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Information on EC 3.4.21.91 - Flavivirin and Organism(s) Dengue virus type 2 and UniProt Accession P29990
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3.4.21.91 FlavivirinSpecify your search results
Word Map
- 3.4.21.91
- polyprotein
- viruses
- ns4a
-
replicons
-
west
- ribavirin
-
anti-hcv
-
direct-acting
-
virologic
-
cirrhosis
-
flaviviral
- boceprevir
- flaviviruses
- encephalitis
-
pegylated
- flaviviridae
- telaprevir
-
subgenomic
-
rna-dependent
-
unwind
-
mosquito-borne
- asunaprevir
-
resistance-associated
-
daclatasvir
- simeprevir
-
hcv-infected
- coronaviruses
-
anti-dengue
- ntpase
-
peptidomimetic
-
nonnucleoside
-
hcv-specific
-
macrodomains
- drug development
-
treatment-naive
-
peginterferon
-
replicase
-
quasispecies
-
interferon-free
-
trans-cleavage
- alphavirus
-
positive-stranded
- sofosbuvir
- analysis
- molecular biology
-
rna-stimulated
- medicine
-
hcv-1b
- synthesis
- gt1
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Selective hydrolysis of -Xaa-Xaa-/-Yaa- bonds in which each of the Xaa can be either Arg or Lys and Yaa can be either Ser or Ala
Synonyms
ns3 protease, nonstructural protein 3, ns2b-ns3 protease, ns3 serine protease, ns2b-ns3pro, ns2b/ns3 protease, ns3 proteinase, ns3pro, ns2b/ns3, zikv protease, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
NS2B-3 proteinase
-
-
-
-
NS2B18NS3
-
NS3 protein covalently attached to 18 residues of the N22B cofactor region
Yellow fever virus (flavivirus) protease
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
154215-26-6
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Abz-AAGRKSLTL-3-nitrotyrosine-amide + H2O
Abz-AAGRK + SLTL-3-nitrotyrosine-amide
-
-
-
-
?
Abz-AGRKGTGN-3-nitrotyrosine-amide + H2O
Abz-AGRKGTGN + 3-nitrotyrosine-amide
-
the poorest substrate
-
-
?
Abz-AGRKS-3-nitrotyrosine-amide + H2O
Abz-AGRKS + 3-nitrotyrosine-amide
-
-
-
-
?
Abz-AGRKSLT-3-nitrotyrosine-amide + H2O
Abz-AGRK + SLT-3-nitrotyrosine-amide
-
-
-
-
?
Abz-ARRKSLTL-3-nitrotyrosine-amide + H2O
Abz-ARRK + SLTL-3-nitrotyrosine-amide
-
-
-
-
?
Abz-FAAGRKSLTL-3-nitrotyrosine-amide + H2O
Abz-FAAGRK + SLTL-3-nitrotyrosine-amide
-
-
-
-
?
Abz-GRKS-3-nitrotyrosine-amide + H2O
Abz-GRK + S-3-nitrotyrosine-amide
-
-
-
-
?
Abz-GRKSLTL-3-nitrotyrosine-amide + H2O
Abz-GRK + SLTL-3-nitrotyrosine-amide
-
-
-
-
?
Abz-KKQR-AGVLnY-amide + H2O
Abz-KKQR + AGVLnY-amide
-
high degree of cleavage efficiency, based on the NS2B/NS3 polyprotein cleavage site
-
-
?
Abz-KKQRAGVL-3-nitrotyrosine-amide + H2O
Abz-KKQR + AGVL-3-nitrotyrosine-amide
-
is based on the NS2B/NS3 polyprotein cleavage site, shows high degree of cleavage efficiency
-
-
?
Abz-KKQRSAGM-3-nitrotyrosine-amide + H2O
Abz-KKQR + SAGM-3-nitrotyrosine-amide
-
substrates containing the capsid protein prime side sequence SAGM are efficiently hydrolysed
-
-
?
