CryoEM guided enhancement of ribosome-targeting antibiotics

CryoEM 引导增强核糖体靶向抗生素

• 批准号: 10219931
• 负责人: David John Lee
• 金额: $ 0.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-08-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10219931
• 关键词: ATP Hydrolysis; Acetylation; Acetyltransferase; Address; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Bar Codes; Binding; Biological Assay; Cellular Structures; Clinical; Collaborations; Complement; Coupled; Cryoelectron Microscopy; Development; Dissociation; Economics; Elements; Engineering; Enterococcus faecalis; Escherichia coli; Evaluation; Fellowship; Future; Goals; Grant; Growth; Hydrogen Bonding; Laboratories; Libraries; Measures; Minimum Inhibitory Concentration measurement; Modern Medicine; Modernization; Modification; Molecular; Molecular Conformation; Mutate; Mutation; Natural Products; Needles; Outcome Study; Peptidyltransferase; Pharmaceutical Preparations; Proteins; RNA; Resistance; Resistance profile; Resolution; Ribosomal Proteins; Ribosomes; Roentgen Rays; Societies; Streptogramins; Streptomyces; Structure; Substrate Specificity; Survivors; Techniques; Translating; Translations; Transmembrane Domain; Ursidae Family; Variant; analog; antimicrobial; appendage; base; clinically relevant; combat; deep sequencing; design; efflux pump; emerging antibiotic resistance; experimental study; inhibitor/antagonist; innovation; insight; mutation screening; next generation; resistance mechanism; resistant strain; screening; small molecule; training opportunity; translation assay

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.

项目摘要/摘要 - 核糖体靶向抗生素的冷冻指导性增强 抗生素耐药性的持续出现对现代医学进行了重大威胁，并且 扩展，现代社会。新开发或发现的抗生素的死亡甚至使情况甚至 随着泛抗菌株的出现，更多的可怕。重新处理和修改现有的抗生素类别 研究和抵消抵抗机制的机会，同时产生真正合理设计的小型 分子药物铅。通过冷冻光学显微镜（Cryoem）的快速结构表征的迭代回合（CRYOEM） 合成修改为这种改装工作提供了合理的方法。最多产的目标之一 对于抗生素是细菌核糖体，该核糖体受到几种抗生素的抑制 链霉菌的种类。链球素A（SA）在核糖体的肽基转移酶中心结合。 链霉菌素在临床上的价值有限，因为抗性机制包括灭活 乙酰转移酶，例如VATA和核糖体保护蛋白的分子干扰，这些蛋白取代 抑制剂。与UCSF的Seiple实验室合作，我们可以访问各种各样的 链球菌类似物。这些是使用模块化合成产生的，以便快速访问变化 结构和氢键元件。最初，我们将使用最小抑制浓度的回合 筛选和结构表征，以提高大肠杆菌链球菌类似物的效率 核糖体。随后将研究调整链霉素A类似物对细菌的活性 表达VATA，乙酰转移酶。具有抑制作用的类似物将以冷冻和 将测量乙酰化速率以补充电阻曲线。探究崛起 然后，我们将基于乙酰化的抗性，然后通过以下SA类似物的大肠杆菌通过大肠杆菌表达VATA 和序列存活。我们将通过深度突变对VATA的全面突变来完成此操作 扫描，进行平行的竞争增长和深度测序。这些方法将在一起 确定充当全球稳定剂和提供底物特异性的突变的突变，并指导 增强SA类似物和“无法实现”之间的空间冲突的努力保留了确定的努力。遵循这些 研究，我们将采用类似的技术来探索核糖体保护蛋白EFRCD的结构基础。 这种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下，核糖体内结构空间的探索将有助于 探究了这种抗药性的理解不足的机制。最终，冷冻的爆炸性增长 结合现代模块化合成提供了一个机会，可以使抗生素类别有限 通过专门修改它们来对抗和理解抗生素耐药性机制的相关性。