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: 10625857
• 负责人: Ya-Ming Hou
• 金额: $ 84.71万
• 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/10625857
• 关键词: Acceleration; 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; 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; Natural Selections; Noise; Penetration; Permeability; Pharmaceutical Preparations; Phenotype; Population; Production; Proline; Protein Biosynthesis; Public Health; Pump; Readiness; Reading Frames; Research; Research Institute; Resistance; Ribosomes; Risk; Risk Assessment; S-Adenosylhomocysteine; S-Adenosylmethionine; Side; Signal Transduction; Silver; Structure; Testing; Time; Transfer RNA; Transferase; Translations; bactericide; combat; drug discovery; efficacy study; efflux pump; genome database; genome sequencing; high throughput screening; in vivo; microbial; mortality; next generation; novel; pandemic disease; posttranscriptional; 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。发现针对革兰氏阴性菌的新抗生素具有独特的挑战性，因为它们 双膜结构，充当药物的渗透屏障和外排泵的锚。努力 由于阻力迅速上升，一次针对一种膜蛋白或一个外排泵的方法无效 突变。我们将靶向 tRNA 的 TrmD 催化的 m1G37 甲基化来抑制多种生物合成 一类膜蛋白，具有加速杀菌作用的潜力。 TrmD 是一种细菌特异性 S- 控制蛋白质合成准确性的腺苷甲硫氨酸 (AdoMet) 依赖性甲基转移酶 阅读框架。 TrmD 的缺失会增加 +1 移码并导致细胞死亡。我们已经证明基因 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- 靠近阅读框起点的依赖密码子。我们假设靶向 TrmD 会减少蛋白质 合成所有这些基因，从而为未满足的需求提供新的解决方案。而阿斯利康 (AZ)、葛兰素史克 (GSK)、 学术实验室尝试通过筛选小分子化合物库来靶向 TrmD，分离出 命中缺乏表现出抗菌作用所需的细胞渗透性。在这里，我们建议大屏 收集细胞渗透性和 TrmD 靶向天然产物 (NP) 的微生物提取物和级分， 比人类对应的 Trm5 更有效且具有选择性。我们将使用基于细胞的检测，包括 1:1 大肠杆菌 (Ec) TrmDmCh 菌株的混合物（依赖于 trmD 生存并表达 mCh (mCherry) 作为 荧光标记）和 Ec Trm5YFP 菌株（依赖于 trm5 生存并表达 YFP），以高 通量筛选 (HTS) 活动旨在分离选择性抑制 TrmDmCh 菌株的 NP。我们执行这个 在 Ec tolC+ 细胞中进行测定，该细胞维持整个革兰氏阴性流出机制，包括主要流出 由 tolC 编码的泵，用于筛选具有细胞渗透性和抗外排性的 NP。带有此的试点屏幕 tolC+ 基于细胞的测定已确定了一个有吸引力的命中，证明了该测定的 HTS 就绪性。在目标 1 中， 我们将使用这种基于 tolC+ 细胞的测定来筛选 74,770 种放线菌提取物和级分，这些提取物和级分可在 The 斯克里普斯研究所 (TSRI)。我们将评估二次测定中的命中，消除误报，评估 它们在全细胞水平上的活性，并在一组革兰氏阴性细菌中测试它们的渗透性和外排性。 在目标 2 中，我们将对前 20 个命中进行去重复，以分离活性 NP，确定其结构，并使用 结合基因组测序和挖掘来识别其生物合成基因簇（BGC）以进行开发 生物技术平台扩大生产规模。在目标 3 中，我们将测试活性 NP 是否授予 TrmD- 全细胞测定中的缺陷表型，确定其效力、选择性、作用机制并评估 他们抵抗的风险。这些纳米粒子代表了抗生素发现新范式的新先导，解决了 革兰氏阴性菌的多重耐药问题。