Discovery of Boronic Acids-Based β-Lactamase Inhibitors Through In Situ Click Chemistry

In this study, we evaluated in situ click chemistry as a platform for discovering boronic acid-based beta-lactamase inhibitors (BLIs). Unlike conventional drug discovery approaches requiring multi-step synthesis, protection strategies, and extensive screening, the in situ method can allow for the generation and identification of potent beta-lactamase inhibitors in a rapid, economic, and efficient way. Using KPC-2 (class A carbapenemase) and AmpC (class C cephalosporinase) as templates, we demonstrated their ability to catalyse azide-alkyne cycloaddition, facilitating the formation of triazole-based beta-lactamase inhibitors. Initial screening of various beta-lactamases and boronic warheads identified compound 3 (3-azidomethylphenyl boronic acid) as the most effective scaffold for kinetic target-guided synthesis (KTGS). KTGS experiments with AmpC and KPC-2 yielded triazole inhibitors with Ki values as low as 140 nM (compound 10a, AmpC) and 730 nM (compound 5, KPC-2). Competitive inhibition studies confirmed triazole formation within the active site, while an LC-MS analysis verified that the reversible covalent interaction of boronic acids did not affect detection of the in situ-synthesised product. While KTGS successfully identified potent inhibitors, limitations in amplification coefficients and spatial constraints highlight the need for optimised warhead designs. This study validates KTGS as a promising strategy for BLI discovery and provides insights for further refinement in fighting beta-lactamase-mediated antibiotic resistance.