Discovery of Gram-negative permeable chemical probes for tRNA methylation

项目摘要。革兰氏阴性(革兰氏(-))细菌对药物具有内在抗药性，原因是双重耐药 膜结构，充当药物的渗透性屏障和外排泵的锚。多克 细菌已经产生了多重抗药性，这构成了现代人类最紧迫的问题之一 医药。抗生素被阻挡并从细胞中排出，无法达到足够高的细胞内浓度 发挥治疗效果的浓度。虽然人们的努力集中在一次针对一种膜蛋白 随着时间的推移，抗药性突变可能会迅速发展。我们建议以m1G37-tRNA甲基化为靶点 通过TrmD同时抑制多个膜蛋白的生物合成，从而减少药物屏障和 外排和加速杀菌作用。TrmD是一种细菌特异性S-腺苷-蛋氨酸(ADOMet)- 依赖甲基转移酶，控制蛋白质合成阅读框的准确性。TrmD的丢失 增加+1帧移位并过早终止蛋白质合成。我们已经发现， 大肠杆菌和其他革兰氏(-)细菌的多个膜蛋白和外排泵含有TrmD依赖 阅读框开头附近的密码子。我们假设靶向TrmD会减少蛋白质的合成 所有这些基因。通过一次减少多个膜蛋白和外排蛋白，我们提出了靶向 TrmD为未得到满足的需求提供了一种新的解决方案。虽然阿斯利康(AZ)试图瞄准TrmD，但 单独的HITS缺乏显示抗菌效果所需的细胞渗透性。我们假设 成功的靶向必须识别对人TrmD具有细胞渗透性和选择性的化合物 对应的Trm5。为了验证这一假设，我们开发并优化了一种基于细胞的荧光分析方法。 对于E.coliTrmD(EcTrmD)，我们将在其中混合1：1比例的E.colimCherry(MCH)表达菌株 依赖TrmD生存，另一株YFP表达菌株依赖Trm5生存 发现选择性抑制TrmD依赖但不抑制Trm5依赖的细胞通透性化合物 紧张。在目标1中，我们将使用这种基于细胞的分析，它是高通量筛选(HTS)就绪的，在一个大型的 开展大规模活动，以发现ECTrmD的细胞渗透性和选择性抑制剂。我们将放映一部多元化的 收集约180,000种化合物和10,000种天然产品，以识别抑制剂并清除 假阳性。在目标2中，我们将在二次检测中评估HITS，以确定它们的效力和机制 行动。我们将把天然产物分离成活性化合物。我们还将测试革兰氏(-)细菌的命中率 沙门氏菌和铜绿假单胞菌。在目标3中，我们将使用全细胞分析来识别抑制 细胞生长，并显示TrmD缺乏表型。我们将评估初始结构-活性关系(SAR) 通过分析从我们的TrmD晶体结构的Silico建模中选择的~20个类似物，对每一簇HITS进行分析 结合tRNA和辛芬净(非反应性的ADOMet类似物)。这些最初的热门歌曲将成为强大的 探索一种新的抗生素发现范例，抑制革兰氏(-)细菌的药物屏障和外排。

Project Summary. Gram-negative (Gram (-)) bacteria are intrinsically resistant to drugs, due to a double membrane structure that acts as a permeability barrier to drugs and as an anchor for efflux pumps. Many Gram (-) bacteria have developed multi-drug resistance, which poses one of the most pressing issues in modern medicine. Antibiotics are barred and extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect. While efforts have focused on targeting one membrane protein at a time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit biosynthesis of multiple membrane proteins simultaneously, thus reducing drug barrier and efflux and accelerating 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 frameshifts and terminates protein synthesis prematurely. We have discovered that genes for multiple membrane proteins and efflux pumps in E. coli and other Gram (-) 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. By reducing multiple membrane- and efflux-proteins at once, we propose that targeting TrmD offers a novel solution to an unmet need. While AstraZeneca (AZ) has attempted to target TrmD, the isolated hits lacked the cell-permeability needed to exhibit an antibacterial effect. We hypothesize that successful targeting must identify compounds that are cell-permeable and selective for TrmD over the human counterpart Trm5. To test this hypothesis, we have developed and optimized a cell-based fluorescence assay for E. coli TrmD (EcTrmD), in which we will mix a 1:1 ratio of an E. coli mCherry (mCh)-expressing strain dependent on TrmD for survival and a separate YFP-expressing strain dependent on Trm5 for survival to discover cell-permeable compounds that selectively inhibit the TrmD-dependent but not the Trm5-dependent strain. In Aim 1, we will use this cell-based assay, which is high-throughput screening (HTS)-ready, in a large- scale campaign to discover cell-permeable and selective inhibitors of EcTrmD. We will screen a diverse collection of ~180,000 compounds and a collection of 10,000 natural products to identify inhibitors and remove false positives. In Aim 2, we will assess hits in secondary assays to determine their potency and mechanism of action. We will fractionate natural products to active compounds. We will also test hits on Gram (-) bacteria Salmonella and Pseudomonas aeruginosa. In Aim 3, we will use whole-cell assays to identify hits that inhibit cell growth and display TrmD-deficient phenotypes. We will assess initial structure-activity relationship (SAR) of each cluster of hits by analysis of ~20 analogs selected from in silico modeling in our TrmD crystal structure with a bound tRNA and sinefungin (non-reactive AdoMet analog). These initial hits will serve as powerful probes in a new paradigm of antibiotic discovery that inhibits the drug barrier and efflux of Gram (-) bacteria.