Citation: Matthew Brecher, Jing Zhang, Hongmin Li. The Flavivirus Protease As a Target for Drug Discovery .VIROLOGICA SINICA, 2013, 28(6) : 326-336.  http://dx.doi.org/10.1007/s12250-013-3390-x

The Flavivirus Protease As a Target for Drug Discovery

  • Corresponding author: Hongmin Li, lih@wadsworth.org
  • Received Date: 08 October 2013
    Accepted Date: 01 November 2013
    Published Date: 14 November 2013
    Available online: 01 December 2013
  • Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.

  • 加载中
    1. Ackermann M, and Padmanabhan R. 2001. De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. J Biol Chem, 276: 39926-39937.
        doi: 10.1074/jbc.M104248200

    2. Aleshin A, Shiryaev S, Strongin A, and Liddington R. 2007. Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold. Protein Sci., 16: 795-806.
        doi: 10.1110/ps.072753207

    3. Aravapalli S, Lai H, Teramoto T, Alliston K R, Lushington G H, Ferguson E L, Padmanabhan R, and Groutas W C. 2012. Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold. Bioorg Med Chem, 20: 4140-4148.
        doi: 10.1016/j.bmc.2012.04.055

    4. Arias C F, Preugschat F, and Strauss J H. 1993. Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology, 193: 888-899.
        doi: 10.1006/viro.1993.1198

    5. Ashour J, Laurent-Rolle M, Shi P Y, and Garcia-Sastre A. 2009. NS5 of dengue virus mediates STAT2 binding and degradation. J Virol, 83: 5408-5418.
        doi: 10.1128/JVI.02188-08

    6. Asnis D S, Conetta R, Waldman G, and Teixeira A A. 2001. The West Nile virus encephalitis outbreak in the United States (1999-2000): from Flushing, New York, to beyond its borders. Ann N Y Acad Sci, 951: 161-171.

    7. Asnis D S, Conetta R, Teixeira A A, Waldman G, and Sampson B A. 2000. The West Nile Virus outbreak of 1999 in New York: the Flushing Hospital experience. Clin Infect Dis, 30: 413-418.
        doi: 10.1086/313737

    8. Assenberg R, Mastrangelo E, Walter T S, Verma A, Milani M, Owens R J, Stuart D I, Grimes J M, and Mancini E J. 2009. Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol, 83: 12895-12906.
        doi: 10.1128/JVI.00942-09

    9. Bazan J F, and Fletterick R J. 1989. Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology, 171: 637-639.
        doi: 10.1016/0042-6822(89)90639-9

    10. Best S M, Morris K L, Shannon J G, Robertson S J, Mitzel D N, Park G S, Boer E, Wolfinbarger J B, and Bloom M E. 2005. Inhibition of interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist. J. Virol., 79: 12828-12839.
        doi: 10.1128/JVI.79.20.12828-12839.2005

    11. Bodenreider C, Beer D, Keller T H, Sonntag S, Wen D, Yap L, Yau Y H, Shochat S G, Huang D, Zhou T, Caflisch A, Su X C, Ozawa K, Otting G, Vasudevan S G, Lescar J, and Lim S P. 2009. A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease. Anal Biochem, 395: 195-204.
        doi: 10.1016/j.ab.2009.08.013

    12. Bressanelli S, Stiasny K, Allison S L, Stura E A, Duquerroy S, Lescar J, Heinz F X, and Rey F A. 2004. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J., 23: 728-738.
        doi: 10.1038/sj.emboj.7600064

    13. Brinton M A. 1981. Isolation of a replication-efficient mutant of West Nile virus from a persistently infected genetically resistant mouse cell culture. J Virol, 39: 413-421.

    14. Brinton M A. 2002. THE MOLECULAR BIOLOGY OF WEST NILE VIRUS: A New Invader of the Western Hemisphere. Annu Rev Microbiol, 56: 371-402.
        doi: 10.1146/annurev.micro.56.012302.160654

    15. Burke D S, and Monath T P. 2001. Flaviviruses. Lippincott William & Wilkins.

    16. CDC. 2010. CDC West Nile virus homepage. http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount03.htm.

    17. Chambers T J, Grakoui A, and Rice C M. 1991. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J. Virol., 65: 6042-6050.

    18. Chambers T J, Hahn C S, Galler R, and Rice C M. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol, 44: 649-688.
        doi: 10.1146/annurev.mi.44.100190.003245

    19. Chambers T J, Nestorowicz A, Amberg S M, and Rice C M. 1993. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J Virol, 67: 6797-6807.

    20. Chambers T J, Droll D A, Tang Y, Liang Y, Ganesh V K, Murthy K H, and Nickells M. 2005. Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities. J Gen Virol, 86: 1403-1413.
        doi: 10.1099/vir.0.80427-0

    21. Chandramouli S, Joseph J S, Daudenarde S, Gatchalian J, Cornillez-Ty C, and Kuhn P. 2010. Serotype-specific structural differences in the protease-cofactor complexes of the dengue virus family. J Virol, 84: 3059-3067.
        doi: 10.1128/JVI.02044-09

    22. Chanprapaph S, Saparpakorn P, Sangma C, Niyomrattanakit P, Hannongbua S, Angsuthanasombat C, and 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.
        doi: 10.1016/j.bbrc.2005.03.107

    23. Chappell K J, Stoermer M J, Fairlie D P, and Young P R. 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.
        doi: 10.1074/jbc.M607641200

