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

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.

Beta-lactams are the most prescribed antibiotics; carbapenems are the most potent beta-lactams and key drugs for infections by opportunistic Gram-negative bacteria. Carbapenem resistance in Enterobacteriaceae due to beta-lactamase production is a major public health challenge. The Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase is distributed worldwide and is the major cause of carbapenem resistance in this organism. KPC efficiently degrades carbapenems, which inhibit the majority of related enzymes, and slowly degrades inhibitors (diazabicyclooctanes, DBOs). The basis of these activities remain poorly understood. Here we combine molecular simulations (molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) based on recent crystal structures from our group) with structural and kinetic experiments to investigate interactions of KPC with substrates (carbapenems and the cephalosporin ceftazidime) and DBO inhibitors, aiming to identify determinants of activity against each of these ligand classes.

QM/MM simulations of carbapenem breakdown will focus on degradation of the acylenzyme intermediate, aiming to establish the relationship between carbapenem turnover, interactions of the deacylating water molecule, and tautomerization of the carbapenem pyrroline ring. Extended MD and QM/MM simulations will investigate ceftazidime:KPC interactions, aiming to identify why this is a relatively poor substrate. QM/MM simulations will identify pathways by which the DBO acylenzyme is either recycled to generate intact active inhibitor or desulphated and inactivated. In each case we will investigate how these activities are affected by point variants of KPC identified in the clinic. The results will establish the basis for the unique properties of KPC enzymes and their capacity for mutation, enabling identification and evaluation of routes to carbapenems and DBOs with enhanced activity against bacteria carrying this key resistance determinant.

The project investigates the basis for activity of the Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase against substrates- carbapenems and the cephalosporin ceftazidime, and inhibitors- diazabicyclooctanes (DBO) such as avibactam. We further seek to understand how variants of the KPC enzyme carrying point mutations differ in their reactivity towards these different ligands, and to test methods of applying this new knowledge to identify iterations of the carbapenem and DBO scaffolds resistant to KPC-mediated degradation.

This work has potential commercial (biotechnology and pharmaceutical) impact. Specific materials we identify (new carbapenem antibiotics or KPC inhibitors) may either themselves be suitable for development or may represent and validate approaches to molecules that are. Our data (experimental crystal structures of KPC complexes with beta-lactams and DBOs, as well as models of such complexes from simulations, and reaction mechanisms) will, as new information on KPC molecular recognition, be applicable in others' studies aimed at combatting KPC. Synthetic approaches to C2 substituted carbapenems will aid those seeking to generate such materials, whilst application of high-level (quantum mechanics/molecular mechanics) simulations to evaluate compounds in silico should be of general interest the wider drug discovery community. Although antibiotic development is a long-term process (10+ years), impacts should begin as soon as our results are made available through publication or presentation. Aside from the public health impact (see below) economic impact of our findings would stem from the investment and employment generated by antibiotic development activities as well as revenue from any product reaching market.

Public health benefits will accrue from improved understanding of KPC-mediated resistance, that may result in more effective antibiotic selection and use for patients infected with KPC-producing bacteria. Identifying specific resistance phenotypes conferred by individual KPC variants will guide prescribing when this information is available- increasingly the case as genomic or nucleic acid based methods reach the clinic. Use of appropriate antibiotics (ie selected using knowledge-based prescribing) will improve outcomes for secondary care patients with resistant infections. Such impact may occur within 5 years as technology capable of identifying specific KPC variants is adopted. In the longer term (10+ years) clinical impact may also arise as new beta-lactams and DBOs, whose development has involved application of our findings, enter medical use. The economic impact of better treatments for infection will be felt both within the NHS, where resources will be freed as patient outcomes improve, and more widely as recovered patients, their family members and social circle engaged in their care and support, regain their ability to contribute economically. Societal benefits will also accrue from renewed social participation of these individuals; and increased public awareness of antibiotic resistance and routes to overcoming it; and increased awareness among policymakers of the existence of such strategies and routes and barriers to their implementation.

Postdoctoral staff will gain a range of specific skills applicable in the bioscience and technology sectors. The interdisciplinarity of this work requires all of those involved to be literate in antimicrobial resistance microbiology, KPC structural biology and computational chemistry. This broad skill set will be applicable across many projects seeking small molecule ligands of protein targets, making these researchers employable in a wide range of drug discovery projects. Researchers will also acquire training and experience in a range of transferable skills including written, visual and oral presentations, team working and supervision (typically of undergraduate students) that equip them for a wide range of professional environments.