PROBING DRUG RESISTANCE IN B-LACTAMASE CRYSTALS BY RAMAN

描述（由申请方提供）：本项目的主要目标是阐明导致β-内酰胺酶家族耐药性的分子因素。β-内酰胺酶是由细菌产生的，它们在杀死其细菌靶标之前破坏青霉素类分子。因此，β-内酰胺酶本身是抑制剂的靶标。临床分离株证明，β-内酰胺酶通过点突变对其药物抑制剂产生耐药性。该提案将阐明SHV-1 B-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;这些化合物充当“自杀抑制剂”。“这些反应将通过拉曼晶体学来表征--通过使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-内酰胺酶晶体的母液中，抑制剂在不到一分钟的时间内完全扩散到晶体中，并且可以通过拉曼差光谱跟踪活性位点中的后续反应。使用合适形式的酶，米氏络合物、酰基酶和最终产物的结构和群体可以从拉曼数据在晶体中确定。通过比较野生型酶和对抑制剂产生耐药性的酶的这些性质，将获得对耐药性潜在分子机制的独特见解。B-内酰胺酶的耐药形式是M691、M69 L和M69 V，根据其临床相关性选择。拉曼方法表征单晶中中间体群体的能力将用于选择快速冷冻的最佳时间。然后用X射线晶体学表征含有捕获的反应中间体的晶体。对于D类B-内酰胺酶OXA-10和OXA-1，最近的研究表明氨甲酰化赖氨酸在活性位点化学中起关键作用。拉曼晶体学将被用于证实这一新颖而有争议的发现。

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.