Design,Activity Validation And Molecular Mechanism Of Novel DPP-Ⅳ Inhibitory Peptides

Diabetes is one of the major chronic diseases.DPP-Ⅳ enzyme is an effective target in the treatment of diabetes.Inhibition of DPP-Ⅳ can reduce blood glucose.DPP-Ⅳ inhibitors on the market are almost synthetic drugs,which have many side effects on human body.And DPP-Ⅳ inhibitory peptides come from a wide range of sources.They are easy to be absorbed orally with little side effects.DPP-Ⅳ inhibitory peptides have a good effect on the prevention and treatment of diabetes.The number of DPP-Ⅳ inhibitory peptides found is small.Traditional methods to extract DPP-Ⅳ inhibitory peptides from animals,plants and microorganisms cost a lot of money.The methods aren’t purposeful and the efficiency is low.Based on the above problems,computer-aided drug design methods were used to establish accurate DPP-Ⅳ inhibitory peptide models and some novel high-activities DPP-Ⅳ inhibitory peptides are designed.Meanwhile,in vitro activity experiments were used to verify the activities of the designed high-activity peptides.The molecular mechanism of DPP-Ⅳ inhibitors not only reveal the nature of diabetes,but also greatly improve the success rate of designing inhibitors.The mechanism of DPP-Ⅳ inhibitors can be divided into competitive inhibition and non-competitive inhibition.Especially,the non-competitive mechanism remains unclear so far.Based on this,we used molecular docking and molecular dynamics simulations to conduct in-depth studies on the effects of novel high-activity DPP-Ⅳ inhibitory peptides.Comparing the results of DPP-Ⅳ inhibitory peptides to investigated their specific inhibitory mechanisms.In this thesis,six DPP-Ⅳ inhibitory peptides with high activity were screened according to the 3D-QSAR models.The activities of six new peptides were further verified by in vitro activity experiments.At the same time,the mechanism of competitive and non-competitive inhibition of DPP-Ⅳ inhibitory peptides was studied.The thesis can be divided into three parts.In the first part,3D-QSAR models were established based on 50dipeptide and tripeptide databases.A Co MFA model（Q²=0.837,R²=0.991）and a Co MSIA model（Q²=0.726,R²=0.990）were built.According to the two models,six DPP-Ⅳ inhibitory peptides with high activity were designed and screened.The results showed that the established Co MFA model and Co MSIA model had good predictive ability and are very stable.In the second part,the activities of six new peptides were verified by in vitro assay.The IC₅₀ of LPI,MPL,LPT,LPE,WPV and CPW were7.51μM,20.73μM,22.88μM,31.27μM,30.31μM and 27.29μM,respectively.Their activities were higher than that of most DPP-Ⅳ inhibitory peptides reported.And their activities were almost consistent with that predicted activity values by the models.The results verified the stability and high predictive ability of the models.In the third part,molecular docking and molecular dynamics simulation were adopted to explore the molecular mechanism of six novel peptides.The results of competitive inhibitory peptides and non-competitive inhibitory peptide were compared to reveal the mechanism.It was found that Glu-205 and Glu-206 were the key residues to inhibit DPP-Ⅳ activity.Competitive inhibitory peptides bind to substrates in the same way,and they competed with substrates for binding sites to inhibit DPP-Ⅳ activity.However,there were two kinds of inhibitory effects of the non-competitive inhibitory peptides.One was that the peptides bound to DPP-Ⅳ and destroyed the structure of active pocket.The other was that the binding reduced the entrance to the active pocket on the side of the DPP-Ⅳ,which prevented the substrate from entering and thus acts as a depressant.All in all,we established a set of research methods for designing novel DPP-Ⅳ inhibitory peptides,verifying the activity and revealing the molecular mechanism of peptides.The methods combined theory with experiment and provided a new way for the discovery of active peptides.