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:10307014
- 负责人:
- 金额:$ 84.55万
- 依托单位:
- 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:
- 关键词:AddressAnabolismAnti-Bacterial AgentsAntibioticsAnticodonBacteriaBindingBiochemistryBiological AssayBiologyBiotechnologyCell DeathCell FractionCellsCellular AssayClinicalCodon NucleotidesCollectionComplementComplexDoseDrug Binding SiteDrug TargetingEnzymesEscherichia coliExhibitsFluorescenceFundingGene ClusterGenesGenetic TranscriptionGram-Negative BacteriaGrowthHomo sapiensHumanInitiator CodonInvadedLibrariesMembraneMembrane ProteinsMessenger RNAMethodsMethylationMiningModelingModificationMolecularMulti-Drug ResistanceNatural ProductsNoisePenetrationPermeabilityPharmaceutical PreparationsPhenotypePopulationProductionProlineProtein BiosynthesisPublic HealthPumpReadinessReading FramesResearchResearch InstituteResistanceRibosomesRiskS-AdenosylhomocysteineSideSignal TransductionSilverStructureTestingTimeTransfer RNATransferaseTranslationsbactericidebasecombatdrug discoveryefficacy studyefflux pumpgenome databasegenome sequencinghigh throughput screeningin vivomicrobialmortalitynext generationnovelpandemic diseaseprematureprogramsresistance mechanismresistance mutationresponsescaffoldscale upscreeningsmall 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.
项目摘要。发现革兰氏阴性菌的新抗生素具有独特的挑战性,因为它们
作为药物的渗透屏障和作为外排泵的锚的双膜结构。努力
一次靶向一个膜蛋白或一个外排泵是无效,这是由于抗性的快速上升
突变。我们将靶向TrmD催化的tRNA的m1 G37甲基化,以抑制多种生物合成。
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-
腺苷甲硫氨酸依赖性甲基转移酶,控制蛋白质合成的准确性
阅读帧。TrmD的缺失增加+1移码并导致细胞死亡。我们已经证明,
多个膜蛋白和外排泵。大肠杆菌和其他革兰氏阴性细菌中含有TrmD-
依赖性密码子靠近阅读框的起始。我们假设靶向TrmD将减少蛋白质
合成所有这些基因,从而为未满足的需求提供了一种新的解决方案。阿斯利康(AstraZeneca)、GSK、
和学术实验室试图通过筛选小分子化合物文库,
HITS缺乏显示抗菌效果所需的细胞渗透性。在这里,我们建议筛选一个大的
收集用于细胞可渗透和TrmD靶向天然产物(NP)的微生物提取物和级分,
比人类的Trm 5更有效更有选择性。我们将使用基于细胞的测定,包括1:1的
E的混合物。大肠杆菌(Ec)TrmDmCh菌株(依赖于trmD存活并将mCh(mCherry)表达为
荧光标记物)和Ec Trm 5 YFP菌株(依赖于trm 5存活并表达YFP),在高表达水平下,
通量筛选(HTS)活动以分离选择性抑制TrmDmCh菌株的NP。我们执行这个
Ec tolC+细胞中的测定,其维持整个革兰氏阴性外排机制,包括主要外排
由tolC编码的泵,以筛选细胞可渗透且耐外排的NP。一个试点屏幕与此
基于tolC+细胞的测定已经鉴定出有吸引力的命中,证明了测定的HTS准备。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,
斯克里普斯研究所(TSRI)。我们将在二次检测中评估命中率,消除假阳性,评估
它们在全细胞水平的活性,并测试它们在一组革兰氏阴性细菌中的渗透性和流出性。
在目标2中,我们将去复制前20个命中以分离活性NP,确定它们的结构,并使用
基因组测序和挖掘相结合,以确定其生物合成基因簇(BGC),
生物技术平台来扩大其生产规模。在目标3中,我们将测试用于赋予TrmD的活性NP。
在全细胞试验中缺乏表型,确定其效力,选择性,作用机制,并评估
抵抗的风险。这些纳米颗粒代表了抗生素发现的新范式中的新线索,
革兰阴性菌的多重耐药问题。
项目成果
期刊论文数量(0)
专著数量(0)
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会议论文数量(0)
专利数量(0)
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Ya-Ming Hou其他文献
Ya-Ming Hou的其他文献
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{{ truncateString('Ya-Ming Hou', 18)}}的其他基金
A cell model of YARS2-associated childhood-onset mitochondrial disease
YARS2 相关的儿童期发病线粒体疾病的细胞模型
- 批准号:
10575369 - 财政年份:2023
- 资助金额:
$ 84.55万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 84.55万 - 项目类别:
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:
10438880 - 财政年份:2021
- 资助金额:
$ 84.55万 - 项目类别:
Exploring 3Dpol for RNA sequencing in real time
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10166895 - 财政年份:2020
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Exploring 3Dpol for RNA sequencing in real time
探索 3Dpol 实时 RNA 测序
- 批准号:
9974889 - 财政年份:2020
- 资助金额:
$ 84.55万 - 项目类别:
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