Raman spectroscopic analysis of drug beta-lactamase interacteractions

描述(由申请人提供)：本提案旨在研究具有良好抑制活性的化合物与β-内酰胺酶之间的化学反应。在临床上，细菌感染对以抗生素为基础的治疗越来越耐药。耐药的一个主要来源是细菌产生的一类β-内酰胺酶。这些酶在青霉素类分子攻击细菌之前对其进行水解，从而使其失活。在这里，我们开始确定由基于机制的抑制剂形成的中间体，这些中间体阻止了临床上有问题的内酰胺酶的作用。这项研究的重点将是阐明铅抑制剂与作为碳青霉烯酶(KPC-2和OXA-24)的A类和D类β-内酰胺酶以及超广谱β-内酰胺酶(SHV-2，SHV-5和OXA 10)的反应。一个主要的推动力涉及到拉曼结晶学的使用，其中使用拉曼显微镜实时跟踪酶单晶内的抑制剂的反应。这提供了关于反应路径上中间体的身份和构象的信息以及关于其种群的动力学数据。拉曼数据还通过其详细的结构洞察提供了与X射线结晶学的直接联系，这两种方法具有高度的协同性。此外，拉曼分析将扩展到溶液研究，将我们对晶体中反应的知识与水相中的相应反应联系起来。这一点很重要，因为对于一些缓蚀剂来说，溶液研究的结果与晶体中的结果不同，这一点变得越来越明显。最初，我们的研究将使用具有良好IC50值的6-(吡啶亚甲基)苯胺砜的先导化合物。我们的结果将确定在生物相关条件下阻断活性部位的反应中间体，并为设计更好的抑制剂提供输入，从而提供潜在的治疗化合物。 公共卫生相关性：抗药性对人类健康造成的威胁是一个很少出现在新闻头条上的话题。细菌正变得越来越不能被“经典的”基于抗生素的疗法消灭。该项目是一个团队努力的一部分，目的是识别化合物，这些化合物可能作为设计有效对抗新出现的耐药细菌的新药的基础。我们的贡献包括使用新开发的技术来表征先导化合物和导致大量“抗药性”爆发的酶之间的化学反应。通过表征在这些反应中形成的中间体，我们可以提出能够更有效地阻断酶的活性部位的化合物，并作为药物设计的起点。这项研究的重点将是阐明铅抑制剂与临床上有问题的碳青霉烯酶(KPC-2和OXA-24)和超广谱β-内酰胺酶(SHV-2，SHV-5和OXA 10)的反应。一个主要的推动力涉及到拉曼结晶学的使用，其中使用拉曼显微镜实时跟踪酶单晶内的抑制剂的反应。这提供了关于反应路径上中间体的身份和构象的信息以及关于其种群的动力学数据。

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.