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.