Raman spectroscopic analysis of drug beta-lactamase interacteractions

描述（由申请人提供）：本提案旨在研究良好抑制剂候选化合物与？内酰胺酶的酶。在临床上，细菌感染对抗生素治疗的耐药性越来越强。抵抗的一个主要来源是？细菌产生的-内酰胺酶。这些酶在类青霉素分子攻击细菌之前水解并使其失活。在这里，我们开始确定由机制抑制剂形成的中间体，阻断临床有问题的？-lactamases。该研究的重点将是阐明铅抑制剂与A类和D类的反应。-内酰胺酶作为碳青霉烯酶（KPC-2和OXA-24）和扩展谱？-内酰胺酶（SHV-2， SHV-5和oxa10）。拉曼晶体学的一个主要推动力是利用拉曼显微镜实时跟踪酶单晶中抑制剂的反应。这提供了有关中间体在反应途径上的身份和构象的信息以及它们种群的动力学数据。拉曼数据还提供了与x射线晶体学的直接联系及其详细的结构见解，这两种方法是高度协同的。此外，拉曼分析将扩展到溶液研究，将我们对晶体反应的知识与水相中的相应反应联系起来。这很重要，因为越来越清楚的是，对于一些抑制剂，溶液研究的结果与晶体研究的结果不同。最初，我们的研究将使用具有良好IC50值的6-（吡啶基甲基）苯胺砜作为先导化合物。我们的研究结果将确定在生物学相关条件下阻断活性位点的反应中间体，并为设计更好的抑制剂和潜在的治疗性化合物提供输入。

DESCRIPTION (provided by applicant): This proposal sets out to study the chemical reactions between compounds that are good inhibitor candidates and ?-lactamase enzymes. In the clinic, bacterial infections are becoming increasingly resistant to antibiotic-based treatments. A major source of resistance is the class of ? -lactamase enzymes produced by the bacteria. These enzymes hydrolyze, and thus inactivate, penicillin-like molecules before they can attack the bacteria. Here we set out to identify the intermediates formed by mechanism-based inhibitors that block the action of clinically problematic ? -lactamases. A focal point of the study will be the elucidation of the reactions of lead inhibitors with class A and class D ? -lactamases that function as carbapenemases (KPC-2 and OXA-24) and extended spectrum ? -lactamases (SHV-2, SHV-5 and OXA 10). A major thrust involves the use of Raman crystallography, where the reactions of the inhibitors within single crystals of the enzyme are followed in real time using a Raman microscope. This provides information on the identity and conformation of intermediates on the reaction pathway as well as kinetic data on their populations. The Raman data also provide an immediate link to X-ray crystallography with its detailed structural insights, and the two approaches are highly synergistic. In addition, the Raman analysis will be extended to solution studies, linking our knowledge of the reactions in crystals to the corresponding reaction in the aqueous phase. This is important because it is becoming clear that, for some inhibitors, the results from solution studies differs from those in crystals. Initially, our studies will use lead compounds that are 6-(pyridylmethylidene) penam sulfones possessing favorable IC50 values. Our results will identify reaction intermediates that block the active site under biologically relevant conditions and provide input into the design of better inhibitors, and thence potential therapeutic compounds. PUBLIC HEALTH RELEVANCE: The threat to human health caused by drug resistance is a topic seldom out of the news' headlines. Bacteria are becoming increasingly resistant to elimination by "classical" antibiotic-based therapies. This project is part of a team-based effort to identify compounds that might serve as a basis for designing novel drugs effective against emerging resistant bacteria. Our contribution involves using newly-developed technology to characterize chemical reactions between lead compounds and the enzymes that are responsible for a large number of "drug resistance" outbreaks. By characterizing intermediates formed in these reactions we can propose compounds that can more effectively block the active sites of the enzymes and serve as a starting point for drug design. A focal point of the study will be the elucidation of the reactions of lead inhibitors with clinically problematic ? -lactamases that function as carbapenemases (KPC-2 and OXA-24) and extended spectrum ? -lactamases (SHV-2, SHV-5 and OXA 10). A major thrust involves the use of Raman crystallography, where the reactions of the inhibitors within single crystals of the enzyme are followed in real time using a Raman microscope. This provides information on the identity and conformation of intermediates on the reaction pathway as well as kinetic data on their populations.