Disarming carbapenemase-producing <i>Acinetobacter baumannii</i> : high potency of the novel therapeutic combination of meropenem and the innovative diazabicyclooctane β-lactamase inhibitor pilabactam (formerly ANT3310)

Carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAB) represents an urgent global health threat, with resistance primarily driven by carbapenem-hydrolyzing class D β-lactamases (CHDLs) such as OXA-23. Therapeutic options remain limited due to the scarcity of effective β-lactam/β-lactamase inhibitor combinations. Pilabactam (formerly ANT3310) is a novel diazabicyclooctane (DBO) β-lactamase inhibitor featuring a fluorine substituent that extends its activity spectrum, relative to approved DBOs like avibactam and relebactam, to include CHDLs. Pilabactam is currently in phase I clinical trials in combination with meropenem, and its activity and mechanism against CRAB remain incompletely defined. Using engineered <i>A. baumannii</i> strains producing individual β-lactamases, we show that pilabactam restores meropenem activity against serine β-lactamase producers, including difficult-to-inhibit CHDLs. This was corroborated in 68 whole-genome-sequenced meropenem-resistant clinical isolates, yielding MIC₅₀ and MIC₉₀ values for meropenem/pilabactam of 1 and 2 mg/L, respectively. Frequency of resistance studies in representative CHDL producers demonstrated suppression of resistance selection at 4× MIC. Kinetic analyses revealed that pilabactam inhibits OXA-23 via a two-step tight binding mechanism, with slightly higher inactivation rates (1.7 × 10⁴ M⁻¹s⁻¹) than that of durlobactam (3.5 × 10³ M⁻¹s⁻¹). Pilabactam also yielded a low dissociation constant (<i>K_d</i> ≈ 4 nM) and slow off-rate, indicating durable inhibition. Molecular dynamics simulations revealed the critical role of the fluorine substituent in forming stabilizing hydrogen-bonding and CH-F interactions within the tunnel-like OXA-23 active site. These findings identify pilabactam as a potent novel DBO supporting its development with meropenem for treating CRAB infections.