CryoEM guided enhancement of ribosome-targeting antibiotics

项目摘要/摘要-冷冻EM引导加强核糖体靶向抗生素 抗生素耐药性的持续出现对现代医学构成了实质性威胁， 延伸，现代社会。缺乏新开发或发现的抗生素使情况雪上加霜。 随着泛耐药菌株的出现，情况变得更加可怕。重组和修改现有类别的抗生素产品 一个研究和抵消阻力机制的机会，同时生产真正合理设计的小 分子药物线索。通过低温电子显微镜(CryoEM)快速表征结构的迭代循环 而合成改装为这种重组努力提供了一种合理的方法。最多产的目标之一 因为抗生素是细菌的核糖体，它被几种链球菌素类抗生素所抑制 链霉菌​Streptomyces​.的种类链霉素A(SA)结合在核糖体的肽基转移酶中心。 链球菌的临床价值有限，因为耐药机制包括 VATA等乙酰转移酶与取代的核糖体保护蛋白的分子干扰 抑制剂。与加州大学旧金山分校的Seiple实验室合作，我们可以访问各种 链球菌素类似物。它们是使用模块合成生成的，以允许快速访问 结构元素和氢键元素。最初，我们将使用轮次最低抑菌浓度 提高链球菌素类似物对​大肠杆菌疗效的筛选和结构鉴定 核糖体。随后将进行调整链菌素A类似物抗细菌活性的研究 表达乙酰转移酶VATA。具有抑制作用的类似物将通过CryoEM和 乙酰化速率将被测量，以补充阻力曲线。探索……的兴起 基于乙酰化的抗性，然后我们将在亚MIC水平的SA类似物中传代表达VATA的​大肠杆菌​ 和序列幸存者。我们将通过深度突变全面突变VATA来补充这一点 扫描，执行并行竞争增长和深度测序。加在一起，这些方法将 识别起全局稳定剂作用的突变和提供底物特异性的突变，并指导 努力加强SA类似物和已确定的“不变”残基之间的空间碰撞。在此之后 在研究中，我们将应用类似的技术来探索核糖体保护蛋白EfrCD的结构基础。 这些蛋白质与外排泵具有同源性，但缺乏细胞外排所需的跨膜结构域。 探索核糖体内存在这样的保护蛋白的结构空间将会有所帮助 探索这种抗性的鲜为人知的机制。最终，低温EM的爆炸性增长 与现代模块化合成相结合，提供了一种在有限的临床条件下重组抗生素类别的机会 通过具体修改它们来对抗和了解抗生素耐药性的机制。

Project Summary/Abstract - CryoEM Guided Enhancement of Ribosome-Targeting Antibiotics The continued emergence of antibiotic resistance materially threatens modern medicine and by extension, modern society. A dearth of newly developed or discovered antibiotics has made the situation even more dire, as pan-resistant strains have emerged. Retooling and modifying existing classes of antibiotics offers an opportunity to study and counteract resistance mechanisms while producing truly rationally-designed small molecule drug leads. Iterative rounds of rapid structural characterization via cryoelectron microscopy (CryoEM) and synthetic modification offers a rational approach to such retooling efforts. One of the most prolific targets for antibiotics is the bacterial ribosome, which is inhibited by streptogramin antibiotics produced by several species of ​Streptomyces​. Streptogramin A (SA) binds at the peptidyl transferase center of the ribosome. Streptogramins are of limited value clinically because of resistance mechanisms including inactivation by acetyltransferases such as VatA and molecular interference by ribosomal protection proteins which displace the inhibitor. In collaboration with the Seiple laboratory at UCSF, we have access to a wide variety of streptogramin analogs. These are produced using modular synthesis, to allow rapid access to variations in structural and hydrogen bonding elements. Initially, we will use rounds of Minimum Inhibitory Concentration screening and structural characterization to increase the efficacy of streptogramin analogs for the ​E. coli ribosome. These will be followed by studies tuning the activity of streptogramin A analogs against bacteria expressing VatA, the acetyltransferase. Analogs with inhibitory effects will be characterized by CryoEM and acetylation rates will be measured to complement the resistance profiles. To probe the rise of acetylation-based resistance, we will then passage ​E. coli​ expressing VatA in sub-MIC levels of SA analogs and sequence survivors. We will complement this by comprehensive mutation of VatA by deep mutational scanning, performing parallel competitive growth and deep sequencing. Together, these approaches will identify mutations that act as global stabilizers and mutations that provide substrate specificity, and guide efforts to enhance steric clashes between the SA analog and “unmutable” residues identified. Following these studies, we will apply similar techniques to explore the structural basis of a ribosomal protection protein, EfrCD. Such proteins bear homology to efflux pumps, but lack the transmembrane domains required for cellular efflux. Explorations of the structural space within the ribosome in the presence of such protection proteins will help probe the poorly understood mechanisms of such resistance. Ultimately, the explosive growth of cryoEM coupled to modern modular synthesis provides an opportunity to retool antibiotic classes with limited clinical relevance by specifically modifying them to combat and understand mechanisms of antibiotic resistance.