3D-QSAR,in Vitro Evaluation And Mechanism Of Novel Antioxidant Peptides

The damages induced by oxidative stress are the major consequence of the pathological changes in the body.Masses of illnesses such as heart disease,arteriosclerosis and diabetes are related to oxidation.Due to the safety performance,antioxidant peptides(AOPs)as the ideal supplements for reducing oxidative stress have become the hotspots.However,most antioxidant peptides from natural products need separation,depuration and determination,wasting massive material resources and even time.In addition,conventional studies are concentrated on the free radical scavenging activity of the antioxidative peptides,which ignored the signaling pathways and targets.Thus,an effective way to obtain AOPs and verify their activity is sufficient.The researches of action mechanism of AOPs are required as well.Considering with the problems,we have established reliable 3D-QSAR models based on the Keap1-Nrf2 pathway.According to the models,novel antioxidative peptides were designed and identified in vitro assay.Meanwhile,molecular docking and molecular dynamics simulation were combined to investigate the effect of AOPs on Keap1-Nrf2.A set of complete strategy for investigating AOPs is established by theory and experiments.The thesis is divided into the following four parts:In the first part,a database containing 54 antioxidant tripeptides with tryptophan as the skeleton was collected basing on the TEAC activity.And the accurate CoMFA/CoMSIA models were established by 3D-QSAR method with the target of Keap1.According to the established models,the antioxidative peptides with high predicted TEAC values were screened out.In the second part,the modified TEAC method(also called "ABTS method ")was combined with the data source method to estimate and screen the designed antioxidant tripeptides GWY(Gly-Trp-Tyr),QWY(Gly-TrpTyr)and KWF(Lys-Trp-Phe).Tripeptides GWY and QWY with significant antioxidant activities were screened out successfully.The antioxidant capacities of the two tripeptides were stronger than that of water-soluble vitamin E.It implied that the established models possessed good abilities to predict high-active antioxidant tripeptides.Therefore,the models can be used for designing and screening novel antioxidant peptides as well.In the third part,the two novel tripeptide GWY and QWY with high activities were further evaluated with the APPH-induced oxidative damage of erythrocyte assay.The results exhibited that both GWY and QWY possessed excellent antioxidant capacity.They protected erythrocytes from hemolysis and lipid peroxidation.What’s more,the two peptides can promote the activities of antioxidant enzymes SOD/CAT and up regulate the GSH content in the cells.Potentials of the two novel antioxidant peptides to prevent human erythrocytes from oxidative injury were confirmed.In the fourth part,molecular docking and MD simulations were combined to reveal the action mechanism of antioxidative peptides GWY and QWY on the interaction of Keap1 and Nrf2.The results of molecular docking exhibited that tripeptides GWY and QWY could occupy the important sites of Nrf2.Additionally,the results of molecular dynamics simulations showed that the two antioxidant tripeptides could bind with the key residues of Keap1 firmly through hydrogen bonding,hydrophobicity and van der Waals forces.This indicated that the antioxidant peptides GWY and QWY possessed potential to compete with Nrf2 for binding sites and promote the expression of the antioxidant reaction system in the body.Finally,our project has successfully screened out novel antioxidant tripeptides with excellent activities and established reliable CoMFA/CoMSIA models with 3D-QSAR method.And activities of the novel peptides were confirmed in vitro.Meanwhile,molecular docking and molecular dynamics simulation of the two novel peptides were carried out based on the Keap1-Nrf2 pathway.A set of systematic methods for the design,activity verification and mechanism analysis of novel antioxidant peptides have been established,which might provide strong support for the research of novel antioxidant peptides.