An Empirical Study of Amide-Heteroarene π-Stacking Interactions Using Reversible Inhibitors of a Bacterial Serine Hydrolase

Compared to aryl–aryl π-stacking interactions, the analogous stacking of heteroarenes on amide π systems is less well understood and vastly underutilized in structure-based drug design. Recent theoretical studies have delineated the important geometric coordinates of the interaction, some of which have been confirmed with synthetic model systems based on Rebek imides. Unfortunately, a broadly useful and tractable protein–ligand model system of this interaction has remained elusive. Here we employed a known inhibitor scaffold to study π-stacking of diverse heteroarene substituents on the amide face of Gly238 in the cephalosporinases CTX-M-14 and CTX-M-27. Biochemical inhibition constants (K(i)) and biophysical binding constants (K(d)) were determined for nineteen new analogues against both enzymes, while multiple high-resolution co-crystal structures revealed remarkably consistent placement of the probe heteroarene on Gly238. The data presented support the predicted importance of opposing dipoles in amide-heteroarene interactions and should be useful for evaluating other theoretical predictions concerning these interactions.