Establishment Of High-throughput Screening System For Anti-influenza And Its Application In Screening Inhibitors Of Traditional Chinese Medicine

ObjectiveIn this study,a high-throughput phenotype screening system for antiviral agents was established by constructing influenza virus A/Puerto Rico/8/1934(H1N1)with Goussia(Glue)luciferase reporter gene.Based on natural products and their derivatives,highthroughput screening and mechanism of action are carried out to provide leading compounds for new drug development.Method1.Rescue of influenza virus with luciferase reporter gene.Based on reverse genetics,8 plasmids pDZ-PB2-Gluc,pDZ-PB1,pDZ-PA,pDZ-HA,pDZ-NP,pDZ-NA,pDZ-M,pDZ-NS were transfected into co-incubated 293T and MDCK cells to rescue the virus,and 9-day-old chicken embryos were used to amplify the virus.2.Establishment and verification of high-throughput screening system.MDCK cells were infected with different multiplicity of infection(MOI),and the optimal MOI was determined according to luciferase level.The signal-to-noise ratio,variation factor,Z’Factor indicators were calculated to evaluate the applicability of the high-throughput screening system,and the sensitivity of the system was verified with four positive drugs with different targets(compound 16,baloxavir,oseltamivir,ribavirin).3.Screening of active compounds.The high-throughput phenotype screening system was used to screen 12 compounds derived from ripe pods of T.chebula,800 natural products and 110 derivatives of natural products to find primary hits.The inhibitory effect and cytotoxicity of primary hits at different concentrations were further tested,and the 50%inhibitory concentration(IC50)and 50%cytotoxic concentration(CC50)of active compounds were fitted,and the selection index(SI)was calculated.4.Inhibitory effects of two active compounds on various subtypes of influenza virus.In order to explore the broad-spectrum anti-influenza effect of the active compounds,the virus titer reduction assay was used to detect the inhibitory effects against influenza A/PR/8/34,Oseltam i vir-resi stance strains A(H1N1)pdm09,A/Brisbane/10/2007(H3N2),A/Wyoming/3/03(H3N2)and two influenza B virus Yamagata and Victoria strains.5.Mechanism of action of punicalagin.Single-cycle replication assay was performed to determine the action stage of punicalagin;virus release inhibition experiment and neuraminidase inhibition experiment were used to determine whether punicalagin acts on the virus release stage mediated by neuraminidase.6.Mechanism of action of nardosinone.Firstly,the action stage of the compound was determined by time-of-addition assay;immunofluorescence experiments were used to further confirmed the results.Finally,RNA dependent RNA polymerase experiment was demonstrated whether nardosinone affected the replication stage of influenza viruses.7.In vivo anti-influenza effect of chalcone derivative A9.BALB/c female mice were intranasally infected with PR8 virus,and 10 mg/kg/day and 30 mg/kg/day were intraperitoneally injected 2 h before virus infection.Viral loads in mouse lung tissue were detected on the second and fifth days,respectively.8.Study on the mechanism of action of chalcone derivatives A9.Firstly,the action stage of the compound was determined by time-of-addition assay;immunofluorescence experiments were used to evaluate the effects of compounds on the amount and localization of influenza virus NP proteins;the nucleus and cytoplasm were separated,and Western Blotting was performed to quantitative the NP protein in the nucleoplasm;the specific binding sites of the compounds were determined by drug-resistant strain mutation experiments.9.Activity of chalcone derivatives A9 series of structural modifiers.The A and B rings of the A9 were substituted,and 34 compounds were modified and synthesize.The antiinfluenza effects of these 34 compounds were screened,and the results were verified by immunofluorescence experiments.Result1.Successful establishment and verification of high-throughput screening system.In this study,the influenza virus PR8-PB2-Gluc with Gaussia luciferase reporter gene was successfully rescued and a high-throughput phenotype screening system was established.Evaluation indicators(signal to noise ratio=45.09,coefficient of variation=9.51%,Z’ Factor=0.70)indicated that the system was suitable for high-throughput screening;the four drugs or compounds with different mechanisms of action were all existed dose-dependent inhibitory effect in this system,proving that the system is sensitive to agents with different targets.2.Punicalagin and chalcone derivatives A9 exhibited antiviral effect.Using the highthroughput screening system,punicalagin and chalcone derivatives A9 identified could significantly inhibit the replication of influenza virus,which IC50 values were 1.25±0.06 μM and 3.51±0.37 μM;CC50 values were>100.00 and 41.46 ± 9.35 μM,and SI values were>80.00 and 11.81,respectively.3.Punicalagin and chalcone derivatives A9 existed broad-spectrum anti-influenza activity.Virus titer reduction experiment showed that punicalagin and chalcone derivatives A9 were effective against