    24. Chappell K J, Stoermer M J, Fairlie D P, and Young P R. 2008. West Nile Virus NS2B/NS3 protease as an antiviral target. Curr Med Chem, 15: 2771-2784.
        doi: 10.2174/092986708786242804

    25. Chappell K J, Stoermer M J, Fairlie D P, and Young P R. 2008. Mutagenesis of the West Nile virus NS2B cofactor domain reveals two regions essential for protease activity. J Gen Virol, 89: 1010-1014.
        doi: 10.1099/vir.0.83447-0

    26. Cleaves G R, and Dubin D T. 1979. Methylation status of intracellular dengue type 2 40 S RNA. Virology, 96: 159-165.
        doi: 10.1016/0042-6822(79)90181-8

    27. Cregar-Hernandez L, Jiao G S, Johnson A T, Lehrer A T, Wong T A, and Margosiak S A. 2011. Small molecule pan-dengue and West Nile virus NS3 protease inhibitors. Antivir Chem Chemother, 21: 209-217.
        doi: 10.3851/IMP1767

    28. Daffis S, Szretter K J, Schriewer J, Li J, Youn S, Errett J, Lin T Y, Schneller S, Zust R, Dong H, Thiel V, Sen G C, Fensterl V, Klimstra W B, Pierson T C, Buller R M, Gale M, Jr., Shi P Y, and Diamond M S. 2010. 2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature, 468: 452-456.
        doi: 10.1038/nature09489

    29. Deng J, Li N, Liu H, Zuo Z, Liew O W, Xu W, Chen G, Tong X, Tang W, Zhu J, Zuo J, Jiang H, Yang C G, Li J, and Zhu W. 2012. Discovery of novel small molecule inhibitors of dengue viral NS2B-NS3 protease using virtual screening and scaffold hopping. J Med Chem, 55: 6278-6293.
        doi: 10.1021/jm300146f

    30. Dong H, Chang D C, Hua M H, Lim S P, Chionh Y H, Hia F, Lee Y H, Kukkaro P, Lok S M, Dedon P C, and Shi P Y. 2012. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase. PLoS pathogens, 8: e1002642.
        doi: 10.1371/journal.ppat.1002642

    31. Egloff M P, Benarroch D, Selisko B, Romette J L, and Canard B. 2002. An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. Embo J, 21: 2757-2768.
        doi: 10.1093/emboj/21.11.2757

    32. Ekonomiuk D, Su X C, Ozawa K, Bodenreider C, Lim S P, Otting G, Huang D, and 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.
        doi: 10.1021/jm900448m

    33. Ekonomiuk D, Su X C, Ozawa K, Bodenreider C, Lim S P, Yin Z, Keller T H, Beer D, Patel V, Otting G, Caflisch A, and 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.
        doi: 10.1371/journal.pntd.0000356

    34. Erbel P, Schiering N, D'Arcy A, Renatus M, Kroemer M, Lim S, Yin Z, Keller T, Vasudevan S, and 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.
        doi: 10.1038/nsmb1073

    35. Ezgimen M, Lai H, Mueller N H, Lee K, Cuny G, Ostrov D A, and Padmanabhan R. 2012. Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease. Antiviral Res, 94: 18-24.
        doi: 10.1016/j.antiviral.2012.02.003

    36. Falgout B, Miller R H, and Lai C J. 1993. Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity. J Virol, 67: 2034-2042.

    37. Falgout B, Bray M, Schlesinger J J, and Lai C J. 1990. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J Virol, 64: 4356-4363.

    38. Falgout B, Pethel M, Zhang Y M, and Lai C J. 1991. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol, 65: 2467-2475.

    39. Ganesh V K, Muller N, Judge K, Luan C H, Padmanabhan R, and Murthy K H. 2005. Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases. Bioorg Med Chem, 13: 257-264.
        doi: 10.1016/j.bmc.2004.09.036

    40. Gao Y, Cui T, and Lam Y. 2010. Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease. Bioorg Med Chem, 18: 1331-1336.
        doi: 10.1016/j.bmc.2009.12.026

    41. Gao Y, Samanta S, Cui T, and Lam Y. 2013. Synthesis and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 1, 3, 4, 5-Tetrasubstituted 1H-Pyrrol-2(5H)-one Scaffold. ChemMedChem, 8: 1554-1560.
        doi: 10.1002/cmdc.201300244

    42. Gouvea I E, Izidoro M A, Judice W A, Cezari M H, Caliendo G, Santagada V, dos Santos C N, Queiroz M H, Juliano M A, Young P R, Fairlie D P, and Juliano L. 2007. Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates. Arch Biochem Biophys, 457: 187-196.
        doi: 10.1016/j.abb.2006.11.005

    43. Grant D, Tan G K, Qing M, Ng J K, Yip A, Zou G, Xie X, Yuan Z, Schreiber M J, Schul W, Shi P Y, and Alonso S. 2011. A Single Amino Acid in Nonstructural Protein NS4B Confers Virulence to Dengue Virus in AG129 Mice through Enhancement of Viral RNA Synthesis. J Virol, 85: 7775-7787.
        doi: 10.1128/JVI.00665-11

    44. Guirakhoo F, Bolin R A, and Roehrig J T. 1992. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology, 191: 921-931.
        doi: 10.1016/0042-6822(92)90267-S

