Mechanistic diversity, post-translational carbamylation, and inhibitor susceptibility in the OXA beta-lactamase family
Lead Research Organisation:
University of Bristol
Department Name: Cellular and Molecular Medicine
Abstract
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.
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.
Technical Summary
Beta-lactams are the dominant antibiotics, making up over half of prescriptions and sales. In Gram-negative bacteria (GNB) most beta-lactam resistance is due to beta-lactamases, that hydrolyse the beta-lactam ring. Of 4 beta-lactamase classes, OXA (class D) enzymes are uniquely distinguished by use of a carbamylated lysine general base for both acylation and deacylation reactions; insusceptibility to many inhibitors; and activity of some family members against carbapenems, the most potent beta-lactams and key antibiotics for GNB infections. OXA-48 is the dominant carbapenemase in U.K. Enterobacterales, while OXA-23 and OXA-24/40 cause transmissible carbapenem resistance in Acinetobacter baumannii.
We propose to explore the relationship between enzyme architecture and reaction chemistry in the OXA beta-lactamases. Preliminary studies demonstrate that lysine carbamylation can be monitored by 13C- and 19F-NMR, and that complexes with diazabicyclooctanone (DBO) and boronate inhibitors differ in the degree of carbamylation. Furthermore, we have recently shown multiple OXAs can rearrange carbapenems to give beta-lactones, in addition to well-established hydrolysis products. Based on these findings, we will investigate the relationships between active site structure/lysine carbamylation; and carbapenem turnover/lactone formation/inhibitor susceptibility; in three clinically relevant OXA groups. We will use solution (enzyme kinetics, NMR, mass spectrometry) and crystallographic (including room-temperature fixed-target experiments on microcrystal slurries) approaches, and include natural variants differing in spectrum of activity. We will synthesise new carbapenems and lactones to explore the effects of substitution on OXA interactions, and test our conclusions by evaluating mutants and variants. The mechanistic results will identify determinants of carbapenemase activity, inhibitor susceptibility, and lactone formation, and identify new types of inhibitor templates.
We propose to explore the relationship between enzyme architecture and reaction chemistry in the OXA beta-lactamases. Preliminary studies demonstrate that lysine carbamylation can be monitored by 13C- and 19F-NMR, and that complexes with diazabicyclooctanone (DBO) and boronate inhibitors differ in the degree of carbamylation. Furthermore, we have recently shown multiple OXAs can rearrange carbapenems to give beta-lactones, in addition to well-established hydrolysis products. Based on these findings, we will investigate the relationships between active site structure/lysine carbamylation; and carbapenem turnover/lactone formation/inhibitor susceptibility; in three clinically relevant OXA groups. We will use solution (enzyme kinetics, NMR, mass spectrometry) and crystallographic (including room-temperature fixed-target experiments on microcrystal slurries) approaches, and include natural variants differing in spectrum of activity. We will synthesise new carbapenems and lactones to explore the effects of substitution on OXA interactions, and test our conclusions by evaluating mutants and variants. The mechanistic results will identify determinants of carbapenemase activity, inhibitor susceptibility, and lactone formation, and identify new types of inhibitor templates.
