Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG pathway. In our previous study, the C128 site of FBPase has been identified as a new allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity. Herein, a series of nitrostyrene derivatives were further synthesized, and their inhibitory activities against FBPase were investigated in vitro. Most of the prepared nitrostyrene compounds exhibit potent FBPase inhibition (IC < 10 μM). Specifically, when the substituents of F, Cl, OCH, CF, OH, COOH, or 2-nitrovinyl were installed at the R (meta-) position of the benzene ring, the FBPase inhibitory activities of the resulting compounds increased 4.5-55 folds compared to those compounds with the same groups at the R (para-) position. In addition, the preferred substituents at the R position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory activity (IC = 0.15 μM). The molecular docking and site-directed mutation suggest that C128 and N125 are essential for the binding of HS36 and FBPase, which is consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction enthalpy calculations show that the order of the reactions of compounds with thiol groups at the R position is Cl > H > CH. CoMSIA analysis is consistent with our proposed binding mode. The effect of compounds HS12 and HS36 on glucose production in primary mouse hepatocytes were further evaluated, showing that the inhibition was 71% and 41% at 100 μM, respectively.