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

开发针对临床相关病原体必需毒力因子的新型治疗前药物的组合方法

• 批准号: 10681469
• 负责人: Hanh Ngoc Lam
• 金额: $ 24.07万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-09 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10681469
• 关键词: Active Sites; Affinity; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Aspartate; Bacteria; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biology; C-terminal; Calorimetry; Cause of Death; Cell Death; Cell membrane; Cells; Cessation of life; Chemicals; Collaborations; Communicable Diseases; Computer Assisted; Consumption; Data; Development; Drug resistance; ESKAPE pathogens; Enzyme Activation; Fluorescent Probes; Foundations; Future; Goals; Gram-Negative Bacteria; Health; Health Care Costs; Host Defense Mechanism; Hybrids; Infection; Inflammation; Knowledge; Learning; Legal patent; Libraries; Medicine; Membrane; Mentors; Methods; Microbe; Molecular Target; Mus; N-substituted Glycines; Natural Products; Nosocomial Infections; Outcome; Pathogenesis; Pathogenicity; Pathology; Peptides; Peptoids; Periodicity; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phenotype; Phospholipase A2; Process; Productivity; Proteins; Pseudomembranous Colitis; Pseudomonas; Pseudomonas aeruginosa; Regulation; Research; Resistance; Resolution; Series; Serine; Serine Hydrolase; Specificity; Structure; Structure-Activity Relationship; Techniques; Therapeutic; Time; Titrations; Type III Secretion System Pathway; United States; Virulence; Virulence Factors; Virulent; Work; X-Ray Crystallography; acute infection; algorithm development; antibiotic resistant infections; antimicrobial; antimicrobial drug; appendage; clinically relevant; cofactor; combat; combinatorial; commensal microbes; cost; cost effective; design; drug candidate; drug discovery; extracellular; genetic approach; global health; healthcare-associated infections; high throughput screening; inhibitor; innovation; insight; lead optimization; macromolecule; marine; microbial; microbiome; microbiota; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; pressure; prevent; priority pathogen; screening; skills; small molecule; targeted treatment; therapeutic target; trait; virtual screening

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.

项目摘要/摘要 抗生素耐药性是严重的全球健康威胁。当前的抗生素靶向必需细菌过程 并施加强大的选择性压力。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，例如艰难梭菌 结肠炎。此外，可以将抗性性状转移到其他微生物中，从而导致抗生素扩散 反抗。使用病毒阻滞剂靶向特定的致病机制，同时离开菌群 完整，是减少抵抗力的承诺策略。该建议将确定新的分子靶 发现了III型分泌系统（T3SS）抑制剂，并探索其行动方式以进一步 优化和发展。我将评估结构 - 活动关系以优化T3SS抑制剂， 循环纤维素体，并使用基于亲和力的方法鉴定其分子靶标在假单胞菌中 铜绿。此外，基于目标的药物发现具有低成本和更少的时间的优势 消费。随着高分辨率结构的可用性和算法的发展以预测结合 小分子与其蛋白质靶标的亲和力和姿势，虚拟筛选可以为优化提供铅。 Exou是一种具有磷脂酶A2活性的效应子，是铜绿假单胞菌的主要效应子，是六个Eskape之一 引起美国大多数医院感染和“逃脱”抗菌药物的病原体。 Exou具有连续/天冬氨酸的催化二元组，并且需要一个单独的辅因子结合域激活 酶。 Exou具有剧毒，与急性感染，抗生素耐药性和严重预后有关 专利。延迟外来表达可以增加小鼠的存活。我们着手寻找海外抑制剂 治疗急性感染并降低抗药性的策略。我们将确定抑制酶的抑制剂 通过靶向其催化退休丝氨酸的活动，或2）结合膜定位域，该结构域将 通过虚拟筛选防止Exou的激活。在等温中表现出与exou具有结合亲和力的抑制剂 将选择滴定量热法评估，并预防由Exou引起的细胞死亡进行优化。结构 抑制剂结合的蛋白将使用X射线晶体学求解。我相信我的导师团队（博士。 Stone和Ottemann），顾问（X射线晶体学专家Rubin；雅各布森博士，计算机专家 - 辅助药物发现）和合作者（洛基（Lokey）博士，大分子合成专家，船员和 自然产品化学家Linington）将为我提供支持，以成功地执行该项目。与 我将学习的生化技术，在K99阶段将获得的结构和新抑制剂，然后我将 与Shaw博士（医学化学家）和Jacobson博士合作，以优化我的候选人热门 独立阶段。该项目扩展了我以生化方法设定的技能，并有可能提供 朝着药物发现和发育发展的巨大动力。这些将作为R01的基础 在完成本研究计划的主要阶段完成后准备的建议。