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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

基本信息

批准号：
10438880
负责人：
Ya-Ming Hou
金额：
$ 83.15万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
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/10438880
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

项目摘要

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.

项目总结。发现针对革兰氏阴性菌的新抗生素是一项独特的挑战，因为它们