Structural and Kinetic Basis for the Rational Design of Next-Generation β-Lactamase Inhibitors.

The global spread of β-lactamase-mediated resistance poses a threat to β-lactam antibiotics. Boron-based β-lactamase inhibitors (BLIs) represent a promising class of reversible covalent inhibitors, yet the molecular basis of their recognition and dissociation remains poorly understood. Using Pseudomonas-derived cephalosporinase-3 (PDC-3) as a model, we employed enhanced sampling strategies with machine learning and steady-state kinetic assays to investigate the binding and unbinding dynamics of LP06, a boronate BLI. We identify three binding pathways, governed by hydrophobic recognition motifs and a conserved arginine anchor that together steer the ligand toward the precovalent state. Sequence alignment of nearly 7000 class C β-lactamases supports the conservation of these determinants, and structural analyses suggest that R349 may act as a shared anchoring point across serine β-lactamases. Additionally, hydrogen-bonding interactions were found to delay productive binding by stabilizing nonproductive conformations. Our findings provide fundamental insights into β-lactamase inhibition and establish design principles for next-generation β-lactamase inhibitors.