Structure Kinetics Relationships and Molecular Dynamics Show Crucial Role for Heterocycle Leaving Group in Irreversible Diacylglycerol Lipase Inhibitors

Drug discovery programs of covalent
irreversible, mechanism-based
enzyme inhibitors often focus on optimization of potency as determined
by IC50-values in biochemical assays. These assays do not
allow the characterization of the binding activity (Ki) and reactivity (kinact)
as individual kinetic parameters of the covalent inhibitors. Here,
we report the development of a kinetic substrate assay to study the
influence of the acidity (pKa) of heterocyclic
leaving group of triazole urea derivatives as diacylglycerol lipase
(DAGL)-α inhibitors. Surprisingly, we found that the reactivity
of the inhibitors did not correlate with the pKa of the leaving group, whereas the position of the nitrogen
atoms in the heterocyclic core determined to a large extent the binding
activity of the inhibitor. This finding was confirmed and clarified
by molecular dynamics simulations on the covalently bound Michaelis–Menten
complex. A deeper understanding of the binding properties of covalent
serine hydrolase inhibitors is expected to aid in the discovery and
development of more selective covalent inhibitors.