Discovery of novel West Nile Virus protease inhibitor based on isobenzonafuranone and triazolic derivatives of eugenol and indan-1,3-dione scaffolds

Autoři: André S. de Oliveira aff001;  Poliana A. R. Gazolla aff002;  Ana Flávia C. da S. Oliveira aff001;  Wagner L. Pereira aff002;  Lívia C. de S. Viol aff002;  Angélica F. da S. Maia aff002;  Edjon G. Santos aff001;  Ítalo E. P. da Silva aff001;  Tiago A. de Oliveira Mendes aff003;  Adalberto M. da Silva aff004;  Roberto S. Dias aff001;  Cynthia C. da Silva aff001;  Marcelo D. Polêto aff001;  Róbson R. Teixeira aff004;  Sergio O. de Paula aff001
Působiště autorů: Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil aff001;  Instituto Federal de Educação, Ciência e Tecnologia do Norte de Minas Gerais, Fazenda Biribiri, MG, Brazil aff002;  Departamento de Bioquímica Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brazil aff003;  Departamento de Química, Universidade Federal de Viçosa, Viçosa, MG, Brazil aff004;  Instituto Federal de Educação, Ciência e Tecnologia Catarinense, Araquari, SC, Brazil aff005
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0223017


The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC50 of 6.86 μmol L-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His51 and Ser135, which are members of the catalytic triad of the WNV NS2B-NS3 protease.

Klíčová slova:

Cytotoxicity – Enzyme inhibitors – Gels – NMR spectroscopy – Proteases – Column chromatography – Thin-layer chromatography – West Nile virus


1. Martin-Acebes MA, Vazquez-Calvo A, Saiz JC. Lipids and flaviviruses, present and future perspectives for the control of dengue, Zika, and West Nile viruses. Prog Lipid Res. 2016;64:123–37. doi: 10.1016/j.plipres.2016.09.005 27702593.

2. Bhakat S, Karubiu W, Jayaprakash V, Soliman ME. A perspective on targeting non-structural proteins to combat neglected tropical diseases: Dengue, West Nile and Chikungunya viruses. Eur J Med Chem. 2014;87:677–702. doi: 10.1016/j.ejmech.2014.10.010 25305334.

3. Chaskopoulou A, Dovas CI, Chaintoutis SC, Kashefi J, Koehler P, Papanastassopoulou M. Detection and early warning of West Nile Virus circulation in Central Macedonia, Greece, using sentinel chickens and mosquitoes. Vector Borne Zoonotic Dis. 2013;13(10):723–32. doi: 10.1089/vbz.2012.1176 23919609.

4. Chaskopoulou A, L'Ambert G, Petric D, Bellini R, Zgomba M, Groen TA, et al. Ecology of West Nile virus across four European countries: review of weather profiles, vector population dynamics and vector control response. Parasit Vectors. 2016;9(1):482. doi: 10.1186/s13071-016-1736-6 27590848; PubMed Central PMCID: PMC5009705.

5. Morales MA, Barrandeguy M, Fabbri C, Garcia JB, Vissani A, Trono K, et al. West Nile virus isolation from equines in Argentina, 2006. Emerg Infect Dis. 2006;12(10):1559–61. doi: 10.3201/eid1210.060852 17176571; PubMed Central PMCID: PMC3290965.

6. Kleinschmidt-DeMasters BK, Beckham JD. West Nile Virus Encephalitis 16 Years Later. Brain Pathol. 2015;25(5):625–33. doi: 10.1111/bpa.12280 26276026.

7. Ometto T, Durigon EL, de Araujo J, Aprelon R, de Aguiar DM, Cavalcante GT, et al. West Nile virus surveillance, Brazil, 2008–2010. Trans R Soc Trop Med Hyg. 2013;107(11):723–30. doi: 10.1093/trstmh/trt081 24008895.

8. Flatau E, Kohn D, Daher O, Varsano N. West Nile fever encephalitis. Isr J Med Sci. 1981;17(11):1057–9. 6274825.

9. Bakonyi T, Hubalek Z, Rudolf I, Nowotny N. Novel flavivirus or new lineage of West Nile virus, central Europe. Emerg Infect Dis. 2005;11(2):225–31. doi: 10.3201/eid1102.041028 15752439; PubMed Central PMCID: PMC3320449.

10. Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses. J Gen Virol. 1997;78 (Pt 9):2293–7. doi: 10.1099/0022-1317-78-9-2293 9292017.

11. Costa SM, Azevedo AS, Paes MV, Sarges FS, Freire MS, Alves AM. DNA vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice. Virology. 2007;358(2):413–23. doi: 10.1016/j.virol.2006.08.052 17020777.

12. Teo KF, Wright PJ. Internal proteolysis of the NS3 protein specified by dengue virus 2. J Gen Virol. 1997;78 (Pt 2):337–41. doi: 10.1099/0022-1317-78-2-337 9018055.

13. Oliveira AS, Silva ML, Oliveira AFCS, Silva CC, Teixeira RR, Paula SOD. NS3 and NS5 proteins: important targets for anti-dengue drug design. J Braz Chem Soc. 2014;25(10):1759–69.

14. Noble CG, Chen YL, Dong H, Gu F, Lim SP, Schul W, et al. Strategies for development of Dengue virus inhibitors. Antiviral Res. 2010;85(3):450–62. doi: 10.1016/j.antiviral.2009.12.011 20060421.

15. Lim SP, Wang QY, Noble CG, Chen YL, Dong H, Zou B, et al. Ten years of dengue drug discovery: progress and prospects. Antiviral Res. 2013;100(2):500–19. doi: 10.1016/j.antiviral.2013.09.013 24076358.

16. Xu T, Sampath A, Chao A, Wen D, Nanao M, Chene P, et al. Structure of the Dengue virus helicase/nucleoside triphosphatase catalytic domain at a resolution of 2.4 A. J Virol. 2005;79(16):10278–88. doi: 10.1128/JVI.79.16.10278-10288.2005 16051821; PubMed Central PMCID: PMC1182654.

17. Lescar J, Luo D, Xu T, Sampath A, Lim SP, Canard B, et al. Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from Dengue virus as a target. Antiviral Res. 2008;80(2):94–101. doi: 10.1016/j.antiviral.2008.07.001 18674567.

18. Yoganathan K, Rossant C, Ng S, Huang Y, Butler MS, Buss AD. 10-Methoxydihydrofuscin, fuscinarin, and fuscin, novel antagonists of the human CCR5 receptor from Oidiodendron griseum. J Nat Prod. 2003;66(8):1116–7. doi: 10.1021/np030146m 12932138.

19. Aravapalli S, Lai H, Teramoto T, Alliston KR, Lushington GH, Ferguson EL, et al. Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold. Bioorg Med Chem. 2012;20(13):4140–8. doi: 10.1016/j.bmc.2012.04.055 22632792; PubMed Central PMCID: PMC3563422.

20. Tiew KC, Dou D, Teramoto T, Lai H, Alliston KR, Lushington GH, et al. Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives. Bioorg Med Chem. 2012;20(3):1213–21. doi: 10.1016/j.bmc.2011.12.047 22249124; PubMed Central PMCID: PMC3279297.

21. Tragoolpua Y, Jatisatienr A. Anti-herpes simplex virus activities of Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison and essential oil, eugenol. Phytother Res. 2007;21(12):1153–8. doi: 10.1002/ptr.2226 17628885.

22. Benencia F, Courreges MC. In vitro and in vivo activity of eugenol on human herpesvirus. Phytother Res. 2000;14(7):495–500. 11054837.

23. Serkedjieva J, Ivancheva S. Antiherpes virus activity of extracts from the medicinal plant Geranium sanguineum L. J Ethnopharmacol. 1999;64(1):59–68. doi: 10.1016/s0378-8741(98)00095-6 10075123.

24. Dai JP, Zhao XF, Zeng J, Wan QY, Yang JC, Li WZ, et al. Drug screening for autophagy inhibitors based on the dissociation of Beclin1-Bcl2 complex using BiFC technique and mechanism of eugenol on anti-influenza A virus activity. PLoS One. 2013;8(4):e61026. doi: 10.1371/journal.pone.0061026 23613775; PubMed Central PMCID: PMC3628889.

