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）感染了世界上至少30%的人口，每年造成约180万人死亡。耐药结核分枝杆菌菌株的出现，占以前治疗的结核病病例的20%，加剧了这一本已令人震惊的流行病。目前结核病治疗方法的不足要求发现治疗结核分枝杆菌感染的新药物。我们已经发现结核分枝杆菌蛋白RelMtb在小鼠急性和慢性结核分枝杆菌感染中所起的作用。RelMtb合成并水解一种重要的细菌信号分子，称为(p)ppGpp。我们已经证明，在结核分枝杆菌感染的所有阶段，RelMtb的(p)ppGpp水解酶活性都是必需的。这表明活动性和慢性结核病都可以通过

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.