CryoEM guided enhancement of ribosome-targeting antibiotics

项目摘要/摘要- CryoEM引导增强核糖体靶向抗生素 抗生素耐药性的持续出现严重威胁着现代医学， 延伸，现代社会。由于缺乏新开发或发现的抗生素， 更可怕的是，随着泛耐药菌株的出现。重组和修改现有的抗生素类别， 一个机会，研究和抵消阻力机制，同时生产真正合理设计的小， 分子药物先导。通过冷冻电子显微镜（CryoEM）进行迭代循环的快速结构表征 而合成修饰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 用于抗生素的是细菌核糖体，它被几种抗生素产生的链阳性抗生素抑制， 链霉菌属Streptomyces链阳性菌素A（SA）结合在核糖体的肽基转移酶中心。 链阳性菌素在临床上的价值有限，因为耐药机制，包括失活， 乙酰转移酶如VatA和核糖体保护蛋白的分子干扰， 抑制剂。通过与加州大学旧金山分校的Seiple实验室合作，我们可以获得各种各样的 链阳性菌素类似物。这些是使用模块化合成产生的，以允许快速访问的变化， 结构和氢键元素。最初，我们将使用几轮最低抑制浓度 筛选和结构表征以增加链阳性菌素类似物对大肠杆菌的功效。杆菌 核糖体随后将进行调整链阳性菌素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 将测量乙酰化速率以补充抗性谱。探究…的兴起 基于乙酰化的抗性，然后我们将传代E。以SA类似物的亚MIC水平表达VatA的大肠杆菌 和序列幸存者。我们将通过深度突变VatA的综合突变来补充这一点。 扫描、并行竞争生长和深度测序。总之，这些方法将 鉴定作为全局稳定剂的突变和提供底物特异性的突变，并指导 努力增强SA类似物和鉴定的“不可变”残基之间的空间冲突。遵循这些 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。 在这种保护蛋白存在的情况下，对核糖体内部结构空间的探索将有助于 探索这种耐药性的机制。最终，冷冻电镜的爆炸性增长 与现代模块化合成相结合，为临床应用有限的抗生素类别提供了重组的机会 相关性通过专门修改它们来对抗和了解抗生素耐药性机制。

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.