Abz-NRRRRSAGM-3-nitrotyrosine-amide + H2O
Abz-NRRRR + SAGM-3-nitrotyrosine-amide
-
-
-
-
?
Abz-RKS-3-nitrotyrosine-amide + H2O
Abz-RK + S-3-nitrotyrosine-amide
-
-
-
-
?
Abz-RKSLTL-3-nitrotyrosine-amide + H2O
Abz-RK + SLTL-3-nitrotyrosine-amide
-
-
-
-
?
Abz-RRRR-3-nitrotyrosine-amide + H2O
Abz-RRRR + 3-nitrotyrosine-amide
-
marginal activity
-
-
?
Abz-RRRR-SAGnY-amide + H2O
Abz-RRRR + SAGnY-amide
-
efficient substrate of the NS3 protease, based on the Dengue virus capsid protein processing site
-
-
?
Abz-RRRRS-3-nitrotyrosine-amide + H2O
Abz-RRRR + S-3-nitrotyrosine-amide
-
-
-
-
?
Abz-RRRRSAG-3-nitrotyrosine-amide + H2O
Abz-RRRR + SAG-3-nitrotyrosine-amide
-
most efficiently hydrolysed substrate, which contains the tetrabasic non-prime side sequence RRRR of the dengue virus capsid protein
-
-
?
Abz-RRRRSAGM-3-nitrotyrosine-amide + H2O
Abz-RRRR + SAGM-3-nitrotyrosine-amide
-
-
-
-
?
Abz-RRRRSLTL-3-nitrotyrosineamide + H2O
Abz-RRRR + SLTL-3-nitrotyrosineamide
-
-
-
-
?
Abz-SKKRAGVL-3-nitrotyrosine-amide + H2O
Abz-SKKR + AGVL-3-nitrotyrosine-amide
-
-
-
-
?
Abz-SKKRSAGM-3-nitrotyrosine-amide + H2O
Abz-SKKR + SAGM-3-nitrotyrosine-amide
-
-
-
-
?
Ac-Thr-Arg-Arg-7-amido-4-methylcoumarin + H2O
Ac-Thr-Arg-Arg + 7-amino-4-methylcoumarin
-
-
-
-
?
Boc-Gly-Arg-Arg-7-amido-4-methylcoumarin + H2O
Boc-Gly-Arg-Arg + 7-amino-4-methylcoumarin
-
-
-
-
?
Gly-Arg-Arg-4-methylcoumarin 7-amide + H2O
Gly-Arg-Arg + 7-amino-4-methylcoumarin
-
-
-
-
?
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin + H2O
N-acetyl-Gly-Arg-Arg + 7-amino-4-methylcoumarin
-
-
-
?
pyroglutamic acid-Arg-Thr-Lys-Arg-7-amido-4-methylcoumarin + H2O
?
-
-
-
-
?
additional information
?
-
-
the recombinant NS2B-NS3 protease is subject to autolytic cleavage
-
-
?
additional information
?
-
-
preferred substrate size for the dengue virus NS3 protease encompasses the residues from P4 to P3'. The presence of basic residues at the P3 and P4 positions significantly promotes reactivity. Serine is the preferred P1' residue
-
-
?
additional information
?
-
-
preferred substrate size for the Dengue virus NS3 protease encompasses the residues from P4 to P3'. Serine is the preferred P1' residue
-
-
?
additional information
?
-
molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P1 and P1' subsites) of DENV type 2 to compare the specificity of the protein-substrate binding recognition
-
-
?
additional information
?
