Skip to main content

Advertisement

SpringerLink
Log in

Structure-guided fragment-based in silico drug design of dengue protease inhibitors

  • Published:
Journal of Computer-Aided Molecular Design Aims and scope Submit manuscript

Abstract

An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC50 = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC50 = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Division of Vector-Borne Infectious Disease. Centers for Disease Control and Prevention, Atlanta, USA (2010) http://www.cdc.gov/Dengue/ Accessed 23 Sept 2010

  2. World Health Organization - Dengue (2010) Geneva, Switzerland. http://www.who.int/topics/dengue/en/ Accessed 23 Sept 2010

  3. 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. Antiviral Res 80:94–101

    Article  CAS  Google Scholar 

  4. Luo D, Wei N, Doan DN, Paradkar PN, Chong Y, Davidson AD, Kotaka M, Lescar J, Vasudevan SG (2010) Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications. J Biol Chem 285:18817–18827

    Article  CAS  Google Scholar 

  5. Aleshin AE, Shiryaev SA, Strongin AY, Liddington RC (2007) Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold. Prot Sci 16:795–806

    Article  CAS  Google Scholar 

  6. 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 13:372–373

    Article  CAS  Google Scholar 

  7. Robin G, Chappell K, Stoermer MJ, Hu S, Young PR, Fairlie DP, Martin JL (2009) Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conformation. J Mol Biol 385:1568–1577

    Article  CAS  Google Scholar 

  8. Chandramouli S, Joseph JS, Daudenarde S, Gatchalian J, Cornillez-Ty C, Kuhn P (2010) Serotype-specific structural differences in the protease-cofactor complexes of the dengue virus family. J Virol 84:3059–3067

    Article  CAS  Google Scholar 

  9. Luo D, Xu T, Hunke C, Grüber G, Vasudevan SG, Lescar J (2008) Crystal structure of the NS3 protease-helicase from dengue virus. J Virol 82:173–183

    Article  CAS  Google Scholar 

  10. Su X, 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:e561

    Article  Google Scholar 

  11. Tomlinson SM, Malmstrom RD, Russo A, Mueller N, Pang Y, Watowich SJ (2009) Structure-based discovery of dengue virus protease inhibitors. Antiviral Res 82:110–114

    Article  CAS  Google Scholar 

  12. Yin Z, Patel SJ, Wang W, Wang G, Chan W, Rao KR, 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 16:36–39

    Article  CAS  Google Scholar 

  13. Yin Z, Patel SJ, Wang W, Chan W, Ranga Rao K, 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 16:40–43

    Article  CAS  Google Scholar 

  14. Knox JE, Ma NL, Yin Z, Patel SJ, Wang W, Chan W, 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 49:6585–6590

    Article  CAS  Google Scholar 

  15. Stoermer MJ, Chappell KJ, Liebscher S, Jensen CM, Gan CH, Gupta PK, Xu W, 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 51:5714–5721

    Article  CAS  Google Scholar 

  16. 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 19:5773–5777

    Article  CAS  Google Scholar 

  17. Ekonomiuk D, Su X, Ozawa K, Bodenreider C, Lim SP, Otting G, Huang D, Caflisch A (2009) Flaviviral protease inhibitors identified by fragment-based library docking into a structure generated by molecular dynamics. J Med Chem 52:4860–4868

    Article  CAS  Google Scholar 

  18. Ekonomiuk D, Su X, Ozawa K, Bodenreider C, Lim SP, Yin Z, Keller TH, Beer D, Patel V, Otting G, Caflisch A, Huang D (2009) Discovery of a non-peptidic inhibitor of West Nile virus NS3 protease by high-throughput docking. PLoS Negl Trop Dis 3:e356

    Article  Google Scholar 

  19. Ganesh VK, Muller N, Judge K, Luan C, Padmanabhan R, Murthy KHM (2005) Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases. Bioorg Med Chem 13:257–264

    Article  CAS  Google Scholar 

  20. Leung D, Schroder K, White H, Fang N, Stoermer MJ, Abbenante G, Martin JL, Young PR, Fairlie DP (2001) Activity of recombinant dengue 2 Virus NS3 protease in the presence of a truncated NS2B Co-factor, small peptide substrates, and inhibitors. J Biol Chem 276:45762–45771

    Article  CAS  Google Scholar 

  21. Nall TA, Chappell KJ, Stoermer MJ, Fang N, Tyndall JDA, Young PR, Fairlie DP (2004) Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease. J Biol Chem 279:48535–48542