    45. Guo J, Hayashi J, and Seeger C. 2005. West nile virus inhibits the signal transduction pathway of alpha interferon. J. Virol., 79: 1343-1350.
        doi: 10.1128/JVI.79.3.1343-1350.2005

    46. Guyatt K J, Westaway E G, and Khromykh A A. 2001. Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin.J Virol Methods, 92: 37-44.
        doi: 10.1016/S0166-0934(00)00270-6

    47. Hammamy M Z, Haase C, Hammami M, Hilgenfeld R, and Steinmetzer T. 2013. Development and characterization of new peptidomimetic inhibitors of the West Nile virus NS2B-NS3 protease. ChemMedChem, 8: 231-241.
        doi: 10.1002/cmdc.201200497

    48. Issur M, Geiss B J, Bougie I, Picard-Jean F, Despins S, Mayette J, Hobdey S E, and Bisaillon M. 2009. The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure. Rna, 15: 2340-2350.
        doi: 10.1261/rna.1609709

    49. Jia F, Zou G, Fan J, and Yuan Z. 2010. Identification of palmatine as an inhibitor of West Nile virus. Arch Virol, 155: 1325-1329.
        doi: 10.1007/s00705-010-0702-4

    50. Johnston P A, Phillips J, Shun T Y, Shinde S, Lazo J S, Huryn D M, Myers M C, Ratnikov B I, Smith J W, Su Y, Dahl R, Cosford N D, Shiryaev S A, and Strongin A Y. 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.
        doi: 10.1089/adt.2007.101

    51. Kiat T S, Pippen R, Yusof R, Ibrahim H, Khalid N, and Rahman N A. 2006. Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease. Bioorg Med Chem Lett, 16: 3337-3340.

    52. Knehans T, Schuller A, Doan D N, Nacro K, Hill J, Guntert P, Madhusudhan M S, Weil T, and Vasudevan S G. 2011. Structure-guided fragment-based in silico drug design of dengue protease inhibitors. J Comput Aided Mol Des, 25: 263-274.
        doi: 10.1007/s10822-011-9418-0

    53. Knox J E, Ma N L, Yin Z, Patel S J, Wang W L, Chan W L, Ranga Rao K R, Wang G, Ngew X, Patel V, Beer D, Lim S P, Vasudevan S G, and Keller T H. 2006. Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors. J Med Chem, 49: 6585-6590.
        doi: 10.1021/jm0607606

    54. Koonin E V. 1993. Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. J Gen Virol, 74: 733-740.
        doi: 10.1099/0022-1317-74-4-733

    55. Kramer L D, and Bernard K A. 2001. West Nile virus infection in birds and mammals. Ann N Y Acad Sci, 951: 84-93.

    56. Kramer L D, Li J, and Shi P Y. 2007. West Nile virus. Lancet Neurol, 6: 171-181.
        doi: 10.1016/S1474-4422(07)70030-3

    57. Kummerer B M, and Rice C M. 2002. Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J. Virol., 76: 4773-4784.
        doi: 10.1128/JVI.76.10.4773-4784.2002

    58. Lai H, Sridhar Prasad G, and Padmanabhan R. 2013. Characterization of 8-hydroxyquinoline derivatives containing aminobenzothiazole as inhibitors of dengue virus type 2 protease in vitro. Antiviral Res, 97: 74-80.
        doi: 10.1016/j.antiviral.2012.10.009

    59. Lai H, Dou D, Aravapalli S, Teramoto T, Lushington G H, Mwania T M, Alliston K R, Eichhorn D M, Padmanabhan R, and Groutas W C. 2013. Design, synthesis and characterization of novel 1, 2-benzisothiazol-3(2H)-one and 1, 3, 4-oxadiazole hybrid derivatives: potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases. Bioorg Med Chem, 21: 102-113.
        doi: 10.1016/j.bmc.2012.10.058

    60. Leung D, Schroder K, White H, Fang N-X, Stoermer M, Abbenante G, Martin J, PR Y, and Fairlie D. 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.
        doi: 10.1074/jbc.M107360200

    61. Leung J Y, Pijlman G P, Kondratieva N, Hyde J, Mackenzie J M, and Khromykh A A. 2008. Role of nonstructural protein NS2A in flavivirus assembly. J Virol, 82: 4731-4741.
        doi: 10.1128/JVI.00002-08

    62. Li H, Clum S, You S, Ebner K E, and Padmanabhan R. 1999. The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. J Virol, 73: 3108-3116.

    63. Li J, Lim S P, Beer D, Patel V, Wen D, Tumanut C, Tully D C, Williams J A, Jiricek J, Priestle J P, Harris J L, and Vasudevan S G. 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.
        doi: 10.1074/jbc.M500588200

    64. Li L, Lok S M, Yu I M, Zhang Y, Kuhn R J, Chen J, and Rossmann M G. 2008. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science, 319: 1830-1834.
        doi: 10.1126/science.1153263

    65. Lin C, Kwong A D, and Perni R B. 2006. Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3.4A serine protease. Infect Disord Drug Targets, 6: 3-16.

    66. Lin K, Perni R B, Kwong A D, and Lin C. 2006. VX-950, a novel hepatitis C virus (HCV) NS3-4A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells. Antimicrob Agents Chemother, 50: 1813-1822.
        doi: 10.1128/AAC.50.5.1813-1822.2006

    67. Lindenbach B D, and Rice C M. 1997. trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J. Virol., 71: 9608-9617.

    68. Lindenbach B D, and Rice C M. 1999. Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J Virol, 73: 4611-4621.

    69. Lindenbach B D, Thiel H J, and Rice C M. 2007. Flaviviridae: The Virus and Their Replication, Fourth ed. Lippincott William & Wilkins.

