Strategies Towards Protease Inhibitors for Emerging Flaviviruses

  • Christoph Nitsche
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1062)


Infections with flaviviruses are a continuing public health threat. In addition to vaccine development and vector control, the search for antiviral agents that alleviate symptoms in patients are of considerable interest. Among others, the flaviviral protease NS2B-NS3 is a promising drug target to inhibit viral replication. Flaviviral proteases share a high degree of structural similarity and substrate-recognition profile, which may facilitate a strategy towards development of pan-flaviviral protease inhibitors. However, the success of various drug discovery attempts during the last decade has been limited by the nature of the viral enzyme as well as a lack of robust structural templates. Small-molecular, structurally diverse protease inhibitors have been reported to reach affinities in the lower micromolar range. Peptide-based, substrate-derived compounds are often nanomolar inhibitors, however, with highly compromised drug-likeness. With some exceptions, the antiviral cellular activity of most of the reported compounds have been patchy and insufficient for further development. Recent progress has been made in the elucidation of inhibitor binding using different structural methods. This will hopefully lead to more rational attempts for the identification of various lead compounds that may be successful in cellular assays, animal models and ultimately translated to patients.


Protease Inhibitor Peptides Small-molecular Flavivirus Dengue West Nile Zika 



Funding by the Alexander von Humboldt Foundation is gratefully acknowledged.


  1. 1.
    Balasubramanian A, Manzano M, Teramoto T, Pilankatta R, Padmanabhan R (2016) High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease. Antivir Res 134:6–16CrossRefPubMedGoogle Scholar
  2. 2.
    Bastos Lima A, Behnam MA, El Sherif Y, Nitsche C, Vechi SM, Klein CD (2015) Dual inhibitors of the dengue and West Nile virus NS2B-NS3 proteases: synthesis, biological evaluation and docking studies of novel peptide-hybrids. Bioorg Med Chem 23(17):5748–5755CrossRefPubMedGoogle Scholar
  3. 3.
    Behnam MA, Graf D, Bartenschlager R, Zlotos DP, Klein CD (2015) Discovery of nanomolar dengue and West Nile virus protease inhibitors containing a 4-benzyloxyphenylglycine residue. J Med Chem 58(23):9354–9370CrossRefPubMedGoogle Scholar
  4. 4.
    Behnam MA, Nitsche C, Boldescu V, Klein CD (2016) The medicinal chemistry of dengue virus. J Med Chem 59(12):5622–5649CrossRefPubMedGoogle Scholar
  5. 5.
    Behnam MA, Nitsche C, Vechi SM, Klein CD (2014) C-terminal residue optimization and fragment merging: discovery of a potent peptide-hybrid inhibitor of dengue protease. ACS Med Chem Lett 5(9):1037–1042CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bodenreider C, Beer D, Keller TH, Sonntag S, Wen D, Yap L, Yau YH, Shochat SG, Huang D, Zhou T, Caflisch A, Su X-C, Ozawa K, Otting G, Vasudevan SG, Lescar J, Lim SP (2009) A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease. Anal Biochem 395(2):195–204CrossRefPubMedGoogle Scholar
  7. 7.
    Castanha PMS, Nascimento EJM, Braga C, Cordeiro MT, de Carvalho OV, de Mendonça LR, Azevedo EAN, França RFO, Dhalia R, Marques ETA (2017) Dengue virus–specific antibodies enhance Brazilian Zika virus infection. J Infect Dis 215(5):781–785PubMedGoogle Scholar
  8. 8.
    Chan JF-W, Chik KK-H, Yuan S, Yip CC-Y, Zhu Z, Tee K-M, Tsang JO-L, Chan CC-S, Poon VK-M, Lu G, Zhang AJ, Lai K-K, Chan K-H, Kao RY-T, Yuen K-Y (2017) Novel antiviral activity and mechanism of bromocriptine as a Zika virus NS2B-NS3 protease inhibitor. Antivir Res 141:29–37CrossRefPubMedGoogle Scholar
  9. 9.
    Chappell KJ, Stoermer MJ, Fairlie DP, Young PR (2008) West Nile virus NS2B/NS3 protease as an antiviral target. Curr Med Chem 15(27):2771–2784CrossRefPubMedGoogle Scholar
  10. 10.
    Chen WN, Nitsche C, Pilla KB, Graham B, Huber T, Klein CD, Otting G (2016) Sensitive NMR approach for determining the binding mode of tightly binding ligand molecules to protein targets. J Am Chem Soc 138(13):4539–4546CrossRefPubMedGoogle Scholar
  11. 11.