-
-
NS2B-NS3 protease is a trypsin-like protease with a serine protease catalytic triad (His51, Asp75, Ser135) that has a polybasic substrate recognition profile
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
characterization of structural determinants necessary for activation of the enzyme by the NS2B cofactor. Alanine substitutions at residues Trp62, Leu75, and Ile79 in the NS2B cofactor result in marked effects on autoprocessing at the NS2B/NS3 site. The activity of the mutant NS3 protease with the synthetic peptide substrate Gly-Arg-Arg-4-methylcoumarin 7-amide is mainly affected by significantly reduced kcat values
-
NS2B cofactor
N-terminal region of NS3 and its cofactor NS2B constitute the protease. To function as an active enzyme, the NS3 protease requires the NS2B cofactor. NS2B is an integral membrane protein of 14 kDa that contains three domains: two trans-membrane segments located at the N- and C-termini and a central region of 47 amino acids (spanning amino-acids 49-96) that acts as an essential protein cofactor of the NS3 protease
-
NS2B cofactor
-
The dengue virus NS2B-NS3 protease (NS2B-NS3p), an important antiviral target for drug development, has been reported to adopt an open or closed conformation in crystal structures with different NS2B C-terminus (NS2Bc) positioning. NS2Bc departs from NS3p in the open conformation, but it forms tight interactions with NS3p in the closed conformation
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(1'R,2R,4'S,6S,8'R,10'E,12'S,14'E,16'E,21'R)-6-[(2S)-butan-2-yl]-12'-[[(2R,4S,5R,6R)-5-[[(2S,4S,5R,6S)-4,5-dihydroxy-6-methyltetrahydro-2H-pyran-2-yl]oxy]-4,6-dihydroxytetrahydro-2H-pyran-2-yl]oxy]-21',24'-dihydroxy-11',22'-dimethyl-3,4,5,6-tetrahydro-2'H-spiro[pyran-2,6'-[3,7,19]trioxatetracyclo[15.6.1.14,8.020,24]pentacosa[10,14,16,22]tetraen]-2'-one
-
-
(2aE,4E,5'S,6S,6'S,7S,8E,11R,13S,15S,17aS,20Z,20aR)-6'-cyclohexyl-20b-hydroxy-5',6,8,11,19-pentamethyl-20-(methylimino)-17-oxo-3',4',5',6,6',10,11,14,15,17,17a,20,20a,20b-tetradecahydro-2H,7H-spiro[11,15-methanofuro[4,3,2-pq][2,6]benzodioxacyclooctadecine
-
-
(3R,7aS)-2,3-bis(4-nitrophenyl)hexahydro-1H-pyrrolo-[1,2-c]imidazol-1-one
-
crystal structure analysis
(3R,7aS)-2-(4-nitrophenyl)-3-(4-(trifluoromethyl)-phenyl)-hexahydro-1H- pyrrolo[1,2-c]-imidazol-1-one
-
below 50% inhibition
(3R,7aS)-2-(4-nitrophenyl)-3-phenylhexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
67% inhibition
(3R,7aS)-3-(3-nitrophenyl)-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
below 50% inhibition
(3R,7aS)-3-(4-chlorophenyl)-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]-imidazol-1-one
-
below 50% inhibition, crystal structure analysis
(3R,7aS)-3-(4-isopropylphenyl)-2-(4-nitrophenyl)-hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
below 50% inhibition
(3R,7aS)-3-(4-methoxyphenyl)-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
below 50% inhibition
(3R,7aS)-3-(4-nitrobenzyl)-2-(4-nitrophenyl)hexahydro-1Hpyrrolo[1,2-c]-imidaz-ol-1-one
-
below 50% inhibition
(3R,7aS)-3-benzyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
42% inhibition
(3R,7aS)-3-ethyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
60% inhibition
(3R,7aS)-3-isopropyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
34% inhibition
(3R,7aS)-3-methyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
42% inhibition
(3S,7aS)-2,3-bis(4-nitrophenyl)hexahydro-1H-pyrrolo-[1,2-c]imidazol-1-one
-
below 50% inhibition
(3S,7aS)-2-(4-nitrophenyl)-3-(4-(trifluoromethyl)-phenyl)hexahydro-1H- pyrrolo[1,2-c]-imidazol-1-one
-
below 50% inhibition
(3S,7aS)-3-(4-chlorophenyl)-2-(4-nitrophenyl)hexa-hydro-1H-pyrrolo[1,2-c]imidazol-1-one
-
below 50% inhibition
(S)-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imida-zol-1-one
-
39% inhibition
1-(3-[[2-(1H-benzimidazol-2-yl)hydrazinyl]methyl]-4-hydroxy-5-[(E)-[2-(3H-indol-2-yl)hydrazinylidene]methyl]phenyl)butane-1,2-dione
-
-
1-[(4-[2-[5-(4-fluorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4-yl]phenyl)sulfonyl]piperidine
-
-
1-[5-[(4-chloro-5-methyl-3-nitro-1H-pyrazol-1-yl)methyl]furan-2-yl]-2-[1-[2-(diethylamino)ethyl]-1H-benzimidazol-2-yl]ethanone
-
-
2-[(E)-[2-(3-bromophenyl)hydrazinylidene]methyl]-N'-[(1E)-2-phenylethylidene]quinoline-4-carbohydrazide
-
-
2-[1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl]-1,2-benzothiazol-3(2H)-one
-
-
3-[(8-benzoyl-1-methyl[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino]benzoic acid
-
-
3-[[6-(3-fluorophenyl)pyridazin-3-yl]amino]-N-[2-(piperidin-1-yl)ethyl]benzamide
