T3SS needle protein inhibitors for the treatment of P. aeruginosa infection

T3SS针蛋白抑制剂用于治疗铜绿假单胞菌感染

• 批准号: 9046046
• 负责人: Joan C Mecsas
• 金额: $ 30.3万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-19 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9046046
• 关键词: Actins; Acute; Address; Affect; Animal Model; Animals; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteremia; Bacteria; Binding Sites; Biological Assay; Cell membrane; Cells; Cessation of life; Clinical; Communities; Critical Illness; Development; Drug Efflux; Drug resistance; Endocarditis; Ensure; Exhibits; Fluorescence; Genetic study; Goals; Gram-Negative Bacterial Infections; Growth; Health; Human; Immune; Immune response; In Vitro; Infection; Intensive Care Units; Intoxication; Lead; Libraries; Lung; Mammalian Cell; Mediating; Medical; Membrane; Methods; Modeling; Monitor; Needles; Nosocomial pneumonia; Outcome; Patients; Phagocytes; Pharmaceutical Preparations; Phase; Pneumonia; Prevalence; Property; Proteins; Pseudomonas aeruginosa; Resistance; Screening Result; Series; Structure; System; Therapeutic; Therapeutic Agents; Thick; Toxicity Tests; Toxin; Urinary tract; Virulence; Virulence Factors; Wound Infection; Yersinia pestis; analog; appendage; bacterial resistance; base; combat; cytotoxic; drug development; efficacy testing; efflux pump; extracellular; high throughput screening; inhibitor/antagonist; novel strategies; novel therapeutics; pathogen; polymerization; public health relevance; resistance factors; resistance mechanism; resistant strain; response; scaffold; screening; self assembly; small molecule; small molecule inhibitor; weapons

 DESCRIPTION (provided by applicant): The inability to treat many Gram-negative bacterial infections effectively with existing antibiotics is a major medical crisis. Pseudomonas aeruginosa is a prime example: 30% of clinical isolates from critically ill patients are resistant to three or more drugs. The overall goal of this project is to address the critical medical need by a novel approach of identifying specific inhibitors of the type-three secretion system (T3SS) targeting the extracellular T3SS needle and developing them into novel therapeutic agents against P. aeruginosa. T3SS is the major virulence factor contributing to the establishment and dissemination of P. aeruginosa infections and is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes and weaken the host's innate immune response. The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients and markedly reduces survival in animal infection models. T3SS inhibitors will be administered therapeutically and prophylactically in combination with anti-pseudomonal agents to inhibit the T3SS, potentiate a robust host innate immune response, and enhance the antibacterial activity of co-administered antibiotics. The strategy is to identify and optimize small molecules that interfere with the extracellular T3SS needle polymerization or stability. Such therapeutics will by-pass P. aeruginosa intrinsic resistance mechanisms caused by a poorly permeable outer membrane and efflux pumps. Preliminary studies revealed a putative binding site for the phenoxyacetamide series of T3SS inhibitors in the polymeric form of the needle protein PscF, indicating that the needle is a target of the P. aeruginosa T3SS for small molecule inhibition. The strategy of screening directly for compounds that alter the needle assembly or stability will capitalize on this newfound vulnerability and provide additional chemotypes of needle inhibitors for the drug development pipeline. In other preliminary studies, we developed methods for the purification of PscF and demonstrated a fluorescence-based assay to monitor the polymerization of a purified T3SS needle protein. In Phase I, a high-throughput screen using purified PscF will be developed, optimized and implemented to identify small molecules that inhibit needle polymerization or stability. Diverse compound libraries will be screened, and resulting 'hits' will be confirmed in the screening assay in replicate, prioritized b potency, and selectivity by eliminating compounds that alter actin polymerization or stability or are promiscuous in multiple screens. Confirmed potent, selective 'hits' will be validated as T3SS inhibitors by determining their ability to inhibit effector secretion and translocation from P. aeruginosa, and by ensuring that they are not cytotoxic, do not disrupt mammalian cell membranes, and do not affect bacterial growth or viability in vitro. Preliminary SAR and in vitro ADME assays and will be used to prioritize analogs. In Phase II, the most promising of these T3SS inhibitors will be optimized to develop lead compounds for efficacy and toxicity testing in animal models.

 描述（由申请人提供）：现有抗生素无法有效治疗许多革兰氏阴性细菌感染是一个重大的医疗危机。铜绿假单胞菌是一个很好的例子：30%的重症患者的临床分离株对三种或三种以上的抗生素耐药。 更多的药物。该项目的总体目标是通过一种新的方法来解决关键的医疗需求，该方法确定了针对细胞外T3SS针的第三型分泌系统（T3SS）的特异性抑制剂，并将其开发为针对铜绿假单胞菌的新型治疗药物。T3SS是促成铜绿假单胞菌感染的建立和传播的主要毒力因子，并且被细菌利用以分泌毒素效应物并将毒素效应物转运到宿主吞噬细胞中，并削弱宿主的先天免疫应答。功能性T3SS的存在与患者的不良临床结局和死亡显著相关，并显著降低动物感染模型的存活率。T3SS抑制剂将与抗假单胞菌剂组合治疗性和抗感染性地施用，以抑制T3SS，增强稳健的宿主先天免疫应答，并增强共同施用的抗生素的抗菌活性。该策略是鉴定和优化干扰细胞外T3SS针聚合或稳定性的小分子。这种治疗剂将绕过由渗透性差的外膜和外排泵引起的铜绿假单胞菌固有抗性机制。初步研究揭示了在针蛋白PscF的聚合形式中T3SS抑制剂的苯氧基乙酰胺系列的推定结合位点，表明针是铜绿假单胞菌T3SS用于小分子抑制的靶标。直接筛选改变针组装或稳定性的化合物的策略将利用这种新发现的脆弱性，并为药物开发管道提供额外的针抑制剂化学型。在其他的初步研究中，我们开发了PscF的纯化方法，并证明了一种基于荧光的测定来监测纯化的T3SS针蛋白的聚合。在第一阶段，将开发、优化和实施使用纯化PscF的高通量筛选，以识别抑制针状聚合或稳定性的小分子。多样化的复合图书馆将成为 筛选，并通过消除改变肌动蛋白聚合或稳定性或在多次筛选中混杂的化合物，在重复、优先B效力和选择性的筛选测定中确认所得“命中物”。通过确定其抑制铜绿假单胞菌的效应物分泌和易位的能力，并通过确保其无细胞毒性、不破坏哺乳动物细胞膜以及不影响体外细菌生长或活力，将确认有效的选择性“命中物”作为T3SS抑制剂。初步SAR和体外ADME试验，并将用于优先考虑类似物。在第二阶段，这些T3SS抑制剂中最有前途的将被优化，以开发用于动物模型中的功效和毒性测试的先导化合物。