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

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.