Structure-Guided Design of AMP Mimics That Inhibit\nFructose-1,6-bisphosphatase with High Affinity and Specificity

AMP binding sites are commonly used by nature for allosteric regulation of enzymes controlling\nthe production and metabolism of carbohydrates and lipids. Since many of these enzymes represent potential\ndrug targets for metabolic diseases, efforts were initiated to discover AMP mimics that bind to AMP-binding\nsites with high affinity and high enzyme specificity. Herein we report the structure-guided design of potent\nfructose 1,6-bisphosphatase (FBPase) inhibitors that interact with the AMP binding site on FBPase despite\ntheir structural dissimilarity to AMP. Molecular modeling, free-energy perturbation calculations, X-ray\ncrystallography, and enzyme kinetic data guided our redesign of AMP, which began by replacing the 5‘-phosphate with a phosphonic acid attached to C8 of the adenine base via a 3-atom spacer. Additional\nbinding affinity was gained by replacing the ribose with an alkyl group that formed van der Waals interactions\nwith a hydrophobic region within the AMP binding site and by replacing the purine nitrogens N1 and N3\nwith carbons to minimize desolvation energy expenditures. The resulting benzimidazole phosphonic acid,\n<b>16</b>, inhibited human FBPase (IC₅₀ = 90 nM) 11-fold more potently than AMP and exhibited high specificity\nfor the AMP binding site on FBPase. <b>16</b> also inhibited FBPase in primary rat hepatocytes and correspondingly\nresulted in concentration-dependent inhibition of the gluconeogenesis pathway. Accordingly, these results\nsuggest that the AMP site of FBPase may represent a potential drug target for reducing the excessive\nglucose produced by the gluconeogenesis pathway in patients with type 2 diabetes.