Enzymatic Characterization and Homology Model of a Catalytically Active Recombinant West Nile Virus NS3 Protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 ∼1 μm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease. West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 ∼1 μm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease. The West Nile Virus (WNV) 1The abbreviations used are: WNV, West Nile Virus; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; ESMS, electrospray mass spectrometry; HCV, hepatitis C virus; HRMS, High Resolution electrospray mass spectrometry; IPTG, isopropyl-d-thiogalactopyranose; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; Rt, retention time; TLCK, tosyl-l-lysine chloromethyl ketone; HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; pNA, para-nitroanilide. was first detected in a woman living in the West Nile region of Uganda almost 70 years ago (1Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar). WNV is a member of the Flaviviridae family that also includes hepatitis C, Kunjin, yellow fever, Dengue, St. Louis, and Japanese encephalitis viruses (1Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar). WNV is transmitted via mosquitoes from avian reservoir hosts to vertebrate dead-end hosts that include humans and horses. Endemic in humans in parts of Africa, Europe, and the Middle East, previous viral outbreaks were mostly detected as asymptomatic infections or mild febrile disease states (2Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (168) Google Scholar). However, recent outbreaks in Israel (1998), Romania (1996), and the United States (1999) (3Lanciotti R.S. Roehrig J.T. Deubel V. Smith J. Parker M. Steele K. Crise B. Volpe K.E. Crabtree M.B. Scherret J.H. Hall R.A. MacKenzie J.S. Cropp C.B. Panigrahy B. Ostlund E. Schmitt B. Malkinson M. Banet C. Weissman J. Komar N. Savage H.M. Stone W. McNamara T. Gubler D.J. Science. 1999; 286: 2333-2337Crossref PubMed Scopus (1296) Google Scholar) have been associated with more serious neurological pathology and fatal infections. During the last 5 years, WNV has spread rapidly throughout the United States (4,156 infections and 284 deaths in 44 states in 2002, 9862 infections and 264 deaths in 2003), 2CDC (2000) Division of Vector-Borne Infectious Disease: West Nile Virus; Centre for Disease Control and Prevention, Atlanta, GA, www.cdc.gov/ncidod/dvbid/westnile/index.htm. Canada, and Mexico, as well as appearing recently in the United Kingdom (5Buckley A. Dawson A. Moss S.R. Hinsley S.A. Bellamy P.E. Gould E.A. J. Gen. Virol. 2003; 84: 2807-2817Crossref PubMed Scopus (178) Google Scholar). This rapid global spread, even through developed countries, has prompted widespread implementation of prevention strategies. No vaccines or therapeutic treatments for WNV infections are yet available. Like other members of the Flaviviridae family, WNV contains a single-stranded, positive-sense RNA genome, which encodes three structural proteins (capsid (C), membrane (M), envelope (E)), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (1Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar, 3Lanciotti R.S. Roehrig J.T. Deubel V. Smith J. Parker M. Steele K. Crise B. Volpe K.E. Crabtree M.B. Scherret J.H. Hall R.A. MacKenzie J.S. Cropp C.B. Panigrahy B. Ostlund E. Schmitt B. Malkinson M. Banet C. Weissman J. Komar N. Savage H.M. Stone W. McNamara T. Gubler D.J. Science. 1999; 286: 2333-2337Crossref PubMed Scopus (1296) Google Scholar). The genome is initially translated as a single, large polyprotein precursor. Post-translational cleavages of the viral polyprotein precursor in the cytoplasm of host-infected cells are necessary to produce the separate structural and functional viral proteins that are essential for assembly of new viral progeny. These cleavages are performed by both host enzymes (including signalases and furin) and by a viral protease encoded by the N-terminal-third of NS3 (Fig. 1). This NS3 viral protease, which is a trypsin-like serine protease with a functional catalytic triad (His51, Asp75, Ser135), is absolutely essential (along with viral-encoded cofactor NS2B) for viral replication and is itself autocatalytically cleaved from the polypeptide precursor. By analogy with the homologous Dengue virus NS3 protease, for which our truncation studies had previously shown that a 40-residue hydrophilic domain of NS2B is necessary for catalytic activity of the NS3 protease, (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) we infer below that a similar sequence within the WNV NS2B is likely necessary for catalytic activity of the WNV NS3 protease. The remainder of NS2B encodes 3 hydrophobic domains, believed to be involved in membrane association and localization of the enzyme to convoluted membrane regions on the endoplasmic reticulum (7Clum S. Ebner K.E. Padmanabhan R. J. Biol. Chem. 1997; 272: 30715-30723Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The structure of WNV NS3 protease is unknown, but there are reported crystal structures for related NS3 proteases of hepatitis C virus (HCV) (8Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and Dengue virus (DEN2) (9Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The structure of the latter protease was however determined in the of the NS2B cofactor and was By of its essential in post-translational processing of the viral WNV NS3 protease is as a potential for of that viral for inhibitors of viral proteases potential in the of inhibitors of proteases of the human immunodeficiency viruses the treatments for humans with Clin. Microbiol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. P.R. E. R. J. Biol. Chem. Full Text PDF PubMed Google and in the shown by inhibitors of the NS3 protease of hepatitis C virus C. K. H.M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This article describes the design and of a catalytically active recombinant WNV protease enzyme the characterization of that enzymatic processing of synthetic chromogenic substrates para-nitroanilide the first reported inhibitor of WNV protease, and the of a three-dimensional structure of the WNV NS3 protease based on the crystal structures of Dengue NS3 protease and NS3 protease with cofactor is that the recombinant protease cofactor chromogenic and homology structure be for structure-based design of even more potent and selective inhibitors of the WNV NS3 protease. inhibitors the for for the treatment of WNV infections. and were from and DIPEA, DCM, and were from other were were by on a using a of and was performed on a with a and a using a 5 were by mass and The of the was determined by electrospray mass on a mass were on mm in on a K. were determined by and using the K. of and Google Scholar). were on or using of RNA was by R. A. Hall and D. J. of was using the WNV are which was used as in the of the WNV and the WNV WNV was by a using the WNV and WNV WNV was in separate the WNV and WNV to the essential cofactor domain of NS2B and the 184 with These were by using the WNV and WNV The WNV and the 184 NS3 protease domain and the essential cofactor domain of NS2B to the 184 NS3 protease via a nonapeptide These were with and and into The of WNV and WNV was by sequence and WNV was used to high of N-terminal recombinant of with the were in of and of the recombinant was by the of to a of mm and for 3 The cells were by and the were and in 5 of mm mm mm 1 of cleavage of and the protease inhibitors were to the of 1 1 1 1 mm PMSF, and The cells were by three through a a of and for The recombinant proteases, the by a were by on a A was with of mm mm mm and the was and with the of the These were on a for to the to to the The were and with of mm the into 1 with mm The and and and the were by substrates to the WNV NS3 protease cleavage and were by a reported G. D. T. Fairlie D.P. 2001; Scholar). m, for m, for m, for m, for was by the of and PubMed Scopus Google Scholar). for recombinant WNV protease was hexapeptide substrates to of cleavage with a chromogenic in the of the from the substrates by the WNV protease a protease activity to be The assay was on a with a of and for optimal recombinant protease, and substrate in separate was by substrate with the enzyme by The was for in a and the in was was determined the using substrate for mm (pH (pH (pH (pH glycerol, was determined by of from to in the of with glycerol, substrate 5 in a was determined for mm in glycerol, CHAPS), substrate in the of were on a and using the both from for were performed optimal and conditions as determined by of the previous glycerol, and via and to be 30% glycerol, 9.5, were used to and for the substrates from to were using for and from from are reported as the of Nile Virus NS3 protease homology were using the structures of the Dengue virus NS3 protease (9Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and with inhibitor (9Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and hepatitis C virus NS3 protease with cofactor (8Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). were using the and the within the modeling R. N. J. Chem. PubMed Scopus Google Scholar). modeling was performed using were using and PubMed Scopus Google Scholar). structure were using for of the three NS3 protease J. Biol. 1999; PubMed Scopus Google Scholar). potential was performed on the WNV NS3 protease using the in E. H. 2003; PubMed Scopus Google Scholar). A substrate was into the active of a homology of the WNV NS3 protease using G. J. Biol. PubMed Scopus Google Scholar). A substrate and WNV protease as well as and substrate and WNV protease were used based on our of D.P. Abbenante G. M.J. J. Chem. PubMed Scopus Google Scholar, D. Abbenante G. Fairlie D.P. J. Chem. PubMed Scopus Google Scholar). D. A. T. and D. for The structures were for using G. J. Biol. PubMed Scopus Google Scholar). of a WNV serine protease within the WNV RNA genome provides and therapeutic for into the design and of a WNV serine protease The first in a catalytically active WNV protease was based on our previous on the Dengue-2 viral protease, for which we a sequence of 184 residues from the NS3 protease and residues from the NS2B cofactor that are necessary for catalytic processing of substrates (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, Fairlie D.P. D. Young P.R. J. Gen. Virol. 