Mechanistic diversity, post-translational carbamylation, and inhibitor susceptibility in the OXA beta-lactamase family

β-内酰胺类抗生素，如青霉素和相关化合物，是最重要的抗生素，占人类使用量的一半以上，反过来又推动了耐药性的出现和传播。细菌通过各种机制抵抗β-内酰胺;在“革兰氏阴性”细菌（包括导致例如手术伤口、通气患者和血流的医疗保健相关感染的病原体）中，最重要的是产生β-内酰胺酶，其破坏环状β-内酰胺结构中的特定化学键，完全消除抗菌活性。超过4000种已知的β-内酰胺酶形成四类（A-D），它们的组成和用于打开β-内酰胺环的化学机制不同。β-内酰胺酶可以使用阻断其活性的药物（抑制剂）与β-内酰胺抗生素一起对抗，但尽管最近取得了进展，但这只是部分有效，因为抑制剂通常仅对特定的β-内酰胺酶起作用。此外，β-内酰胺酶进化为作用于更广泛的β-内酰胺，并逃避抑制剂。了解β-内酰胺酶如何发挥作用，以及变异如何影响与β-内酰胺类药物和抑制剂的相互作用，可为开发新的β-内酰胺类抗生素和β-内酰胺酶抑制剂提供信息。本提案重点关注D类（OXA）β-内酰胺酶，特别是那些导致对碳青霉烯类耐药的酶，碳青霉烯类是用于严重感染的最有效的β-内酰胺类药物。其中一组被称为OXA-48，是大肠杆菌及其亲属（血液感染的常见原因）中碳青霉烯耐药的最常见原因;另外两种（OXA-23和OXA-24/40）引起鲍曼不动杆菌耐药，这是一种对大多数替代抗生素耐药的生物体。除了它们能够分解碳青霉烯类之外，大多数可用的β-内酰胺酶抑制剂对OXA β-内酰胺酶的有效性不足以使它们用于治疗由产生它们的生物体引起的感染。OXA β-内酰胺酶在化学上是不寻常的，因为它们需要通过与二氧化碳反应进行修饰才能分解β-内酰胺。我们最近开发了光谱方法来检测这一点，使我们能够监测β-内酰胺酶与抗生素/抑制剂反应时修饰程度的变化。此外，我们最近发现OXA β-内酰胺酶，包括OXA-23和OXA-48，可以分解碳青霉烯类形成新的结构（β-内酯），而不是简单地打开β-内酰胺环。令人兴奋的是，内酯产物可逆地与OXA β-内酰胺酶结合，以防止与β-内酰胺的进一步反应，这表明内酯可能形成新的β-内酰胺酶抑制剂的基础。基于这些发现，我们将研究OXA β-内酰胺酶如何与碳青霉烯类，内酯和抑制剂相互作用;以及这些相互作用如何受到不同OXA组内变化的影响。我们将测量碳青霉烯通过目标OXA酶的分解，其活性如何受到内酯和其他抑制剂的影响，并通过NMR监测这些相互作用如何影响二氧化碳的修饰。除了商业上可用的化合物，我们将设计和合成新的碳青霉烯和内酯，以探索修饰这些分子如何影响与OXA酶的反应。我们将使用X射线可视化，在近原子的细节，如何OXA β-内酰胺酶结合碳青霉烯，内酯和抑制剂。利用这些信息，我们将通过测试我们的目标酶的特定变化的影响，并通过调查新发现的酶从患者样本中确定这些相互作用的OXA结构的最重要的元素。这些发现将确定不同的OXA酶如何分解碳青霉烯类，对抑制剂的敏感性不同，以及与内酯相互作用;这些信息可用于改善β-内酰胺抗生素并扩大β-内酰胺酶抑制剂的范围。

Beta-lactams, such as penicillins and related compounds, are the most important antibiotics, accounting for over half of human usage, in turn driving emergence and spread of resistance. Bacteria resist beta-lactams by various mechanisms; in 'Gram-negative' bacteria (including pathogens responsible for healthcare-associated infections of e.g. surgical wounds, ventilated patients and the bloodstream) the most important is production of beta-lactamase enzymes that break a specific chemical bond in the ring-like beta-lactam structure, completely abolishing antibacterial activity. The over 4000 known beta-lactamases form four classes (A - D) differing in their composition and in the chemical mechanism used to break open the beta-lactam ring. Beta-lactamases can be countered using drugs that block their activity (inhibitors) alongside beta-lactam antibiotics, but despite recent advances this is only partially effective as inhibitors generally act only against specific groups of beta-lactamases. Moreover, beta-lactamases evolve both to act on a wider range of beta-lactams, and to evade inhibitors. Understanding how beta-lactamases function, and how variations affect interactions with beta-lactams and inhibitors, provides information exploitable in new beta-lactam antibiotics and beta-lactamase inhibitors.This proposal focuses on class D (OXA) beta-lactamases, specifically those causing resistance to carbapenems, the most powerful beta-lactams used for severe infections. One such group, termed OXA-48, are the commonest cause of carbapenem resistance in Escherichia coli and its relatives (frequent causes of bloodstream infections); two others (OXA-23 and OXA-24/40) cause resistance in Acinetobacter baumannii, an organism resistant to most alternative antibiotics. In addition to their ability to break down carbapenems, most available beta-lactamase inhibitors are not sufficiently effective against OXA beta-lactamases to make them useful treatments for infections by organisms that produce them.OXA beta-lactamases are chemically unusual as they require modification by reaction with carbon dioxide before they can break down beta-lactams. We have recently developed spectroscopic methods to detect this, enabling us to monitor how the degree of modification changes as the beta-lactamase reacts with antibiotics/inhibitors. Moreover, we have recently discovered that OXA beta-lactamases, including OXA-23 and OXA-48, can break down carbapenems to form new structures (beta-lactones) rather than simply opening the beta-lactam ring. Excitingly, the lactone products bind reversibly to the OXA beta-lactamases to prevent further reaction with beta-lactams, suggesting that lactones may form the basis for new beta-lactamase inhibitors.Based on these findings, we will investigate how OXA beta-lactamases interact with carbapenems, lactones and inhibitors; and how these interactions are affected by variations within the different OXA groups. We will measure carbapenem breakdown by target OXA enzymes, how their activity is affected by lactones and other inhibitors, and monitor by NMR how these interactions affect modification by carbon dioxide. Alongside commercially available compounds, we will design and synthesise new carbapenems and lactones to explore how modifying these molecules affects reaction with OXA enzymes. We will use X-rays to visualise, in near atomic detail, how OXA beta-lactamases bind carbapenems, lactones and inhibitors. Using this information, we will identify elements of the OXA structure of greatest importance to these interactions by testing the effects of specific changes in our target enzymes, and by investigating newly identified enzymes from patient samples. These findings will establish how different OXA enzymes break down carbapenems, differ in susceptibility to inhibitors, and interact with lactones; information that may be applied to improve beta-lactam antibiotics and expand the range of beta-lactamase inhibitors.