A Mechanism-Based Approach to Metallo-beta-Lactamase Inhibition

描述(申请人提供)：碳青霉烯耐药革兰氏阴性病原体的全球传播是发病率和死亡率的重要来源。碳青霉烯酶，或碳青霉烯类水解酶，严重限制了由拥有这些酶的细菌引起的感染的治疗。在碳青霉烯酶中，金属β-内酰胺酶(MBLS)迅速出现，最受关注。虽然有关MBLs的流行病学报道越来越多，但关于MBLs的重要反应机制和结构-功能特性的了解却很少。在丝氨酸碳青霉烯酶和MBLS的集体经验的指导下，我们独特的合作伙伴关系正在启动深入研究，以解决这一重要的知识差距，并获得最终支持新药设计努力的机械性见解。我们认为MBLS在碳青霉烯类抗生素的水解过程中形成了一种共同的阴离子中间体。认识这种常见的阴离子中间体是从机理上理解MBLS反应途径的重要的第一步。因此，我们的研究将检验以下假设：1)结构上不同的MBLS通过共同的反应中间体水解β-内酰胺类药物，特别是碳青霉烯类；2)MBLS活性中心两侧的移动环对于识别广泛的底物很重要；3)模拟共同反应中间体并与移动环相互作用的化合物将作为反应机制的抑制剂。为了检验这些假设，我们将努力实现以下特定目标：1)表征临床相关MBLS(即NDM-1、VIM-2、IMP-1和SPM-1)中填充的反应中间体；2)确定普通MBLS活性位点两侧的移动环的生化特征，帮助识别广泛的底物库，并评估这些环作为潜在的药效团用于抑制剂开发；以及3)设计模拟常见阴离子中间体的化合物，以深入了解抑制机制。在这一提议中，我们将提供强有力的证据，证明所有亚类的MBLS在反应途径上都存在共同的中间体，这一物种将使我们对反应机理有更深入的了解。我们的联盟在许多临床重要的β-内酰胺酶的结构研究以及临床微生物学、结晶学、核磁共振、稳态和稳态前动力学、质谱学和β-内酰胺酶抑制剂测试方面经验丰富。不同的人才和人员的结合将确保从这些研究中获得的结果将促进这项工作的翻译性质。

DESCRIPTION (provided by applicant): The global dissemination of carbapenem-resistant Gram-negative pathogens is a significant source of morbidity and mortality. Carbapenemases, or carbapenem-hydrolyzing b-lactamases, are severely limiting the treatment of infections caused by bacteria possessing these enzymes. Among the carbapenemases, metallo- b-lactamases (MBLs) are rapidly emerging and are the most concerning. Although reports of the epidemiology of MBLs are increasing, little knowledge is available regarding the important reaction mechanisms and structure-function properties of MBLs. Guided by the collective experience with serine carbapenemases and MBLs, our unique partnership is initiating in-depth studies to address this important knowledge gap and to acquire mechanistic insights that will ultimately support novel drug design efforts. We propose that a common anionic intermediate is formed in the hydrolysis of carbapenems by MBLs. Recognizing this common anionic intermediate is an important "first step" in the mechanistic understanding of the reaction pathway of MBLs. Therefore, our investigations will test the following hypotheses: 1) structurally divergent MBLs hydrolyze b-lactams, especially carbapenems, by proceeding through common reaction intermediates; 2) the mobile loops flanking the active sites of MBLs are important in recognizing a broad repertoire of substrates; and 3) compounds that mimic the common reaction intermediates and interact with the mobile loops will serve as inhibitors of the reaction mechanism. To test these hypotheses we will endeavor to accomplish the following specific aims: 1) characterizing the populated reaction intermediates in clinically relevant MBLs (i.e., NDM-1, VIM-2, IMP-1, and SPM-1); 2) identify biochemical features of the mobile loops flanking the active sites of common MBLs that assist in recognizing a broad repertoire of substrates and evaluate these loops as potential pharmacophores for inhibitor development; and 3) design compounds that will mimic the common anionic intermediates to give insight into the mechanism of inhibition. In this proposal, we will provide strong evidence that a common intermediate exists along the reaction pathway of MBLs from all subclasses and that this species will lead us to a deeper understanding of the reaction mechanism. Our consortium is experienced in structural studies of many clinically important b- lactamases, as well as in clinical microbiology, crystallography, NMR, steady-state and pre-steady state kinetics, mass spectrometry and b-lactamase inhibitor testing. The joining together of diverse talents and personnel will ensure the findings obtained from these studies will advance the translational nature of this work.