How Clavulanic Acid Inhibits Serine β‐Lactamases

• Published in: Chembiochem : a European journal of chemical biology 2024-11, Vol.25 (22), p.e202400280-n/a
• Main Authors: Lang, Pauline A., Munnik, Mariska, Oluwole, Abraham O., Claridge, Timothy D. W., Robinson, Carol V., Brem, Jürgen, Schofield, Christopher J.
• Format: Article
• Language: English
• Subjects: Acids; Antimicrobial resistance; beta-Lactamase Inhibitors - chemical synthesis; beta-Lactamase Inhibitors - chemistry; beta-Lactamase Inhibitors - pharmacology; beta-Lactamases - chemistry; beta-Lactamases - metabolism; Clavulanic acid; Clavulanic Acid - chemistry; Clavulanic Acid - pharmacology; Complex formation; E coli; Escherichia coli - drug effects; Escherichia coli - enzymology; Fragmentation; In vivo methods and tests; Mass spectrometry; Mass spectroscopy; Mechanism-based inhibition; Oxazolidine; Penam sulfone; Ring opening; Scaffolds; Serine; Serine - chemistry; Serine - metabolism; Serine β-lactamase inhibitor; Sulfones; β Lactamase
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202400280

Clavulanic acid is a medicinally important inhibitor of serine β‐lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM‐116), C (Escherichia coli AmpC), and D (OXA‐10) SBLs using denaturing and non‐denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC–MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM‐116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest. Inhibition of serine β‐lactamases by clavulanic acid occurs via a bifurcating mechanism. The formation of an acyl‐enzyme complex may be followed by hydrolysis, or oxazolidine ring opening and subsequent decarboxylation to give more stable species. Further fragmentation is slow and is less likely to be biologically relevant in inhibition.