R21-3使用小型集中化合物文库进行中试筛选，以证明高通量筛选试验的适用性。该项目第二阶段的目标是1）鉴定先导化合物，2）优化先导化合物，3）靶向RelMtb介导的（p）ppGpp水解以抑制Mtb活力和感染。具体而言，我们将追求以下三个目标：R33-1。在我们的体外和体内测定中筛选选择的化合物文库。 R33-2基于成功抑制剂的支架、对RelMtb的选择性、代谢稳定性和RelMtb蛋白结构，设计化学抑制剂以优化对RelMtb的活性。R33-3展示临床前概念验证的抑制剂，以打击结核分枝杆菌感染和耐药性问题。这些目标的成功实现将导致迫切需要的结核病治疗新战略的制定。RelMtb同源物在所有细菌中是保守的，但在动物中不是，因此我们的发现可能会影响其他致病性和众所周知的耐药细菌的治疗，包括粪肠球菌，化脓性链球菌和金黄色葡萄球菌。我们的目标的实现将表征和验证（p）ppGpp水解酶作为针对耐药细菌病原体的治疗干预的靶标。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) infects at least 30% of the world's population and causes an estimated 1.8 million deaths a year. The emergence of drug-resistant Mtb strains, which constitute 20% of previously treated tuberculosis (TB) cases, has exacerbated this already alarming epidemic. The inadequacies of present TB therapies demand the discovery of new agents to treat Mtb infection. We have discovered a role for the Mtb protein RelMtb that is essential for acute and chronic Mtb infection in mice. RelMtb both synthesizes and hydrolyzes an important bacterial signaling molecule termed (p)ppGpp. We have shown that it is specifically the (p)ppGpp hydrolase activity of RelMtb that is required for all stages of Mtb infection. This indicates that both active and chronic TB could be treated by inhibiting the RelMtb hydrolase domain with a small molecule antibiotic. Importantly, RelMtb has not yet been exploited as an antibacterial target and, therefore, drug-resistant Mtb strains with mutations in other drug targets will still be susceptible to chemical inhibitors of RelMtb. The objectives of the first phase of this project are to 1) develop assays to screen for inhibitors of RelMtb-mediated (p)ppGpp hydrolysis and 2) validate RelMtb as a druggable target. Specifically, we will pursue the following 3 Aims: R21-1. Develop non-radioactive high-throughput in vitro assays of RelMtb (p)ppGpp hydrolase activity. R21-2. Develop in vivo mycobacterial cell-based assays to screen for inhibitors of RelMtb activity. R21-3. Conduct pilot screens with small focused compound libraries to demonstrate suitability of assays for high throughput screening. The objectives of the second phase of this project are to 1) identify a lead compound, 2) optimize the lead compound, and 3) target RelMtb-mediated (p)ppGpp hydrolysis to inhibit Mtb viability and infection. Specifically, we will pursue the following 3 Aims: R33-1. Screen select compound libraries in our in vitro and in vivo assays. R33-2. Design chemical inhibitors to optimize activity against RelMtb based on the scaffold of successful inhibitors, selectivity against RelMtb, metabolic stability, and the RelMtb protein structure. R33-3. Demonstrate preclinical proof-of-concept for inhibitors to combat Mtb infection and the drug-resistance problem. Successful completion of these aims will lead to the development of a critically needed new strategy for TB therapy. RelMtb homologs are conserved in all bacteria, but not in animals, and thus our findings could impact the treatment of other pathogenic and notoriously drug-resistant bacteria including Enterococcus faecalis, Streptococcus pyogenes, and Staphylococcus aureus. Achievement of our aims will characterize and validate (p)ppGpp hydrolases as a target for therapeutic intervention against drug-resistant bacterial pathogens.