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

OXA β-内酰胺酶家族的机制多样性、翻译后氨甲酰化和抑制剂敏感性

• 批准号: BB/W001187/1
• 负责人: James Spencer
• 金额: $ 103.29万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW001187%2F1
• 关键词: Mechanistic; diversity; post; translational; carbamylation

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.

-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，反过来又推动耐药性的出现和传播。细菌通过多种机制抵抗β -内酰胺；在“革兰氏阴性”细菌（包括导致医疗保健相关感染的病原体，例如外科伤口、通气病人和血液）中，最重要的是产生β -内酰胺酶，这种酶会破坏环状β -内酰胺结构中的特定化学键，从而完全取消抗菌活性。超过4000种已知的β -内酰胺酶分为四类（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）和β -内酰胺抗生素来对抗，但尽管最近取得了进展，但这只是部分有效，因为抑制剂通常只对特定的β -内酰胺酶起作用。此外，β -内酰胺酶进化为既能作用于更广泛的β -内酰胺，又能逃避抑制剂。了解β -内酰胺酶的功能，以及变化如何影响β -内酰胺和抑制剂的相互作用，为开发新的β -内酰胺类抗生素和β -内酰胺酶抑制剂提供了可利用的信息。这项建议的重点是D类(OXA) β -内酰胺酶，特别是那些对碳青霉烯类产生耐药性的酶，碳青霉烯类是用于严重感染的最有效的β -内酰胺类。其中一组称为OXA-48，是大肠杆菌及其近亲（血液感染的常见原因）中碳青霉烯类耐药的最常见原因；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。除了分解碳青霉烯类的能力外，大多数现有的β -内酰胺酶抑制剂对OXA β -内酰胺酶的作用不够有效，因此无法有效治疗产生OXA β -内酰胺酶的生物体造成的感染。OXA -内酰胺酶在化学上是不寻常的，因为它们需要与二氧化碳反应才能分解-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、内酯类和抑制剂相互作用；以及这些相互作用如何受到不同OXA组内变化的影响。我们将测量目标OXA酶对碳青霉烯的分解，它们的活性如何受到内酯和其他抑制剂的影响，并通过核磁共振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类和内酯，以探索如何修饰这些分子影响与OXA酶的反应。我们将使用x射线在接近原子的细节上可视化OXA -内酰胺酶是如何结合碳青霉烯类、内酯类和抑制剂的。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，以及通过研究从患者样本中新发现的酶，来确定OXA结构中对这些相互作用最重要的元素。这些发现将确定不同的OXA酶如何分解碳青霉烯类，对抑制剂的易感性不同，以及与内酯相互作用；可用于改进-内酰胺类抗生素和扩大-内酰胺酶抑制剂范围的信息。