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

## TL;DR

This study explores how boron-based inhibitors bind to β-lactamase enzymes, revealing key molecular interactions that could guide the design of better antibiotics.

## Contribution

The study identifies conserved binding motifs and a shared anchoring point in β-lactamases, offering new design principles for inhibitors.

## Key findings

- Three binding pathways were identified, guided by hydrophobic motifs and a conserved arginine anchor.
- R349 is suggested as a shared anchoring point across serine β-lactamases.
- Hydrogen-bonding interactions delay productive binding by stabilizing nonproductive conformations.

## Abstract

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.