    Chen X, Yang K, Wu C, Chen C, Hu C, Buzovetsky O, Wang Z, Ji X, Xiong Y, Yang H (2016) Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease. Cell Res 26(11):1260–1263CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    de la Cruz L, Chen WN, Graham B, Otting G (2014) Binding mode of the activity-modulating C-terminal segment of NS2B to NS3 in the dengue virus NS2B-NS3 protease. FEBS J 281(6):1517–1533CrossRefPubMedGoogle Scholar
  13. 13.
    Dejnirattisai W, Supasa P, Wongwiwat W, Rouvinski A, Barba-Spaeth G, Duangchinda T, Sakuntabhai A, Cao-Lormeau VM, Malasit P, Rey FA, Mongkolsapaya J, Screaton GR (2016) Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with Zika virus. Nat Immunol 17(9):1102–1108CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Erbel P, Schiering N, D’Arcy A, Renatus M, Kroemer M, Lim SP, Yin Z, Keller TH, Vasudevan SG, Hommel U (2006) Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 13(4):372–373CrossRefPubMedGoogle Scholar
  15. 15.
    Ezgimen M, Lai H, Mueller NH, Lee K, Cuny G, Ostrov DA, Padmanabhan R (2012) Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease. Antivir Res 94(1):18–24CrossRefPubMedGoogle Scholar
  16. 16.
    Gupta G, Lim L, Song J (2015) NMR and MD studies reveal that the isolated dengue NS3 protease is an intrinsically disordered chymotrypsin fold which absolutely requests NS2B for correct folding and functional dynamics. PLoS One 10(8):e0134823CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hammamy MZ, Haase C, Hammami M, Hilgenfeld R, Steinmetzer T (2013) Development and characterization of new peptidomimetic inhibitors of the West Nile virus NS2B-NS3 protease. ChemMedChem 8(2):231–241CrossRefPubMedGoogle Scholar
  18. 18.
    Johnston PA, Phillips J, Shun TY, Shinde S, Lazo JS, Huryn DM, Myers MC, Ratnikov BI, Smith JW, Su Y, Dahl R, Cosford ND, Shiryaev SA, Strongin AY (2007) HTS identifies novel and specific uncompetitive inhibitors of the two-component NS2B-NS3 proteinase of West Nile virus. Assay Drug Dev Technol 5(6):737–750CrossRefPubMedGoogle Scholar
  19. 19.
    Kato F, Ishida Y, Oishi S, Fujii N, Watanabe S, Vasudevan SG, Tajima S, Takasaki T, Suzuki Y, Ichiyama K, Yamamoto N, Yoshii K, Takashima I, Kobayashi T, Miura T, Igarashi T, Hishiki T (2016) Novel antiviral activity of bromocriptine against dengue virus replication. Antivir Res 131:141–147CrossRefPubMedGoogle Scholar
  20. 20.
    Knehans T, Schuller A, Doan DN, Nacro K, Hill J, Guntert P, Madhusudhan MS, Weil T, Vasudevan SG (2011) Structure-guided fragment-based in silico drug design of dengue protease inhibitors. J Comput Aided Mol Des 25(3):263–274CrossRefPubMedGoogle Scholar
  21. 21.
    Knox JE, Ma NL, Yin Z, Patel SJ, Wang WL, Chan WL, Ranga Rao KR, Wang G, Ngew X, Patel V, Beer D, Lim SP, Vasudevan SG, Keller TH (2006) Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors. J Med Chem 49(22):6585–6590CrossRefPubMedGoogle Scholar
  22. 22.
    Koh-Stenta X, Joy J, Wang SF, Kwek PZ, Wee JL, Wan KF, Gayen S, Chen AS, Kang C, Lee MA, Poulsen A, Vasudevan SG, Hill J, Nacro K (2015) Identification of covalent active site inhibitors of dengue virus protease. Drug Des Devel Ther 9:6389–6399CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Kuiper BD, Slater K, Spellmon N, Holcomb J, Medapureddy P, Muzzarelli KM, Yang Z, Ovadia R, Amblard F, Kovari IA, Schinazi RF, Kovari LC (2017) Increased activity of unlinked Zika virus NS2B/NS3 protease compared to linked Zika virus protease. Biochem Biophys Res Commun 492:668–673.CrossRefPubMedGoogle Scholar
  24. 24.
    Lee H, Ren J, Nocadello S, Rice AJ, Ojeda I, Light S, Minasov G, Vargas J, Nagarathnam D, Anderson WF, Johnson ME (2017) Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus. Antivir Res 139:49–58CrossRefPubMedGoogle Scholar
  25. 25.
    Lei J, Hansen G, Nitsche C, Klein CD, Zhang L, Hilgenfeld R (2016) Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor. Science 353(6298):503–505CrossRefPubMedGoogle Scholar
  26. 26.
    Lescar J, Luo D, Xu T, Sampath A, Lim SP, Canard B, Vasudevan SG (2008) Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from dengue virus as a target. Antivir Res 80(2):94–101CrossRefPubMedGoogle Scholar
  27. 27.
