1887

Abstract

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

Funding
This study was supported by the:
  • National Key Research and Development Program of China (Award 2017YFD0501605)
    • Principle Award Recipient: QuanZhang
  • Priority Academic Program Development of Jiangsu Higher Education Institutions
    • Principle Award Recipient: XiulongXu
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/content/journal/jgv/10.1099/jgv.0.001534
2021-01-08
2021-08-02
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References

  1. Kennedy JM, Earle JAP, Omar S, Abdullah Hani'ah, Nielsen O et al. Canine and Phocine distemper viruses: global spread and genetic basis of jumping species barriers. Viruses 2019; 11:944 [View Article][PubMed]
    [Google Scholar]
  2. Loots AK, Mitchell E, Dalton DL, Kotzé A, Venter EH. Advances in canine distemper virus pathogenesis research: a wildlife perspective. J Gen Virol 2017; 98:311–321 [View Article][PubMed]
    [Google Scholar]
  3. Buczkowski H, Muniraju M, Parida S, Banyard AC. Morbillivirus vaccines: recent successes and future hopes. Vaccine 2014; 32:3155–3161 [View Article][PubMed]
    [Google Scholar]
  4. Trejo-Avila LM, Morales-Martínez ME, Ricque-Marie D, Cruz-Suarez LE, Zapata-Benavides P et al. In vitro anti-canine distemper virus activity of fucoidan extracted from the brown alga Cladosiphon okamuranus. Virusdisease 2014; 25:474–480 [View Article][PubMed]
    [Google Scholar]
  5. Xue X, Zhu Y, Yan L, Wong G, Sun P et al. Antiviral efficacy of favipiravir against canine distemper virus infection in vitro. BMC Vet Res 2019; 15:316 [View Article][PubMed]
    [Google Scholar]
  6. Elia G, Belloli C, Cirone F, Lucente MS, Caruso M et al. In vitro efficacy of ribavirin against canine distemper virus. Antiviral Res 2008; 77:108–113 [View Article][PubMed]
    [Google Scholar]
  7. Gallina L, Dal Pozzo F, Galligioni V, Bombardelli E, Scagliarini A. Inhibition of viral RNA synthesis in canine distemper virus infection by proanthocyanidin A2. Antiviral Res 2011; 92:447–452 [View Article][PubMed]
    [Google Scholar]
  8. Krumm SA, Yan D, Hovingh ES, Evers TJ, Enkirch T et al. An orally available, small-molecule polymerase inhibitor shows efficacy against a lethal morbillivirus infection in a large animal model. Sci Transl Med 2014; 6:232ra252 [View Article]
    [Google Scholar]
  9. Rendon-Marin S, da Fontoura Budaszewski R, Canal CW, Ruiz-Saenz J. Tropism and molecular pathogenesis of canine distemper virus. Virol J 2019; 16:30 [View Article][PubMed]
    [Google Scholar]
  10. Takeda M, Seki F, Yamamoto Y, Nao N, Tokiwa H. Animal morbilliviruses and their cross-species transmission potential. Curr Opin Virol 2020; 41:38–45 [View Article][PubMed]
    [Google Scholar]
  11. Breedveld FC, Dayer JM. Leflunomide: mode of action in the treatment of rheumatoid arthritis. Ann Rheum Dis 2000; 59:841–849 [View Article][PubMed]
    [Google Scholar]
  12. Xu X, Blinder L, Shen J, Gong H, Finnegan A et al. In vivo mechanism by which leflunomide controls lymphoproliferative and autoimmune disease in MRL/MpJ-lpr/lpr mice. J Immunol 1997; 159:167–174[PubMed]
    [Google Scholar]
  13. Xu X, Shen J, Mall JW, Myers JA, Huang W et al. In vitro and in vivo antitumor activity of a novel immunomodulatory drug, leflunomide: mechanisms of action. Biochem Pharmacol 1999; 58:1405–1413 [View Article][PubMed]
    [Google Scholar]
  14. Xu X, Williams JW, Gong H, Finnegan A, Chong AS. Two activities of the immunosuppressive metabolite of leflunomide, A77 1726. Inhibition of pyrimidine nucleotide synthesis and protein tyrosine phosphorylation. Biochem Pharmacol 1996; 52:527–534 [View Article][PubMed]
    [Google Scholar]
  15. Xu X, Williams JW, Bremer EG, Finnegan A, Chong AS. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem 1995; 270:12398–12403 [View Article][PubMed]
    [Google Scholar]
  16. Rückemann K, Fairbanks LD, Carrey EA, Hawrylowicz CM, Richards DF et al. Leflunomide inhibits pyrimidine de novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans. J Biol Chem 1998; 273:21682–21691 [View Article][PubMed]
    [Google Scholar]
  17. Elder RT, Xu X, Williams JW, Gong H, Finnegan A et al. The immunosuppressive metabolite of leflunomide, A77 1726, affects murine T cells through two biochemical mechanisms. J Immunol 1997; 159:22–27[PubMed]