25. Bondre VP, Jadi RS, Mishra AC, Yergolkar PN, Arankalle VA. West Nile virus isolates from India: evidence for a distinct genetic lineage. J Gen Virol. 2007;88(Pt 3):875–84. doi: 10.1099/vir.0.82403-0 17325360.

26. Behnam MA, Nitsche C, Boldescu V, Klein CD. The Medicinal Chemistry of Dengue Virus. J Med Chem. 2016;59(12):5622–49. doi: 10.1021/acs.jmedchem.5b01653 26771861.

27. Weigel LF, Nitsche C, Graf D, Bartenschlager R, Klein CD. Phenylalanine and Phenylglycine Analogues as Arginine Mimetics in Dengue Protease Inhibitors. J Med Chem. 2015;58(19):7719–33. doi: 10.1021/acs.jmedchem.5b00612 26367391.

28. Timiri AK, Sinha BN, Jayaprakash V. Progress and prospects on DENV protease inhibitors. Eur J Med Chem. 2016;117:125–43. doi: 10.1016/j.ejmech.2016.04.008 27092412.

29. Huang XZ, Zhu Y, Guan XL, Tian K, Guo JM, Wang HB, et al. A novel antioxidant isobenzofuranone derivative from fungus Cephalosporium sp.AL031. Molecules. 2012;17(4):4219–24. doi: 10.3390/molecules17044219 22481542.

30. Strobel G, Ford E, Worapong J, Harper JK, Arif AM, Grant DM, et al. Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities. Phytochemistry. 2002;60(2):179–83. doi: 10.1016/s0031-9422(02)00062-6 12009322.

31. Peng Y, Zeng X, Feng Y, Wang X. Antiplatelet and antithrombotic activity of L-3-n-butylphthalide in rats. J Cardiovasc Pharmacol. 2004;43(6):876–81. doi: 10.1097/00005344-200406000-00018 15167282.

32. Yang H, Hu GY, Chen J, Wang Y, Wang ZH. Synthesis, resolution, and antiplatelet activity of 3-substituted 1(3H)-isobenzofuranone. Bioorg Med Chem Lett. 2007;17(18):5210–3. doi: 10.1016/j.bmcl.2007.06.082 17632002.

33. Ma F, Gao Y, Qiao H, Hu X, Chang J. Antiplatelet activity of 3-butyl-6-bromo-1(3H)-isobenzofuranone on rat platelet aggregation. J Thromb Thrombolysis. 2012;33(1):64–73. doi: 10.1007/s11239-011-0647-9 22057435.

34. Cardozo JAB-F, Raimudo; Javier Rincón-Velandia; Guerrero-Pabón, Mario F. 3-Buthyl-isobenzofuranone: a compound isolated from Apium graveolens with anticonvulsant activity. Rev Colomb Ciencias Quim Farm. 2005;34(1):69–76.

35. Teixeira RR, Bressan GC, Pereira WL, Ferreira JG, de Oliveira FM, Thomaz DC. Synthesis and antiproliferative activity of C-3 functionalized isobenzofuran-1(3H)-ones. Molecules. 2013;18(2):1881–96. doi: 10.3390/molecules18021881 23377131.

36. Artico M, Di Santo R, Costi R, Novellino E, Greco G, Massa S, et al. Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling. J Med Chem. 1998;41(21):3948–60. doi: 10.1021/jm9707232 9767632.

37. Goudreau N, Cameron DR, Deziel R, Hache B, Jakalian A, Malenfant E, et al. Optimization and determination of the absolute configuration of a series of potent inhibitors of human papillomavirus type-11 E1-E2 protein-protein interaction: a combined medicinal chemistry, NMR and computational chemistry approach. Bioorg Med Chem. 2007;15(7):2690–700. doi: 10.1016/j.bmc.2007.01.036 17306550.