1999; PubMed Scopus Google Scholar). that we created a catalytically active protease by to by the active a nonapeptide as a for the cleavage of the WNV and Dengue virus (Fig. sequence and sequence within the 184 residues of This catalytic residues and and in our in WNV NS3 was in Dengue-2 virus NS3. of the NS2B cofactor sequence and sequence and These sequence by analogy with the protease, a catalytically active sequence for WNV protease residues from the NS2B cofactor and 184 residues from NS3, via a nonapeptide as in of the cleavage the putative catalytic protease as WNV A recombinant was also consisting of the 184 NS3 protease domain in the of the NS2B cofactor domain to that activity was on the association of the as was previously shown for the Dengue virus (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). WNV and of WNV and WNV first was to and a recombinant WNV NS3 protease domain and a catalytically active recombinant WNV protease based on the recombinant Dengue-2 virus NS3 protease (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). The homologous WNV recombinant and WNV were and into the The sequence of and into was by sequence The was used a high of expression, with the of The of N-terminal by The of WNV and WNV are shown in A and and the of WNV is shown in a in the with This was to recombinant WNV as is the of the and with the of WNV by of we had previously with Dengue virus (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) the of the was in the with in the a in the with This was to recombinant WNV as is the of the and with the of WNV by of The of was in the that the recombinant was likely to be were from the by of the of recombinant WNV in the were however that the WNV recombinant was and The in and C was likely to the of and The were to of were in the of recombinant enzyme with on These were to have on processing of synthetic substrates as the of cleavage is of that of substrate cleavage and the substrate is in of enzyme there are seven post-translational cleavage that are by WNV protease in the cytoplasm of host cells (Fig. 1). residues to cleavage are in from of to putative substrate in a The putative substrate were and These potential substrates were to be used to activity of WNV a of assay in of conditions that could be used for more and for inhibitor A of substrates that was and with the that substrate was used in the of that substrate processing by the protease. WNV was to its enzymatic and was the to the that the protease is in the of the NS2B of on WNV NS3 protease was a from using a of for (pH (pH (pH (pH The optimal for cleavage was determined to be (Fig. the of the substrate was to the optimal processing of 9.5, and was The optimal processing of the WNV protease (pH is to the optimal processing that we previously for the Dengue-2 virus NS3 protease (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). of on had previously that catalytic processing by Dengue NS3 protease (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google we to enzyme Fang N.X. Fairlie D.P. Young P.R. Martin J.L. 2002; PubMed Scopus Google Scholar). the of in the assay on catalytic of processing of was a on (Fig. with of enzyme and of the enzyme active to of the assay to processing of the of the assay to 30% in the assay of on of was by to the The (Fig. that the processing of the High be by with substrate and for of the substrate active substrate is to in and of substrates in to the protease. has been that proteases substrates and inhibitor in D.P. Abbenante G. M.J. J. Chem. PubMed Scopus Google Scholar, Fairlie D.P. J. 1999; PubMed Scopus Google Scholar). of substrates in a the catalytic as and to the cleavage of the WNV RNA genome, were for to be cleaved by the WNV protease the optimal processing conditions mm 30% 1 mm CHAPS, The assay was using substrate from to The were to (Fig. in to the and the in processing the with the cleavage and the cleavage The substrate was the sequence to the cleavage The for catalytic of substrates was By processing of Dengue substrates by Dengue-2 (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) and that were for the WNV This catalytic for substrate processing by the WNV NS3 protease with the recombinant Dengue NS3 protease for the of synthetic substrates by in a new on for the substrate sequence to we also with a and (Fig. to be a potent competitive inhibitor ∼1 of catalytic processing of by WNV This is the that the in as (Fig. with a in which the of has with and with through of the The is likely to be the active of inhibitor R. A. J. Biol. Chem. Full Text PDF PubMed Google but is in the in the of the in the of the and in (Fig. The for inhibitor be a of the of with of the active of the inhibitor for with the active serine The of protease inhibitors on processing of the substrate by WNV was also (Fig. serine protease inhibitors as PMSF, and TLCK, to protease inhibition of the conditions was a inhibitor