-
-
4-guanidino-benzoic acid 4-nitrophenyl ester
-
the fold of Dengue NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance spectroscopy is analyzed. Multiple paramagnetic lanthanide tags are attached to different sites to generate pseudocontact shifts (PCS). The PCSs show, that in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the West Nile virus protease. To assess the open state, a binding site for a Gd3+ complex is created and paramagnetic relaxation enhancements is measured. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution
4-hydroxypanduratin A
-
cyclohexenyl chalcone derivative, competetive inhibitor, compound from Boesenbergia rotunda (L.) Mansf. Kulturpfl.
4-[3-acetyl-5-(2-phenylquinolin-4-yl)-2,3-dihydro-1,3,4-oxadiazol-2-yl]benzoic acid
-
-
4-[[3-(azepan-1-yl)-6-oxo-6H-anthra[1,9-cd][1,2]oxazol-5-yl]amino]butanoic acid
-
-
5-methyl-2-[4-[(4-[[4-(4-methyl-4,5-dihydro-1H-imidazol-3-ium-2-yl)phenyl]amino]-4a,8a-dihydrophthalazin-1-yl)amino]phenyl]-4,5-dihydro-1H-imidazol-3-ium
-
-
benzoyl-Nle-KRR
homology model of the DV2 NS2B/NS3pro complexed with the peptidic inhibitor based on West nile virus structure
cardamonin
-
chalcone, non-competetive inhibitor, compound from Boesenbergia rotunda (L.) Mansf. Kulturpfl.
cyclo(D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-arginyl)
-
-
cyclo(D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-phenylalanyl)
-
-
cyclo(L-alanylglycyl-L-alanyl-L-arginyl-L-lysyl-L-serylglycyl-L-cysteinyl)
-
-
cyclo(L-alanylglycyl-L-lysyl-L-alanyl-L-lysyl-L-serylglycyl-L-cysteinyl)
-
-
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-alanyl-L-serylglycyl-L-cysteinyl)
-
-
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-alanylglycyl-L-cysteinyl)
-
-
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-L-phenylalanyl-L-cysteinyl)
-
-
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-serylglycyl-L-alanyl)
-
-
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-serylglycyl-L-cysteinyl)
-
-
cyclo(L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-phenylalanyl-D-prolyl-L-arginyl)
-
-
cyclo(L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-phenylalanyl-D-prolyl-L-lysyl)
-
-
cyclo-[L-Pro-L-Phe-L-Leu-L-Lys-Gly-L-Val-L-Tyr-L-Glu-L-Asp-L-Phe-L-Phe-Gly]
-
-
cyclo[3-(1-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-alanyl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[3-(1-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[3-(1-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-N6-[CH2NH]-L-alanyl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[3-(2-naphthyl)-D-alanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-3-(2-naphthyl)-D-alanyl-(2S)-2-amino-4-phenylbutanoyl-L-phenylalanyl]
-
-
cyclo[3-(2-naphthyl)-D-alanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-D-phenylalanyl-(2S)-2-amino-4-phenylbutanoyl-L-phenylalanyl]
-
-
cyclo[3-(2-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[3-(2-naphthyl)-L-alanyl-L-seryl-D-phenylalanyl-D-prolyl-L-lysyl-L-arginyl-L-lysyl-L-seryl]
-
-
cyclo[D-alanyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl]
-
-
cyclo[L-alany-(2R)-2-phenylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[L-alany-(2S)-2-phenylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[L-alanyl-(2R)-2-amino-4-phenylbutanoylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[L-alanyl-(2S)-2-amino-4-phenylbutanoylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[L-alanyl-D-phenylalanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[L-alanyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl]
-
-
cyclo[L-alanyl-L-phenylalanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
-
-
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2R)-2-amino-4-phenylbutanoyl-L-cysteinyl]
-
-
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-alanyl]
-
-
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-cysteinyl]
-
-
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-phenylglycyl-L-cysteinyl]
-
-
cyclo[L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl-D-prolyl-L-lysyl]
-
-
cyclo[L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl-L-prolyl-L-lysyl]
-
-
cyclo[L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanylglycyl-L-lysyl]
-
-
diphenyl [2-(4-aminophenyl)-1-[[(benzyloxy)carbonyl]amino]ethyl]phosphonate
-
-
N'-(2-ethylhexyl)-N'-(6-[N'-[N-(3-methylheptan-2-yl)carbamimidoyl]carbamimidamido]hexyl)imidodicarbonimidic diamide (non-preferred name)
-
-
N-benzyl-N,N-dimethyl-2-[2-[4-(2,2,3-trimethylpentan-3-yl)phenoxy]ethoxy]ethanaminium