    Article  CAS  Google Scholar 

  22. Johnston PA, Phillips J, Shun TY, Shinde S, Lazo JS, Huryn DM, Myers MC, Ratnikov BI, Smith JW, Su Y, Dahl R, Cosford NDP, 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 5:737–750

    Article  CAS  Google Scholar 

  23. Chanprapaph S, Saparpakorn P, Sangma C, Niyomrattanakit P, Hannongbua S, Angsuthanasombat C, Katzenmeier G (2005) Competitive inhibition of the dengue virus NS3 serine protease by synthetic peptides representing polyprotein cleavage sites. Biochem Biophys Res Commun 330:1237–1246

    Article  CAS  Google Scholar 

  24. 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 52:3385–3393

    Article  CAS  Google Scholar 

  25. Shiryaev S, Ratnikov B, Chekanov A, Sikora S, Rozanov D, Godzik A, Wang J, Smith J, Huang Z, Lindberg I, Samuel M, Diamond M, Strongin A (2006) Cleavage targets and the d-arginine-based inhibitors of the West Nile virus NS3 processing proteinase. Biochem J 393:503

    Article  CAS  Google Scholar 

  26. Tomlinson SM, Watowich SJ (2008) Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease. Biochemistry 47:11763–11770

    Article  CAS  Google Scholar 

  27. Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27:157–162

    Article  CAS  Google Scholar 

  28. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815

    Article  CAS  Google Scholar 

  29. Marti-Renom MA, Madhusudhan MS, Sali A (2004) Alignment of protein sequences by their profiles. Protein Sci 13:1071–1087

    Article  CAS  Google Scholar 

  30. Shen M, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Sci 15:2507–2524

    Article  CAS  Google Scholar 

  31. DeLano W (2009) The PyMOL molecular graphics system. DeLano Scientific, Palo Alto

    Google Scholar 

  32. Chemical Computing Group (2009) MOE—the molecular operating environment. Montreal, Canada

    Google Scholar 

  33. Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963) Stereochemistry of polypeptide chain configurations. J Mol Biol 7:95–99

    Article  CAS  Google Scholar 

  34. Irwin JJ, Shoichet BK (2005) ZINC—a free database of commercially available compounds for virtual screening. J Chem Inf Model 45:177–182

    Article  CAS  Google Scholar 

  35. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 46:3–26

    Article  CAS  Google Scholar 

  36. Lewell XQ, Judd DB, Watson SP, Hann MM (1998) RECAP—retrosynthetic combinatorial analysis procedure: a powerful new technique for identifying privileged molecular fragments with useful applications in combinatorial chemistry. J Chem Inf Comput Sci 38:511–522

    CAS  Google Scholar 

  37. Congreve M, Carr R, Murray C, Jhoti H (2003) A ‘Rule of Three’ for fragment-based lead discovery? Drug Discov Today 8:876–877

    Article  Google Scholar 

  38. Gasteiger J, Marsili M (1980) Iterative partial equalization of orbital electronegativity - a rapid access to atomic charges. Tetrahedron 36:3219–3228

    Article  CAS  Google Scholar 

  39. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791

    Article  CAS  Google Scholar 

  40. Markley JL, Westler WM (1996) Protonation-state dependence of hydrogen bond strengths and exchange rates in a serine protease catalytic triad: bovine chymotrypsinogen A. Biochem 35:11092–11097

    Article  CAS  Google Scholar 

  41. Wang J, Cieplak P, Kollman PA (2000) How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comput Chem 21:1049–1074

    Article  CAS  Google Scholar 

  42. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461

    CAS  Google Scholar 

  43. Weininger D (1988) SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. J Chem Inf Comput Sci 28:31–36

    CAS  Google Scholar 

  44. Accelrys Inc. (2007) Pipeline Pilot. Accelrys Inc., San Diego

    Google Scholar 

  45. Rogers D, Hahn M (2010) Extended-connectivity fingerprints. J Chem Inf Model 50:742–754

    Article  CAS  Google Scholar 

  46. Tanimoto T (1957) IBM internal report. 1957

  47. Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267:727–748

    Article  CAS  Google Scholar 

  48. Eldridge MD, Murray CW, Auton TR, Paolini GV, Mee RP (1997) Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes. J Comput Aided Mol Des 11:425–445

    Article  CAS  Google Scholar 

  49. Li J, Lim SP, Beer D, Patel V, Wen D, Tumanut C, Tully DC, Williams JA, Jiricek J, Priestle JP, Harris JL, Vasudevan SG (2005) Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries. J Biol Chem 280:28766–28774

