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。为革兰氏阴性细菌发现新的抗生素具有独特的挑战性，因为它们 双层膜结构，充当药物的渗透性屏障和外排泵的锚。努力 一次靶向一个膜蛋白或一个外排泵是无效的，因为阻力迅速上升。 突变。我们将靶向TrmD催化的tRNA的m1G37甲基化，以抑制 膜蛋白类，具有加速杀菌作用的潜力。TrmD是一种细菌特异性S- 腺苷蛋氨酸(ADO-Met)依赖的甲基转移酶控制蛋白质合成的准确性 阅读框。TrmD的丢失增加了+1移码，并导致细胞死亡。我们已经证明了 大肠杆菌和其他革兰氏阴性菌的多个膜蛋白和外排泵含有TrmD- 阅读框起始处附近的依赖密码子。我们假设靶向TrmD会减少蛋白质 所有这些基因的合成，从而为未得到满足的需求提供了一种新的解决方案。而阿斯利康(AZ)、葛兰素史克、 学术实验室试图通过筛选分离的小分子化合物文库来靶向TrmD HITS缺乏展示抗菌效果所需的细胞渗透性。在这里，我们建议放映一部大型 收集可穿透细胞和靶向TrmD的天然产品(NPs)的微生物提取物和馏分 比人类的对应物Trm5更有效和有选择性。我们将使用基于细胞的分析，由1：1组成 混合大肠杆菌(EC)TrmDmCH菌株(依赖trmD生存并将MCH(MCherry)表达为 荧光标记)和一株EC Trm5YFP菌株(依赖trm5存活和表达YFP)，在 吞吐量筛选(HTS)活动，以分离选择性抑制TrmDmCH菌株的NPs。我们执行这项任务 在EC-TolC+细胞中检测，维持整个革兰氏阴性外排机制，包括主要外排 TolC编码的泵，用于筛选细胞透过性和抗外排的NPs。一个Pilot屏幕上有这个 基于TolC+细胞的检测已经确定了一个有吸引力的打击，证明了该检测的HTS就绪状态。在目标1中， 我们将使用这种基于tolC+细胞的分析方法来筛选74,770个放线菌提取物和可在 斯克里普斯研究所(TSRI)。我们将在二次化验中评估命中情况，删除假阳性，评估 在全细胞水平上测试它们的活性，并测试它们在一组革兰氏阴性细菌中的渗透性和外排。 在目标2中，我们将取消复制前20个命中，以分离活动的NP，确定它们的结构，并使用 基因组测序和挖掘相结合识别其生物合成基因簇(BGC)用于开发 生物技术平台扩大其生产规模。在目标3中，我们将测试授予TrmD- 全细胞分析中的缺陷表型，确定它们的效力、选择性、作用机制，并评估 他们抵抗的风险。这些NPs代表了抗生素发现新范式的新线索， 革兰氏阴性菌的多重耐药问题。