Electrostatic interactions define nacubactam potency against OXA-48-like β-lactamases

Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, severely challenge treatment of multi-drug resistant bacterial infections. OXA-48 is among the most widely disseminated carbapenemases in Enterobacterales , consequently new treatment options for OXA-48 producers are urgently required. Development of diazabicyclooctane (DBO) inhibitors to overcome β-lactamase-mediated resistance is one attractive option, due to their efficacy against a wide range of β-lactamases. The DBO avibactam is already licensed for clinical use, with the related compound nacubactam currently in phase III trials for carbapenem-resistant infections. Here we investigate the activities of avibactam and nacubactam towards OXA-48 and two variants, OXA-163 and OXA-405, that contain deletions in the β5 - β6 loop adjacent to the active site and show modified activity towards different β-lactam classes. Compared to avibactam, nacubactam is c. 80-fold less potent towards OXA-48, but this difference is reduced in OXA-163 and OXA-405. Crystal structures of the respective avibactam and nacubactam complexes, and molecular dynamics simulations based upon these, reveal residue Arg214 on the OXA-48 β5 - β6 active-site loop to be electrostatically repelled by nacubactam, but not avibactam, binding. This increases flexibility of the OXA-48 β5 – β6 loop, as well as neighbouring active site loops, in simulations of the OXA-48:nacubactam, compared to the avibactam, complex. Such effects are not observed in simulations of the respective complexes of OXA-163 and OXA-405, which lack Arg214. These data indicate that interactions with Arg214 can determine DBO potency towards OXA-48-like enzymes, and suggest that sequence variation in this β-lactamase family affects reactivity towards inhibitors as well as β-lactam substrates.