Probes for a tRNA modification enzyme

tRNA 修饰酶探针

基本信息

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
项目类别：
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

项目摘要

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.

描述：虽然细菌感染对人类健康构成了主要威胁，但抗菌发现很困难，尤其是基于单酶的策略。两个主要原因是：单个酶靶标的抗性突变的发生，以及用于筛选单个酶靶标的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排名为主要抗菌靶标，因为它对于细菌生长至关重要，在细菌种类中广泛保守，与其人类对应物不同，并且具有模仿s-腺苷蛋白酶（Adome）的药物样分子的“可药物”位点可以结合。 TRMD与临床抗生素的靶标不同（核糖体，DNA陀螺酶和拓扑异构酶以及细胞壁生物合成酶）。取而代之的是，TRMD是一种使用ADOMEM作为甲基供体的tRNA酶，可将G37转换为M1G37。我们假设TRMD对基于酶的药物发现具有吸引力。因为靶向TRMD会减少细菌外排，从而使多种药物的细胞内积累在发生抗性之前快速杀死细胞。我们还建议针对TRMD的药物必须探索新颖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）（GSK）已做出了巨大的努力，以作为细菌中成长的酶成员的成员瞄准TRMD，但其抗TRMD程序却停滞不前，部分原因是在高发射率筛选（HTS）中使用了放射性的3h-podote。相反，我们建议开发和优化一种更强大和更具成本效益的新型荧光测定，并且基于与3H分析的原理不同。这种荧光测定法的发展将使新型抑制剂类别发现靶向TRMD如何在创新的生长停滞机制中与临床使用中的抗生素机制不同，靶向TRMD如何对细菌外排造成附带损害。使用大肠杆菌TRMD（ECTRMD）作为模型，初步工作已验证了荧光测定的鲁棒性和休息性对HTS格式。 AIM 1将进一步改善分析的参数。 AIM 2将通过与哈佛大学的NSRB/ICCB-Longwood（NSRB/ICCB-L）筛选设施进行合作来验证HTS准备的测定。验证后，我们将在哈佛大学发起一个大规模的筛选活动，以筛选来自不同化学库的500,000种化合物。假阳性将在柜台屏幕上删除，并将使用次级，第三级和表型测定法进行筛选。 AIM 3将验证能力的命中基于我们最近开发的TRNA结合晶体结构的TRMD在整个细胞中靶向ECTRMD，并将通过化学优化提高命中质量，与Sininefungin（ADOMEM的非反应性类似物）中的复合物中的TRMD结构。带有所需标准的命中将测试是否有生长停滞和体内功效。已确定的命中将作为了解靶向TRMD的生长危机机制的化学探针，并作为抗生素发现的引导，以应对细菌传染病的全球负担。