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

The global spread of β-lactamase–mediated resistance poses a severe threat to β-lactam antibiotics. Boronic acid derivatives have emerged as a promising class of reversible covalent inhibitors; however, the molecular determinants governing their recognition and dissociation remains poorly understood. Using Pseudomonas-derived cephalosporinase-3 (PDC-3) as a model, we combined enhanced sampling simulations with machine learning self-organizing maps to investigate the binding and unbinding dynamics of the potent boronic acid inhibitor LP06. Our analysis reveals three distinct binding pathways driven by hydrophobic recognition motifs and a conserved arginine anchor (R349), which collectively guide the ligand toward the pre-covalent state. Unexpectedly, hydrogen-bonding interactions were found to stabilize non-reactive conformations, delaying productive binding. Site-directed mutagenesis of R349, supported by steady-state kinetic assays across varying pH, confirmed its critical role in efficient catalysis. Comparative sequence and structural analyses of >6600 serine β-lactamases, demonstrate that this hydrophobic recognition and arginine anchoring mechanism is broadly conserved. These findings provide mechanistic insights into β-lactamase inhibition and establish design principles for next-generation inhibitors targeting β-lactamase inhibitors.