even a of (Fig. to a structural for of the WNV protease, for which crystal structure a homology structural was based on the crystal structures of flavivirus proteases, the Dengue virus NS3 protease and (9Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (115) Google and the hepatitis C virus NS3 protease (8Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The Dengue virus NS3 and hepatitis C virus proteases were as a for the WNV protease for sequence homology Dengue and structural homology with sequence a for a WNV NS3 protease homology the NS3 proteases a cofactor for the protease structure (8Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) the cofactor was used for the WNV protease homology into the of the WNV NS2B cofactor in the of the WNV NS3 protease. sequence the protease and WNV protease or protease and Dengue viral protease was and the of high structural homology based on a previous of and Dengue virus proteases structural for the of the by Fairlie D.P. D. Young P.R. J. Gen. Virol. 1999; PubMed Scopus Google Scholar) a of Dengue virus NS3 protease based on the NS3 protease crystal sequence This has been by a crystal structure for Dengue virus NS3 protease used in the WNV homology of the protease and WNV protease or protease and Dengue virus protease and into the structural the three The with cofactor the of a WNV protease and potential cofactor in the Dengue virus crystal The Dengue NS3 crystal structure in with inhibitor the in the active of the Dengue NS3 protease. The Dengue NS3 structure a for the of WNV of cleavage was used in substrate and active with a to inhibitor of the West Nile Virus protease. were based on the structures and using sequence in to for the WNV protease The used for of putative substrate in the active was based on the Dengue NS3 protease with the inhibitor The of the WNV and the Dengue-2 viral protease crystal structure were on the cleavage of the WNV NS3 protease, and a substrate was into the active of the WNV protease in to the the substrate and WNV protease (Fig. the is into the protease residues and are in (Fig. likely that with in the The substrate with protease residues Asp75, and (Fig. The is but a be and The catalytic triad is also and to the The a potential with protease residues and (Fig. The with protease by to and is in to (Fig. of and of the active residues and J. T. A. M. J. J. D. A. D. and R. in West Nile Virus infections have the and the associated and have the for R.S. Gubler D.J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). treatments for WNV infections also for for other flavivirus infections. A protease enzyme within the WNV genome is to be for post-translational cleavages of the polypeptide the structural and functional viral proteins that the for virus and assembly into new virus of that protease has the to viral The was and the first catalytically active recombinant WNV protease. a recombinant West Nile Virus protease, we of the sequence homology WNV and Dengue-2 virus proteins (Fig. and our previous that to a recombinant Dengue virus protease (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). we the WNV of the putative NS2B cofactor domain and of the NS3 protease domain and with a nonapeptide (G4SG4) for the cleavage The was a catalytically active recombinant WNV protease, WNV which is active with the cofactor the enzyme processing of the recombinant WNV protease, optimal and to The the of hexapeptide substrates to of cleavage within the WNV genome to a chromogenic has the potential for rapidly inhibitors of the based on of the substrates we also the first inhibitor 1 of the WNV protease. A of the enzymatic activity was the catalytic of the recombinant proteases of Dengue virus and West Nile The high (8Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, Clin. Microbiol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) for processing is and yet be but a of processing be of the The cleavage that are by WNV protease in (Fig. residues and and a a sequence for the cleavage of the WNV genome by its protease. This sequence is almost throughout the proteases and is in with host we have shown for both the Dengue-2 (6Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) and WNV virus proteases (Fig. that a of serine protease inhibitors was that protease. This for the design of potent inhibitors that be selective for flavivirus proteases host of a potent and selective inhibitor of WNV protease in for of treatment for WNV, and with potential for inhibition of in even more potent and selective inhibitors of WNV protease as we also created a three-dimensional structural of the protease. in the protease for both the NS2B cofactor and that the active and putative that valuable insights for inhibitor This could be with the in protease active of the D.P. Abbenante G. M.J. J. Chem. PubMed Scopus Google Scholar, Fairlie D.P. J. 1999; PubMed Scopus Google and the of Fairlie D.P. Rev. Chem. 2002; PubMed Scopus Google Scholar, Fairlie D.P. Chem. Google Scholar) as for design of inhibitors of

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