38. Davidson W, McGibbon GA, White PW, Yoakim C, Hopkins JL, Guse I, et al. Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry. Anal Chem. 2004;76(7):2095–102. doi: 10.1021/ac035335o 15053675.

39. Yoakim C, Ogilvie WW, Goudreau N, Naud J, Hache B, O'Meara JA, et al. Discovery of the first series of inhibitors of human papillomavirus type 11: inhibition of the assembly of the E1-E2-Origin DNA complex. Bioorg Med Chem Lett. 2003;13(15):2539–41. doi: 10.1016/s0960-894x(03)00510-9 12852961.

40. Liu Y, Saldivar A, Bess J, Solomon L, Chen CM, Tripathi R, et al. Investigating the origin of the slow-binding inhibition of HCV NS3 serine protease by a novel substrate based inhibitor. Biochemistry. 2003;42(29):8862–9. doi: 10.1021/bi034661v 12873147.

41. Oliveira A, de Souza APM, de Oliveira AS, da Silva ML, de Oliveira FM, Santos EG, et al. Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease. Eur J Med Chem. 2018;149:98–109. Epub 2018/03/03. doi: 10.1016/j.ejmech.2018.02.037 29499491.

42. Perrin DD, Armarego WLF. Purification of Laboratory Chemicals. 3rd edn ed. Oxford: Pergamon; 1988.

43. da Silva Maia AF, Siqueira RP, de Oliveira FM, Ferreira JG, da Silva SF, Caiuby CAD, et al. Synthesis, molecular properties prediction and cytotoxic screening of 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones. Bioorg Med Chem Lett. 2016;26(12):2810–6. Epub 2016/05/09. doi: 10.1016/j.bmcl.2016.04.065 27155902.

44. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. doi: 10.1016/0022-1759(83)90303-4 6606682.

45. LigPrep SR. 3.4.014 ed. New York: Schrodinger, LLC; 2016.

46. Aleshin AE, Shiryaev SA, Strongin AY, Liddington RC. Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold. Protein Sci. 2007;16(5):795–806. doi: 10.1110/ps.072753207 17400917; PubMed Central PMCID: PMC2206648.

47. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem. 2004;47(7):1739–49. doi: 10.1021/jm0306430 15027865.

48. Schrödinger. Glide. New York: Schrödinger, LLC; 2016.

49. Sherman W, Day T, Jacobson MP, Friesner RA, Farid R. Novel procedure for modeling ligand/receptor induced fit effects. J Med Chem. 2006;49(2):534–53. doi: 10.1021/jm050540c 16420040.

50. Schrödinger. Schrödinger Release. New york: Schrödinger, LLC; 2016.

51. Schrödinger. Maestro. 10.6 ed. New York: Schrödinger, LLC; 2016.

52. Borgati TF, Alves RB, Teixeira RR, Freitas RPd, Perdigão TG, Silva SFd, et al. Synthesis and phytotoxic activity of 1,2,3-triazole derivatives. J Braz Chem Soc. 2013;24:953–61.

53. Balasubramanian A, Manzano M, Teramoto T, Pilankatta R, Padmanabhan R. High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease. Antiviral Res. 2016;134:6–16. doi: 10.1016/j.antiviral.2016.08.014 27539384; PubMed Central PMCID: PMC5065773.

54. Luo D, Vasudevan SG, Lescar J. The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development. Antiviral Res. 2015;118:148–58. doi: 10.1016/j.antiviral.2015.03.014 25842996.

55. Xu M, Lee EM, Wen Z, Cheng Y, Huang WK, Qian X, et al. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat Med. 2016;22(10):1101–7. doi: 10.1038/nm.4184 27571349.

56. Khalilzadeh E, Hazrati R, Saiah GV. Effects of topical and systemic administration of Eugenia caryophyllata buds essential oil on corneal anesthesia and analgesia. Res Pharm Sci. 2016;11(4):293–302. doi: 10.4103/1735-5362.189297 27651809; PubMed Central PMCID: PMC5022377.

57. Kildeaa MA, GL GLA, Kearney RE. Accumulation and clearance of the anaesthetics clove oil and AQUI-S from the edible tissue of silver perch (Bidyanus bidyanus) Aquaculture. 2004;232:265–77.

