PROBING DRUG RESISTANCE IN B-LACTAMASE CRYSTALS BY RAMAN

描述(申请人提供)：本项目的主要目标是阐明导致B-内酰胺酶家族耐药的分子因素。(-内酰胺酶是由细菌产生的，它们在杀死细菌靶标之前摧毁青霉素类分子。因此，β-内酰胺酶本身就是抑制剂的靶标。临床分离株表明，B-内酰胺酶通过发生点突变而对其药物抑制剂产生耐药性。这项提议将阐明SHV-1B-内酰胺酶与临床重要药物他唑巴坦、舒巴坦和克拉维酸之间的化学反应；这些化合物起到“自杀抑制剂”的作用。这些反应将用拉曼结晶学来表征--用拉曼显微镜跟踪酶单晶中的反应。将药物分别注入含有β-内酰胺酶结晶的母液中，在不到1分钟的时间内，抑制剂完全扩散到晶体中，并通过拉曼差谱跟踪活性部位的后续反应。使用合适的酶形式，可以从拉曼数据中确定晶体中米氏络合物、酰基酶和最终产物的结构和数量。通过比较野生型酶和对抑制剂产生抗药性的酶的这些性质，将获得对耐药性潜在的分子机制的独特见解。B-内酰胺酶的耐药形式为M691、M69L和M69V，根据其临床相关性进行选择。拉曼方法表征单晶中中间体数量的能力将被用来选择闪速冷冻的最佳时间。含有捕获的反应中间体的晶体将通过X射线结晶学进行表征。对于D类B-内酰胺酶OXA-10和OXA-1，最近的研究表明，氨基甲酰化赖氨酸在活性中心化学中起着关键作用。拉曼结晶学将被用来证实这一新的和有争议的发现。

DESCRIPTION (provided by applicant): The principal goal of this project is to elucidate the molecular factors that lead to drug resistance in the B-lactamase family of enzymes. (-lactamases are produced by bacteria and they destroy penicillin-type molecules before they can kill their bacterial targets. Thus, B-lactamases themselves are targets for inhibitors. Clinical isolates demonstrate that, B-lactamases develop resistance to their drug-inhibitors by undergoing point mutations. This proposal will elucidate the chemical reactions between a SHV-1 B-lactamase 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 B-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 B-lactamase 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 B-lactamases 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.