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Probe discovery for tRNA methylation

tRNA 甲基化探针的发现

基本信息

批准号：
9925237
负责人：
Ya-Ming Hou
金额：
$ 42.33万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-01 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9925237
关键词：
Address Anabolism Anti-Bacterial Agents Antibiotics Anticodon Bacteria Bacterial Infections Binding Biochemistry Biological Assay Biology Catalogs Cell Death Cells Cellular Assay Chemical Structure Chemicals Clinical Codon Nucleotides Collection Complex Computer Assisted Crystallization Dose Drug Binding Site Drug Efflux Drug Targeting Enzymes Escherichia coli Event Exhibits Fluorescence Gene Expression Genes Genetic Transcription Gram-Negative Bacteria Growth Hand Homo sapiens Homologous Gene Human Initiator Codon Libraries Measurement Mediating Medical Membrane Membrane Proteins Messenger RNA Methodology Methylation Modality Modeling Modern Medicine Modification Molecular Molecular Conformation Multi-Drug Resistance Permeability Pharmaceutical Preparations Phenotype Positioning Attribute Powder dose form Protein Biosynthesis Proteins Pump Radioactivity Reading Frames Reagent Reporting Resistance Ribosomes S-Adenosylhomocysteine Series Shapes Side Site Specificity Structure Structure-Activity Relationship Testing Therapeutic Effect Time Transfer RNA Transferase Translations Vendor Work analog bactericide base cell growth chemical property cheminformatics cost effective cross reactivity drug discovery efflux pump gene discovery genome-wide analysis high throughput screening inhibitor/antagonist membrane activity membrane assembly novel preempt premature prevent repository resistance mutation response scaffold screening sinefungin small molecule small molecule libraries

项目摘要

Multi-drug resistance is one of the most pressing issues in treating bacterial infections. Antibiotics are extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect. This problem is most formidable with Gram-negative (Gram (-)) bacteria, due to their double-membrane structure. While efforts have focused on targeting one efflux pump at a time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit protein synthesis of multiple pumps simultaneously, thus reducing drug efflux and accelerating bactericidal action. TrmD is a bacteria-specific S-adenosyl-methionine (AdoMet)-dependent methyl transferase that controls the accuracy of protein-synthesis reading frame. Loss of TrmD increases +1 frameshifts and terminates protein synthesis prematurely. We have discovered that genes for multiple membrane proteins and efflux pumps in E. coli and other Gram (-) bacteria contain TrmD-dependent codons near the start of the reading frame. We hypothesize that targeting TrmD will reduce protein synthesis of all of these genes. By reducing multiple membrane and efflux proteins at once, we propose that targeting TrmD offers a novel solution to an unmet medical need. While AstraZeneca (AZ) has attempted to target TrmD, progress has stalled, because isolated inhibitors lacked both the selectivity against the human counterpart (Trm5) and the activity against bacterial growth. We hypothesize that successful targeting must explore novel chemical space and diversity to capture the unique conformation of AdoMet when bound to TrmD. To test this hypothesis, our multi-PI team will use E. coli TrmD (EcTrmD) as a model and apply a series of high-throughput screening (HTS) assays, each unique to our team, to isolate potent and selective inhibitors. In Aim 1, we will use an enzyme-based fluorescence assay to isolate active inhibitors of EcTrmD. This fluorescence assay is HTS-ready, has all of the required reagents in hand, and exhibits advantages over the radioactivity-based (3H-AdoMet) assay. We will screen the collection of ~370,000 compounds in the NCATS SMR (small molecular repository) at Sanford Burnham Prebys (SBP) and will apply human Trm5 in a counter screen to remove non-selective compounds. In Aim 2, we will use cheminformatics to prioritize hits. We will assess hits in a multitude of secondary assays to determine their inhibition potency and modality. In Aim 3, we will screen hits with our whole-cell assays to isolate compounds that inhibit cell growth and display phenotypes specific to TrmD deficiency, including reduced drug efflux. We will assess the structure-activity relationship of each hit by analysis of ~20 analogs from commercial vendors and determine the binding modality using a computer-aided approach based on our ternary TrmD crystal structure in complex with a bound tRNA and sinefungin (a non-reactive analog of AdoMet). We will determine hits for specificity of targeting EcTrmD inside E. coli cells. These hits will serve as powerful chemical probes in a new paradigm of antibiotic discovery that inhibits Gram (-) bacterial drug efflux by targeting TrmD.

多药耐药性是治疗细菌感染中最紧迫的问题之一。抗生素从细胞中挤出并且不能达到足够高的细胞内浓度以发挥治疗效果。这个问题在革兰氏阴性（革兰氏（-））细菌中最为棘手，因为它们的双膜结构虽然努力集中在一次靶向一个外排泵，但耐药突变可以迅速开发.我们建议靶向TrmD催化的m1 G37-tRNA甲基化，以抑制蛋白质合成，多个泵同时工作，从而减少药物外排并加速杀菌作用。TrmD是一个细菌特异性S-腺苷甲硫氨酸（SNMet）依赖性甲基转移酶，其控制蛋白质合成阅读框架。TrmD的缺失增加+1移码并终止蛋白质合成过早地。我们发现，在大肠杆菌中，多种膜蛋白和外排泵的基因。杆菌和其它革兰氏（-）细菌在靠近阅读框起始处含有TrmD依赖性密码子。我们假设靶向TrmD将减少所有这些基因的蛋白质合成。通过减少多个膜和同时，我们提出靶向TrmD为未满足的医疗需求提供了一种新的解决方案。虽然阿斯利康（AZ）试图靶向TrmD，但进展已经停滞，因为分离的抑制剂缺乏对人对应物（Trm 5）的选择性和对细菌生长的活性。我们假设成功的靶向必须探索新的化学空间和多样性，以捕获独特的当与TrmD结合时，图1示出了C3 Met的构象。为了验证这个假设，我们的多PI团队将使用E。大肠杆菌TrmD （EcTrmD）作为模型，并应用一系列高通量筛选（HTS）测定，每种测定都是我们的独特测定。团队，以分离有效的和选择性的抑制剂。在目标1中，我们将使用基于酶的荧光测定法，分离EcTrmD活性抑制剂。该荧光测定是HTS就绪的，具有所有需要的试剂，手，并表现出优于放射性为基础的（3 H-Met）测定。我们将筛选 Sanford Burnham Prebys（SBP）的NCATS SMR（小分子储存库）中约有370，000种化合物，将在计数器筛选中应用人Trm 5以去除非选择性化合物。在目标2中，我们将使用化学信息学来确定命中的优先级。我们将在众多的二级检测中评估命中率，以确定其抑制效力和形态。在目标3中，我们将使用全细胞检测筛选命中物以分离化合物其抑制细胞生长并显示对TrmD缺乏特异的表型，包括减少的药物外排。我们将通过分析来自商业供应商的约20种类似物来评估每个命中的结构-活性关系并使用基于我们的三元TrmD晶体的计算机辅助方法确定结合方式与结合的tRNA和sinefungin（一种非反应性的蛋氨酸类似物）复合的结构。我们将确定靶向E.大肠杆菌细胞。这些命中将作为强大的化学探针，通过靶向TrmD抑制革兰氏（-）细菌药物外排的抗生素发现的新范例。