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（克（ - ））细菌本质上对药物具有抗药性，这是由于双重的膜结构充当药物的渗透性障碍，也是外排泵的锚点。许多克（ - ）细菌发展了多药抗性，这在现代中提出了最紧迫的问题之一药品。抗生素被禁止并挤出细胞，无法达到足够高的细胞内发挥治疗作用的浓度。努力集中在靶向一个膜蛋白时间，阻力突变会迅速发展。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，从而降低了药物屏障和外排和加速杀菌作用。 TRMD是细菌特异性的S-腺苷甲硫代氨酸（ADOMET） - 控制蛋白质合成阅读框的准确性的依赖甲基转移酶。损失TRMD 增加+1移料器并过早终止蛋白质合成。我们发现了该基因大肠杆菌和其他克（ - ）细菌中的多种膜蛋白和外排泵含有TRMD依赖性密码子接近阅读框的开始。我们假设靶向TRMD将减少所有这些基因。通过一次减少多种膜和外排 - 蛋白质，我们提出了靶向 TRMD为未满足的需求提供了一种新颖的解决方案。阿斯利康（AZ）试图瞄准TRMD，而孤立的命中缺乏表现出抗菌作用所需的细胞渗透性。我们假设这一点成功的靶向必须识别可渗透细胞的化合物，并且对人类的TRMD有选择性对应物TRM5。为了检验该假设，我们开发并优化了基于细胞的荧光测定法对于大肠杆菌TRMD（ECTRMD），我们将混合表达大肠杆菌（MCH）的1：1比率取决于TRMD的生存和单独的表达YFP应变，取决于TRM5的生存发现有选择地抑制TRMD依赖性但不依赖TRM5的细胞渗透化合物拉紧。在AIM 1中，我们将使用此基于细胞的测定法，即高通量筛选（HTS） - 在大型中已经准备就绪比例曲运动，以发现ECTRMD的细胞渗透和选择性抑制剂。我们将筛选多样的收集了约180,000种化合物和10,000种天然产品，以识别抑制剂并删除误报。在AIM 2中，我们将评估次要测定的命中率，以确定其效力和机制行动。我们将对活性化合物分馏。我们还将在克（ - ）细菌上测试命中沙门氏菌和铜绿假单胞菌。在AIM 3中，我们将使用全细胞测定法来识别抑制的命中细胞生长和显示TRMD缺陷的表型。我们将评估初始结构 - 活性关系（SAR）通过分析在我们的TRMD晶体结构中选择的〜20个类似物的分析，通过分析〜20个类似物的分析具有结合的tRNA和SinineFungin（非反应性ADOMET类似物）。这些最初的命中将是强大的在新的抗生素发现范式中的探针抑制了克（ - ）细菌的药物屏障和外排。