PROBING DRUG RESISTANCE IN B-LACTAMASE CRYSTALS BY RAMAN

通过拉曼探测 B-内酰胺酶晶体的耐药性

• 批准号: 7594860
• 负责人: PAUL R CAREY
• 金额: $ 1.52万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7594860
• 关键词: Active Sites; Acylation; Amino Acids; Bacteria; Chemistry; Class; Clavulanic Acid; Clavulanic Acids; Clinical; Complex; Condition; Crystallography; Data; Dependence; Diffuse; Drug resistance; Enzymes; Family; Freezing; Goals; Hour; Investigation; Kinetics; Laboratories; Lactamase; Lead; Lysine; Maps; Mass Spectrum Analysis; Mechanics; Methionine; Methods; Microscope; Molecular; Mothers; Mutation; Outcome; Pathway interactions; Penicillins; Pharmaceutical Preparations; Play; Point Mutation; Population; Property; Proteolysis; Reaction; Resistance; Resistance development; Roentgen Rays; Role; Serine; Side; Spectrum Analysis; Structure; Sulbactam; Tazobactam; Techniques; Testing; Time; X-Ray Crystallography; acyl group; beta-lactamase PSE-2; carboxylate; chemical reaction; clinically relevant; deacylation; distilled alcoholic beverage; enzyme structure; inhibitor/antagonist; insight; interest; killings; mutant; novel; oxytocin, Asp(5)-; protonation; quantum; research study; suicide inhibitor

PROVIDED. The principal goal of this project is to elucidate the molecular factors that lead to drug resistance in the ,8- lactamase family of enzymes. ,8-1actamases are produced by bacteria and they destroy penicillin-type molecules before they can kill their bacterial targets. Thus, ,8-1actamases themselves are targets for inhibitors. Clinical isolates demonstrate that ,8-1actamases develop resistance to their drug-inhibitors by undergoing point mutations. This proposal will elucidate the chemical reactions between a SHV-1 ,8-1actamase and the clinically important drugs, tazobactam, sulbactam and clavulanic acid; these compounds act as "suicide inhibitors". The reactions will be characterized by Raman crystallography - by following the reactions in single crystals of the enzyme using a Raman microscope. The drugs are injected separately into the mother liquor containing a crystal of p-lactamase, the inhibitors diffuse fully into the crystal in less than one minute and the subsequent reaction in the active site can be followed via the Raman difference spectrum. Using suitable forms of the enzyme, the structures and populations of the Michaelis complexes, acyl enzymes and final products can be defined in the crystals from the Raman data. By comparing these properties for the wild-type enzyme, and the enzyme that has developed a resistance to the inhibitors, unique insight into the molecular mechanisms underlying resistance will be gained. The resistant forms of the ,8-1actamase are M691, M69L and M69V, selected for their clinical relevance. The ability of the Raman method to characterize populations of intermediates in single crystals will be used to select optimal times for flash freezing. The crystals containing the trapped reaction intermediates will then be characterized by X-ray crystallography. For the class D ,8-1actamases OXA-10 and OXA-1 recent studies have indicated that a carbamylated lysine plays a key role in active site chemistry. Raman crystallography will be used to confirm this novel and controversial finding.

假如。 该项目的主要目标是阐明导致8--的耐药性的分子因素 酶酶家族。 ，8-1个乳酶是由细菌产生的，它们破坏了青霉素型 分子可以杀死其细菌靶标。因此，8-1个分酶本身是抑制剂的靶标。 临床分离株表明，8-1个撒氨酶通过关注点对其药物抑制剂产生抗性 突变。该提案将阐明SHV-1，8-1个酶和临床上的化学反应 重要的药物，tazobactam，sulbactam和clavulanic酸；这些化合物充当“自杀抑制剂”。这 反应将以拉曼晶体学为特征 - 通过遵循在单晶中的反应 使用拉曼显微镜的酶。这些药物分别注射到含有晶体的母液中 P-内酰胺酶，抑制剂在不到一分钟的时间内完全扩散到晶体中，随后的反应 可以通过拉曼差异频谱跟随活动位点。使用合适形式的酶， 可以在Michaelis综合体，酰基酶和最终产品的结构和种群中定义 拉曼数据的晶体。通过比较这些特性的野生型酶和具有的酶 对抑制剂产生了抗性，对抗性的分子机制的独特见解将 获得。以临床相关性选择了8-1 actamase的抗性形式是M691，M69L和M69V。 拉曼法表征单晶中间体种群的能力将用于 选择最佳闪光冻结时间。然后，包含被困反应中间体的晶体将是 以X射线晶体学为特征。 对于D级，8-1分酶OXA-10和OXA-1最近的研究表明甲酰化赖氨酸的作用 在主动部位化学中的关键作用。拉曼晶体学将用于确认这一小说和有争议的 发现。