-
-
panduratin A
-
cyclohexenyl chalcone derivative, competetive inhibitor, compound from Boesenbergia rotunda (L.) Mansf. Kulturpfl.
pinostrobin
-
flavanone, non-competetive inhibitor, compound from Boesenbergia rotunda (L.) Mansf. Kulturpfl.
additional information
-
discovery of cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity. The introduction of aromatic residues at the appropriate positions and conformational restriction generates the most promising cyclic peptide. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibit antiviral activity against DENV. Replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilizes the cyclic peptides against hydrolysis by NS2BNS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity
-
additional information
-
identification of fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors. The preliminary structure-activity relationship reveals that a substituent and its stereochemistry at C-3 position, substitution (X) at N-2 arene and a linker (Y) between C-3 position and its attached arene are important for the fused-ring scaffold of pyrrolidino [1,2-c]imidazolidinone to block the active site of NS2B-NS3 protease. The linear dipeptide L-methionyl-N-(4-nitrophenyl)-L-prolinamide) and the non-peptidic fused ring L-methionyl-N-(4-nitrophenyl)-L-prolinamide) show comparable activities against DENV-2 NS2B-NS3 protease and wild-type DENV-2 virus in a viral replication assay
-
additional information
-
in silico screening, alanine mutation, and density functional theory approaches for identification of NS2B/NS3 protease inhibitors, using molecular docking, free energy calculations, and simulation protocols in computational inhibitor screening, determination of ligand binding affinitites and protein interactions, overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
presence of basic residues at the P3 and P4 positions significantly promotes reactivity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.146
Gly-Arg-Arg-4-methylcoumarin 7-amide
-
pH 9.0, 37°C, wild-type NS3 protease with wild-tyüe NS2B cofactor
additional information
additional information
kinetics of cysteine-substituted mutant variants
-
0.03
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant A125C/V162C
0.03
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant S75C/K117C
0.04
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant I73C/P106C
0.04
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117A
0.04
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant S75C
0.04
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant S75C/K117C
0.04
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form wild-type enzyme
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117C
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117R
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant A125C/V162C
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant I73C/P106C
0.07
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form wild-type enzyme
0.08
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant K117A
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.02
Gly-Arg-Arg-4-methylcoumarin 7-amide
-
pH 9.0, 37°C, wild-type NS3 protease with wild-tyüe NS2B cofactor
0.006
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117A
0.01
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant A125C/V162C
0.02
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant A125C/V162C
0.02
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117C
0.02
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant S75C/K117C
0.03
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant I73C/P106C
0.04
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117R
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant S75C
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form wild-type enzyme
0.05
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant K117A
0.07
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant S75C/K117C
0.08
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant I73C/P106C
0.09
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form wild-type enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.44
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form wild-type enzyme
0.57
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant S75C/K117C
0.625
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant I73C/P106C
0.71
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form wild-type enzyme
