High Throughput Screening to Discover Novel Toxin Inhibitors Relevant to the Treatment of Otitis Media

高通量筛选发现与中耳炎治疗相关的新型毒素抑制剂

• 批准号: 9310342
• 负责人: Dayle Anita Daines
• 金额: $ 40.64万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-17 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9310342
• 关键词: Active Sites; Acute Disease; Affect; Affinity; Air; Antibiotic Therapy; Antibiotics; Antitoxins; Bacteria; Bacterial Genome; Binding; Biological Assay; Cell Line; Cell physiology; Cells; Characteristics; Chemicals; Childhood; Chinchilla (genus); Chronic Bronchitis; Chronic Obstructive Airway Disease; Cleaved cell; Clinical; Complex; Detection; Disease; Dose; Effectiveness; Engineering; Epithelial; Experimental Models; Fibroblasts; Fluorescence; Genes; Goals; Gram-Negative Bacteria; Growth; Homologous Protein; Human; In Vitro; Individual; Infection; Influenza; Lead; Library Science; Liquid substance; Lung; Measures; Mediating; Messenger RNA; Modeling; Molecular; Nontypable Haemophilus influenza; Nutrient; Operon; Organism; Otitis Media; Pancreatic ribonuclease; Parents; Pathway interactions; Patients; Peptide Hydrolases; Primary Care Physician; Primary Infection; Proteins; Public Health; RNA Degradation; Recurrence; Recurrent disease; Ribonucleases; Specificity; Stress; Targeted Toxins; Testing; Tissues; Toxic effect; Toxicity Tests; Toxin; Translational Research; United States National Institutes of Health; Vaccines; Validation; alpha Toxin; antimicrobial; counterscreen; cytotoxicity; design; efficacy study; high throughput screening; improved; in vivo; inhibitor/antagonist; innovation; killings; miniaturize; mutant; new therapeutic target; novel; pathogen; prevent; public health relevance; respiratory; response; screening; small molecule; small molecule libraries

 DESCRIPTION (provided by applicant): Otitis media (OM) is the second most common disease of childhood, and an important OM pathogen that can cause both acute and recurrent disease is nontypeable Haemophilus influenzae (NTHi). We have identified a toxin-antitoxin protein pair in NTHi, VapBC-1, as being crucial for the ability of the organism to survive during experimental OM. Upon encountering stressful conditions, the VapB-1 antitoxin is degraded and the VapC-1 ribonuclease toxin is freed to cleave mRNA, arresting bacterial growth. This state of dormancy facilitates nonspecific antibiotic tolerance, as most antimicrobials are designed to inhibit essential cellular functions necessary for replication. Further, it allows a subpopulation f the infecting bacteria to survive treatment and resume growth, resulting in recurrent disease. Homologues of this protein pair are very highly conserved in numerous bacterial genomes. We intend to target this protease-mediated molecular switch and block the ability of the organism to go into stasis using a high-throughput screening approach to discover and validate novel chemical probes that interfere with VapC-1 toxin activity. These small molecule probes will inhibit the ability of the organism to enter into a bacteriostatic state, allowing antimicrobial therapy to kill the bacteria that were previously tolerant due to dormancy. We propose the following Specific Aims: 1) Use our innovative miniaturized and validated fluorescence-quenching ribonuclease interference assay to screen the NCATS library of small molecules for toxin-targeted compounds (TTCs); 2) perform secondary screens against the hits including a different detection wavelength, a specificity assay and a cytotoxicity assay; 3) validate the specificity of the non-cytotoxic TTCs in vitro using an engineered bacterial strain with inducible VapC-1 toxin expression, and 4) determine the effectiveness of the TTCs to treat NTHi infections of primary human respiratory tissues held at the air-liquid interface. Treatment with the chemical probes validated in this study could be used in conjunction with antibiotics to achieve a complete eradication of infecting organisms, giving clinicians a novel adjunctive therapy for their patients with recurrent disease.

 描述（由申请人提供）：中耳炎（OM）是儿童中第二常见的疾病，可引起急性和复发性疾病的重要OM病原体是不可分型流感嗜血杆菌（NTHi）。我们已经鉴定出一种毒素-抗毒素蛋白。 NTHi 中的 VapBC-1 配对，对于实验 OM 期间生物体的生存能力至关重要，在遇到应激条件时，VapB-1 抗毒素会被降解，并且VapC-1 核糖核酸酶毒素被释放以裂解 mRNA，抑制细菌生长，这种休眠状态有利于非特异性抗生素耐受，因为大多数抗菌药物旨在抑制复制所需的基本细胞功能。此外，它还允许感染细菌的亚群存活。治疗并恢复生长，导致该蛋白质对的同源物在许多细菌基因组中高度保守，我们打算针对这种蛋白酶介导的分子开关并阻断其能力。使用高通量筛选方法发现和验证干扰 VapC-1 毒素活性的新型化学探针，使生物体进入停滞状态，这些小分子探针将抑制生物体进入抑菌状态的能力，从而允许抗菌治疗。杀死以前因休眠而耐受的细菌，我们提出以下具体目标：1) 使用我们创新的小型化且经过验证的荧光淬灭核糖核酸酶干扰测定来筛选细菌。毒素靶向化合物 (TTC) 的 NCATS 小分子库；2) 对命中结果进行二次筛选，包括不同的检测波长、特异性测定和细胞毒性测定；3) 使用体外方法验证非细胞毒性 TTC 的特异性具有可诱导的 VapC-1 毒素表达的工程细菌菌株，以及 4) 确定 TTC 治疗气液界面处原发性人类呼吸组织 NTHi 感染的有效性。本研究中验证的化学探针可以与抗生素结合使用，以实现彻底根除感染微生物，为复发性疾病患者提供一种新的辅助疗法。