Structural basis of Cfr-mediated resistance to antibiotics targeting the bacterial ribosome

Cfr介导的针对细菌核糖体的抗生素耐药性的结构基础

• 批准号: 10282911
• 负责人: YURY POLIKANOV
• 金额: $ 23.28万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10282911
• 关键词: Adenine; Affect; Affinity; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Cells; Chemicals; Chloramphenicol; Clinical; Cloning; Complex; Crystallization; Data; Development; Drug Binding Site; Drug Design; Drug Targeting; Drug resistance; Engineering; Enzymes; Essential Drugs; Exhibits; FDA approved; Foundations; Genes; Goals; Growth; Healthcare; High temperature of physical object; Infection; Knowledge; Learning; Linezolid; Macrolides; Mediating; Medical; Methylation; Methyltransferase; Methyltransferase Gene; Modification; Molecular; Molecular Machines; Multi-Drug Resistance; Nitrogen; Nucleotides; Oxazolidinones; Peptides; Peptidyltransferase; Pharmaceutical Preparations; Phenotype; Positioning Attribute; Problem Solving; Protein Biosynthesis; Research Personnel; Residual state; Resistance; Resistance development; Resolution; Ribosomal RNA; Ribosomes; Roentgen Rays; Site; Solid; Source; Streptogramins; Structure; Suggestion; Temperature; Thermus; Thermus thermophilus; Work; bacterial resistance; base; clinically relevant; cold temperature; design; experience; experimental study; hygromycin A; inhibitor/antagonist; knowledge base; lincosamide; methyl group; next generation; novel therapeutics; pathogen; pathogenic bacteria; pleuromutilin; prevent; resistance mechanism; success; thermophilic bacteria

SUMMARY The ribosome is an essential drug target as many classes of clinically important antibiotics bind to its functional centers and interfere with different aspects of protein synthesis. Out of several functional centers, the catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically relevant mechanisms of resistance to such PTC-acting drugs is based on the methylation of the C8-position of an adenine residue 2503 (A2503) of the 23S rRNA by Cfr methyltransferase. This modification confers resistance to a wide range of PTC-targeting antibiotics, including phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramins A (PhLOPSa phenotype), as well as 16-membered macrolides and hygromycin A. The Cfr-mediated drug resistance is an alarming healthcare threat because of the rapid spread of the cfr gene among pathogenic bacteria renders many clinically important antibiotics ineffective for anti-infection therapies. Thus, developing next-generation PTC-targeting inhibitors acting against the Cfr-positive pathogens is of utmost medical importance. While this problem could be tackled by structure-driven rational drug design, a structure of the Cfr-modified ribosome is currently unknown. In the proposed project, we will solve this problem by determining the first high-resolution X-ray crystal structure of the Cfr-modified bacterial ribosome in isolation or in complex with antibiotics exhibiting residual activity against the A2503-methylated ribosome. To achieve this goal, we will optimize the expression of functionally-active Cfr methyltransferases in the cells of thermophilic bacterium Thermus thermophilus, which has been widely used as the source of ribosomes suitable for crystallographic studies. We have found that in spite of growing optimally at 65-72°C, T. thermophilus can also grow at notably lower temperatures (48°C). By cloning and expressing Cfr- methyltransferase genes from moderately thermophilic bacteria in T. thermophilus at high temperatures (65- 72°C) or by expressing the erm genes from the mesophilic bacteria in the T. thermophilus host grown at reduced temperatures (48-65°C), we will obtain crystallizable ribosomes with methylated A2503. Once the structure of the A2503-methylated ribosome is determined, we will solve the structures of this Cfr-modified ribosome in complex with several PTC-acting drugs exhibiting residual binding affinity for the modified ribosome. The resulting information will be instrumental for understanding the molecular principles of Cfr-mediated resistance and will instruct the subsequent rational design of new antibacterials active against Cfr-positive pathogens. Moreover, upon successful completion of the proposed project, we shall answer a long-standing fundamental and medically relevant question: How the addition of a methyl group to a single nucleotide in the 2.5-MDa ribosome results in resistance to many classes of chemically-unrelated PTC-targeting antibiotics?

总结 核糖体是一种重要的药物靶标，因为许多类别的临床重要抗生素与其功能性结合。 中心和干扰蛋白质合成的不同方面。在几个功能中心中，催化剂 肽基转移酶中心（PTC）是属于多种化学物质的最广泛的抑制剂的靶点 班对此类PTC作用药物的耐药的最丰富和临床相关机制之一 基于Cfr对23 S rRNA的腺嘌呤残基2503（A2503）的C8位的甲基化 甲基转移酶。这种修饰赋予对广泛的PTC靶向抗生素的抗性，包括 酚类、林可酰胺类、恶唑烷酮类、截短侧耳素和链阳性菌素A（PhLOPSa表型），以及 如16元大环内酯和潮霉素A。Cfr介导的耐药性是一个令人担忧的医疗保健问题 由于cfr基因在致病菌中的快速传播， 抗生素对抗感染治疗无效。因此，开发下一代PTC靶向抑制剂 对抗CFR阳性病原体的作用具有最重要的医学意义。虽然这个问题可以解决 通过结构驱动的合理药物设计，Cfr修饰的核糖体的结构目前是未知的。 在建议的项目中，我们将通过确定第一个高分辨率X射线晶体结构来解决这个问题 Cfr修饰的细菌核糖体的分离或与抗生素的复合物显示出针对以下疾病的残留活性： A2503甲基化核糖体。为了实现这一目标，我们将优化功能活性Cfr的表达， 嗜热菌Thermus thermophilus细胞中的甲基转移酶，其已被广泛用作 适合晶体学研究的核糖体的来源。我们发现，尽管增长最佳， 65-72 ℃，T.嗜热菌也可以在明显较低的温度（48°C）下生长。通过克隆和表达Cfr- 甲基转移酶基因的中度嗜热菌在T。嗜热菌在高温下（65- 72°C）或通过在T.嗜热宿主生长在 在48-65°C的温度下，我们将获得具有甲基化A2503的可结晶核糖体。一旦结构 确定了A2503-甲基化的核糖体，我们将解决这种Cfr-修饰的核糖体的结构， 与几种PTC作用药物形成复合物，这些药物对修饰的核糖体表现出残余的结合亲和力。的 所得到的信息将有助于理解Cfr介导的抗性的分子原理 并将指导后续合理设计针对Cfr阳性病原体的新型抗菌药物。 此外，在成功完成拟议项目后，我们将回答一个长期存在的基本问题， 和医学相关的问题：如何在2.5-MDa中的单个核苷酸上添加甲基 核糖体导致对许多化学上不相关的PTC靶向抗生素的耐药性？