Combinatorial approach to develop novel pre-therapeutic agents targeting virulence factors essential to clinically relevant pathogens

项目摘要/摘要 抗生素耐药性是一个严重的全球健康威胁。目前的抗生素针对的是必要的细菌过程 并对抵抗施加强大的选择压力。在抗生素治疗后，健康的微生物群是 数量和多样性都减少，导致严重的健康后果，如艰难梭菌 结肠炎。此外，抗药性特征可能会转移到其他微生物身上，导致抗生素的传播。 抵抗。使用毒力阻滞剂靶向特定的致病机制，同时离开微生物区系 完好无损，是一种很有希望的减少阻力的策略。这项提议将识别一种新的分子靶标 发现了III型分泌系统(T3SS)抑制物，并进一步探讨了它们的作用模式 优化发展。我将评估构效关系以优化T3SS抑制剂， 利用亲和力方法鉴定其在假单胞菌中的分子靶标 铜绿假单胞菌。此外，基于靶点的药物发现具有成本低、时间短的优点。 在消费。随着高分辨率结构的可用性和预测结合的算法的发展 将小分子的亲和力和姿态作为其蛋白质靶标，虚拟筛选可以为优化提供线索。 ExoU是一种具有磷脂酶A2活性的效应器，是六种ESKAPE之一的铜绿假单胞菌的主要效应器 病原体是导致美国大多数医院感染的病原体，它们“逃脱”了抗菌药物的治疗。 ExoU有一个丝氨酸/天冬氨酸催化二联体和一个单独的辅因子结合结构域，需要激活 这种酶。ExoU具有高度毒性，与急性感染、抗生素耐药性和严重后果有关 专利。延迟ExoU的表达可以提高小鼠的存活率。因此我们开始寻找ExoU抑制剂作为一种 治疗急性感染和减少耐药性的策略。我们将确定ExoU抑制剂：1)抑制酶 活性通过靶向其催化残基丝氨酸，或2)结合到膜的定位结构域，这将 通过虚拟筛选阻止ExoU的激活。等温条件下与ExoU具有结合亲和力的抑制剂 滴定量热法和防止ExoU引起的细胞死亡将被选为优化方法。的结构 抑制剂结合的蛋白质将使用X射线结晶学进行解析。我相信我的导师团队(Dr。 斯通和奥特曼)，顾问(鲁宾博士，X射线结晶学专家；雅各布森博士，计算机专家- 辅助药物发现)和合作者(洛基博士，高分子合成专家，克鲁斯博士和 林宁顿，天然产品化学家)将为我提供支持，以成功地开展这个项目。与 我将学习生化技术，我将在K99阶段获得结构和新的抑制剂，然后我将 与Shaw博士(药物化学家)和Jacobson博士合作，优化我的 独立阶段。这个项目扩展了我在生化方法方面的技能，并有可能提供 药物发现和开发的巨大势头。这些将作为R01的基础 将在本研究计划的主要阶段完成后编写的建议。

Project Summary/Abstract Antibiotic resistance is a serious global health threat. Current antibiotics target essential bacterial processes and impose strong selective pressure for resistance. Upon antibiotic treatment, the healthy microbiota are reduced in both number and diversity, leading to serious health consequences such as Clostridium difficile colitis. Moreover, resistant traits can be transferred to other microbes, leading to the spread of antibiotic resistance. Using virulence blockers to target specific pathogenicity mechanisms, while leaving the microbiota intact, is a promising strategy to reduce resistance. This proposal will identify molecular target of a newly discovered the type III secretion system (T3SS) inhibitor, and explore their modes of action for further optimization and development. I will assess structure – activity relationship to optimize the T3SS inhibitors, cyclic pepeptomers, and use affinity based method to identify their molecular targets in Pseudomonas aeruginosa. Besides, target-based drug discovery offers the advantage of being low cost and less time consuming. With the availability of high-resolution structure and development of algorithms to predict binding affinity and poses of small molecules to its protein target, virtual screening can provide lead for optimization. ExoU, an effector with phospholipase A2 activity, is the major effector in P. aeruginosa, one of six ESKAPE pathogens which cause the majority of nosocomial infections in the U.S. and “escape” antimicrobial drugs. ExoU has a serine/aspartate catalytic dyad and a separate cofactor-binding domain required for activation of the enzyme. ExoU is highly toxic, associated with acute infection, antibiotic resistance and severe outcome in patents. Delay ExoU expression can increase mice survival. Thus we set out to find ExoU inhibitors as a strategy to treat acute infection and reduce resistance. We will identify ExoU inhibitors that 1) inhibit enzymatic activity by targeting its catalytic residue serine, or 2) bind to the membrane localization domain which will prevent ExoU's activation by virtual screening. The inhibitors that show binding affinity to ExoU in isothermal titration calorimetry assay, and prevent cell death caused by ExoU will be chosen for optimization. Structure of inhibitor-bound proteins will be solved using X-ray crystallography. I believe that my team of mentors (Drs. Stone and Ottemann), advisors (Dr. Rubin, expert in X-ray crystallography; Dr. Jacobson, expert in computer- aided drug discovery) and collaborators (Dr. Lokey, an expert in macromolecule synthesis, Drs. Crews and Linington, natural product chemists) will provide me support to successfully carry out this project. With the biochemical techniques I will learn, the structures and new inhibitors I will obtain in the K99 phase, I will then collaborate with Dr. Shaw (medicinal chemist) and Dr. Jacobson to optimize the candidate hits in my independent phase. This project extends my skill set in biochemical methods and has the potential to provide substantial momentum towards drug discovery, and development. These will serve as the foundation of a R01 proposal to be prepared upon the completion of the main stages of this research plan.