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？-内酰胺酶在临床上，细菌感染对基于抗生素的治疗越来越耐药。一个主要的阻力来源是类？- 由细菌产生的内酰胺酶。这些酶在攻击细菌之前水解青霉素样分子，从而使青霉素样分子变性。在这里，我们着手确定中间体形成的机制为基础的抑制剂，阻止行动的临床问题？- 内酰胺酶。研究的一个重点将是阐明铅抑制剂与A类和D类？- 具有碳青霉烯酶（KPC-2和OXA-24）和超广谱功能的内酰胺酶？- 内酰胺酶（SHV-2、SHV-5和OXA 10）。一个主要的推动力涉及使用拉曼晶体学，其中使用拉曼显微镜真实的跟踪酶单晶内抑制剂的反应。这提供了关于反应途径上中间体的身份和构象的信息以及关于其群体的动力学数据。拉曼数据还提供了一个直接的链接到X射线晶体学与其详细的结构见解，这两种方法是高度协同。此外，拉曼分析将扩展到溶液研究，将我们对晶体中反应的了解与水相中相应的反应联系起来。这一点很重要，因为越来越清楚的是，对于某些抑制剂，溶液研究的结果与晶体中的结果不同。最初，我们的研究将使用具有良好IC 50值的先导化合物6-（吡啶亚甲基）青霉烷砜。我们的研究结果将确定在生物相关条件下阻断活性位点的反应中间体，并为更好的抑制剂的设计提供输入，从而提供潜在的治疗化合物。 公共卫生相关性：耐药性对人类健康的威胁是一个很少出现在新闻头条中的话题。细菌对“经典”的基于抗生素的治疗的消除越来越有抵抗力。该项目是一个以团队为基础的努力的一部分，以确定可能作为设计有效对抗新出现的耐药细菌的新药的基础的化合物。我们的贡献包括使用新开发的技术来表征导致大量“耐药性”爆发的先导化合物和酶之间的化学反应。通过表征这些反应中形成的中间体，我们可以提出可以更有效地阻断酶活性位点的化合物，并作为药物设计的起点。研究的重点将是阐明铅抑制剂的反应与临床问题？- 具有碳青霉烯酶（KPC-2和OXA-24）和超广谱功能的内酰胺酶？- 内酰胺酶（SHV-2、SHV-5和OXA 10）。一个主要的推动力涉及使用拉曼晶体学，其中使用拉曼显微镜真实的跟踪酶单晶内抑制剂的反应。这提供了关于反应途径上中间体的身份和构象的信息以及关于其群体的动力学数据。

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.