Discovery of Gram-negative permeable chemical probes for tRNA methylation

发现用于 tRNA 甲基化的革兰氏阴性渗透性化学探针

基本信息

批准号：
10092920
负责人：
Ya-Ming Hou
金额：
$ 68.42万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10092920
关键词：
Address Anabolism Anti-Bacterial Agents Antibiotics Anticodon Bacteria Binding Biological Assay Cell Death Cell Density Cells Cellular Assay Cessation of life Chemical Structure Chemicals Clinical Codon Nucleotides Collection Crystallization Dose Drug Binding Site Drug Efflux Drug Industry Drug Targeting Drug resistance Drug usage Escherichia coli Event Exhibits Fluorescence Gene Expression Genes Genetic Transcription Gram-Negative Bacteria Growth Homo sapiens Human Infection Initiator Codon Ligand Binding Membrane Membrane Proteins Messenger RNA Methylation Modeling Modern Medicine Modification Multi-Drug Resistance Natural Products Noise Permeability Pharmaceutical Chemistry Pharmaceutical Preparations Phenotype Positioning Attribute Protein Biosynthesis Proteins Pseudomonas aeruginosa Public Health Reading Frames Resistance Ribosomes S-Adenosylhomocysteine Salmonella Salmonella enterica Series Shapes Side Signal Transduction Site Specificity Structure Structure-Activity Relationship Synthesis Chemistry Testing Therapeutic Effect Time Transfer RNA Transferase Translations analog antimicrobial antimicrobial drug bactericide base cell growth chemical property clinically relevant drug discovery efflux pump gene discovery genome-wide analysis high throughput screening improved in silico inhibitor/antagonist membrane activity minimal inhibitory concentration novel pathogen preempt premature resistance mutation response scaffold screening sinefungin small molecule success

项目摘要

Project Summary. Gram-negative (Gram (-)) bacteria are intrinsically resistant to drugs, due to a double membrane structure that acts as a permeability barrier to drugs and as an anchor for efflux pumps. Many Gram (-) bacteria have developed multi-drug resistance, which poses one of the most pressing issues in modern medicine. Antibiotics are barred and extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect. While efforts have focused on targeting one membrane protein at a time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit biosynthesis of multiple membrane proteins simultaneously, thus reducing drug barrier and efflux and accelerating bactericidal action. TrmD is a bacteria-specific S-adenosyl-methionine (AdoMet)- dependent methyl transferase that controls accuracy of the 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 need. While AstraZeneca (AZ) has attempted to target TrmD, the isolated hits lacked the cell-permeability needed to exhibit an antibacterial effect. We hypothesize that successful targeting must identify compounds that are cell-permeable and selective for TrmD over the human counterpart Trm5. To test this hypothesis, we have developed and optimized a cell-based fluorescence assay for E. coli TrmD (EcTrmD), in which we will mix a 1:1 ratio of an E. coli mCherry (mCh)-expressing strain dependent on TrmD for survival and a separate YFP-expressing strain dependent on Trm5 for survival to discover cell-permeable compounds that selectively inhibit the TrmD-dependent but not the Trm5-dependent strain. In Aim 1, we will use this cell-based assay, which is high-throughput screening (HTS)-ready, in a large- scale campaign to discover cell-permeable and selective inhibitors of EcTrmD. We will screen a diverse collection of ~180,000 compounds and a collection of 10,000 natural products to identify inhibitors and remove false positives. In Aim 2, we will assess hits in secondary assays to determine their potency and mechanism of action. We will fractionate natural products to active compounds. We will also test hits on Gram (-) bacteria Salmonella and Pseudomonas aeruginosa. In Aim 3, we will use whole-cell assays to identify hits that inhibit cell growth and display TrmD-deficient phenotypes. We will assess initial structure-activity relationship (SAR) of each cluster of hits by analysis of ~20 analogs selected from in silico modeling in our TrmD crystal structure with a bound tRNA and sinefungin (non-reactive AdoMet analog). These initial hits will serve as powerful probes in a new paradigm of antibiotic discovery that inhibits the drug barrier and efflux of Gram (-) bacteria.

项目摘要。革兰氏阴性 (Gram (-)) 细菌本质上对药物具有耐药性，这是由于双重膜结构，充当药物的渗透屏障和外排泵的锚。许多克 (-) 细菌已经产生了多重耐药性，这是现代社会最紧迫的问题之一药品。抗生素被细胞阻挡和挤出，无法到达细胞内足够高的位置浓度才能发挥治疗作用。虽然工作重点是针对一种膜蛋白随着时间的推移，耐药性突变会迅速发展。我们建议靶向 m1G37-tRNA 甲基化催化通过TrmD同时抑制多种膜蛋白的生物合成，从而降低药物屏障和外排并加速杀菌作用。 TrmD 是一种细菌特异性 S-腺苷甲硫氨酸 (AdoMet)- 控制蛋白质合成阅读框准确性的依赖甲基转移酶。 TrmD 丢失增加+1移码并过早终止蛋白质合成。我们发现基因大肠杆菌和其他革兰氏 (-) 细菌中的多种膜蛋白和外排泵含有 TrmD 依赖性靠近阅读框起点的密码子。我们假设靶向 TrmD 将减少所有这些基因。通过同时减少多种膜蛋白和外排蛋白，我们建议靶向 TrmD 为未满足的需求提供了新颖的解决方案。虽然阿斯利康 (AZ) 试图瞄准 TrmD，但孤立的命中缺乏表现出抗菌作用所需的细胞渗透性。我们假设成功的靶向必须识别出具有细胞渗透性且对 TrmD 的选择性高于人类的化合物对应的Trm5。为了检验这一假设，我们开发并优化了基于细胞的荧光测定对于大肠杆菌 TrmD (EcTrmD)，我们将以 1:1 的比例混合表达大肠杆菌 mCherry (mCh) 的菌株依赖于 TrmD 生存，而单独的表达 YFP 的菌株则依赖于 Trm5 生存发现可选择性抑制 TrmD 依赖性而非 Trm5 依赖性的细胞渗透性化合物拉紧。在目标 1 中，我们将在大规模实验中使用这种基于细胞的检测方法，该检测方法已做好高通量筛选 (HTS) 准备。大规模活动以发现 EcTrmD 的细胞渗透性和选择性抑制剂。我们将筛选多元化的收集约 180,000 种化合物和 10,000 种天然产物，用于识别抑制剂并去除误报。在目标 2 中，我们将评估二次测定中的命中，以确定它们的效力和机制行动。我们将天然产物分馏为活性化合物。我们还将测试对革兰氏 (-) 细菌的命中沙门氏菌和铜绿假单胞菌。在目标 3 中，我们将使用全细胞分析来识别抑制的命中细胞生长并表现出 TrmD 缺陷表型。我们将评估初始构效关系 (SAR) 通过分析从我们的 TrmD 晶体结构中的计算机建模中选择的约 20 个类似物来分析每个命中簇与结合的 tRNA 和 sinefungin（非反应性 AdoMet 类似物）。这些最初的打击将发挥强大的作用探索抗生素发现的新范式，抑制革兰氏 (-) 细菌的药物屏障和外流。