The Continuing Challenge of Carbapenemases in K. pneumoniae: KPC-2 & NDM-1

在使用β-内酰胺酶抑制剂(BLIS)治疗患者30多年后，迫切的问题 仍然站在我们临床工作的前沿。尽管如此，β-内酰胺/BLI组合的值(例如， 哌拉西林/他唑巴坦)在20世纪90年代得到很好的确立，β-内酰胺酶的迅速出现 水解性广谱头孢菌素、碳青霉烯类抗生素和BLIS产生的抗灭活作用 一场全球性的抗菌化疗危机，并推动了对一类新型抑制剂的探索 二氮杂双环辛烷(DBO)。除了超广谱β-内酰胺酶(ESBL S)外，主要挑战 在肺炎克雷伯菌中要克服的是丝氨酸碳青霉烯酶(如KPC-2，OXA-48)和金属... β-内酰胺酶(MBLS)(如NDM-1、VIM和IMP)。幸运的是，DBO BLI的阿维巴坦(AVI)可以使KPC-2失活 和OXA-48；AVI对MBLS缺乏疗效。 我们在前一个赠款周期的调查使我们获得了前所未有的、令人恐惧的见解。 我们痛苦地被提醒，我们不能预测β-内酰胺酶的进化；氨基酸的多样性 自然界可以产生的序列及其对催化活性和抗药性的影响是不可预测的。我们发现 令我们惊愕的是，Kpc-2β-内酰胺酶变异体在大肠杆菌DH10B中表达的氨基酸 S130G、K234R和R220M突变使其对氨苄西林-AVI产生抗性。此外，我们发现， VIM-24(R228L)的单一氨基酸替换可增强对头孢他啶和头孢吡肟的耐药性。 我们得出了一个不可避免的结论：i)在BLI发布之前就已经出现了对AVI的抵抗；以及ii) 与A类和C类头孢菌素类似，MBLs可以扩大其底物谱并增强头孢菌素耐药性 超广谱β-内酰胺酶(ESBL)。在这个优点应用程序中，我们的目标是：1)继续探索 KPC对AVI抗性的结构和机制基础；2)了解新的底物专一性是如何进化的 KPC和NDM碳青霉烯酶。我们将首次应用互补结构方法(x射线 结晶学、核磁共振和双电子共振，帮助我们了解结构 活性关系是在碳青霉烯酶中定义的，目的是这些新的见解将导致新的 BLI设计中的方法。此外，最近对KpC-2β-内酰胺酶分子动力学的计算分析 预测“疏水网络”有助于KPC-2和变构信号的结构完整性和变构信号 其他A类β-内酰胺酶。这一新颖的见解定义了我们的第三个目标：3)如果这些疏水网络有助于 对于变构信号转导，变构抑制剂为BLI的设计提供了新的机遇。坚持不懈的步伐 耐药性使得我们必须了解基本的生化相互作用才能发现BLIS 通过新的机制发挥作用。 我们研究碳青霉烯酶的多学科方法将使我们能够预测新的突变 将影响BLI和头孢菌素的催化，并预测新的耐药表型。我们还提议，我们将 发现AVI结合的重要构象变化，并发现可能涉及的网络 变构信号。如果这一点得到证实，这些“疏水网络”的破坏将开启另一种选择 克服阻力并导致新的BLIS的方法。

After more than 30 years of using β-lactamase inhibitors (BLIs) in the treatment of patients, pressing questions still remain at the forefront of our clinical efforts. Although, the value of β-lactam/BLI combinations (e.g., piperacillin/tazobactam) was well-established by the 1990s, the rapid emergence of β-lactamases that hydrolyzed expanded spectrum cephalosporins, carbapenems and were resistant to inactivation by BLIs created a global crisis in antimicrobial chemotherapy and propelled the quest for a novel class of inhibitors, the diazabicyclooctanes (DBOs). In addition to the extended spectrum β-lactamases (ESBLs), the major challenges to overcome in Klebsiella pneumoniae are the serine carbapenemases (e.g., KPC-2, OXA-48) and the metallo- β-lactamases (MBLs) (e.g., NDM-1, VIM and IMP). Fortunately, avibactam (AVI), a DBO BLI, inactivates KPC-2 and OXA-48; the efficacy of AVI against MBLs is absent. Our investigations in the previous grant cycle awarded us with unprecedented and frightening insights. We were painfully reminded that we cannot anticipate β-lactamase evolution; the diversity in amino acid sequences that nature can yield and their impact on catalytic activity and resistance are unpredictable. We found to our dismay that KPC-2 β-lactamase variants expressed in Escherichia coli DH10B with the amino acid substitutions, S130G, K234R, and R220M conferred resistance to ampicillin-AVI. Moreover, we discovered that a single amino acid substitution in VIM-24 (R228L) confers enhanced resistance to ceftazidime and cefepime. We came to the inevitable conclusion that: i) resistance to AVI was present before this BLI was released; and ii) MBLs can expand their substrate profile and enhance cephalosporin resistance much like the class A and C extended-spectrum β-lactamases (ESBLs). In this Merit application, our goals are to: 1) continue to probe the structural and mechanistic basis for resistance to AVI in KPC; 2) learn how novel substrate specificity evolves in KPC and NDM carbapenemases. We will apply for the first time complementary structural methods (x-ray crystallography, NMR, and Double Electron Electron Resonance, DEER) to help us understand how structure activity relationship are defined in carbapenemases with the intent that these new insights will lead to new approaches in BLI design. In addition, recent computational analyses of KPC-2 β-lactamase molecular dynamics predict that “hydrophobic networks” contribute to the structural integrity and allosteric signaling of KPC-2 and other class A β-lactamases. This novel insight defines our third goal: 3) if these hydrophobic networks contribute to allosteric signaling, can allosteric inhibitors offer new opportunities for BLI design. The unrelenting pace of resistance makes it imperative that we understand fundamental biochemical interactions in order to find BLIs that act by novel mechanisms. Our multidisciplinary approach to studying carbapenemases will allow us to anticipate how new mutations will effect BLI and cephem catalysis and anticipate novel resistance phenotypes. We also propose that we will discover important conformational changes upon AVI binding and uncover networks that may be involved in allosteric signaling. If this is confirmed, disruption of these “hydrophobic networks” will open an alternative approach to overcoming resistance and lead to new BLIs.