    Article  CAS  Google Scholar 

  50. GraphPad Software Inc. (2009) GraphPad prism. GraphPad Software Inc., La Jolla

    Google Scholar 

  51. Wichapong K, Pianwanit S, Sippl W, Kokpol S (2010) Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction. J Mol Recognit 23:283–300

    CAS  Google Scholar 

  52. Chappell KJ (2007) Structure-function relationships of the West Nile virus protease NS3 and its cofactor NS2B. PhD thesis, University of Queensland, Australia

  53. Chappell KJ, Stoermer MJ, Fairlie DP, Young PR (2006) Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies. J Biol Chem 281:38448–38458

    Article  CAS  Google Scholar 

  54. Shiryaev SA, Ratnikov BI, Aleshin AE, Kozlov IA, Nelson NA, Lebl M, Smith JW, Liddington RC, Strongin AY (2007) Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis. J Virol 81:4501–4509

    Article  CAS  Google Scholar 

  55. Kolb P, Caflisch A (2006) Automatic and efficient decomposition of two-dimensional structures of small molecules for fragment-based high-throughput docking. J Med Chem 49:7384–7392

    Article  CAS  Google Scholar 

  56. Majeux N, Scarsi M, Caflisch A (2001) Efficient electrostatic solvation model for protein-fragment docking. Proteins 42:256–268

    Article  CAS  Google Scholar 

  57. Budin N, Majeux N, Caflisch A (2001) Fragment-based flexible ligand docking by evolutionary optimization. Biol Chem 382:1365–1372

    Article  CAS  Google Scholar 

  58. Bemis GW, Murcko MA (1996) The properties of known drugs. 1. Molecular frameworks. J Med Chem 39:2887–2893

    Article  CAS  Google Scholar 

  59. Xu Y, Johnson M (2001) Algorithm for naming molecular equivalence classes represented by labeled pseudographs. J Chem Inf Comput Sci 41:181–185

    CAS  Google Scholar 

  60. Schuffenhauer A, Ertl P, Roggo S, Wetzel S, Koch MA, Waldmann H (2007) The scaffold tree—visualization of the scaffold universe by hierarchical scaffold classification. J Chem Inf Model 47:47–58

    Article  CAS  Google Scholar 

  61. Xu Y, Johnson M (2002) Using molecular equivalence numbers to visually explore structural features that distinguish chemical libraries. J Chem Inf Comput Sci 42:912–926

    CAS  Google Scholar 

  62. Hopkins AL, Groom CR, Alex A (2004) Ligand efficiency: a useful metric for lead selection. Drug Discov Today 9:430–431

    Article  Google Scholar 

Download references

Acknowledgments

We are grateful to Mr. Kok Soon Lai and Ms. Si Fang Wang for expert technical assistance. The National Cancer Institute is thanked for providing three compounds free of charge. Funding was provided by Duke-NUS Signature Research Program (funded by the Agency for Science, Technology and Research, Singapore and the Ministry of Health, Singapore) as a startup grant to SV.

Author information

Authors and Affiliations

  1. Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance and Frankfurt Institute for Advanced Studies, J.W. Goethe-University, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany

    Tim Knehans & Peter Güntert

  2. Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Road, Singapore, 169857, Singapore

    Tim Knehans, Andreas Schüller, Danny N. Doan & Subhash G. Vasudevan

  3. Experimental Therapeutic Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos Level 3, Singapore, 138669, Singapore

    Kassoum Nacro & Jeffrey Hill

  4. Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore, 138671, Singapore

    M. S. Madhusudhan

  5. Institute of Organic Chemistry III/Macromolecular Chemistry, University of Ulm, Albert-Einstein-Allee 11, 89069, Ulm, Germany

    Tanja Weil

Authors
  1. Tim Knehans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Schüller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Danny N. Doan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kassoum Nacro
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter Güntert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. S. Madhusudhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tanja Weil
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Subhash G. Vasudevan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Subhash G. Vasudevan.

Additional information

Tim Knehans and Andreas Schüller have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 198 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Knehans, T., Schüller, A., Doan, D.N. et al. Structure-guided fragment-based in silico drug design of dengue protease inhibitors. J Comput Aided Mol Des 25, 263–274 (2011). https://doi.org/10.1007/s10822-011-9418-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10822-011-9418-0

Keywords

Search

Navigation