Computational prediction and experimental validation of the activator function of 2C-β-d-glucopyranosyl-1，3，6，7-tetrahydroxyxanthone on pancreatic and hepatic hexokinase.

This study identifies and validates hexokinase type 4 (HK4)， an isozyme of hexokinase in the liver and pancreas， as an important target of 2C-β-d-glucopyranosyl-1，3，6，7-tetrahydroxyxanthone (βdGT)， a xanthone glucoside suggested to have antidiabetic property. In the study， we applied the computational pipeline of molecular docking followed by the molecular dynamics simulations to shortlist potential βdGT protein targets. The analysis of protein dynamics and the binding free energy (ΔG) led us to the identification of HK4 as a key βdGT target; whereby the binding mode and domain dynamics suggested the activator function of βdGT. βdGT bound to the allosteric site of the isozyme ∼13Å away from the substrate (glucose)-binding site. The binding free energy of the ligand-protein complex was energetically feasible (ΔG， -41.61 kcal/mol) and the cleft angle deviation between the two (small and large) domains of HK4 revealed differential HK4 dynamics in response to βdGT binding. 3D Structure analysis of the isozyme-ligand complex highlighted the role of Arg63， Glu67 and Lys458 in ligand stabilization and hydrophobic interactions mediated by Tyr214 and Met235. Experimental validation of the results of computational analysis confirmed the activator function of βdGT on HK4. The study has implication in diabetes as βdGT may be used to lower the blood glucose level by activating hepatic and pancreatic hexokinase without the risk of hypoglycemia.