H1N1 influenza A/PR/8/34(H1N1),oseltamivirresistant strain A(H1N1)pdm09,A/Brisbane/10/2007(H3N2),A/Wyoming/3/03(H3N2)and two influenza B virus strains(Yamagata and Victoria)with a dosedependent manner.4.Mechanism of action of punicalagin.Single-cycle replication assay showed that punicalagin did not affect the entry and replication stages of influenza virus.Virus release inhibition assay and neuraminidase inhibition assay suggested that the punicalagin blocked the release of viruses by inhibiting the activity of neuraminidase;Molecular docking further speculated the possible binding sites of punicalagin and neuraminidase.5.Mechanism of action of nardosinone.The aristolonc and fangchinolinc had antiinfluenza activity.The time-of-addition experiment proved that nardosinonc acted on the invasion stage of influenza virus,and the immunofluorescence experiment further confirmed this result.Finally,RNA dependent RNA polymerase experiments showed that nardosinone did not affect the replication stage of influenza virus.6.In vivo antiviral activity of chalcone derivative A9.Inhibition rate of chalcone derivative A9 on the second and fifth day of viral loads in lung tissue at a dose of 10 and 30 mg/kg/day were 38.60%and 61.40%,respectively.The result indicated that A9 could significantly reduce the viral loads in lung tissue,and thus it had anti-influenza activity in vivo.7.The mechanism of action of chalcone derivatives A9.Time-of-addition assay showed that A9 acted on the post-entry stage of influenza virus.Immunofluorescence experiments were conducted to investigate the effect of A9 on the nuclear export of the viral ribonucleoprotein complex,and it was clarified that A9 inhibited the nuclear export of RNP.To further quantify this results,MDCK and A549 cells were infected with virus and treated with A9,the nucleus and cytoplasm were separated,and the NP protein in the nucleoplasm was quantified by western blotting,which further confirmed that A9 can inhibit RNP complex in both MDCK and A549 cells.Finally,the A9resistant strain was isolated by the drug-resistant strain mutation experiment,the NP genome was sequenced to identify the mutation site N397S,and reverse genetic technology was used to rescue the mutant virus PR8 N397S.Compared with the effect of A9 on wild-type PR8,the inhibitory effect of A9 on the mutant strain was significantly reduced,suggesting that the binding site of A9 may be related to the 397 site of the NP protein in the RNP complex.8.Activity of chalcone derivatives A9 series of structural modifiers.In this study,the A and B ring structures of A9 were modified.Firstly,the two methoxy groups of the B ring were substituted,and a total of 22 compounds were synthesized.The screening results of 22 compounds indicated that compound F11 had the best activity,with IC50 and CC50 values of 13.63±9.06 and 59.54 ± 12.42 μM,respectively.The activity of F11 was about 4 times weaker than that of A9.It is speculated that the B-ring structure of A9 plays a key role in the anti-influenza activity.Therefore,the B-ring structure of A9 was retained and the ligustrazine ring A of A9 is replaced.A total of 12 compounds were synthesized and screened.The screening results showed that JZ-57 and JZ-63 had the best anti-influenza effects,with IC50 values of 2.96±0.17 and 2.46±0.54 μM,and CC50 values of 16.65±5.70 and 11.34 ± 1.10 μM,respectively.The activity was slightly better than that of A9.Immunofluorescence experiments confirmed that the action mechanism of the three modifiers was the same as that of A9,which could inhibit the nuclear export of influenza virus NP protein and exist anti-influenza effects.Conclusion1.In this study,a high-throughput phenotype screening system for anti-influenza drugs was successfully constructed using the influenza virus PR8-PB2-Gluc.The system could screen and identify inhibitors of different targets,and has the advantages of sensitivity,high efficiency,and high signal-to-noise ratio.2.Using the anti-influenza high-throughput phenotype screening system,it was found that punicalagin possesses anti-influenza activity.Punicalagin exists a dose-dependent inhibitory effect on influenza A viruses(including H274Y oseltamivir-resistant strain)and influenza B viruses,and its mechanism of action is to interfere with the neuraminidase activity,indicating that punicalagin is a broad-spectrum anti-influenza inhibitor targeting neuraminidase.3.In this study,it was found that nardosinone and its analogues aristolone and fangchinoline have anti-influenza activity.Glyptostrone can inhibit the invasion process of influenza virus without affecting the transcription and replication stage of influenza virus.4.In this study,it was found that four chalcone derivatives(A9,F11,JZ-57 and JZ-63)could inhibit the nuclear export of influenza virus ribonucleoprotein,and A9 interact with the 397 site of NP protein.The chalcone derivatives have inhibitory effects on both influenza A viruses(including H274Y oseltamivir-resistant strain)and influenza B viruses,suggesting that the derivatives are broad-spectrum anti-influenza inhibitors targeting the NP protein.