TrmD-targeting actinobacterial natural products as next generation antibiotics

TrmD靶向放线菌天然产物作为下一代抗生素

• 批准号: 10307014
• 负责人: Ya-Ming Hou
• 金额: $ 84.55万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-25 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10307014
• 关键词: Address; Anabolism; Anti-Bacterial Agents; Antibiotics; Anticodon; Bacteria; Binding; Biochemistry; Biological Assay; Biology; Biotechnology; Cell Death; Cell Fraction; Cells; Cellular Assay; Clinical; Codon Nucleotides; Collection; Complement; Complex; Dose; Drug Binding Site; Drug Targeting; Enzymes; Escherichia coli; Exhibits; Fluorescence; Funding; Gene Cluster; Genes; Genetic Transcription; Gram-Negative Bacteria; Growth; Homo sapiens; Human; Initiator Codon; Invaded; Libraries; Membrane; Membrane Proteins; Messenger RNA; Methods; Methylation; Mining; Modeling; Modification; Molecular; Multi-Drug Resistance; Natural Products; Noise; Penetration; Permeability; Pharmaceutical Preparations; Phenotype; Population; Production; Proline; Protein Biosynthesis; Public Health; Pump; Readiness; Reading Frames; Research; Research Institute; Resistance; Ribosomes; Risk; S-Adenosylhomocysteine; Side; Signal Transduction; Silver; Structure; Testing; Time; Transfer RNA; Transferase; Translations; bactericide; base; combat; drug discovery; efficacy study; efflux pump; genome database; genome sequencing; high throughput screening; in vivo; microbial; mortality; next generation; novel; pandemic disease; premature; programs; resistance mechanism; resistance mutation; response; scaffold; scale up; screening; small molecule

Project Summary. Discovering new antibiotics for Gram-negative bacteria is uniquely challenging, due to their double-membrane structure that acts as a permeability barrier to drugs and as an anchor for efflux pumps. Efforts that target one membrane protein or one efflux pump at a time are ineffective, due to rapid rise of resistance mutations. We will target the TrmD-catalyzed m1G37 methylation of tRNA to inhibit biosynthesis of multiple classes of membrane proteins, with the potential to accelerate 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 frameshifting and causes cell death. We have shown that genes for multiple membrane proteins and efflux pumps in E. coli and in other Gram-negative 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, thus offering a novel solution to an unmet need. While AstraZeneca (AZ), GSK, and academic labs have attempted to target TrmD by screening small molecular compound libraries, isolated hits lack the cell-permeability needed to exhibit an antibacterial effect. Here, we propose to screen a large collection of microbial extracts and fractions for cell-permeable and TrmD-targeting natural products (NPs) that are potent and selective over the human counterpart Trm5. We will use a cell-based assay, consisting of a 1:1 mix of an E. coli (Ec) TrmDmCh strain (dependent on trmD for survival and expressing mCh (mCherry) as a fluorescence marker) and an Ec Trm5YFP strain (dependent on trm5 for survival and expressing YFP), in a high- throughput screening (HTS) campaign to isolate NPs that selectively inhibit the TrmDmCh strain. We perform this assay in Ec tolC+ cells, which maintain the entire Gram-negative efflux machinery including the major efflux pump encoded by tolC, to screen for NPs that are cell-permeable and resistant to efflux. A pilot screen with this tolC+ cell-based assay has identified an attractive hit, demonstrating the HTS-readiness of the assay. In Aim 1, we will use this tolC+ cell-based assay to screen 74,770 actinobacterial extracts and fractions available at The Scripps Research Institute (TSRI). We will assess hits in secondary assays, remove false positives, evaluate their activity at the whole-cell level, and test them for permeability and efflux in a panel of Gram-negative bacteria. In Aim 2, we will de-replicate the top 20 hits to isolate the active NPs, determine their structures, and use a combination of genome sequencing and mining to identify their biosynthetic gene clusters (BGCs) for developing biotechnology platforms to scale up their production. In Aim 3, we will test active NPs for conferring TrmD- deficient phenotypes in whole-cell assays, determine their potency, selectivity, mechanism of action, and assess their risk of resistance. These NPs represent novel leads in a new paradigm of antibiotic discovery that addresses the multi-drug resistance problem of Gram-negative bacteria.

项目摘要。发现革兰氏阴性细菌的新抗生素，由于它们 双膜结构是对药物的渗透性障碍，也是外排泵的锚点。努力 该靶向一种膜蛋白或一次外排泵，由于电阻的迅速上升而无效 突变。我们将靶向tRNA的TRMD催化M1G37甲基化，以抑制多重的生物合成 膜蛋白的类别，有可能加速杀菌作用。 TRMD是细菌特异性的S- 腺苷甲硫代（Adomet）依赖性甲基转移酶，该酶控制蛋白质合成的准确性 阅读框架。 TRMD的损失增加+1帧速率并导致细胞死亡。我们已经证明了用于的基因 大肠杆菌和其他革兰氏阴性细菌中的多种膜蛋白和外排泵含有TRMD- 读取框架开始附近的依赖密码子。我们假设靶向TRMD会减少蛋白质 所有这些基因的合成，从而为未满足的需求提供了一种新颖的解决方案。而阿斯利康（AZ），gsk， 学术实验室试图通过筛选小分子化合物库来靶向TRMD 点击缺乏表现出抗菌作用所需的细胞渗透性。在这里，我们建议筛选一个大的 收集微生物提取物和分数，用于细胞可渗透和靶向TRMD的天然产品（NP） 对人类对应物TRM5具有有力和选择性。我们将使用基于细胞的测定法，由1：1组成 大肠杆菌（EC）TRMDMCH菌株的混合（取决于TRMD生存和表达MCH（MCHERRY）作为A 在高 - 吞吐量筛选（HTS）运动，以分离有选择性抑制TRMDMCH菌株的NP。我们执行此操作 在EC TOLC+细胞中的测定，该分析维持整个革兰氏阴性外排机器，包括主要外排 由TOLC编码的泵，以筛选可渗透且对外排抗性的NP。一个试点屏幕 基于TOLC+细胞的测定已确定有吸引力的命中，证明了该测定法的HTS准备度。在AIM 1中， 我们将使用此基于TOLC+细胞的测定法进行74,770个阳光细菌提取物和分数 Scripps研究所（TSRI）。我们将在次要测定中评估命中，删除误报，评估 它们在整个细胞水平上的活性，并在一组革兰氏阴性细菌中测试它们的渗透性和外排。 在AIM 2中，我们将删除前20个命中以隔离活性NP，确定其结构并使用 基因组测序和采矿的结合，以鉴定其生物合成基因簇（BGC）用于发展 生物技术平台扩大生产。在AIM 3中，我们将测试主动NP，以授予TRMD- 全细胞测定中缺乏表型，确定其效力，选择性，作用机理并评估 他们的抵抗风险。这些NP代表了新的抗生素发现范式的小说。 革兰氏阴性细菌的多药抗性问题。