Structure–activity relationshipinvestigations conductedat the 5-position of the N-pyridine ring of a seriesof N-arylsulfonyl-N′-2-pyridinyl-piperazinesled to the identification of a novel bis-pyridinyl piperazine sulfonamide(51) that was a potent disruptor of the glucokinase–glucokinaseregulatory protein (GK–GKRP) interaction. Analysis of the X-raycocrystal of compound 51bound to hGKRP revealed thatthe 3-pyridine ring moiety occupied a previously unexplored bindingpocket within the protein. Key features of this new binding mode includedforming favorable contacts with the top face of the Ala27-Val28-Pro29(“shelf region”) as well as an edge-to-face interactionwith the Tyr24 side chain. Compound 51was potent inboth biochemical and cellular assays (IC50= 0.005 μMand EC50= 0.205 μM, respectively) and exhibitedacceptable pharmacokinetic properties for in vivo evaluation. Whenadministered to db/dbmice (100 mg/kg, po), compound 51demonstrated a robust pharmacodynamic effect and significantlyreduced blood glucose levels up to 6 h postdose. [ABSTRACT FROM AUTHOR]