Tetrahydroisoquinoline CompoundYX-99-4-S1 Targets The PB2 Cap Bindingdomain To Inhibit Influenza A Virus Replication

Background:Influenza virus is a highly infectious pathogen of human respiratory diseases,which has the characteristics of strong transmission,wide range,high morbidity and high mortality.It is also a human medical,health and economic and social problem,but at present there is no satisfactory treatment and prevention method.Vaccination is the cornerstone of seasonal influenza control and rapid containment of influenza pandemic.However,due to the wide variation in vaccine efficiency caused by antigenic drift and antigenic transformation of influenza viruses,vaccination alone is not sufficient.With appropriate antigen by the vaccines induce different specificity and immunogenicity,antiviral drugs can not limit the antigenicity of circumstances to protect the host from influenza virus infection and in a short period of time the antiviral effect has a great advantage,especially in the case of an emergency,such as human not familiar with the subtypes of zoonotic infections and new subtype strains causing a pandemic.Tetrahydroisoquinoline ring has a wide range of pHarmacological properties and plays an important role in drug development.Tetrahydroisoquinoline conpounds synthesized by researchers after structural transformation has anti-tumor,antibacterial,antiviral,anti-inflammatory,anticoagulant,bronchiectasis and central nervous system activities.Objectives:In this study,a series of compounds with tetrahydroisoquinoline ring structure were synthesized and screened for anti-influenza virus activity in vitro,so as to further study their antiviral activity and mechanism.The study provides new ideas for further development of new anti-influenza drugs.Methods:1.Influenza virus was amplified by chicken embryo allantoic cavity method,and TCID50 and PFU were detected by CPE effect test and plaque formation test respectively.2.The cytotoxicity and anti-influenza A virus activity of tetrahydroisoquinoline compounds were determined by MTT colorimetry.3.MTT colorimetric assay was used to detect the cytotoxicity of YX-99-4-S1 to MDCK cells,A549 cells and Beas-2B cells,and to determine the antiviral effect against different virus subtypes.4.Western Blotting,RT-qPCR and indirect immunofluorescence method were used to evaluate the effect of YX-99-4-S1 on gene expression of IAV virus.5.The effect of YX-99-4-S1 on viral load of progeny was investigated by plaque formation experiment.6.Hemagglutination inhibition assay,hemolysis assay and H5N1 pseudovirus inhibition assay were used to investigate the effect of YX-99-4-S1 on virus entry into cells,and the effect of neuraminidase inhibition assay was used to detect the release of progeny virus particles.The specific funtional stage of YX-99-4-S1 was determined by dosing mode experiment,single-round replication administration experiment.7.The target of YX-99-4-S1 was determined by mini-replicon experiment,specific reverse transcription experiment protein purification technology,fluorescence polarization experiment,surface plasmon resonance technology and molecular docking technology.8.Antiviral activity of YX-99-4-S1 against Zika virus,Coronavirus,Herpes virus,Dengue virus and HIV clones was detected by MTT colorimetry,CPE effect test,Western Blotting,RT-qPCR pseudovirus encapsulation technology and single luciferase gene test.Results:1.The TCID50 of A/WSN/1933(H1N1)is 105.5TCID50/100 μl,and the PFU is 1.9×10-6 PFU/ml.2.18 tetrahydroisoquinoline compounds were screened for antiviral effects with IC50 values ranging from 0.76 μM to 8.60 μM.3.YX-99-4-S1 inhibits the infection of A/WSN/1933(H1N1)in MDCK cells,A549 cells and Beas-2B cells,and reduces viral load in progeny.Moreover,there was no toxicity to the three cell lines in the range of detected concentration.4.YX-99-4-S1 does not inhibit the hemagglutination and hemolysis induced by the virus,and is not effective against H5N1 pseudovirus infection.5.YX-99-4-S1 does not inhibit neuraminidase activity.6.YX-99-4-S1 is effective at 0-8 h after virus infection.7.YX-99-4-S1 could inhibit the activity of viral RNA-dependent RNA polymerase complex and inhibit the expression of vRNA and cRNA.8.YX-99-4-S1 affects cap capturing activity by specific binding to PB2 cap binding domain.9.YX-99-4-S1 shows antiviral activity against HSV-1,HSV-2,ZIKA and HCOVOC43,but has no inhibitory activity against DENV-2 and HIV clone virus.Conclusions:1.Tetrahydroisoquinoline YX-99-4-S1 has potential inhibitory activity against different subtypes of influenza A virus,and has basically no toxicity to cells within the range of detected activity concentration.2.YX-99-4-S1 inhibits the protein and mRNA expression of influenza A virus in a concentration-dependent manner,and reduce viral load of progeny.3.YX-99-4-S1 acts on viral genome replication and transcriptional translation stage,and influences viral RNA polymerase activity through interaction with PB2 cap binding domain,thereby inhibiting vRNA replication and transcription to exert antiviral effect.4.YX-99-4-S1 has broad spectrum antiviral ability,it has inhibitory activity against HSV,ZIKA and HCoV-OC43.