Design,Dynthesis,Activity And Mechanism Studies Of Dual Inhibitors Of ?-glucosidase And PTP1B

Objective: Type 2 diabetes mellitus(T2DM)is a series of metabolic disorders syndrome characterized by insulin resistance and/or islet cell failure.T2 DM is often associated with a variety of acute and chronic complications,which has become one of the biggest burden on the Chinese health system.The new treatment for T2 DM is imminent,since the present anti-diabetic drugs used in clinic are not effective to stop the further necrosis of islet ? cells and are associated with multiple side effects.Multi-target drug therapeutics could improve the curative effect and avoid the side effects produced by combination of multiple drugs,which makes great sense for the treatment of T2 DM.?-Glucosidase and protein tyrosine phosphatase 1B(PTP1B)are two key target enzymes associated with T2 DM.If both of ?-glucosidase and PTP1 B are inhibited simultaneously,we can reduce postprandial blood glucose,on one hand would enhance insulin sensitivity and on the other hand would reduce pancreatic ?-cell load and help T2 DM patients reduce their body weight.In this study,novel dual ?-glucosidase/PTP1 B inhibitors were designed,synthesized,assayed for bioactivities and analyzed for their mechanism,hoping to provide new ideas for the development of multi-target hypoglycemic agents.Methods:(1)In the part of drug design,by targeting both ?-glucosidase and PTP1 B,the ZINC small molecule database was screened to find the lead compound using high-throughput virtual screening method.Based on the structure of the active sites of ?-glucosidase and PTP1 B,the lead compound was optimized by the method of Core-hopping,and design a series of compounds as the dual ?-glucosidase/PTP1 B inhibitors.(2)In the part of chemical synthesis,the synthetic routes of the above compounds were designed according to the related literature.(3)In the part of activity test,the recombinant overexpression plasmid containing the target gene PTPN1 was constructed and transferred into competent cells to express PTP1 B protein.The PTP1 B protein was purified by GST fusion protein purification method.The inhibitory activities of the synthetic compounds against both ?-glucosidase and recombinant human PTP1 B were then assayed,and the structure-activity relationship was discussed.(4)In the study of drug-protein mechanism,the mechanism of action between the inhibitor and ?-glucosidase was studied by inhibition kinetics and dialysis test.In addition,the binding mode at the molecular level between the inhibitor and its two targets ?-glucosidase and PTP1 B was investigated respectively by molecular docking method.Results:(1)In the part of drug design,ZINC62431 was found as the lead compound of dual inhibitors of ?-glucosidase/PTP1 B by the method of high-throughput virtual screening.The structure of ZINC62431 was divided into three parts: the catalytic site binder,linker and the second site binder.Finally,we got 32 molecules with high binding energy and reasonable ligand-bound conformation through rationally modifying three parts of the lead compound by Core-hopping method.(2)In the part of chemical synthesis,we employed two step nucleophilic substitution reaction to get the target compounds by referring to the relevant information.The structure of the target compounds was confirmed by 1H NMR,13 C NMR,ESI-MS and HRMS-ESI.Finally,32 target compounds were successfully synthesized.(3)In the part of activity test,the recombinant overexpression plasmid p GEX-4T-1-PTPN1 was successfully constructed and human recombinant PTP1 B protein was purified.Both the recombinant PTP1 B protein and the purchased ?-glucosidase were used as target proteins,and the in vitro inhibitory activity of 32 synthetic compounds was assayed against these two targets.Compound 5j showed the most potent ?-glucosidase inhibitory activity with an IC50 value of 10.11 ?M,which was about 5-fold stronger than that of the positive control acarbose(IC50 = 51.32 ?M).With an IC50 value of 13.46 ?M,the PTP1 B inhibitory activity of 5j was good as well,stronger than its positive control ursolic acid(IC50 = 14.50 ?M).Studies on the structure-activity relationship revealed that,benzoxazole derivatives and benzothiazole derivatives were more beneficial to ?-glucosidase/PTP1 B dual inhibition than corresponding thiazoline derivatives;aromatic primary amines were more favorable to improve dual ?-glucosidase/PTP1 B inhibition than aliphatic primary amines;the introduction of electron-withdrawing substituents improved the inhibitory activity towards ?-glucosidase compared with phenylamine without substituent;extending the linker appropriately was favorable to ?-glucosidase inhibition.(4)In the study of drug-protein mechanism,the kinetic analysis and dialysis experiment revealed compound 5j inhibited ?-glucosidase in a reversible and mixed manner.Molecular docking studies suggested that compound 5j spirally matched well with the active pockets of ?-glucosidase and PTP1 B,and involved respectively in intense interactions(hydrogen bonds,van der Waals,charge interactions and Pi-cation interactions,etc.)with both receptors ?-glucosidase and PTP1 B.Conclusion: In this paper,with the method of computer-aided drug design,a series of compounds with ?-glucosidase/PTP1 B dual-target inhibitory activities was successfully obtained using the inhibitory activities of ?-glucosidase and PTP1B(two therapeutic targets of T2DM)as the detection index.The bioactivity of compound 5j in vitro was better than that of the positive control against ?-glucosidase and PTP1 B,respectively.The inhibition kinetics analysis showed that 5j was a reversible and mixed inhibitor of ?-glucosidase.The study of structure-activity relationship and docking analysis will guide us to perform structural modification of these compounds to further improve their activity and reduce side effects.This study will provide an effective method to develop multi-target therapeutic drugs and provide new ideas to treat T2 DM.