    70. Luo D, Xu T, Hunke C, Gruber G, Vasudevan S G, and Lescar J. 2008. Crystalstructure of the NS3 protease-helicase from dengue virus. J Virol, 82: 173-183.
        doi: 10.1128/JVI.01788-07

    71. Luo D, Wei N, Doan D N, Paradkar P N, Chong Y, Davidson A D, Kotaka M, Lescar J, and Vasudevan S G. 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.
        doi: 10.1074/jbc.M109.090936

    72. Luo D, Xu T, Watson R P, Scherer-Becker D, Sampath A, Jahnke W, Yeong S S, Wang C H, Lim S P, Strongin A, Vasudevan S G, and Lescar J. 2008. Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein. Embo J, 27: 3209-3219.
        doi: 10.1038/emboj.2008.232

    73. Mangano D T, Tudor I C, and Dietzel C. 2006. The risk associated with aprotinin in cardiac surgery. N Engl J Med, 354: 353-365.
        doi: 10.1056/NEJMoa051379

    74. Mangano D T, Miao Y, Vuylsteke A, Tudor I C, Juneja R, Filipescu D, Hoeft A, Fontes M L, Hillel Z, Ott E, Titov T, Dietzel C, and Levin J. 2007. Mortality associated with aprotinin during 5 years following coronary artery bypass graft surgery. JAMA, 297: 471-479.
        doi: 10.1001/jama.297.5.471

    75. Marianneau P, Steffan A M, Royer C, Drouet M T, Jaeck D, Kirn A, and Deubel V. 1999. Infection of primary cultures of human Kupffer cells by Dengue virus: no viral progeny synthesis, but cytokine production is evident. J Virol, 73: 5201-5206.

    76. Menendez-Arias L. 2010. Molecular basis of human immunodeficiency virus drug resistance: an update. Antiviral Res, 85: 210-231.
        doi: 10.1016/j.antiviral.2009.07.006

    77. Miller S, Kastner S, Krijnse-Locker J, Buhler S, and Bartenschlager R. 2007. The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner. J Biol Chem, 282: 8873-8882.
        doi: 10.1074/jbc.M609919200

    78. Modis Y, Ogata S, Clements D, and Harrison S C. 2004. Structure of the dengue virus envelope protein after membrane fusion. Nature, 427: 313-319.
        doi: 10.1038/nature02165

    79. Mueller N H, Yon C, Ganesh V K, and Padmanabhan R. 2007. Characterization of the West Nile virus protease substrate specificity and inhibitors. Int J Biochem Cell Biol, 39: 606-614.
        doi: 10.1016/j.biocel.2006.10.025

    80. Mueller N H, Pattabiraman N, Ansarah-Sobrinho C, Viswanathan P, Pierson T C, and 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.
        doi: 10.1128/AAC.01508-07

    81. Munoz-Jordan J L, Sanchez-Burgos G G, Laurent-Rolle M, and Garcia-Sastre A. 2003. Inhibition of interferon signaling by dengue virus. Proc. Natl. Acad. Sci. USA, 100: 14333-14338.
        doi: 10.1073/pnas.2335168100

    82. Munoz-Jordan J L, Laurent-Rolle M, Ashour J, Martinez-Sobrido L, Ashok M, Lipkin W I, and Garcia-Sastre A. 2005. Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses. J. Virol., 79: 8004-8013.
        doi: 10.1128/JVI.79.13.8004-8013.2005

    83. Muylaert I R, Galler R, and Rice C M. 1997. Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation. J. Virol., 71: 291-298.

    84. Nall T A, Chappell K J, Stoermer M J, Fang N X, Tyndall J D, Young P R, and Fairlie D P. 2004. Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease. J Biol Chem, 279: 48535-48542.
        doi: 10.1074/jbc.M406810200

    85. Nitsche C, Steuer C, and Klein C D. 2011. Arylcyanoacrylamides as inhibitors of the Dengue and West Nile virus proteases. Bioorg Med Chem, 19: 7318-7337.
        doi: 10.1016/j.bmc.2011.10.061

    86. Nitsche C, Behnam M A, Steuer C, and Klein C D. 2012. Retro peptide-hybrids as selective inhibitors of the Dengue virus NS2B-NS3 protease. Antiviral Res, 94: 72-79.
        doi: 10.1016/j.antiviral.2012.02.008

    87. Niyomrattanakit P, Winoyanuwattikun P, Chanprapaph S, Angsuthanasombat C, Panyim S, and Katzenmeier G. 2004. Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. J Virol, 78: 13708-13716.
        doi: 10.1128/JVI.78.24.13708-13716.2004

    88. Noble C G, Seh C C, Chao A T, and Shi P Y. 2012. Ligand-bound structures of the dengue virus protease reveal the active conformation. Journal of Virology, 86: 438-446.
        doi: 10.1128/JVI.06225-11

    89. Noble C G, Chen Y L, Dong H, Gu F, Lim S P, Schul W, Wang Q Y, and Shi P Y. 2010. Strategies for development of Dengue virus inhibitors. Antiviral Res, 85: 450-462.
        doi: 10.1016/j.antiviral.2009.12.011

    90. Pambudi S, Kawashita N, Phanthanawiboon S, Omokoko M D, Masrinoul P, Yamashita A, Limkittikul K, Yasunaga T, Takagi T, Ikuta K, and Kurosu T. 2013. A Small Compound Targeting the Interaction between Nonstructural Proteins 2B and 3 Inhibits Dengue Virus Replication. Biochem Biophys Res Commun: in press (doi: 10.1016/j.bbrc.2013.1009.1078).