    Lim SP, Shi PY (2013) West Nile virus drug discovery. Virus 5(12):2977–3006CrossRefGoogle Scholar
  28. 28.
    Lim SP, Wang QY, Noble CG, Chen YL, Dong H, Zou B, Yokokawa F, Nilar S, Smith P, Beer D, Lescar J, Shi PY (2013) Ten years of dengue drug discovery: progress and prospects. Antivir Res 100(2):500–519CrossRefPubMedGoogle Scholar
  29. 29.
    Liu H, Wu R, Sun Y, Ye Y, Chen J, Luo X, Shen X, Liu H (2014) Identification of novel thiadiazoloacrylamide analogues as inhibitors of dengue-2 virus NS2B/NS3 protease. Bioorg Med Chem 22(22):6344–6352CrossRefPubMedGoogle Scholar
  30. 30.
    Mahawaththa MC, Pearce BJG, Szabo M, Graham B, Klein CD, Nitsche C, Otting G (2017) Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease. Antivir Res 142:141–147CrossRefPubMedGoogle Scholar
  31. 31.
    Mueller NH, Pattabiraman N, Ansarah-Sobrinho C, Viswanathan P, Pierson TC, Padmanabhan R (2008) Identification and biochemical characterization of small-molecule inhibitors of West Nile virus serine protease by a high-throughput screen. Antimicrob Agents Chemother 52(9):3385–3393CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Nitsche C, Behnam MA, Steuer C, Klein CD (2012) Retro peptide-hybrids as selective inhibitors of the dengue virus NS2B-NS3 protease. Antivir Res 94(1):72–79CrossRefPubMedGoogle Scholar
  33. 33.
    Nitsche C, Holloway S, Schirmeister T, Klein CD (2014) Biochemistry and medicinal chemistry of the dengue virus protease. Chem Rev 114(22):11348–11381CrossRefPubMedGoogle Scholar
  34. 34.
    Nitsche C, Otting G (2017) Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags. Prog Nucl Magn Reson Spectrosc 98–99:20–49CrossRefPubMedGoogle Scholar
  35. 35.
    Nitsche C, Schreier VN, Behnam MA, Kumar A, Bartenschlager R, Klein CD (2013) Thiazolidinone-peptide hybrids as dengue virus protease inhibitors with antiviral activity in cell culture. J Med Chem 56(21):8389–8403CrossRefPubMedGoogle Scholar
  36. 36.
    Nitsche C, Zhang L, Weigel LF, Schilz J, Graf D, Bartenschlager R, Hilgenfeld R, Klein CD (2017) Peptide–boronic acid inhibitors of flaviviral proteases: medicinal chemistry and structural biology. J Med Chem 60(1):511–516CrossRefPubMedGoogle Scholar
  37. 37.
    Noble CG, Seh CC, Chao AT, Shi PY (2012) Ligand-bound structures of the dengue virus protease reveal the active conformation. J Virol 86(1):438–446CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Phoo WW, Li Y, Zhang Z, Lee MY, Loh YR, Tan YB, Ng EY, Lescar J, Kang C, Luo D (2016) Structure of the NS2B-NS3 protease from Zika virus after self-cleavage. Nat Commun 7:13410CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Priyamvada L, Quicke KM, Hudson WH, Onlamoon N, Sewatanon J, Edupuganti S, Pattanapanyasat K, Chokephaibulkit K, Mulligan MJ, Wilson PC, Ahmed R, Suthar MS, Wrammert J (2016) Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proc Natl Acad Sci U S A 113(28):7852–7857CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Prusis P, Junaid M, Petrovska R, Yahorava S, Yahorau A, Katzenmeier G, Lapins M, Wikberg JES (2013) Design and evaluation of substrate-based octapeptide and non substrate-based tetrapeptide inhibitors of dengue virus NS2B–NS3 proteases. Biochem Biophys Res Commun 434(4):767–772CrossRefPubMedGoogle Scholar
  41. 41.
    Schüller A, Yin Z, Brian Chia CS, Doan DN, Kim HK, Shang L, Loh TP, Hill J, Vasudevan SG (2011) Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease. Antivir Res 92(1):96–101CrossRefPubMedGoogle Scholar
  42. 42.
    Shiryaev SA, Cheltsov AV, Gawlik K, Ratnikov BI, Strongin AY (2011) Virtual ligand screening of the National Cancer Institute (NCI) compound library leads to the allosteric inhibitory scaffolds of the West Nile virus NS3 proteinase. Assay Drug Dev Technol 9(1):69–78CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Shiryaev SA, Farhy C, Pinto A, Huang CT, Simonetti N, Ngono AE, Dewing A, Shresta S, Pinkerton AB, Cieplak P, Strongin AY, Terskikh AV (2017) Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists. Antivir Res 143:218–229CrossRefPubMedGoogle Scholar
  44. 44.