    [Google Scholar]
  18. Siemasko K, Chong AS, Jäck HM, Gong H, Williams JW et al. Inhibition of JAK3 and STAT6 tyrosine phosphorylation by the immunosuppressive drug leflunomide leads to a block in IgG1 production. J Immunol 1998; 160:1581–1588[PubMed]
    [Google Scholar]
  19. Siemasko KF, Chong AS, Williams JW, Bremer EG, Finnegan A. Regulation of B cell function by the immunosuppressive agent leflunomide. Transplantation 1996; 61:635–642 [View Article][PubMed]
    [Google Scholar]
  20. Doscas ME, Williamson AJ, Usha L, Bogachkov Y, Rao GS et al. Inhibition of p70 S6 kinase (S6K1) activity by A77 1726 and its effect on cell proliferation and cell cycle progress. Neoplasia 2014; 16:824–834 [View Article][PubMed]
    [Google Scholar]
  21. Bernhoff E, Tylden GD, Kjerpeseth LJ, Gutteberg TJ, Hirsch HH et al. Leflunomide inhibition of BK virus replication in renal tubular epithelial cells. J Virol 2010; 84:2150–2156 [View Article]
    [Google Scholar]
  22. Liacini A, Seamone ME, Muruve DA, Tibbles LA. Anti-BK virus mechanisms of sirolimus and leflunomide alone and in combination: toward a new therapy for BK virus infection. Transplantation 2010; 90:1450–1457 [View Article][PubMed]
    [Google Scholar]
  23. Waldman WJ, Knight DA, Blinder L, Shen J, Lurain NS et al. Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressive agent leflunomide. Intervirology 1999; 42:412–418 [View Article][PubMed]
    [Google Scholar]
  24. Waldman WJ, Knight DA, Lurain NS, Miller DM, Sedmak DD et al. Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide. Transplantation 1999; 68:814–825 [View Article][PubMed]
    [Google Scholar]
  25. Chacko B, John GT. Leflunomide for cytomegalovirus: bench to bedside. Transpl Infect Dis 2012; 14:111–120 [View Article][PubMed]
    [Google Scholar]
  26. Wang Y, Wang W, Xu L, Zhou X, Shokrollahi E et al. Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis E virus replication. Antimicrob Agents Chemother 2016; 60:2834–2848 [View Article][PubMed]
    [Google Scholar]
  27. Chen S, Ding S, Yin Y, Xu L, Li P et al. Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication. Antiviral Res 2019; 167:35–44 [View Article][PubMed]
    [Google Scholar]
  28. Dunn MCC, Knight DA, Waldman WJ. Inhibition of respiratory syncytial virus in vitro and in vivo by the immunosuppressive agent leflunomide. Antivir Ther 2011; 16:309–317 [View Article][PubMed]
    [Google Scholar]
  29. Wang J, Sun J, Hu J, Wang C, Prinz RA et al. A77 1726, the active metabolite of the anti-rheumatoid arthritis drug leflunomide, inhibits influenza A virus replication in vitro and in vivo by inhibiting the activity of Janus kinases. FASEB J 2020; 34:10132–10145 [View Article][PubMed]
    [Google Scholar]
  30. Li X, Sun J, Prinz RA, Liu X, Xu X. Inhibition of porcine epidemic diarrhea virus (PEDV) replication by A77 1726 through targeting JAK and Src tyrosine kinases. Virology 2020; 551:75–83 [View Article][PubMed]
    [Google Scholar]
  31. Xiong R, Zhang L, Li S, Sun Y, Ding M et al. Correction to: novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2. Protein Cell 2020 [View Article]
    [Google Scholar]
  32. Hu K, Wang M, Zhao Y, Zhang Y, Wang T et al. A small-scale medication of leflunomide as a treatment of COVID-19 in an open-label Blank-Controlled clinical trial. Virol Sin 2020; 59: [View Article]
    [Google Scholar]
  33. Yi L, Cheng S. A monoclonal antibody against truncated N protein (AA 277-471) of canine distemper virus. Monoclon Antib Immunodiagn Immunother 2014; 33:52–56 [View Article][PubMed]
    [Google Scholar]
  34. Yi L, Cheng Y, Zhang M, Cao Z, Tong M et al. Identification of a novel canine distemper virus B-cell epitope using a monoclonal antibody against nucleocapsid protein. Virus Res 2016; 213:1–5 [View Article][PubMed]
    [Google Scholar]
  35. Li S, Yi L, Cao Z, Cheng Y, Tong M et al. Identification of linear B-cell epitopes on the phosphoprotein of canine distemper virus using four monoclonal antibodies. Virus Res 2018; 257:52–56 [View Article][PubMed]
    [Google Scholar]
  36. Bi Z, Wang Y, Pan Q, Xia X, Xu L. Development of CDV-specific monoclonal antibodies for differentiation of variable epitopes of nucleocapsid protein. Vet Microbiol 2017; 211:84–91 [View Article][PubMed]