    91. Phong W Y, Moreland N J, Lim S P, Wen D, Paradkar P N, and Vasudevan S G. 2011. Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target. Bioscience reports.

    92. Radichev I, Shiryaev S A, Aleshin A E, Ratnikov B I, Smith J W, Liddington R C, and Strongin A Y. 2008. Structure-based mutagenesis identifies important novel determinants of the NS2B cofactor of the West Nile virus two-component NS2B-NS3 proteinase. J Gen Virol, 89: 636-641.
        doi: 10.1099/vir.0.83359-0

    93. Ray D, Shah A, Tilgner M, Guo Y, Zhao Y, Dong H, Deas T, Zhou Y, Li H, and Shi P. 2006. West nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5. J. Virol., 80: 8362-8370.
        doi: 10.1128/JVI.00814-06

    94. Rice C M, Lenches E M, Eddy S R, Shin S J, Sheets R L, and Strauss J H. 1985. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science, 229: 726-733.
        doi: 10.1126/science.4023707

    95. Robin G, Chappell K, Stoermer M J, Hu S H, Young P R, Fairlie D P, and Martin J L. 2009. Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conformation. J Mol Biol, 385: 1568-1577.
        doi: 10.1016/j.jmb.2008.11.026

    96. Romano K P, Ali A, Royer W E, and Schiffer C A. 2010. Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding. Proc Natl Acad Sci U S A, 107: 20986-20991.
        doi: 10.1073/pnas.1006370107

    97. Roosendaal J, Westaway E G, Khromykh A, and Mackenzie J M. 2006. Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein. J Virol, 80: 4623-4632.
        doi: 10.1128/JVI.80.9.4623-4632.2006

    98. Rothan H A, Han H C, Ramasamy T S, Othman S, Rahman N A, and Yusof R. 2012. Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1. BMC Infect Dis, 12: 314.
        doi: 10.1186/1471-2334-12-314

    99. Samanta S, Cui T, and Lam Y. 2012. Discovery, synthesis, and in vitro evaluation of West Nile virus protease inhibitors based on the 9, 10-dihydro-3H, 4aH-1, 3, 9, 10a-tetraazaphenanthren-4-one scaffold. ChemMedChem, 7: 1210-1216.
        doi: 10.1002/cmdc.v7.7

    100. Sarrazin C, Rouzier R, Wagner F, Forestier N, Larrey D, Gupta S K, Hussain M, Shah A, Cutler D, Zhang J, and Zeuzem S. 2007. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology, 132: 1270-1278.
        doi: 10.1053/j.gastro.2007.01.041

    101. Schuller A, Yin Z, Brian Chia C S, Doan D N, Kim H K, Shang L, Loh T P, Hill J, and Vasudevan S G. 2011. Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease. Antiviral Res, 92: 96-101.
        doi: 10.1016/j.antiviral.2011.07.002

    102. Shi P Y, Tilgner M, and Lo M K. 2002. Construction and characterization of subgenomic replicons of New York strain of West Nile virus. Virology, 296: 219-233.
        doi: 10.1006/viro.2002.1453

    103. Shi P Y, Tilgner M, Lo M K, Kent K A, and Bernard K A. 2002. Infectious cDNA clone of the epidemic west nile virus from New York City. J Virol, 76: 5847-5856.
        doi: 10.1128/JVI.76.12.5847-5856.2002

    104. Shi P Y, Kauffman E B, Ren P, Felton A, Tai J H, Dupuis A P, 2nd, Jones S A, Ngo K A, Nicholas D C, Maffei J, Ebel G D, Bernard K A, and Kramer L D. 2001. High-throughput detection of West Nile virus RNA. J Clin Microbiol, 39: 1264-1271.

    105. Shiryaev S, Ratnikov B, Chekanov A, Sikora S, Rozanov D, Godzik A, Wang J, Smith J, Huang Z, Lindberg I, Samuel M, Diamond M, and Strongin A. 2006. Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase. Biochem J., 393: 503-511.
        doi: 10.1042/BJ20051374

    106. Sidique S, Shiryaev S A, Ratnikov B I, Herath A, Su Y, Strongin A Y, and Cosford N D. 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.
        doi: 10.1016/j.bmcl.2009.07.150

    107. Steuer C, Gege C, Fischl W, Heinonen K H, Bartenschlager R, and Klein C D. 2011. Synthesis and biological evaluation of alpha-ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture. Bioorg Med Chem, 19: 4067-4074.
        doi: 10.1016/j.bmc.2011.05.015

    108. Stoermer M J, Chappell K J, Liebscher S, Jensen C M, Gan C H, Gupta P K, Xu W J, Young P R, and Fairlie D P. 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.
        doi: 10.1021/jm800503y

    109. Tan B H, Fu J, Sugrue R J, Yap E H, Chan Y C, and Tan Y H. 1996. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. Virology, 216: 317-325.
        doi: 10.1006/viro.1996.0067

