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 (HuFBPase) bound to a sulfonylurea-based inhibitor (compound 5, IC50 = 2.93 μM) at a previously unrecognized cryptic
allosteric pocket. Compound 5 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 29 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 in vivo, providing the first evidence that targeting this
pocket confers therapeutic benefit. Collectively, our findings establish
a novel, druggable allosteric site on HuFBPase for
T2DM treatment.