Discovery of Gram-negative permeable chemical probes for tRNA methylation

项目摘要。革兰氏阴性（革兰氏（-））细菌本质上对药物具有耐药性，这是由于双 作为药物渗透屏障和外排泵的锚的膜结构。许多革兰氏 (-)细菌已经产生了多重耐药性，这是现代医学中最紧迫的问题之一。 药抗生素被阻止并从细胞中挤出，不能到达足够高的细胞内 浓度以发挥治疗效果。虽然努力集中在靶向一种膜蛋白， 随着时间的推移，耐药性突变会迅速发展。我们建议靶向m1 G37-tRNA甲基化催化 通过TrmD同时抑制多种膜蛋白的生物合成，从而降低药物屏障， 外排和加速杀菌作用。TrmD是细菌特异性S-腺苷甲硫氨酸（S-腺苷甲硫氨酸）。 控制蛋白质合成阅读框准确性的依赖性甲基转移酶。TrmD丢失 增加+1移码并过早终止蛋白质合成。我们发现， 多个膜蛋白和外排泵。大肠杆菌和其他革兰氏（-）细菌含有TrmD依赖性 靠近阅读框开始的密码子。我们假设靶向TrmD将减少蛋白质合成， 所有这些基因。通过同时减少多种膜蛋白和外排蛋白，我们提出靶向 TrmD为未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， 分离的命中物缺乏表现出抗菌效果所需的细胞渗透性。我们假设 成功的靶向必须鉴定出细胞可渗透的化合物，并且对TrmD的选择性超过对人的选择性。 对应物Trm 5。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检测方法， 大肠coli TrmD（EcTrmD）中，将1：1比例的E.大肠杆菌mCherry（mCh）表达菌株 依赖TrmD生存，而另一种表达YFP的菌株依赖Trm 5生存， 发现选择性抑制TrmD依赖性但不抑制Trm 5依赖性的细胞渗透性化合物 株在目标1中，我们将使用这种基于细胞的测定，其是高通量筛选（HTS）就绪的，在大规模的 大规模的运动，以发现细胞渗透性和选择性抑制剂的EcTrmD。我们将筛选一个多样化的 收集约180，000种化合物和10，000种天然产物，以识别抑制剂并去除 假阳性在目标2中，我们将在二级试验中评估命中，以确定其效力和机制。 行动上我们将把天然产物分馏成活性化合物。我们还将测试革兰氏（-）细菌的命中 沙门氏菌和绿脓杆菌。在目标3中，我们将使用全细胞测定来鉴定抑制 细胞生长并显示TrmD缺陷表型。我们将评估初始构效关系（SAR） 通过分析从我们的TrmD晶体结构中的计算机模拟中选择的约20种类似物， 与结合的tRNA和sinefungin（非反应性的抗甲硫氨酸类似物）。这些最初的打击将作为强大的 在抗生素发现的新范例中，探针抑制革兰氏（-）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.