    110. Tassaneetrithep B, Burgess T H, Granelli-Piperno A, Trumpfheller C, Finke J, Sun W, Eller M A, Pattanapanyasat K, Sarasombath S, Birx D L, Steinman R M, Schlesinger S, and Marovich M A. 2003. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med, 197: 823-829.
        doi: 10.1084/jem.20021840

    111. Tiew K C, Dou D, Teramoto T, Lai H, Alliston K R, Lushington G H, Padmanabhan R, and Groutas W C. 2012. Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives. Bioorg Med Chem, 20: 1213-1221.
        doi: 10.1016/j.bmc.2011.12.047

    112. Tomlinson S M, and Watowich S J. 2011. Anthracene-based inhibitors of dengue virus NS2B-NS3 protease. Antiviral Res, 89: 127-135.
        doi: 10.1016/j.antiviral.2010.12.006

    113. Tomlinson S M, and Watowich S J. 2012. Use of parallel validation high-throughput screens to reduce false positives and identify novel dengue NS2B-NS3 protease inhibitors. Antiviral Res, 93: 245-252.
        doi: 10.1016/j.antiviral.2011.12.003

    114. Tomlinson S M, Malmstrom R D, Russo A, Mueller N, Pang Y P, and Watowich S J. 2009. Structure-based discovery of dengue virus protease inhibitors. Antiviral Res, 82: 110-114.
        doi: 10.1016/j.antiviral.2009.02.190

    115. Umareddy I, Chao A, Sampath A, Gu F, and Vasudevan S G. 2006. Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA. J Gen Virol, 87: 2605-2614.
        doi: 10.1099/vir.0.81844-0

    116. USGS. 2010. Disease Maps 2010. http://diseasemaps.usgs.gov/.

    117. Warrener P, Tamura J K, and Collett M S. 1993. RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J Virol, 67: 989-996.

    118. Wegzyn C M, and Wyles D L. 2012. Antiviral drug advances in the treatment of human immunodeficiency virus (HIV) and chronic hepatitis C virus (HCV). Curr Opin Pharmacol, 12: 556-561.
        doi: 10.1016/j.coph.2012.06.005

    119. Wengler G. 1981. Terminal sequences of the genome and replicative-from RNA of the flavivirus West Nile virus: absence of poly(A) and possible role in RNA replication. Virology, 113: 544-555.
        doi: 10.1016/0042-6822(81)90182-3

    120. Wengler G. 1991. The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology, 184: 707-715.
        doi: 10.1016/0042-6822(91)90440-M

    121. Westaway E G, Brinton M A, Gaidamovich S Y, Horzinek M C, Igarashi A, Kaariainen L, Lvov D K, Porterfield J S, Russell P K, and Trent D W. 1985. Flaviviridae. Intervirol., 24: 183-192.
        doi: 10.1159/000149642

    122. WHO. 2009. Immunization, vaccines and biologicals: Japanese encephalitis. <http://www.who.int/nuvi/je/en/>>.

    123. WHO. 2009. Dengue factsheet. <http://www.who.int/mediacentre/factsheets/fs117/en/>.

    124. WHO. 2009. Yellow fever factsheet. <http://www.who.int/mediacentre/factsheets/fs100/en/>.

    125. Wyles D L. 2012. Beyond telaprevir and boceprevir: resistance and new agents for hepatitis C virus infection. Top Antivir Med, 20: 139-145.

    126. Wyles D L. 2013. Antiviral resistance and the future landscape of hepatitis C virus infection therapy. J Infect Dis, 207 Suppl 1: S33-39.

    127. Xu S, Li H, Shao X, Fan C, Ericksen B, Liu J, Chi C, and Wang C. 2012. Critical effect of peptide cyclization on the potency of peptide inhibitors against Dengue virus NS2B-NS3 protease. J Med Chem, 55: 6881-6887.
        doi: 10.1021/jm300655h

    128. Yang C C, Hsieh Y C, Lee S J, Wu S H, Liao C L, Tsao C H, Chao Y S, Chern J H, Wu C P, and Yueh A. 2011. Novel dengue virus-specific NS2B/NS3 protease inhibitor, BP2109, discovered by a high-throughput screening assay. Antimicrob Agents Chemother, 55: 229-238.
        doi: 10.1128/AAC.00855-10

    129. Yin Z, Patel S J, Wang W L, Wang G, Chan W L, Rao K R, Alam J, Jeyaraj D A, Ngew X, Patel V, Beer D, Lim S P, Vasudevan S G, and Keller T H. 2006. Peptide inhibitors of Dengue virus NS3 protease. Part 1: Warhead. Bioorg Med Chem Lett, 16: 36-39.
        doi: 10.1016/j.bmcl.2005.09.062

    130. Yin Z, Patel S J, Wang W L, Chan W L, Ranga Rao K R, Wang G, Ngew X, Patel V, Beer D, Knox J E, Ma N L, Ehrhardt C, Lim S P, Vasudevan S G, and Keller T H. 2006. Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors. Bioorg Med Chem Lett, 16: 40-43.

