Bifunctional inhibitors of β-lactamases and ferroptosis: Design, synthesis and anti-gram-negative bacterial infection evaluation.

The spread of MDR-GNB producing both SBLs and MBLs has created an urgent unmet clinical need, as current therapies fail to address both enzyme-mediated resistance and infection-induced inflammation. Ebselen is a selenium-containing heterocyclic compound and has been identified as a novel covalent inhibitor of MBLs. Guided by structural optimization and SAR analysis, we introduced a boronic acid group as a covalent warhead targeting SBLs and finally obtained E3f. E3f exhibited potent inhibitory activity against NDM-1 (MBLs, IC50 = 1.8 μM) and KPC-2 (SBLs, IC50 = 2.6 μM) by forming covalent adducts with Cys221 of NDM-1 and Ser70 of KPC-2, respectively. In vitro antibacterial assays revealed that E3f exerts synergistic effects with antibiotics and restores the susceptibility of clinically isolated MDR-GNB to antibiotics. The MIC was reduced to as low as 1/128 of the original value. In the murine intra-abdominal infection model, combined administration of E3f and meropenem markedly reduced bacterial loads in the liver and spleen compared with meropenem monotherapy. Meanwhile, the serum levels of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 were decreased, indicating excellent in vivo efficacy. Acute toxicity tests demonstrated that E3f presents low toxicity and favorable overall safety in both Galleria mellonella larvae and mice. Furthermore, E3f effectively mitigated LPS-induced acute lung injury in mice by inhibiting ferroptosis, as evidenced by elevated FTH1 and GPX4 expression and reduced MDA levels in lung tissues. Collectively, these results confirm that E3f is a highly efficacious lead compound. Ebselen derivatives, which dually inhibit β-lactamases and produce ferroptosis-mediated anti-inflammatory activity, hold great promise as therapeutic agents against infections caused by MDR-GNB.