A Mechanism Based Approach to Metallo beta-lactamase Inhibition

产生金属β-内酰胺酶的革兰氏阴性病原菌MBLS严重威胁公众健康。MBLS 是最令人担忧的碳青霉烯酶，能灭活“最后手段”的碳青霉烯类抗生素和大多数β-内酰胺类抗生素 抵抗所有商用的β-内酰胺酶抑制剂(BLI)。MBL抑制剂设计中的主要挑战是 理解反应机理，因为它与3个不同亚类(B1， B2和B3)。在上一个五年筹资周期中，我们取得了重要的里程碑：一)确定了积极的形式 细菌周质中临床相关的MBLs；ii)将NDM-1表征为膜结合蛋白， 确定这种本地化如何赋予NDM-1独特的稳定性；iii)展示了所有3个Mbls 亚类利用碳青霉烯类降解的共同机制建议新的抑制剂方法 开发；iv)设计了一系列新的化合物，双噻唑烷(BTZ)，作为底物模拟物， 包括带有金属结合基团的非β-内酰胺类青霉素核心；以及v)表明BTZ是 无毒、有效的跨类MBL抑制剂，并鉴定其抑制作用的结构基础。 为了应对寻找新疗法的明显紧迫性，我们的团队将在这些成就的基础上确定， 合成、评价和开发新的跨类MBL抑制剂。我们独特的方法是基于 从机理上理解MBL催化，这将被用来激发有效的抑制剂。为此，我们将 合成新化合物作为机械中间体或产物的模拟物[TZS，Δ4- [Δ4-TZs和Δ4-OXZ(Δ4-OXZ)]或碳青霉烯类[Δ4-BTZ(Δ4-BTZ)] 和双环辛烷(BCO)]。我们的第二个具体目标是评估抑制剂对Mbls的体外活性。 所有子类。接下来，我们将分析抑制剂在增强β-内酰胺类药物对抗多发性骨髓瘤疗效中的作用。 生产模型菌株，评估体外测试之间的差异和对细菌的影响，并验证 针对一组具有不同MBL等位基因的临床菌株选择抑制剂。我们的第三个目标将结合核磁共振 和X-射线结晶学研究MBL-抑制剂加合物的结构，旨在为缓蚀剂提供细节 进步。我们还将利用微聚焦光谱学和结晶学进行机理研究。 耦合到XFEL(X射线自由电子激光)以捕捉酶NDM-1中的瞬时β-内酰胺结合物种， L1和Vim.这种使用新技术的“高风险、高影响”的创新方法将提供信息 用于抑制剂的改进。最后，我们将对合成的化合物进行脱靶活性和体外毒性测试。 化合物，在临床菌株中对美罗培南-BLI组合进行时间杀灭试验；并使用小鼠血液 流和肺部感染模型，以评估美罗培南/MBL抑制剂组合的体内效力。这 知识将有助于为设计治疗线索提供信息，以对抗MBL产生细菌。

Gram-negative pathogens producing metallo-β-lactamases, MBLs, seriously threaten the public health. MBLs are the most worrisome carbapenemases, inactivating the “last resort” carbapenems and most β-lactams, and resist all commercially available β-lactamase inhibitors (BLIs). The main challenges in MBL inhibitor design are understanding the reaction mechanism as it relates to the structural diversity of the 3 distinct subclasses (B1, B2, and B3). In the previous 5-year funding cycle, we achieved important milestones: i) identified the active form of clinically relevant MBLs in the bacterial periplasm; ii) characterized NDM-1 as a membrane-bound protein, establishing how this localization endows NDM-1 with unique stability; iii) demonstrated that MBLs of all 3 subclasses utilize a common mechanism for carbapenem hydrolysis suggesting novel approaches for inhibitor development; iv) designed a series of novel compounds, bisthiazolidines (BTZs), as substrate mimics, comprising a non- β-lactam “penicillin core” decorated with metal binding groups; and v) showed that BTZs are non-toxic, effective cross-class MBL inhibitors and identified the structural bases of their inhibitory action. Responding to the clear urgency to find novel therapies, our team will build on these accomplishments to identify, synthesize, evaluate and develop new cross-class MBL inhibitors. Our unique approach is based upon a mechanistic understanding of MBL catalysis which will be utilized to inspire potent inhibitors. To this end, we will synthesize new compounds as mimics of mechanistic intermediates or product mimics [Thiazolidines (TZs), Δ4- Thiazolidines (Δ4-TZs), and Δ4-Oxazolidines (Δ4-OXZs)] or carbapenem mimics [Δ4-Bisthiazolidines (Δ4-BTZs) and Bicyclooctanes (BCOs)]. Our second specific aim will evaluate inhibitors for in vitro activity against MBLs of all subclasses. We will next assay the impact of inhibitors in potentiating β-lactam efficacy against MBL- producing model strains, assess differences between in vitro assays and effect on bacteria, and validate the selected inhibitors against a panel of clinical strains with different MBL alleles. Our third aim will combine NMR and X-ray crystallography to study the structure of MBL-inhibitor adducts aimed to provide details for inhibitor improvement. We will also pursue mechanistic studies using micro-focusing spectroscopy and crystallography coupled to XFEL (X-ray free electron lasers) to trap transient β-lactam-bound species in the enzymes NDM-1, L1, and VIM. This “high-risk, high impact” innovative approach using new technologies will provide information for inhibitor improvement. Lastly, we will assay off-target activity and in vitro toxicity of the synthesized compounds, perform time-kill assays for meropenem-BLI combinations in clinical strains; and use mouse blood stream and lung infection models to assess the in vivo potency of meropenem/MBL-inhibitor combinations. This knowledge will serve to inform the design of therapeutic leads to combat MBL producing bacteria.