    [Google Scholar]
  37. Bi Z, Wang Y, Wang X, Xia X. Phylogenetic analysis of canine distemper virus in domestic dogs in Nanjing, China. Arch Virol 2015; 160:523–527 [View Article][PubMed]
    [Google Scholar]
  38. Zhang J, Ruan T, Sheng T, Wang J, Sun J et al. Role of c-Jun terminal kinase (JNK) activation in influenza A virus-induced autophagy and replication. Virology 2019; 526:1–12 [View Article][PubMed]
    [Google Scholar]
  39. Dibble CC, Asara JM, Manning BD. Characterization of Rictor phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1. Mol Cell Biol 2009; 29:5657–5670 [View Article][PubMed]
    [Google Scholar]
  40. Rosner M, Schipany K, Hengstschläger M. P70 S6K1 nuclear localization depends on its mTOR-mediated phosphorylation at T389, but not on its kinase activity towards S6. Amino Acids 2012; 42:2251–2256 [View Article][PubMed]
    [Google Scholar]
  41. Pearce LR, Alton GR, Richter DT, Kath JC, Lingardo L et al. Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1). Biochem J 2010; 431:245–255 [View Article][PubMed]
    [Google Scholar]
  42. Ben-Sahra I, Howell JJ, Asara JM, Manning BD. Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 2013; 339:1323–1328 [View Article][PubMed]
    [Google Scholar]
  43. Robitaille AM, Christen S, Shimobayashi M, Cornu M, Fava LL et al. Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science 2013; 339:1320–1323 [View Article][PubMed]
    [Google Scholar]
  44. Bilger A, Plowshay J, Ma S, Nawandar D, Barlow EA et al. Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)- induced lymphoproliferative disease and lytic viral replication. Oncotarget 2017; 8:44266–44280 [View Article][PubMed]
    [Google Scholar]
  45. Knight DA, Hejmanowski AQ, Dierksheide JE, Williams JW, Chong AS et al. Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide. Transplantation 2001; 71:170–174 [View Article][PubMed]
    [Google Scholar]
  46. Bernhoff E, Tylden GD, Kjerpeseth LJ, Gutteberg TJ, Hirsch HH et al. Leflunomide inhibition of BK virus replication in renal tubular epithelial cells. J Virol 2010; 84:2150–2156 [View Article][PubMed]
    [Google Scholar]
  47. Hoffmann H-H, Kunz A, Simon VA, Palese P, Shaw ML. Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc Natl Acad Sci U S A 2011; 108:5777–5782 [View Article][PubMed]
    [Google Scholar]
  48. Bonavia A, Franti M, Pusateri Keaney E, Kuhen K, Seepersaud M et al. Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV). Proc Natl Acad Sci U S A 2011; 108:6739–6744 [View Article][PubMed]
    [Google Scholar]
  49. Wang Q-Y, Bushell S, Qing M, Xu HY, Bonavia A et al. Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J Virol 2011; 85:6548–6556 [View Article][PubMed]
    [Google Scholar]
  50. Davis IC, Lazarowski ER, Hickman-Davis JM, Fortenberry JA, Chen F-P et al. Leflunomide prevents alveolar fluid clearance inhibition by respiratory syncytial virus. Am J Respir Crit Care Med 2006; 173:673–682 [View Article][PubMed]
    [Google Scholar]
  51. Mohamad Fairus AK, Choudhary B, Hosahalli S, Kavitha N, Shatrah O. Dihydroorotate dehydrogenase (DHODH) inhibitors affect ATP depletion, endogenous ROS and mediate S-phase arrest in breast cancer cells. Biochimie 2017; 135:154–163 [View Article][PubMed]
    [Google Scholar]
  52. Whitley NT, Day MJ. Immunomodulatory drugs and their application to the management of canine immune-mediated disease. J Small Anim Pract 2011; 52:70–85 [View Article][PubMed]
    [Google Scholar]
  53. Singer LM, Cohn LA, Reinero CR, Papich MG. Leflunomide pharmacokinetics after single oral administration to dogs. J Vet Pharmacol Ther 2011; 34:609–611 [View Article][PubMed]
    [Google Scholar]
  54. Chong AS, Huang W, Liu W, Luo J, Shen J et al. In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression. Transplantation 1999; 68:100–109 [View Article][PubMed]
    [Google Scholar]
  55. Chan V, Charles BG, Tett SE. Population pharmacokinetics and association between A77 1726 plasma concentrations and disease activity measures following administration of leflunomide to people with rheumatoid arthritis. Br J Clin Pharmacol 2005; 60:257–264 [View Article][PubMed]
    [Google Scholar]
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