Probe discovery for tRNA methylation

多药耐药是治疗细菌感染最紧迫的问题之一。抗生素是 从细胞中排出，不能达到足够高的细胞内浓度来发挥治疗效果。 这个问题在革兰氏阴性(革兰氏(-))细菌中最为严重，因为它们的双层膜 结构。虽然努力集中在一次针对一个外排泵，但耐药性突变可能很快 发展。我们建议靶向TrmD催化的m1G37-tRNA甲基化来抑制蛋白质的合成 多个泵同时工作，从而减少药物外流，加速杀菌作用。TrmD是一种 细菌特异性S-腺苷甲硫氨酸(ADOMet)依赖的甲基转移酶控制 蛋白质合成读框。TrmD的丢失增加了+1个移码并终止了蛋白质合成 为时过早。我们已经发现，在大肠杆菌中，多种膜蛋白和外排泵的基因 其他革兰氏(-)细菌在阅读框开始处附近含有依赖TrmD的密码子。我们假设 靶向TrmD将减少所有这些基因的蛋白质合成。通过减少多层膜和 我们认为，靶向TrmD为未得到满足的医疗需求提供了一种新的解决方案。 尽管阿斯利康(AstraZeneca)试图针对TrmD，但进展停滞不前，因为缺乏单独的抑制剂 对人类类似物(Trm5)的选择性和对细菌生长的抑制活性。我们 假设成功的靶向必须探索新的化学空间和多样性来捕捉独特的 当与TrmD结合时，Adobe Met的构象。为了验证这一假设，我们的多PI团队将使用E.coliTrmD (EcTrmD)作为模型，并应用一系列高通量筛选(HTS)分析，每一种分析都是我们独特的 团队，以分离有效和选择性的抑制剂。在目标1中，我们将使用基于酶的荧光分析来 分离ECTrmD的活性抑制剂。这种荧光分析是HTS就绪的，拥有所有需要的试剂 与基于放射性的(~3H-ADOMet)分析相比，显示出更多的优势。我们将筛选收集到的 Sanford Burnham Prebys(SBP)和NCATS SMR(小分子储存库)中的约37万种化合物 将在计数器屏幕上应用人类Trm5来去除非选择性化合物。在目标2中，我们将使用 化学信息学来确定命中的优先顺序。我们将在多种二次化验中评估命中率，以确定其 抑制效力和情态。在目标3中，我们将用我们的全细胞分析来筛选命中结果以分离化合物 抑制细胞生长并表现出TrmD缺乏的特异性表型，包括药物外排减少。我们 将通过分析来自商业供应商的约20个类似物来评估每一次打击的结构-活性关系 并使用基于我们的三元TrmD晶体的计算机辅助方法确定结合方式 结合tRNA和新诺明(一种ADOMet的非反应性类似物)的复合体结构。我们将决定 在大肠杆菌细胞内靶向EcTrmD的特异性。这些命中将作为强大的化学探测器在 通过靶向TrmD抑制革兰氏(-)细菌药物外排的抗生素发现的新范例。

Multi-drug resistance is one of the most pressing issues in treating bacterial infections. Antibiotics are extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect. This problem is most formidable with Gram-negative (Gram (-)) bacteria, due to their double-membrane structure. While efforts have focused on targeting one efflux pump at a time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit protein synthesis of multiple pumps simultaneously, thus reducing drug efflux and accelerating bactericidal action. TrmD is a bacteria-specific S-adenosyl-methionine (AdoMet)-dependent methyl transferase that controls the accuracy of 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 medical need. While AstraZeneca (AZ) has attempted to target TrmD, progress has stalled, because isolated inhibitors lacked both the selectivity against the human counterpart (Trm5) and the activity against bacterial growth. We hypothesize that successful targeting must explore novel chemical space and diversity to capture the unique conformation of AdoMet when bound to TrmD. To test this hypothesis, our multi-PI team will use E. coli TrmD (EcTrmD) as a model and apply a series of high-throughput screening (HTS) assays, each unique to our team, to isolate potent and selective inhibitors. In Aim 1, we will use an enzyme-based fluorescence assay to isolate active inhibitors of EcTrmD. This fluorescence assay is HTS-ready, has all of the required reagents in hand, and exhibits advantages over the radioactivity-based (3H-AdoMet) assay. We will screen the collection of ~370,000 compounds in the NCATS SMR (small molecular repository) at Sanford Burnham Prebys (SBP) and will apply human Trm5 in a counter screen to remove non-selective compounds. In Aim 2, we will use cheminformatics to prioritize hits. We will assess hits in a multitude of secondary assays to determine their inhibition potency and modality. In Aim 3, we will screen hits with our whole-cell assays to isolate compounds that inhibit cell growth and display phenotypes specific to TrmD deficiency, including reduced drug efflux. We will assess the structure-activity relationship of each hit by analysis of ~20 analogs from commercial vendors and determine the binding modality using a computer-aided approach based on our ternary TrmD crystal structure in complex with a bound tRNA and sinefungin (a non-reactive analog of AdoMet). We will determine hits for specificity of targeting EcTrmD inside E. coli cells. These hits will serve as powerful chemical probes in a new paradigm of antibiotic discovery that inhibits Gram (-) bacterial drug efflux by targeting TrmD.