Carbapenem Antibiotic Resistance in Enterobacteriaceae: Understanding Interactions of KPC Carbapenemases with Substrates and Inhibitors

β-内酰胺类(BLS、青霉素及其近亲)是全球使用最多的抗生素。碳青霉烯类是最新和最有效的BLS，对于治疗所谓的机会性革兰氏阴性菌(GNB)感染特别重要。这些微生物通常存在于人体或自然环境(土壤、水)中，不被认为对健康的个人有害，但可能会导致免疫防御受损的患者感染，可能严重，甚至危及生命。这类感染的风险因素包括伤口(手术、烧伤、受伤)、使用医疗设备(导管、呼吸机)以及影响免疫防御的条件(艾滋病毒)或治疗(癌症化疗或防止移植排斥反应的药物)。越来越多的患者属于这些类别。GNB是一个特殊的治疗问题，因为它们的细胞结构阻止了许多杀死其他类型细菌的抗生素达到他们的目标；寻找有效对抗GNB的新抗生素的努力在很大程度上是不成功的。直到最近，碳青霉烯类被认为是治疗GNB感染的最后手段，对其他治疗无效。然而，对其他抗生素日益增长的耐药性使碳青霉烯类抗生素越来越成为怀疑感染GNB时的首选。当碳青霉烯类抗生素失效时，替代品是有限的，而且往往是有毒的，因此碳青霉烯类抗生素的耐药性被视为一项重大的公共卫生挑战。在GNB中，碳青霉烯类耐药主要是由于一种名为碳青霉烯酶的蛋白质，它与碳青霉烯类抗生素结合并降解，从而丧失了它们杀灭细菌的能力。碳青霉烯酶是一大类蛋白质(β-内酰胺酶)的一部分，可以破坏其他类型的BL抗生素，但大多数β-内酰胺酶不能分解碳青霉烯类抗生素。第二组药物(β-内酰胺酶抑制剂)可以对抗β-内酰胺酶，从而使BL抗生素能够用于治疗携带β-内酰胺酶的细菌，但并不是所有的β-内酰胺酶都可以通过这种途径被阻止，一些酶可以突变或进化以逃避抑制剂的作用。这项建议调查了来自GNB肺炎克雷伯菌的碳青霉烯酶KPC(肺炎克雷伯菌碳青霉烯酶)如何降解碳青霉烯类和其他BL抗生素，并与一类特定的抑制剂(双氮杂环辛烷，DBOS)相互作用，以及KPC中的突变如何影响这些活性。肺炎克雷伯菌是与医疗保健相关的感染(泌尿系和呼吸道感染、败血症)的重要原因，而KPC是全球碳青霉烯类耐药的主要原因之一。我们最近首次描述了KPC如何在其分解的关键阶段与碳青霉烯类和其他BL(头孢他啶，一种用于医疗相关感染的抗生素)结合；以及KPC如何结合DBO。基于这些信息，我们将使用最先进的计算方法来构建KPC与这三类分子中的每一种的反应的详细模型，以确定每种反应发生的最可能的途径。这些模型的准确性将通过将每次反应的预测速度与在患者治疗中使用的一系列抗生素的实验测量的实际值进行比较来测试。然后，我们将调查KPC中的特定变化如何影响这些反应，试图了解目前在人类患者细菌中发现的这些变化如何提高KPC分解抗生素的能力，并降低DBO阻止KPC作用的能力。最后，我们将使用这些信息在计算机模型和实验中设计和测试新的碳青霉烯类抗生素，它们抵抗KPC的分解，以及新的DBO，它们是更有效的KPC抑制剂；并且在每种情况下都不受KPC突变的影响。这提供了一条途径，通过了解KPC可以设计出对一组重要的抗生素耐药细菌有效的新治疗方法。

beta-lactams (BLs, penicillin and its relatives) are the most used antibiotics worldwide. Carbapenems are the newest and most potent BLs, and particularly important for treating infections by so-called opportunistic Gram-negative bacteria (GNB). These are organisms, either normally present in the human body or the natural environment (soil, water), that are not considered harmful to healthy individuals but can cause infections, possibly severe and even life-threatening, in patients whose immune defences are compromised. Risk factors for such infections include wounds (surgery, burns, injury), use of medical devices (catheters, ventilators), and conditions (HIV) or treatments (cancer chemotherapy or drugs that prevent transplant rejection) that affect immune defences. Growing numbers of patients fall into these categories. GNB are a particular treatment problem as their cell structure prevents many antibiotics that kill other types of bacteria from reaching their targets; efforts to discover new antibiotics effective against GNB have been largely unsuccessful.Until recently, carbapenems were regarded as "last resort" drugs for infections by GNB unresponsive to other treatments. However, growing resistance to other antibiotics makes carbapenems increasingly a first choice when infection by a GNB is suspected. When carbapenems fail alternatives are limited and often toxic, hence carbapenem resistance is regarded as a major public health challenge. In GNB carbapenem resistance is largely due to proteins called carbapenemases that bind to and degrade carbapenems, removing their ability to kill bacteria. Carbapenemases are part of a larger group of proteins (beta-lactamases) that destroy other types of BL antibiotics, however most beta-lactamases cannot break down carbapenems. beta-lactamases can be countered by a second group of drugs (beta-lactamase inhibitors) that block their activity and enable BL antibiotics to be used to treat bacteria carrying beta-lactamases, but not all beta-lactamases can be blocked by this route and some can mutate or evolve to escape the action of inhibitors.This proposal investigates how one carbapememase from the GNB Klebsiella pneumoniae, KPC (Klebsiella pneumoniae carbapenemase) degrades carbapenems and other BL antibiotics, and interacts with one specific class of inhibitors (diazabicyclooctanes, DBOs) and how these activities are affected by mutations in KPC. Klebsiella pneumoniae is an important cause of infections (urinary and respiratory infections, sepsis) associated with healthcare, and KPC is one of the main causes of carbapenem resistance worldwide.We recently described, for the first time, how KPC binds carbapenems and other BLs (ceftazidime, an antibiotic used for healthcare-associated infections) during a key stage in their breakdown; and how KPC binds DBOs. Based on this information we will use state-of-the-art computational methods to construct detailed models of the reaction of KPC with each of these three classes of molecules, in order to identify the most likely route by which each reaction occurs. The accuracy of these models will be tested by comparing the speed predicted for each reaction with actual values measured in experiments for a range of antibiotics used in patient treatment. We will then investigate how these reactions are affected by specific alterations in KPC, seeking to understand how such changes now identified in bacteria from human patients can improve the ability of KPC to break down antibiotics and reduce the ability of DBOs to block KPC action. Finally we will use this information to design and test, in computer models and experiments, new carbapenems that resist breakdown by KPC and new DBOs that are more effective KPC inhibitors; and that in each case are not affected by KPC mutations. This provides a route by which understanding of KPC can be exploited to design new treatments effective against an important group of antibiotic resistant bacteria.