Targeting a New Essential Virulence Mechanism in Drug-Resistant Mycobacteria

针对耐药分枝杆菌的新基本毒力机制

• 批准号: 8704078
• 负责人: Christina Leigh Stallings
• 金额: $ 22.22万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8704078
• 关键词: Achievement; Acute; Animals; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Biological Assay; Catalytic Domain; Cell surface; Cells; Cessation of life; Chemicals; Chronic; Clinical Trials; Development; Diphosphates; Disease; Drug Resistant Tuberculosis; Drug Targeting; Drug resistance; Drug resistance in tuberculosis; Drug-sensitive; Enterococcus faecalis; Enzymes; Epidemic; Genetic Transcription; Genotoxic Stress; Genus Mycobacterium; Guanine Nucleotides; Guanosine Triphosphate; Health; Homologous Gene; Human; Hydrolase; Hydrolysis; Hypoxia; In Vitro; Infection; Lead; Libraries; Mediating; Metabolic; Molecular; Morphology; Mus; Mutation; Mycobacterium tuberculosis; New Agents; Nucleotides; Oxidative Stress; Pharmaceutical Preparations; Phase; Phosphodiesterase Inhibitors; Population; Production; Proliferating; Proteins; RNA; Reporting; Role; Signaling Molecule; Staging; Staphylococcus aureus; Starvation; Streptococcus pyogenes; Stress; Therapeutic Intervention; Tuberculosis; Virulence; World Health Organization; acid stress; base; biological adaptation to stress; combat; design; drug resistant bacteria; high throughput screening; in vitro Assay; in vivo; inhibitor/antagonist; killings; mutant; mycobacterial; novel therapeutics; pathogen; phosphoric diester hydrolase; pre-clinical; protein structure; public health relevance; research study; resistant strain; response; scaffold; small molecule; tuberculosis treatment

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.

描述(申请人提供)：结核分枝杆菌(Mtb)感染至少30%的世界人口，估计每年导致180万人死亡。耐药结核分枝杆菌菌株的出现加剧了这一本已令人担忧的流行病，该菌株占以前治疗的结核病病例的20%。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。我们已经发现了Mtb蛋白RelMtb的一个作用，它对小鼠的急性和慢性Mtb感染至关重要。RelMtb可以合成和降解一种重要的细菌信号分子，称为(P)ppGpp。我们已经证明，正是RelMtb的(P)ppGpp水解酶活性是结核分枝杆菌感染的所有阶段所必需的。这表明活动性和慢性结核病都可以通过 用小分子抗生素抑制RelMtb水解酶结构域。重要的是，RelMtb尚未被用作抗菌靶点，因此，具有其他药物靶点突变的耐药Mtb菌株仍将对RelMtb的化学抑制剂敏感。该项目第一阶段的目标是：1)开发检测方法，筛选RelMtb介导的(P)ppGpp水解酶的抑制剂；2)确认RelMtb是一个可用药的靶点。具体来说，我们将追求以下3个目标：R21-1。建立RelMtb(P)ppGpp水解酶活性的非放射性高通量体外检测方法。R21-2。开发基于体内分枝杆菌细胞的检测方法来筛选RelMtb活性的抑制物。R21-3。使用小的聚焦化合物文库进行中试筛选，以证明分析方法对高通量筛选的适用性。该项目第二阶段的目标是1)确定一个先导化合物，2)优化先导化合物，以及3)靶向RelMtb介导的(P)ppGpp水解以抑制Mtb的活性和感染。具体来说，我们将追求以下3个目标：R33-1。在我们的体外和体内实验中筛选选定的化合物文库。R33-2。根据成功抑制剂的骨架、对RelMtb的选择性、代谢稳定性和RelMtb的蛋白质结构，设计化学抑制剂以优化抗RelMtb的活性。R33-3。展示用于对抗结核分枝杆菌感染和耐药性问题的抑制剂的临床前概念验证。这些目标的成功实现将导致制定迫切需要的结核病治疗新战略。RelMtb同源物在所有细菌中都是保守的，但在动物中不是，因此我们的发现可能会影响其他致病和臭名昭著的耐药细菌的治疗，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水解酶作为治疗干预耐药细菌病原体的目标。