Mechanistic Analysis of 5-Hydroxy γ-Pyrones as Michael Acceptor Prodrugs

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Substituted 5-hydroxy γ-pyrones have shown promise as covalent inhibitor leads against cysteine proteases and transcription factors, but their hydrolytic instability has hindered optimization efforts. Previous mechanistic proposals have suggested that these molecules function as Michael acceptor prodrugs, releasing a leaving group to generate an o-quinone methide-like structure. Addition to this electrophile of either water or an active site cysteine was purported to lead to inhibitor hydrolysis or enzyme inhibition, respectively. Through the use of kinetic nuclear magnetic resonance experiments, Hammett analysis, kinetic isotope effect studies, and density functional theory calculations, our findings suggest that enzyme inhibition and hydrolysis proceed by distinct pathways and are differentially influenced by substituent electronics. This mechanistic revision helps enable a more rational optimization for this class of promising compounds.