58. Daniel AN, Sartoretto SM, Schmidt G, Caparroz-Assef SM, Bersani-Amado CA, Cuman RKN. Anti- inflammatory and antinociceptive activities A of eugenol essential oil in experimental animal models. Rev Bras Farmacogn. 2009;19:212–7.

59. Kurian R, Arulmozhi DK, Veeranjaneyulu A, Bodhankar SL. Effect of eugenol on animal models of nociception. Indian J of Pharmacol. 2006;38:341–5.

60. Lionnet L, Beaudry F, Vachon P. Intrathecal eugenol administration alleviates neuropathic pain in male Sprague-Dawley rats. Phytother Res. 2010;24(11):1645–53. doi: 10.1002/ptr.3174 21031622.

61. Ohkubo T, Shibata M. The selective capsaicin antagonist capsazepine abolishes the antinociceptive action of eugenol and guaiacol. J Dent Res. 1997;76(4):848–51. doi: 10.1177/00220345970760040501 9126180.

62. Park SH, Sim YB, Lee JK, Kim SM, Kang YJ, Jung JS, et al. The analgesic effects and mechanisms of orally administered eugenol. Arch Pharm Res. 2011;34(3):501–7. doi: 10.1007/s12272-011-0320-z 21547684.

63. Islamuddin M, Chouhan G, Want MY, Ozbak HA, Hemeg HA, Afrin F. Immunotherapeutic Potential of Eugenol Emulsion in Experimental Visceral Leishmaniasis. PLoS Negl Trop Dis. 2016;10(10):e0005011. doi: 10.1371/journal.pntd.0005011 27776125; PubMed Central PMCID: PMC5077126.

64. Fujisawa S, Murakami Y. Eugenol and Its Role in Chronic Diseases. Adv Exp Med Biol. 2016;929:45–66. doi: 10.1007/978-3-319-41342-6_3 27771920.

65. Mahadlek J, Charoenteeraboon J, Phaechamud T. Zinc Oxide Gels for periodontitis treatment. J Metal Mater Mineral. 2010;20:159–63.

66. Tanaka S, Royds C, Buckley D, Basketter DA, Goossens A, Bruze M, et al. Contact allergy to isoeugenol and its derivatives: problems with allergen substitution. Contact Dermatitis. 2004;51(5–6):288–91. doi: 10.1111/j.0105-1873.2004.00446.x 15606655.

67. Lei J, Hansen G, Nitsche C, Klein CD, Zhang L, Hilgenfeld R. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor. Science. 2016;353(6298):503–5. doi: 10.1126/science.aag2419 27386922.

68. Noble CG, Seh CC, Chao AT, Shi PY. Ligand-bound structures of the dengue virus protease reveal the active conformation. J Virol. 2012;86(1):438–46. doi: 10.1128/JVI.06225-11 22031935; PubMed Central PMCID: PMC3255909.

69. Erbel P, Schiering N, D'Arcy A, Renatus M, Kroemer M, Lim SP, et al. Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol. 2006;13(4):372–3. doi: 10.1038/nsmb1073 16532006.

70. Robin G, Chappell K, Stoermer MJ, Hu SH, Young PR, Fairlie DP, et al. Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conformation. J Mol Biol. 2009;385(5):1568–77. Epub 2008/12/09. doi: 10.1016/j.jmb.2008.11.026 19059417.

71. Hammamy MZ, Haase C, Hammami M, Hilgenfeld R, Steinmetzer T. Development and characterization of new peptidomimetic inhibitors of the West Nile virus NS2B-NS3 protease. ChemMedChem. 2013;8(2):231–41. doi: 10.1002/cmdc.201200497 23307694.

72. Nitsche C, Zhang L, Weigel LF, Schilz J, Graf D, Bartenschlager R, et al. Peptide-Boronic Acid Inhibitors of Flaviviral Proteases: Medicinal Chemistry and Structural Biology. J Med Chem. 2017;60(1):511–6. Epub 2016/12/15. doi: 10.1021/acs.jmedchem.6b01021 27966962.

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