A Mechanism Based Approach to Metallo beta-lactamase Inhibition

产金属β-内酰胺酶（MBL）的革兰阴性菌严重威胁着公众健康。MBLs 是最令人担忧的碳青霉烯酶，使“最后手段”碳青霉烯类和大多数β-内酰胺类失活， 抵抗所有市售β-内酰胺酶抑制剂（BLI）。MBL抑制剂设计的主要挑战是 理解与3种不同亚类（B1， B2和B3）。在上一个5年资助周期中，我们实现了重要的里程碑：i）确定了活动形式 细菌周质中临床相关的MBL; ii）将NDM-1表征为膜结合蛋白， 确定这种定位如何赋予NDM-1独特的稳定性; iii）证明所有3种MBL 亚类利用碳青霉烯水解的共同机制，提示抑制剂的新方法 iv）设计了一系列新型化合物，双噻唑烷（BTZ），作为底物模拟物， 包含用金属结合基团修饰的非- β-内酰胺“青霉素核”;和v）表明BTZ是 无毒，有效的跨类MBL抑制剂，并确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、评价和开发新交叉类MBL抑制剂。我们独特的方法基于 对MBL催化的机理理解，其将用于激发有效的抑制剂。为此我们将 合成作为机械中间体模拟物或产物模拟物的新化合物[噻唑烷（TZs），Δ4- 噻唑烷（Δ4-TZs）和Δ4-恶唑烷（Δ4-OXZs）]或碳青霉烯类模拟物[Δ4-双噻唑烷（Δ4-BTZs） 和双环辛烷（BCO）]。我们的第二个具体目标是评估抑制剂对MBL的体外活性， 所有子类我们接下来将测定抑制剂在增强β-内酰胺对MBL-1的功效中的影响。 生产模型菌株，评估体外测定之间的差异和对细菌的影响，并验证 针对具有不同MBL等位基因的一组临床菌株的选择的抑制剂。我们的第三个目标是将联合收割机 和X射线晶体学研究MBL-抑制剂加合物的结构，旨在为抑制剂的研究提供细节 改进.我们还将利用微聚焦光谱学和晶体学进行机理研究 与XFEL（X射线自由电子激光）耦合，以捕获酶NDM-1中的瞬时β-内酰胺结合物质， L1和Vim。这种利用新技术的“高风险、高影响”创新方法将提供信息， 用于抑制剂改进。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组合进行时间杀灭测定;并使用小鼠血液 流和肺感染模型，以评估美罗培南/MBL-抑制剂组合的体内效力。这 这些知识将用于设计治疗性先导物以对抗产生MBL的细菌。

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.