Probes for a tRNA modification enzyme

tRNA 修饰酶探针

• 批准号: 8760580
• 负责人: Ya-Ming Hou
• 金额: $ 29.48万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760580
• 关键词: Address; Anti-Bacterial Agents; Antibiotics; Area; Bacteria; Bacterial Infections; Binding; Biological Assay; Cell Wall; Cells; Chemicals; Clinical; Collaborations; Communicable Diseases; Complex; Cytokine Inducible SH2-Containing Protein; DNA Gyrase; DNA Topoisomerases; Development; Drug Targeting; Enzymes; Escherichia coli; Fluorescence; Genes; Goals; Growth; Health; Homo sapiens; Homologous Gene; Human; Inhibitory Concentration 50; Libraries; Ligand Binding; Link; Measurement; Membrane; Methionine; Methodology; Methylation; Modeling; Modification; Molecular; Molecular Conformation; Noise; Outcome; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Positioning Attribute; Protein Biosynthesis; Public Health; Radioactive; Reaction; Reagent; Relative (related person); Reporting; Reproducibility; Resistance; Ribosomes; Signal Transduction; Site; Structure; Testing; Transfer RNA; Validation; Work; analog; assay development; base; cell growth; cell killing; cost effective; cytotoxicity; design; drug discovery; efflux pump; enzyme biosynthesis; experience; genome-wide analysis; high throughput screening; improved; in vivo; inhibitor/antagonist; innovation; member; novel; programs; protein expression; public health relevance; resistance mutation; screening; sinefungin; small molecule libraries

DESCRIPTION: While bacterial infections present major threats to human health, antibacterial discovery has been difficult, particularly with the single-enzyme-based strategy. Two main reasons stand out: the occurrence of resistance mutations in the single enzyme target, and the insufficient chemical diversity of compound libraries used for screening inhibitors of the single enzyme target. A recent genome-wide analysis has ranked the enzyme TrmD as a leading antibacterial target, because it is essential for bacterial growth, broadly conserved across bacterial species, distinct from its human counterpart, and has a "druggable" site that drug-like molecules mimicking S- adenoscyl methionine (AdoMet) can bind to. TrmD is unlike targets of clinical antibiotics (the ribosome, DNA gyrase and topoisomerases, and cell-wall biosynthesis enzymes). Instead, TrmD is a tRNA enzyme that modifies G37 to m1G37 using AdoMet as the methyl donor. We hypothesize that TrmD is attractive for singe- enzyme-based drug discovery; because targeting TrmD would reduce bacterial efflux, allowing intracellular accumulation of multiple drugs for rapid cell killing before the occurrence of resistance. We also suggest that drugs targeting TrmD must explore novel chemical space and diversity. While pharmaceutical companies AstraZeneca (AZ) and GlaxoSmithKline (GSK) have made intense efforts to target TrmD as a member of growth-essential enzymes in bacteria, their anti-TrmD program has stalled, in part due to the use of radioactive 3H-AdoMet in a high-throughput screening (HTS) assay. We propose instead to develop and optimize a novel fluorescence assay that is more robust and cost-effective and is based on a principle different from that of the 3H assay. The development of this fluorescence assay will enable discovery of novel classes of inhibitors to probe how targeting TrmD can cause collateral damage on bacterial efflux in an innovative growth arrest mechanism distinct from the mechanisms of antibiotics in clinical use. Using E. coli TrmD (EcTrmD) as a model, preliminary work has validated the robustness and amenability of the fluorescence assay to the HTS format. Aim 1 will further improve parameters of the assay. Aim 2 will validate the assay for HTS-ready by collaboration with the NSRB/ICCB-Longwood (NSRB/ICCB-L) screening facility at Harvard. Following the validation, we will launch a large-scale screening campaign at Harvard to screen ~500,000 compounds from diverse chemical libraries. False positives will be removed in counter screens and the hit pool will be screened using secondary, tertiary, and phenotypic assays. Aim 3 will validate hits for the ability of targeting EcTrmD in the whole cell and will improve qualities of hits by chemical optimization based on our recently developed tRNA-bound crystal structure of TrmD in complex with sinefungin (a non-reactive analog of AdoMet). Hits with desired criteria will be tested for growth arrest and in vivo efficacy. The identified hits will serve as chemical probes to understand the growth-arrest mechanism of targeting TrmD and as leads for antibiotic discovery to address the global burden of bacterial infectious disease.

产品说明：虽然细菌感染对人类健康构成重大威胁，但抗菌剂的发现一直很困难，特别是基于单酶的策略。有两个主要原因：在单一酶靶中出现抗性突变，以及用于筛选单一酶靶抑制剂的化合物文库的化学多样性不足。最近的全基因组分析将酶TrmD列为主要的抗菌靶标，因为它是细菌生长所必需的，在细菌物种中广泛保守，与其人类对应物不同，并且具有模拟S-腺苷甲硫氨酸（S-adenosyl methionine，TRMet）的药物样分子可以结合的“可药用”位点。TrmD不同于临床抗生素的靶点（核糖体、DNA促旋酶和拓扑异构酶以及细胞壁生物合成酶）。相反，TrmD是一种tRNA酶，它使用α-Met作为甲基供体将G37修饰为m1 G37。我们假设TrmD对于基于单酶的药物发现是有吸引力的;因为靶向TrmD将减少细菌外排，允许多种药物在耐药性发生之前在细胞内积累以快速杀死细胞。我们还建议靶向TrmD的药物必须探索新的化学空间和多样性。 虽然制药公司阿斯利康（AZ）和葛兰素史克（GSK）已经做出了巨大的努力，将TrmD作为细菌中生长必需酶的成员，但他们的抗TrmD计划已经停滞，部分原因是在高通量筛选（HTS）试验中使用了放射性3 H-Met。相反，我们建议开发和优化一种新的荧光检测方法，该方法更稳健，更具成本效益，并且基于与3 H检测不同的原理。这种荧光测定的开发将能够发现新型抑制剂，以探索靶向TrmD如何在与临床使用的抗生素机制不同的创新生长停滞机制中对细菌外排造成附带损害。 使用大肠coliTrmD（EcTrmD）作为模型，初步工作验证了荧光分析对HTS格式的稳健性和可适应性。目标1将进一步改进测定的参数。目标2将通过与哈佛的NSRB/ICCB-Longwood（NSRB/ICCB-L）筛选机构合作，验证HTS-ready检测试剂盒。在验证之后，我们将在哈佛开展大规模筛选活动，从不同的化学库中筛选约50万种化合物。将在计数器筛选中去除假阳性，并使用二级、三级和表型测定筛选命中池。目标3将验证命中的能力 在整个细胞中靶向EcTrmD，并将通过基于我们最近开发的TrmD与sinefungin（一种非反应性的抗肿瘤类似物）复合的tRNA结合晶体结构的化学优化来提高命中质量。将测试具有所需标准的命中物的生长停滞和体内功效。鉴定的命中将作为化学探针，以了解靶向TrmD的生长停滞机制，并作为抗生素发现的线索，以解决细菌感染性疾病的全球负担。