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

描述（由申请方提供）：碳青霉烯类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水解β-内酰胺酶严重限制了对由具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（MBL）迅速出现并且是最令人关注的。虽然有关MBLs流行病学的报道越来越多，但关于MBLs的重要反应机制和结构-功能特性的知识却很少。在丝氨酸碳青霉烯酶和MBL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将支持新药设计工作的机制见解。我们认为MBLs在水解碳青霉烯类抗生素过程中形成了一个共同的阴离子中间体。认识到这一共同的阴离子中间体是重要的“第一步”的机制理解的反应途径的MBLs。因此，我们的研究将验证以下假设：1）结构不同的MBL通过共同的反应中间体水解β-内酰胺类，特别是碳青霉烯类; 2）MBL活性位点两侧的移动的环在识别广泛的底物库中是重要的;和3）模拟普通反应中间体并与移动的环相互作用的化合物将作为反应机理的抑制剂。为了检验这些假设，我们将奋进实现以下具体目标：1）表征临床相关MBL中的填充反应中间体（即，NDM-1、Vim-2、IMP-1和SPM-1）; 2）鉴定常见MBL活性位点侧翼的移动的环的生物化学特征，这些环有助于识别广泛的底物库，并评估这些环作为抑制剂开发的潜在药效团; 3）设计将模拟常见阴离子中间体的化合物，以深入了解抑制机制。在这个建议中，我们将提供强有力的证据，一个共同的中间体存在沿着的反应途径MBLs从所有的子类，这个物种将导致我们更深入地了解反应机制。我们的联盟在许多临床重要的B-内酰胺酶的结构研究以及临床微生物学、晶体学、NMR、稳态和稳态前动力学、质谱和b-内酰胺酶抑制剂测试方面经验丰富。不同的人才和人员的加入将确保从这些研究中获得的结果将推进这项工作的转化性质。 公共卫生相关性：碳青霉烯酶或碳青霉烯类水解β-内酰胺酶，其潜在地抑制包括碳青霉烯类在内的所有B-内酰胺类，可严重限制革兰氏阴性菌引起的感染的治疗。在碳青霉烯酶中，金属β-内酰胺酶（MBL，如NDM-1、Vim-2、IMP-1和SPM-1）在世界范围内迅速出现，并构成最严重的公共卫生威胁之一。我们试图研究这些临床相关的MBL，以提供证据表明存在一种共同的反应中间体，并可用于制备有效的抑制剂。

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. PUBLIC HEALTH RELEVANCE: Carbapenemases, or carbapenem-hydrolyzing b-lactamases that potentially inactivate all b- lactams including carbapenems, can severely limit the treatment of infections caused by Gram- negative bacteria. Among the carbapenemases, metallo- b-lactamases (MBLs such as NDM-1, VIM-2, IMP-1, and SPM-1) are rapidly emerging world-wide and pose one of the most serious public health threats. We seek to study these clinically relevant MBLs to provide evidence that a common reaction intermediate exists and can be exploited to make an effective inhibitor.