    Shiryaev SA, Ratnikov BI, Chekanov AV, Sikora S, Rozanov DV, Godzik A, Wang J, Smith JW, Huang Z, Lindberg I, Samuel MA, Diamond MS, Strongin AY (2006) Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase. Biochem J 393(Pt 2):503–511CrossRefPubMedGoogle Scholar
  45. 45.
    Sidique S, Shiryaev SA, Ratnikov BI, Herath A, Su Y, Strongin AY, Cosford ND (2009) Structure-activity relationship and improved hydrolytic stability of pyrazole derivatives that are allosteric inhibitors of West Nile virus NS2B-NS3 proteinase. Bioorg Med Chem Lett 19(19):5773–5777CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Stoermer MJ, Chappell KJ, Liebscher S, Jensen CM, Gan CH, Gupta PK, Xu WJ, Young PR, Fairlie DP (2008) Potent cationic inhibitors of West Nile virus NS2B/NS3 protease with serum stability, cell permeability and antiviral activity. J Med Chem 51(18):5714–5721CrossRefPubMedGoogle Scholar
  47. 47.
    Su XC, Ozawa K, Qi R, Vasudevan SG, Lim SP, Otting G (2009) NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile virus NS2B-NS3 protease. PLoS Negl Trop Dis 3(12):e561CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Takagi Y, Matsui K, Nobori H, Maeda H, Sato A, Kurosu T, Orba Y, Sawa H, Hattori K, Higashino K, Numata Y, Yoshida Y (2017) Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity. Bioorg Med Chem Lett 27:3586–3590.CrossRefPubMedGoogle Scholar
  49. 49.
    Weigel LF, Nitsche C, Graf D, Bartenschlager R, Klein CD (2015) Phenylalanine and phenylglycine analogues as arginine mimetics in dengue protease inhibitors. J Med Chem 58(19):7719–7733CrossRefPubMedGoogle Scholar
  50. 50.
    Weng Z, Shao X, Graf D, Wang C, Klein CD, Wang J, Zhou GC (2016) Identification of fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors. Eur J Med Chem 125:751–759CrossRefPubMedGoogle Scholar
  51. 51.
    Wu H, Bock S, Snitko M, Berger T, Weidner T, Holloway S, Kanitz M, Diederich WE, Steuber H, Walter C, Hofmann D, Weissbrich B, Spannaus R, Acosta EG, Bartenschlager R, Engels B, Schirmeister T, Bodem J (2015) Novel dengue virus NS2B/NS3 protease inhibitors. Antimicrob Agents Chemother 59(2):1100–1109CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Yang CC, Hsieh YC, Lee SJ, Wu SH, Liao CL, Tsao CH, Chao YS, Chern JH, Wu CP, Yueh A (2011) Novel dengue virus-specific NS2B/NS3 protease inhibitor, BP2109, discovered by a high-throughput screening assay. Antimicrob Agents Chemother 55(1):229–238CrossRefPubMedGoogle Scholar
  53. 53.
    Yang CC, Hu HS, Wu RH, Wu SH, Lee SJ, Jiaang WT, Chern JH, Huang ZS, Wu HN, Chang CM, Yueh A (2014) A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease. Antimicrob Agents Chemother 58(1):110–119CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Yin Z, Patel SJ, Wang W-L, Chan W-L, Ranga Rao KR, Wang G, Ngew X, Patel V, Beer D, Knox JE, Ma NL, Ehrhardt C, Lim SP, Vasudevan SG, Keller TH (2006) Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors. Bioorg Med Chem Lett 16(1):40–43CrossRefPubMedGoogle Scholar
  55. 55.
    Yin Z, Patel SJ, Wang W-L, Wang G, Chan W-L, Rao KRR, Alam J, Jeyaraj DA, Ngew X, Patel V, Beer D, Lim SP, Vasudevan SG, Keller TH (2006) Peptide inhibitors of dengue virus NS3 protease. Part 1: warhead. Bioorg Med Chem Lett 16(1):36–39CrossRefPubMedGoogle Scholar
  56. 56.
    Zhang Z, Li Y, Loh YR, Phoo WW, Hung AW, Kang C, Luo D (2016) Crystal structure of unlinked NS2B-NS3 protease from Zika virus. Science 354(6319):1597–1600CrossRefPubMedGoogle Scholar
  57. 57.
    Zhou GC, Weng Z, Shao X, Liu F, Nie X, Liu J, Wang D, Wang C, Guo K (2013) Discovery and SAR studies of methionine-proline anilides as dengue virus NS2B-NS3 protease inhibitors. Bioorg Med Chem Lett 23(24):6549–6554CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Research School of ChemistryThe Australian National UniversityCanberraAustralia

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