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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• The rise of beta-lactamase-mediated resistance threatens the efficacy of beta-lactam antibiotics.
• Boron-based inhibitors (BLIs) show promise for reversible covalent inhibition of beta-lactamases.
• The molecular mechanisms of BLI binding and dissociation are not fully understood.

Purpose of the Study:

• To investigate the binding and unbinding dynamics of the boronate BLI, LP06, using Pseudomonas-derived cephalosporinase-3 (PDC-3) as a model.
• To elucidate the molecular basis of recognition and dissociation for boron-based beta-lactamase inhibitors.
• To identify conserved structural determinants for designing next-generation BLIs.

Main Methods:

• Utilized enhanced sampling strategies combined with machine learning.
• Performed steady-state kinetic assays.
• Conducted sequence alignment of approximately 7000 class C beta-lactamases and structural analyses.

Main Results:

• Identified three distinct binding pathways for LP06, driven by hydrophobic motifs and a conserved arginine anchor.
• Found that a conserved arginine (R349) acts as a key anchoring point across serine beta-lactamases.
• Observed that hydrogen bonds can stabilize nonproductive conformations, delaying productive binding.

Conclusions:

• Provided fundamental insights into the inhibition mechanisms of beta-lactamases by boron-based inhibitors.
• Established design principles for developing more effective next-generation beta-lactamase inhibitors.
• Highlighted the importance of conserved structural features and specific interactions in BLI efficacy.