All Title Author
Keywords Abstract

PLOS ONE  2012 

A Drug Screening Method Based on the Autophagy Pathway and Studies of the Mechanism of Evodiamine against Influenza A Virus

DOI: 10.1371/journal.pone.0042706

Full-Text   Cite this paper   Add to My Lib


In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation - fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1β, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound.


[1]  Bai GR, Chittaganpitch M, Kanai Y, Li YG, Auwanit W, et al. (2009) Amantadine- and oseltamivir-resistant variants of influenza A viruses in Thailand. Biochem Biophys Res Commun 390: 897–901.
[2]  Lei H, Sheng Z, Ding Q, Chen J, Wei X, et al. (2011) Evaluation of Oral Immunization with Recombinant Avian Influenza Virus HA1 Displayed on the Lactococcus lactis Surface and Combined with the Mucosal Adjuvant Cholera Toxin Subunit B. Clin Vaccine Immunol. 18: 1046–1051.
[3]  Meng S, Liu Z, Xu L, Li L, Mei S, et al. (2011) Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice. PLoS One 6: e19863.
[4]  Hoelscher M, Gangappa S, Zhong W, Jayashankar L, Sambhara S (2008) Vaccines against epidemic and pandemic influenza. Expert Opin Drug Deliv 5: 1139–1157.
[5]  Wang S, Hackett A, Jia N, Zhang C, Zhang L, et al. (2011) Polyvalent DNA vaccines expressing HA antigens of H5N1 influenza viruses with an optimized leader sequence elicit cross-protective antibody responses. PLoS One 6: e28757.
[6]  Zhou Z, Jiang X, Liu D, Fan Z, Hu X, et al. (2009) Autophagy is involved in influenza A virus replication. Autophagy 5: 321–328.
[7]  Ma J, Sun Q, Mi R, Zhang H (2011) Avian influenza A virus H5N1 causes autophagy-mediated cell death through suppression of mTOR signaling. J Genet Genomics 38: 533–537.
[8]  Kang R, Zeh HJ, Lotze MT, Tang D (2011) The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18: 571–580.
[9]  Shang L, Wang X (2011) AMPK and mTOR coordinate the regulation of Ulk1 and mammalian autophagy initiation. Autophagy 7: 924–926.
[10]  Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13: 132–141.
[11]  Inoki K, Zhu T, Guan KL (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577–590.
[12]  Itakura E, Kishi C, Inoue K, Mizushima N (2008) Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol Biol Cell 19: 5360–5372.
[13]  Itakura E, Mizushima N (2009) Atg14 and UVRAG: mutually exclusive subunits of mammalian Beclin 1-PI3K complexes. Autophagy 5: 534–536.
[14]  Munz C (2011) Beclin-1 targeting for viral immune escape. Viruses 3: 1166–1178.
[15]  Notte A, Leclere L, Michiels C (2011) Autophagy as a mediator of chemotherapy-induced cell death in cancer. Biochemical Pharmacology 82: 427–434.
[16]  Jounai N, Takeshita F, Kobiyama K, Sawano A, Miyawaki A, et al. (2007) The Atg5 Atg12 conjugate associates with innate antiviral immune responses. Proc Natl Acad Sci U S A 104: 14050–14055.
[17]  Lee H, Kim IK, Park TG (2010) Intracellular trafficking and unpacking of siRNA/quantum dot-PEI complexes modified with and without cell penetrating peptide: confocal and flow cytometric FRET analysis. Bioconjug Chem 21: 289–295.
[18]  Sui Y, Wu Z (2007) Alternative statistical parameter for high-throughput screening assay quality assessment. J Biomol Screen 12: 229–234.
[19]  Matarrese P, Nencioni L, Checconi P, Ciarlo L, Gambardella L, et al. (2011) Pepstatin A alters host cell autophagic machinery and leads to a decrease in influenza A virus production. J Cell Physiol 226: 3368–3377.
[20]  Harris J (2011) Autophagy and cytokines. Cytokine 56: 140–144.
[21]  Liu A, Cao H, Du G (2005) Drug screening for influenza neuraminidase inhibitors. Sci China C Life Sci 48: 1–5.
[22]  Giffin K, Rader RK, Marino MH, Forgey RW (1995) Novel assay for the influenza virus M2 channel activity. FEBS Lett 357: 269–274.
[23]  Su CY, Cheng TJ, Lin MI, Wang SY, Huang WI, et al. (2010) High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity. Proc Natl Acad Sci U S A 107: 19151–19156.
[24]  Noah JW, Severson W, Noah DL, Rasmussen L, White EL, et al. (2007) A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals. Antiviral Res 73: 50–59.
[25]  Shih SR, Chu TY, Reddy GR, Tseng SN, Chen HL, et al. (2010) Pyrazole compound BPR1P0034 with potent and selective anti-influenza virus activity. J Biomed Sci 17: 13.
[26]  Maddry JA, Chen X, Jonsson CB, Ananthan S, Hobrath J, et al. (2011) Discovery of novel benzoquinazolinones and thiazoloimidazoles, inhibitors of influenza H5N1 and H1N1 viruses, from a cell-based high-throughput screen. J Biomol Screen 16: 73–81.
[27]  Muller B, Krausslich HG (2009) Antiviral strategies. Handb Exp Pharmacol: 1–24.
[28]  Chattopadhyay S, Marques JT, Yamashita M, Peters KL, Smith K, et al. (2010) Viral apoptosis is induced by IRF-3-mediated activation of Bax. Embo J 29: 1762–1773.
[29]  Chattopadhyay S, Yamashita M, Zhang Y, Sen GC (2011) The IRF-3/Bax-mediated apoptotic pathway, activated by viral cytoplasmic RNA and DNA, inhibits virus replication. J Virol 85: 3708–3716.
[30]  Huang J, Manning BD (2008) The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J 412: 179–190.
[31]  Inoki K, Li Y, Xu T, Guan KL (2003) Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 17: 1829–1834.
[32]  Law AH, Lee DC, Yuen KY, Peiris M, Lau AS (2010) Cellular response to influenza virus infection: a potential role for autophagy in CXCL10 and interferon-alpha induction. Cell Mol Immunol 7: 263–270.
[33]  Tang H, Da L, Mao Y, Li Y, Li D, et al. (2009) Hepatitis B virus X protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression. Hepatology 49: 60–71.
[34]  Dreux M, Chisari FV (2009) Autophagy proteins promote hepatitis C virus replication. Autophagy 5: 1224–1225.
[35]  Yoon SY, Ha YE, Choi JE, Ahn J, Lee H, et al. (2008) Coxsackievirus B4 uses autophagy for replication after calpain activation in rat primary neurons. J Virol 82: 11976–11978.
[36]  Hayden FG, Cote KM, Douglas RG Jr (1980) Plaque inhibition assay for drug susceptibility testing of influenza viruses. Antimicrob Agents Chemother 17: 865–870.
[37]  Dai JP, Chen J, Bei YF, Han BX, Wang S (2009) Influence of borneol on primary mice oral fibroblasts: a penetration enhancer may be used in oral submucous fibrosis. J Oral Pathol Med 38: 276–281.
[38]  Choi HJ, Lim CH, Song JH, Baek SH, Kwon DH (2009) Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses. Phytomedicine 16: 35–39.
[39]  Li W, Wang G, Zhang H, Xin G, Zhang D, et al. (2010) Effects of NS1 variants of H5N1 influenza virus on interferon induction, TNFalpha response and p53 activity. Cell Mol Immunol 7: 235–242.


comments powered by Disqus