    131. Zhang Y, Corver J, Chipman P R, Zhang W, Pletnev S V, Sedlak D, Baker T S, Strauss J H, Kuhn R J, and Rossmann M G. 2003. Structures of immature flavivirus particles. EMBO J., 22: 2604-2613.
        doi: 10.1093/emboj/cdg270

    132. Zhou Y, Ray D, Zhao Y, Dong H, Ren S, Li Z, Guo Y, Bernard K A, Shi P Y, and Li H. 2007. Structure and function of flavivirus NS5 methyltransferase. J Virol, 81: 3891-3903.
        doi: 10.1128/JVI.02704-06

  • 加载中

Figures(1)

Article Metrics

Article views(6388) PDF downloads(19) Cited by()

Related
Proportional views

    The Flavivirus Protease As a Target for Drug Discovery

      Corresponding author: Hongmin Li, lih@wadsworth.org
    • 1. Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany NY 12208, USA
    • 2. Department of Biomedical Sciences, School of Public Health, State University of New York, Empire State Plaza, PO Box 509, Albany, New York 12201-0509, USA

    Abstract: Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.

    • Flaviviruses belong to the viral family Flaviviridae that include about 70 viruses (Brinton M A, 1981; Brinton M A, 2002; Westaway E G, et al., 1985). Many flaviviruses are significant human pathogens. Dengue virus (DENV) serotypes 1-4, Yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis complex virus (TBEV) are categorized as global emerging pathogens and are NIAID Priority Pathogens as well ( Burke DS, et al., 2001). Flaviviruses cause significant human disease, some of which are fatal such as dengue hemorrhagic syndromes and various encephalitides (Asnis D S, et al., 2001; Asnis D S, et al., 2000; Kramer L D, et al., 2001; Shi P Y, et al., 2002; Shi P Y, et al., 2002; Shi P Y, et al., 2001).

      The World Health Organization has estimated annual human cases of 50, 000 for JE (WHO, 2009), 200, 000 for YF (WHO, 2009), and more than 50 million for Dengue fever (WHO, 2009). WNV is now the leading cause of arboviral encephalitis in the US, leading to more than a thousand human deaths (CDC, 2010; USGS, 2010). Morbidity and mortality rates are waning for WNV in the US, but are expected to increase for DENV. Currently, approximately 2.5 billion people are at risk of DENV infection, with an estimated 500, 000 cases in the form of life-threatening disease such as dengue hemorrhagic fever and dengue shock syndrome (WHO, 2009). However, vaccines for humans currently are available only for YFV, JEV, and TBEV ( Burke DS, et al., 2001); and more importantly no clinically approved antiviral therapy is available for treatment of flavivirus infection. Therefore, it is a public health priority to develop antiviral agents for post-infection treatment (Kramer L D, et al., 2007).

      This article will review recent advances in flavivirus drug development targeting the essential viral protease.

    • The NS3 protein (~618 amino acids (aa)) is the second largest protein encoded by flavivirus. The N-terminal 170 aa of NS3 displays protease activity, and a hydrophobic core of about 40 aa in length within NS2B provides an essential cofactor function ( T J, et al., 1991; Chambers T J, et al., 1990; Falgout B, et al., 1991).The NS3 protease belongs to the trypsin serine protease superfamily with a catalytic triad (e.g. His51-Asp75-Ser135 for the DENV NS3) (Bazan J F, et al., 1989). The NS2B/NS3 protease complex prefers a substrate with basic residues (Arg or Lys) at the P1 and P2 sites and a short side-chain amino acid (Gly, Ser, or Ala) at the P1′ site (Chambers T J, et al., 1990; Gouvea I E, et al., 2007). The central function of the NS2B/NS3 protease complex is to process the flavivirus polyprotein precursor. As shown in Fig. 1 , the peptide bonds between capsid, NS2A-NS2B, NS2B-NS3, NS3-NS4A and NS4B-NS5 are cleaved by the NS2B/NS3 protease complex, leading to the release of mature individual NS proteins.

      The NS2B/NS3 protease complex is essential for the flavivirus replication and virion assembly, as evidenced by the lack of production of infectious virions in mutants carrying inactivating viral proteases (Chambers T J, et al., 1993).

    • The development of protease inhibitor began with the determination of the three-dimensional (3D) structures of the flavivirus NS3 protease, the NS2B/NS3 protease complex, and the protease-inhibitor complexes (Aleshin A, et al., 2007; Assenberg R, et al., 2009; Chandramouli S, et al., 2010; Erbel P, et al., 2006; Hammamy M Z, et al., 2013; Luo D, et al., 2008; Luo D, et al., 2010; Luo D, et al., 2008; Noble C G, et al., 2012; Robin G, et al., 2009). Currently, fourteen crystal structures of the NS2B/NS3 protease complex are available for the flavivirus N S2B/NS3 protease complexes, including the apo structures of proteases of WNV, DENV-1, DENV-2, DENV-4, and Murray Valley encephalitis virus (MVEV), the structures of proteases of WNV and DENV3 in complex peptide substrate-based inhibitors, and the broad-spectrum serine protease inhibitor aprotinin-bound structures of proteases of WNV and DENV-3.

