Crystallographic Study Reveals a Cryptic Allosteric Site of FBPase by Sulfonylurea Inhibitors toward T2DM Treatment

Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme and an attractive target for type 2 diabetes mellitus (T2DM); however, clinical progress has been limited by known inhibitor binding sites. Here, we report the cocrystal structure of human FBPase (<i>Hu</i>FBPase) bound to a sulfonylurea-based inhibitor (compound <b>5</b>, IC₅₀ = 2.93 μM) at a previously unrecognized cryptic allosteric pocket. Compound <b>5</b> engages this site via π-π stacking with Y57 and extensive hydrogen-bond interactions, induces conformational rearrangement of K72 and D74, and facilitates the formation of a hydrogen-bond network with S123 that disrupts substrate catalysis. Structure-guided optimization yielded compound <b>29</b> with improved potency (IC₅₀ = 0.75 μM). Cellular target engagement was confirmed by thermal stabilization of FBPase in LO2 cells. Notably, compound 29 exhibited hypoglycemic activity <i>in vivo</i>, providing the first evidence that targeting this pocket confers therapeutic benefit. Collectively, our findings establish a novel, druggable allosteric site on <i>Hu</i>FBPase for T2DM treatment.