Spiranic compounds as novel AMPK paradoxical inhibitors: can we derive new mechanistic insights?

In this study, we synthesized and fully characterized spiranic compounds designed as conformationally restricted mimetics of A-769662 by means of crystallographic, spectroscopic, and computational methods. In vitro enzymatic assays and surface plasmon resonance experiments revealed a mixed-type inhibition mechanism. We developed a stable and equilibrated α1β1 AMPK model featuring the A-loop in a closed conformation, which was used to investigate alternative binding pockets beyond the canonical AMPK sites, namely type II -like pocket. The cell-based and tissue experiments further demonstrated that these compounds act as paradoxical inhibitors, since it enhances downstream signalling by increasing phosphorylation of ACC and eNOS, ultimately activating the AMPK–eNOS–NO pathway and inducing vasodilation in rat aorta. Altogether, our findings offer new insights into AMPK modulation and highlight the potential of spiranic scaffolds as tools for probing alternative regulatory mechanisms.