Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors
SBL inhibitors are drugs used in combination with ß-lactam antibiotics to overcome antimicrobial resistance (AMR). A new study from the IOI clarifies how SBL inhibitors work and paves the way for the development of new and improved drugs to counteract AMR.
Antibiotics are used to control a wide range of bacterial infections, yet their utility is threatened by the spread of antimicrobial resistance (AMR), which has been declared by the World Health Organization (WHO) as one of the top 10 global public health threats facing humanity. For example, multiple bacteria have developed ways to degrade ß-lactams, the most widely used class of antibiotics that includes penicillins. One strategy used by bacteria to destroy these antibiotics involves enzymes called serine ß-lactamases (SBLs), which are capable of hydrolyzing (i.e. breaking up) the ß-lactams. To prevent this, clinical treatment with ß-lactams can be combined with the use of SBL inhibitors, which are drugs based on modified penicillins (or closely related molecules) that bind to the bacterial SBLs and prevent them from degrading the antibiotic. However, exactly how these SBL inhibitors work is still being elucidated. Now, a study led by scientists from the INEOS Oxford Institute (IOI) for Antimicrobial Research at the University of Oxford offers novel insights into how such clinically used SBL inhibitors work, providing new information that may be explored to developed even more effective ways to counteract AMR.
In the study, published in The Proceedings of the National Academy of Sciences (PNAS), the authors start by trying to understand how a new SBL inhibitor that has recently completed clinical trials, called enmetazobactam, interacts with SBLs that are present in different bacteria responsible for human infections. To do this, they used a range of methods to characterize how the inhibitor binds to its target SBL, including mass spectrometry, a technique that enables researchers to identify modifications that drugs induce on proteins. Interestingly, they found that enmetazobactam binds to a range of SBLs from different bacteria and remains intact upon binding to its target, which contrasted with some previous studies that proposed that SBL inhibitors are fragmented upon binding. Intrigued by this finding, the authors then studied two other SBL inhibitors widely used in the clinic, tazobactam and sulbactam, showing that these drugs also mainly remain intact upon binding to their target SBL. To better understand why SBL inhibitors showed fragmentation in some studies but not others, the authors further explored how SBLs and their inhibitors interact under different conditions and using distinct laboratory techniques. These experiments revealed that the various methods used across studies can impact on whether SBL inhibitors are fragmented after binding to their target, with more conventional techniques (such as liquid chromatography–mass spectrometry, or LC-MS analyses) leading to fragmentation, whereas SBL inhibitors remained mostly intact when analyzed with spectroscopic methods and state of the art mass spectrometry techniques. Importantly, these results not only explain the discrepancies observed between previous studies but also clarify how SBL inhibitors work, showing that fragmentation is not essential for their activity.
Pauline Lang, the DPhil student who led the study, stated that:
The work highlights the power of modern techniques, such as cutting edge mass spectrometry, to study biological mechanisms, but also suggests that care must be taken when analysing the complex data they generate.
The researchers then also used another technique, termed X-ray crystallography, to directly visualize how different SBL inhibitors bind to their targets. The structures generated using this technique – including of enmetazobactam bound to AmpCEc (an SBL from antibiotic-resistant E. coli) – showed again no evidence that the inhibitor is fragmented upon binding to the target. Furthermore, the experiments also provided additional information into how the inhibitors inactivate the bacterial SBLs, showing that upon prolonged interaction, the inhibitors can sometimes react to form a cross link in the SBL protein, thereby irreversibly altering its structure to leave the SBL unable to degrade antibiotics. Although this reaction is not important for SBL inhibition by the current drugs, this observation might inspire the development of new types of inhibitors that can induce such permanent alterations of the SBL more efficiently and prevent it from conferring resistance to antibiotics.
Professor Christopher Schofield, who supervised the study, said:
It is fascinating that the most important molecules that combat penicillin resistance are themselves modified penicillins or closely related molecules. Our study sheds some light on why this is and suggests avenues for future work.“
Collectively, the findings of this study provide new information about how some of the most important SBL inhibitors used in the clinic work, including by clarifying that fragmentation of these compounds is not essential for their activity. The results also suggest how new types of SBL inhibitors may be designed, leading to improved drugs that irreversibly inactivate SBLs to further counteract the spread of AMR.
Read the full paper: Lang PA, Raj R, Tumber A, Lohans CT, Rabe P, Robinson CV, Brem J, Schofield CJ. Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors. Proc Natl Acad Sci U S A. 2022;119(18) Available from: https://www.pnas.org/doi/10.1073/pnas.2117310119