The Antiviral Activities Of Two JAK Inhibitors,Leflunomide And Gingereone A,on Influenza A Virus Replication

Influenza is an acute respiratory disease caused by influenza virus(IV)and characterized by high infectivity,rapid transmission,interspecies transmission and so on.Influenza leads high morbidity and significant mortality in humans and animals and causes great damage to global public health and agricultural development.It is imperative to develop new vaccines and antiviral drugs for the prevention and treatment of influenza.Influenza virus infections can be prevented by vaccination and treated by antiviral drugs.However,the vaccines currently used have a low protection rate and little effect on new influenza viruses caused by antigenic drift or antigenic shift.Currently,two classes of drugs are mainly used for treating influenza:M2 ion channel blockers and neuraminidase inhibitors.Recently.RNA polymerse inhibitors have also been approved for treating influenza in some countries.However,they are often associated with adverse side-effects,limited efficacies and emergence of drug resistance etc.Therefore,there is an urgent need for developing novel antivirals with new targets and the potential to reduce drug resistance.There has been growing interest in pursuing the key the cellular factors crucial for virus replication as novel molecular targets for antiviral therapy.High-throughput screening and gene knockout array revealed that Janus kinase(JAK),JAK1 and JAK2,play an important role in influenza virus replication.JAK1 and JAK2 gene silencing and JAK kinase inhibitors inhibit the replication of influenza virus.In addition,the activation of JAK2 is also involved in the assembly of influenza virus particles.These results suggest that JAK kinase can be used as a drug target for the treatment of influenza virus.The purpose of this study is to investigate the antiviral activity of JAK inhibitors from the pool of clinically approved drugs and from the ingredients of Chinese herbal medicine with antiviral activity.Leflunomide is an immunosuppressant and mainly used for treating rheumatoid a rthritis.The active metabolite A77 1726 inhibits the activities of p70 S6 kinase(S6 K1)and protein tyrosine kinases,and inhibit pyrimidine mononucleotide biosynthesis by inhibiting dihydroorotic acid dehydrogenase(DHO-DHase).Recent studies have f ound that leflunomide not only has anti-inflammatory and immunomodulatory function s but also has significant antiviral activity.However,whether leflunomide has anti-in fluenza virus activity has not been reported;The molecular mechanism of leflunomi de-mediated antiviral effect remains controversial.Since preliminary studies have sho wn that A77 1726 inhibits the activity of JAK1 and JAK3,we hypothesize that A771726 can function as a JAK inhibitor to supppress IAV infection.Leflunomide is a synthetic compound and has been approved for long-term use to treat RA,some patients develop untolerable side-effects.The section part of our study is to identify JAK kinase inhibitors from plants with antiviral activity and develop them as antiviral drugs.Ginger exhibits antimicrobial activities on viruses,bacteria,fungi,and nematodes.Ginger extract restricts the replication of herpes virus,rhinovirus,and respiratory syncytial virus.In addition,ginger is an important ingredient in several traditional Chinese medicines prescribed for the treatment of respiratory tract infections.To date,little is known about the properties of compounds in ginger extracts related to their anti-IV activity.Gingerenone A(Gin A),a compound extracted from ginger,is a dual inhibitor of JAK2 and p70 S6 kinase(S6K1).The purpose of this study is to explore whether Gin A can inhibit influenza virus infection by inhibiting the activity of JAK2.1.Effect and mechanism of active metabolite A77 1726 of leflunomide against influenza infection.We first investigated the effect A77 1276 on influenza virus replication.A77 1726 significantly inhibited the replication of IAV including A/mallard/Huadong/S/2005(H5N 1),A/PR/8/34(H1N1),and Ck/SH/F/98(H9N2)virsues in chicken embryo fibroblasts(CEF),A549,and MDCK cells.QPCR and Western blot revealed that A77 1726 reduced the levels of viral matrix protein(M1)mRNA,and viral nuclear proteins(NP),M1 and hemagglutinin(HA)proteins.The mini-genome viral polymerase assay revealed that A771726 reduced the activity of viral RNP(PB2,PB1,PA and NP)polymerase.Consistently,in vivo experiments showed that leflunomide alleviated their body weight loss of mice,prolonged their survival,reduce the virus titer and lesions in lungs tissue caused by IAV infection.To further study the mechanism of influenza virus replication by A77 1726.Western blot analysis showed that the S6K1 inhibitor PF-4708671 and the pyrimidine nucleotide synthesis inhibitor Brequinar Sodium(BQR)did not inhibit influenza virus replication.The JAK specific inhibitor Ruxolitinib(Rux)effectively inhibited the replication of influenza virus.A77 1726 inhibited the phosphorylation of JAK1,JAK2 and STAT3.JAK2 overexpression enhanced H5N1 virus replication and compromised the antiviral activity of A77 1726.These results suggest that the ability of A77 1726 to inhibit JAK2 activity contributes to its antiviral activity.2.Effect and mechanism of gingerenone A against influenza infection.JAK2 plays an important role in IAV replication.Here we investigated if the antiviral activity of Gin A,a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase(S6K1),on influenza virus replication and its molecular mechanism.Here we report that Gin A significantly inhibited the replication of IAV such as A/mallard/Huadong/S/2005(H5N1),A/PR/8/34(H1N1),and Ck/SH/F/98(H9N2)in 293T,MDCK,A549 and DF-1 cells.Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice,alleviated the body weight loss,and prolonged their survival.qPCR and Western blot analysis showed that Gin A decreased the mRNA level of viral matrix protein(M1)and inhibited the synthesis of viral polymerase 2(PB2),NP and M1.Gin A inhibited the activity of virus Ribonucleoproteins(RNP)polymerase in 293T cells.IAV replication was inhibited by Ruxolitinib(Rux),a JAK inhibitor,but not by PF-4708671,a S6K1 inhibitor.Gin A inhibited tyrosine phosphorylation of JAK2 and STAT3.JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity.Our results demonstrate the ability of Gin A to restrict IAV replication by inhibiting JAK2 activity,suggesting that Gin A may have the potential to become a new anti-influenza virus drug.In summary,our present study has verified the role of JAK in IAV replication and validated the feasibility of targeting JAK as antiviral drugs.Our study also clarifies the mechansims of antiviral activity of leflunomide by showing that A77 1726 inhibited IAV replication largely by inhibiting JAK activity.Finally,our study demonstrates the antiviral activity of Gin A and provides novel insights into the mechanisms of actions of Gin A.Since leflunomide is a clinically approved anti-rheumatoid arthritis drug with the proven antiviral activity against CMV and BK virus infections in transplant patients,whereas Gin A,a compound derived from ginger.should have low side-effects,it is anticipated that both of them could be repurposed as novel antiviral agents for treating IAV as well as other virus infections such as SARS-CoV2.