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重大威胁，但抗菌药物的发现一直很困难，特别是基于单酶的策略。两个主要原因很突出：单一酶靶点中出现抗性突变，以及用于筛选单一酶靶点抑制剂的化合物库的化学多样性不足。最近的一项全基因组分析将 TrmD 酶列为主要抗菌靶标，因为它对细菌生长至关重要，在细菌物种中广泛保守，与人类对应物不同，并且具有药物样分子模仿的“可成药”位点S-腺苷甲硫氨酸（AdoMet）可以结合。 TrmD 与临床抗生素的靶标（核糖体、DNA 旋转酶和拓扑异构酶以及细胞壁生物合成酶）不同。相反，TrmD 是一种 tRNA 酶，它使用 AdoMet 作为甲基供体将 G37 修饰为 m1G37。我们假设 TrmD 对于基于单酶的药物发现具有吸引力；因为靶向TrmD会减少细菌外流，使多种药物在细胞内积累，在耐药性出现之前快速杀死细胞。我们还建议针对 TrmD 的药物必须探索新的化学空间和多样性。尽管阿斯利康 (AZ) 和葛兰素史克 (GSK) 制药公司已做出巨大努力，将 TrmD 作为细菌生长必需酶的成员，但他们的抗 TrmD 计划已陷入停滞，部分原因是在细菌中使用了放射性 3H-AdoMet。高通量筛选（HTS）测定。相反，我们建议开发和优化一种新型荧光测定法，该测定法更加稳健且更具成本效益，并且基于与 3H 测定法不同的原理。这种荧光测定的开发将能够发现新型抑制剂，以探究靶向 TrmD 如何在不同于临床使用抗生素机制的创新生长抑制机制中对细菌流出造成附带损害。使用大肠杆菌 TrmD (EcTrmD) 作为模型，初步工作验证了 HTS 格式荧光测定的稳健性和适应性。目标 1 将进一步改进测定的参数。 Aim 2 将与哈佛大学 NSRB/ICCB-Longwood (NSRB/ICCB-L) 筛选机构合作，验证 HTS 就绪检测方法。验证后，我们将在哈佛大学发起大规模筛选活动，从不同的化学库中筛选约 500,000 种化合物。假阳性将在计数器筛选中消除，并且将使用二级、三级和表型测定来筛选命中池。目标 3 将验证该能力的命中靶向全细胞中的 EcTrmD，并将基于我们最近开发的 TrmD 与 sinefungin（AdoMet 的非反应性类似物）复合物的 tRNA 结合晶体结构，通过化学优化来提高命中质量。将测试符合所需标准的命中的生长停滞和体内功效。确定的命中将作为化学探针，以了解针对 TrmD 的生长抑制机制，并作为抗生素发现的线索，以解决全球细菌传染病的负担。