      In general, the flavivirus NS3 proteases display a chymotrypsin-like fold (Erbel P, et al., 2006). In all these structures, a NS2B fragment composed of about 44-47 amino acids, which provides an essential cofactor function (Chambers T J, et al., 1991; Chambers T J, et al., 1990; Falgout B, et al., 1990), is associated with NS3. When no substrate or inhibitor is present, the N-terminal (residues 51-61 in DENV-2) but not the C-terminal portion of NS2B is bound to NS3 (Erbel P, et al., 2006) (Fig. 1A). The central portion of this N-terminal part forms a β-strand and is part of the β-barrel of NS3 (Erbel P, et al., 2006). Consistent with the important structural role of this part of NS2B, structural comparison indicates that the NS2B residues within the N-terminal portion display similar conformations in all structures, regardless of presence or absence of inhibitors (Fig. 1A). It has also been reported that the N-terminal portion of NS2B (aa 49-66 only) is sufficient to bind and stabilize the NS3 conformation (Luo D, et al, 2008; Luo D, et al., 2010), although such a complex lacks protease activity (Luo D, et al., 2008; Luo D, et al., 2010; Phong W Y, et al., 2011). Mutagenesis studies demonstrated that two NS2B regions are critical for the protease function (Chappell K J, et al., 2008; Niyomrattanakit P, et al., 2004; Phong W Y, et al., 2011; Radichev I, et al., 2008) (Fig. 1D). Region one corresponds to the N-terminal region mentioned above, whereas region two is referred to a C-terminal region composed of residues 74-86 of NS2B. Residues within region one show great sequence conservation, especially for several hydrophobic residues at positions 51, 53, 59, and 61 (in DENV-2 order), with Trp61 strictly conserved (Fig. 1D). Functional studies indicated that three of these residues are essential, and the remaining one is also important, for the protease function (Chappell K J, et al., 2008). Structure comparison indicated that these conserved hydrophobic residues bind deeply into several pockets of NS3 (Fig. 1A). In contrast, residues within region two display greater sequence variation than those within region one, which may contribute to their fine substrate specificities as region two is part of the protease active site (see below) (Fig. 1B, 1C). In addition, in contrast to the N-terminal region which shows similar conformations, the C-terminal portion (beyond aa 61) of NS2B displays significantly large conformational differences between inhibitor-bound and inhibitor-free structures, and even between inhibitor-free structures (Fig. 1A). These results suggest that the N-terminal portion, but not the C-terminal portion, of NS2B is essential for NS2B to bind and stabilize NS3.

      The C-terminal portion of NS2B has an integral role in active site formation in WNV and DENV. Although the C-terminal portions of NS2B display significantly different conformation in various apo crystal structures, the C-terminal portions of bound structures show remarkable conformational similarity when the complex is bound either to substrate analogs or the protease inhibitor aprotinin (Fig. 1B). In the structure of inhibitor-bound form, the C-terminal portion of NS2B forms a β-hairpin and "wraps around" the NS3 core, closing the NS3 active site. Several residues within this region make direct interactions, including hydrogen bonds, with substrate analogs or aprotinin inhibitors. Unsurprisingly, results from mutagenesis studies have demonstrated the importance of this region in protease function (Chappell K J, et al., 2008; Niyomrattanakit P, et al., 2004), likely due to its structural role in formation of the protease active site. The active site of the flavivirus NS2B/NS3 protease complex is quite flat and hydrophilic (Fig. 1C) and requires several basic residues as substrates, potentially hampering the development of potent competitive inhibitors.

    • Historically, the most straightforward approach to developing inhibitors of an enzyme target has been to screen for compounds that competitively bind the enzyme's active site and displace native substrate. The advantage of such an approach is that characterization of the properties of a particular enzyme's substrate is often a sufficient starting point for selecting compounds that mimic or exceed the substrate in its affinity for the enzyme. Unfortunately, this approach might be unlikely to yield effective compounds in the case of flavivirus NS2B/NS3 protease for three reasons: First, NS2B/NS3 has a flat and hydrophilic active site which decreases the likelihood that compounds can bind specifically with high affinity. Second, the NS2B/NS3 active site is similar enough to those of host serine proteases that toxic effects in the host are likely for many compounds, as has been observed in the case of aprotinin. Third, the active site preferentially binds positively charged moieties; this charge can have deleterious effects on compound bioavailability.

      In addition, lessons should be learned from the development of active site inhibitors for the HCV protease. Although two HCV protease substrate-based inhibitors were developed, resistant mutations occurred quickly (Wyles D L, 2013). This is because the active site of the HCV protease is shallow and solvent exposed. The featureless property of the active site of the HCV protease implies that inhibitors would rely on relatively few interactions with the enzyme for tight binding, resulting in a low barrier to resistance and extensive cross-resistance (Romano K P, et al., 2010; Wegzyn C M, et al., 2012). It has been reported that as few as a single key mutation resulted in a significant loss of inhibition and cross-resistance (Romano K P, et al., 2010; Wyles D L, 2012; Wyles D L, 2013). Similar to that of the HCV protease, the active site of flavivirus NS2B/NS3 protease complex is also flat and featureless, in addition to the hydrophilic nature. Therefore, potential drug resistance should be taken into account, when development of active-site inhibitors for flavivirus protease complex is considered.

      Fortunately, the solved crystal structures of flavivirus protease in both substrate bound and unbound states has yielded mechanistic insight into protease function. Details of the interaction of the NS2B cofactor, critical for enzyme function, with NS3 have suggested an allosteric approach to inhibition through disruption of NS2B/NS3 binding. Lead compounds developed by this approach are less likely to have the drawbacks observed with active site inhibitors, and are amenable to both computational and HTS screening methods. In the future, this "structure-guided" approach may suggest additional allosteric sites in flavivirus protease and has the potential to open broad avenues to drug discovery in other disease target proteins.

    • This research was partially supported by grants (AI094335) from the National Institute of Health and from the Wadsworth Center Scientific Interaction Group.

    • All authors carried out the work presented here. MB, JZ, and HML wrote the paper the paper. MB and HML defined, reviewed and edited the theme of this review.

    Figure (1)  Reference (132) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return