Structure-Activity Relationship And Mechanism Of Arctigenin Derivatives Against Infectious Hematopoietic Necrosis Virus

As the causative pathogen of infectious hematopoietic necrosis?IHN?,infectious hematopoietic necrosis virus?IHNV?has strong pathogenicity and extensive transmissibility,resulting in a high mortality rate approaching 100%of salmonid species.IHN is designated a notifiable disease by the Office International des Epizooties?OIE?and also listed B in Chinese animal epidemic diseases directory.As the immune system of fish fingerlings is not fully developed,vaccines have failed to protect them effectively and cannot solve the problem of fish fingerling disease.Therefore,the development of related therapeutic drugs seems more urgent.Up to now,there is no licensed antiviral drugs available for the control of IHNV infection.Arctigein,a natural product,has a certain inhibitory effect on IHNV.On this basis,a series of arctigenin derivatives were designed and synthesized.The antiviral activity of the derivatives was evaluated by cell screening model in vitro.The structure-activity relationship of the derivatives was described by Co MFA model.The chemical structure of the optimal derivatives against IHNV was determined and synthesized.Then,the antiviral effect of the optimal derivatives against IHNV was evaluated at the cellular and individual level,and the antiviral molecular mechanism was explored in depth,in order to provide theoretical basis for the development of special aquatic anti-IHNV drugs and the control of IHN.The main results are described as follows:1. Synthesis and identification of arctigenin derivatives.Through a series of synthesis methods such as Wiiliamson etherification and substitution reaction,the structure of the synthesized derivatives was identified by column chromatography and spectroscopy,and 57 arctigenin derivatives were obtained,including 1deoxyarctiin derivative,19 alkylated derivatives,9 esterified derivatives,5 brominated derivatives and 23 nitrogen-containing heterocyclic derivatives.2. Structure-activity relationship of arctigenin derivatives against IHNV.The proliferation of IHNV in host cells was detected by q RT-PCR.Among the screened compounds,the inhibitory percentage of 26 derivatives was examined more than50%,as the effective agents in this test.Among them,derivative 56 had the best antiviral activity,and its IC₅₀was 1.32?M,which was significantly better than that of ribavirin(IC₅₀=1.65?M).Using derivative 56 as a template molecule,a Co MFA model was established based on the structure of 23 active derivatives and their anti-IHNV activity.The model's R²cv is 0.945,ONC is 2,R²ncv is 0.986,SEE is 0.060,F value is 577.417,three-dimensional field coefficient is 0.715,electrostatic field coefficient is 0.285,and R²_pred is 0.988.All these parameters prove that the established Co MFA model has good prediction ability and significant statistical significance.The model shows that the antiviral activity could be improved by increasing the space structure of the end of the carbon chain,introducing a positively charged group,or introducing a negatively charged group at the2nd and 4th positions of the nitrogen heterocycle.3. Design,synthesis and anti-IHNV activity study of potential drugsAccording to the prediction of Co MFA equipotential diagram,eight new arctigenin derivatives were redesigned and synthesized.The results showed that the IC₅₀ of derivatives EOA,59,60 and 62 against IHNV were 0.98,1.12,1.15 and 1.77?M,respectively.Among them,the antiviral activity of derivative EOA,59 and 60 is better than that of derivative 56,indicating that the prediction of the model is reliable and could be used in the design of anti-IHNV drugs.Through the detection of the virus titers of derivative EOA,59,60 and 62,it was found that the derivative EOA could significantly reduce the infectivity of IHNV in EPC cells,stably play an antiviral role.After 72 h of IHNV infection,EPC cells will produce apoptotic bodies and decrease microfilaments in the cytoskeleton,while the cells treated with EOA would be stained normally,apoptotic bodies rarely appear and microfilaments are clear and complete.The ultrastructures of EPC cells were observed by scanning electron microscopy and transmission electron microscopy.It was found that there were a lot of holes on the surface of EPC cells after IHNV infection,the nucleus of EPC cells was shrunk,and the matrix of EPC cells flowed out.After EOA treatment,EPC cells had no significant difference compared with the control group,and maintained a good morphological structure.The results of flow cytometry showed that more than 28.50%of EPC cells were apoptotic after IHNV infection,while the apoptotic rate of cells in EOA group was 22.74%lower than that in virus group,and the number of normal cells was basically the same as that in control group,with 94.24%of normal cells.These results indicate that EOA could effectively inhibit the apoptosis of host cells induced by IHNV.Further in vivo studies found that the cumulative mortality of rainbow trout after EOA treatment was reduced by 32.00%relative to the virus group,indicating that EOA could improve the survival rate of rainbow trout after IHNV infection.q RT-PCR detected a significant decrease in virus expression in spleen and kidney of rainbow trout after EOA treatment,indicating that EOA could effectively control the proliferation of IHNV in spleen and kidney of rainbow trout.In addition,the spleen and kidney of rainbow trout after viral infection were rich in red blood cells with severe congestion,while the spleen and kidney of the drug group had the same morphology of that of normal rainbow trout.At the same time,q RT-PCR results showed that the pro-inflammatory factors IL-8,IL-12p40and TNF-?in spleen and kidney were significantly down regulated after EOA treatment,which were 142.85,6.02,60.07 times and 5.59,22.22 and 37.98 times lower than those in the control group,respectively.These data suggest that EOA could play an antiviral role in vivo and improve the inflammation of spleen and kidney caused by IHNV infection in rainbow trout.Based on the above results,it was determined that the derivative EOA designed and synthesized according to the model has good in vitro and in vivo activity against IHNV and could be used as a potential anti-IHNV drug for aquatic products.4. Study on anti-IHNV mechanism of derivative EOA.After EOA was mixed with IHNV particles for 1,2,and 4 h,the drug was separated from the virus by ultracentrifugation,and the virus reinfected EPC cells.The results showed that the expression of the treated virus in EPC cells and the CPE it caused were basically the same as those of the untreated control group.These data suggest that EOA may not directly affect the antiviral effect of virus particles.The results of virus adsorption test showed that the expression of IHNV after EOA treatment was similar to that of the control group,indicating that it may not play a role in the adsorption of IHNV on EPC cells.Through the results of time-addition test,we could see that EOA could obviously inhibit the virus within 0?4 h after IHNV infecting EPC cells,which indicates that EOA may act on the pre-replication of the virus in the host cells.It is worth noting that rapamycin,an autophagy inducer,inhibited IHNV by 76.23%at 72 h,indicating that autophagy could control IHNV proliferation in host cells to some extent.Autophagy fluorescence detection showed that EPC cells showed a strong autophagy body fluorescence after being treated with derivative EOA.A large number of autophagic vesicles were observed in EOA-treated cells by transmission electron microscopy.Furthermore,eukaryotic expression plasmid of LC3 protein was constructed,and EOA was found to effectively promote the expression of p EGFP-LC3 in EPC cells by cell transfection,fluorescence microscopy and flow cytometry.These results indicate that EOA could specifically activate autophagy.In addition,WB results confirmed that EOA promotes autophagy by activating JNK pathway.The above results indicate that EOA has an antiviral effect by causing autophagy of EPC cells in the early stage of IHNV replicationIn conclusion,the derivative EOA synthesized according to the structure-activity relationship design could inhibit the early replication of IHNV by inducing autophagy of host cells,which could significantly reduce the amount of virus in vitro and in vivo.EOA could effectively maintain the growth activity of host cells,and reduce the mortality of rainbow trout infected with the IHNV.Therefore,the derivative EOA has a broad application prospect in the prevention and control of IHN.