Zwitterionic Penicillin-Derived Sulfone Inhibitor for Combating β-Lactamase-Mediated Antibiotic Resistance.

β-Lactam antibiotics, which account for nearly 70% of all prescriptions for bacterial infections, remain the cornerstone of antimicrobial chemotherapy. However, their clinical effectiveness is increasingly compromised by the widespread emergence of β-lactamase-mediated resistance. These enzymes hydrolyze the β-lactam ring through an addition-elimination mechanism, abolishing antibacterial activity. Preserving the efficacy of β-lactam antibiotics, particularly carbapenems, regarded as the last line of defense against multidrug-resistant (MDR) pathogens, is therefore critical. Here, we report the design and synthesis of compound 3, a zwitterionic penicillin-derived sulfone that integrates two key principles: enhanced permeability of zwitterionic molecules and improved inhibitory potency through (2-pyridyl)methylene introduction within the sulbactam scaffold to achieve an adduct stable against hydrolysis. In vitro assays demonstrated that compound 3 significantly restored β-lactam activity against MDR pathogens producing extended-spectrum β-lactamases (ESBLs) and carbapenem-hydrolyzing class D β-lactamases. It notably improved imipenem and ceftazidime efficacy against strains expressing OXA-48 and PDC-1, whose inhibitory capacity was determined by kinetic analysis with the isolated enzymes. Mechanistic studies using mass spectrometry and molecular dynamics simulations revealed ligand-induced pocket formation, π-stacking modulation, and hydrophobic engagement, supporting a covalent, water-shielded inactivation pathway via indolizine adduct formation. The nonsterically hindered amine in 3 favored PDC-1 binding and overall permeability while exerting a neutral effect on OXA-48 binding. These results underscore the potential of compound 3 as a next-generation β-lactamase inhibitor for combating infections caused by ESBL- and carbapenemase-producing Enterobacterales and Pseudomonas aeruginosa.