Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA.
Louis Stokes Cleveland Veteran's Affairs Medical Center Research Service, Cleveland, Ohio, USA.
mBio. 2021 Feb 16;12(1):e03058-20. doi: 10.1128/mBio.03058-20.
Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is β-lactamase-mediated degradation of β-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as β-lactam "enhancers" due to inhibition of penicillin-binding protein 2 (PBP2), are also class A and C β-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why PBP2 is less susceptible to inhibition by β-lactam antibiotics compared to the PBP2, we determined the crystal structure of PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 β-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent β-lactamase-resistant, non-β-lactam PBP inhibitors. Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by β-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of PBP2 to be inhibited by β-lactam antibiotics, and provide insights that could be used for further antibiotic development.
多药耐药（MDR）病原体对公共健康构成重大威胁。MDR 病原体表达的主要耐药机制是β-内酰胺酶介导的β-内酰胺类抗生素的降解。由于抑制青霉素结合蛋白 2（PBP2），二氮杂二环辛烷（DBO）化合物齐他巴坦和 WCK 5153 被认为是β-内酰胺“增强剂”，也是 A 类和 C 类β-内酰胺酶抑制剂。为了从结构上研究它们抑制 PBP2 的模式，并研究为什么与 PBP2 相比，β-内酰胺类抗生素对 PBP2 的抑制作用较低，我们确定了 PBP2 与 WCK 5153 复合物的晶体结构。WCK 5153 与 PBP2 的催化 S327 形成抑制性共价键。该结构表明，WCK 5153 的二酰肼部分在与 PBP 基序中的天冬氨酸残基的相互作用中起重要作用。在 PBP2 的活性部位对齐他巴坦进行建模揭示了类似的结合模式。两种 DBO 都增加了 PBP2 的熔点，证实了它们的稳定相互作用。为了帮助设计可以抑制多种 PBPs 的 DBO，我们通过晶体学方法研究了三种 DBO 与 PBP3 的相互作用能力。尽管 DBO 与 PBP3 发生共价结合，但它们使 PBP3 不稳定。总体而言，这些研究提供了与 PBP3 相比，齐他巴坦和 WCK 5153 对 PBP2 抑制的见解，以及与进化相关的 KPC-2β-内酰胺酶的抑制。这些关于双重靶向 DBO 的分子见解提高了我们对进一步 DBO 优化以开发新型有效的β-内酰胺酶耐药、非β-内酰胺类 PBP 抑制剂的认识。抗生素耐药性是一个重大的临床问题。开发克服已知耐药机制的新型抗生素是非常需要的。齐他巴坦等二氮杂二环辛烷抑制剂具有这种潜力，因为它们可以轻易失活青霉素结合蛋白，但不能被β-内酰胺酶降解。在这项研究中，我们使用蛋白质晶体学和生物物理分析方法研究了二氮杂二环辛烷对来自的青霉素结合蛋白 PBP2 和 PBP3 的抑制作用。这些结构和分析有助于定义这些抑制剂的抗生素特性，解释为什么 PBP2 对β-内酰胺类抗生素的抑制作用较低，并提供可用于进一步抗生素开发的见解。
Zotero 插件