The Covalent Allosteric Regulation Mechanism Of Disulfiram And Its Derivatives On Hu-FBPase

Diabetes is a global health problem that has been characterised as one of the largest epidemics in history.Type II diabetes mellitus(T2DM),which accounts for approximately 86%of all diabetes cases,is characterized by hyperglycemia due to defective insulin secretion or impaired insulin action.Human liver fructose-1,6-bisphosphatase(Hu-FBPase)is a rate-controlling enzyme of gluconeogenesis.Its role in endogenous glucose overproduction has been widely recognized and has become an important target for the treatment of type II diabetes.Hu-FBPase is a homologous tetramer with a substrate binding site and an AMP allosteric site on each monomer.Currently,many of the inhibitors of Hu-FBPase are currently designed for AMP allosteric sites,the acting mode is reversible and non-covalent,so the effective duration of inhibition effect is short.Moreover,most of these inhibitors are AMP analogues,and AMP is widely involved in important physiological regulation of human body.Therefore,inhibitors acting on AMP allosteric sites are often accompanied by poor selectivity and large toxic and side effects.However,it takes a high cost and a long time to develop a new drug acting on a new site,so we adopted the strategy of drug repurposing to design a new hypoglycemic drug targeting the new site of Hu-FBPase.This paper mainly carried out the following research work:1.The inhibitory activity of disulfiram against Hu-FBPase was tested,and it was found that disulfiram could inhibit Hu-FBPase with IC50=2.65±0.31?M.It is a time dependent inhibitor of Hu-FBPase and has synergistic relationship with the substrate FBP and competitive relationship with Mg2+.And it has obvious hypoglycemic effect on primary liver cells and ICR mice.2.Based on the design and synthesis of Li Jian's group,the inhibitory activity of 70 disulfide derivatives on Hu-FBPase were tested.The acting site and inhibition mechanism are the same as that of disulfiram:they bound to Cys128 covalently and have time-dependent inhibitory activity on Hu-FBPase.They have obvious hypoglycemic effect on primary liver cells of rats and ICR mice.3.By means of crystallography to explore the inhibition mechanism of disulfiram derivatives to Hu-FBPase,we obtained the complex crystal of Hu-FBPase and 71b with diffraction rate of 2 A by soaking.The crystal structure shows the covalent binding inhibition mechanism of disulfide compounds to Hu-FBPase Cysl28 more intuitively,which makes helix nearby Cys128 changes greatly,the residue of S123,which is closely related to Hu-FBPase catalytic activity,is deflected away from the substrate and forms hydrogen bonds with D74 and K71 and water molecules,thus reduce the catalytic activity of Hu-FBPase.