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

在使用β-内酰胺酶抑制剂（BLI）治疗患者30多年后， 仍然是我们临床工作的前沿。虽然，β-内酰胺/BLI组合的价值（例如， 哌拉西林/他唑巴坦）在20世纪90年代得到了很好的确立， 水解的广谱头孢菌素类、碳青霉烯类，对BLI产生的灭活具有抗性 全球性的抗微生物化疗危机，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（ESBLs）， 在肺炎克雷伯氏菌中要克服的是丝氨酸碳青霉烯酶（例如，KPC-2，OXA-48）和金属- β-内酰胺酶（MBL）（例如，NDM-1、Vim和IMP）。幸运的是，阿维巴坦（AVI），一种DBO BLI，可灭活KPC-2 和OXA-48; AVI对MBL的疗效不存在。 我们在上一个资助周期的调查给了我们前所未有的和可怕的见解。 我们痛苦地提醒我们，我们不能预测β-内酰胺酶的进化; 自然界可以产生的序列及其对催化活性和抗性的影响是不可预测的。我们发现 令人沮丧的是，在大肠杆菌DH 10 B中表达的KPC-2 β-内酰胺酶变体具有氨基酸 S130 G、K234 R和R220 M取代赋予对氨苄青霉素-AVI的抗性。此外，我们发现， Vim-24（R228 L）中的单个氨基酸取代赋予对头孢他啶和头孢吡肟增强的抗性。 我们得出了一个不可避免的结论，即：i）在BLI发布之前，对AVI的抵抗力已经存在;以及ii） MBL可以扩大其底物谱，并增强头孢菌素耐药性，就像A类和C类一样 超广谱β-内酰胺酶（ESBLs）。在这个Merit应用程序中，我们的目标是：1）继续探索 KPC中抗AVI结构和机制基础; 2）了解新底物特异性如何在 KPC和NDM碳青霉烯酶。我们将首次应用互补结构方法（X射线 晶体学，NMR和双电子电子共振，DEER）来帮助我们了解结构如何 在碳青霉烯酶中定义活性关系，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KPC-2 β-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的结构完整性和变构信号传导， 其他A类β-内酰胺酶。这一新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 变构抑制剂能否为BLI的设计提供新的机会。无情的步伐 为了找到BLI，我们必须了解基本的生物化学相互作用 通过新的机制运作。 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是如何发生的。 将影响BLI和头孢烯催化作用，并预测新的耐药表型。我们还提议， 发现AVI结合后的重要构象变化，并揭示可能参与的网络 变构信号如果这一点得到证实，这些“疏水网络”的破坏将打开一个替代方案， 克服阻力的方法，并导致新的BLI。

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.