0.8
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant S75C
1.25
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117R
1.33
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant I73C/P106C
1.5
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant A125C/V162C
1.5
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant S75C/K117C
1.6
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant K117A
2.5
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117C
5
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant unlinked form mutant A125C/V162C
6.67
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin
pH 8.5, 37°C, recombinant linked form mutant K117A
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0013
Bz-Nle-(3-amino)-Phe-(3-imino)-Phe-(3,4-dimethoxy)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0018
Bz-Nle-(3-guanidino)-Phe-(4-guanidino)-Phe-(4-guanidino)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0002
Bz-Nle-(4-amino)-Phe-(3-guanidino)-Phe-(4-amino)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.002
Bz-Nle-(4-amino)-Phe-(3-guanidino)-Phe-Arg
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0017
Bz-Nle-(4-amino)-Phe-(3-imino)-Phe-Arg
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0018
Bz-Nle-(4-amino)-Phe-(4-guanidino)-Phe-Trp
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0015
Bz-Nle-Arg-(3-guanidino)-Phe-(4-amino)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0001
Bz-Nle-His-(3-guanidino)-Phe-(4-guanidino)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0008
Bz-Nle-His-(3-guanidino)-Phe-beta-(2-naphthyl)-Ala
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0017
Bz-Nle-His-(3-imino)-Phe-Arg
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0018
Bz-Nle-His-(4-guanidino)-Phe-(4-guanidino)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0009
Bz-Nle-His-(4-guanidino)-Phe-Trp
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0005
Bz-Nle-homo-His-(3-guanidino)-Phe-(4-guanidino)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0019
Bz-Nle-homoHis-(3-guanidino)-Phe-Arg
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0014
Bz-Nle-homoHis-(4-guanidino)-Phe-(4-phenyl)-Phe
-
predicted inhibition constant, pH and temperature not specified in the publication
0.0016
Bz-Nle-Lys-Arg-Trp
-
predicted inhibition constant, pH and temperature not specified in the publication
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0012
(3R,7aS)-2,3-bis(4-nitrophenyl)hexahydro-1H-pyrrolo-[1,2-c]imidazol-1-one
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0023
cyclo(D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-arginyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0037
cyclo(D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-phenylalanyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.2
cyclo(L-alanylglycyl-L-alanyl-L-arginyl-L-lysyl-L-serylglycyl-L-cysteinyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.2
cyclo(L-alanylglycyl-L-lysyl-L-alanyl-L-lysyl-L-serylglycyl-L-cysteinyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.2
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-alanyl-L-serylglycyl-L-cysteinyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.2
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-alanylglycyl-L-cysteinyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0393
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-L-phenylalanyl-L-cysteinyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0574
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-serylglycyl-L-alanyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.114
cyclo(L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-serylglycyl-L-cysteinyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0038
cyclo(L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-phenylalanyl-D-prolyl-L-arginyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0038
cyclo(L-arginyl-L-lysyl-L-seryl-4-phenyl-L-phenylalanyl-L-seryl-D-phenylalanyl-D-prolyl-L-lysyl)
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0017
cyclo[3-(1-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-alanyl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0016
cyclo[3-(1-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0021
cyclo[3-(1-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-N6-[CH2NH]-L-alanyl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0011
cyclo[3-(2-naphthyl)-D-alanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-3-(2-naphthyl)-D-alanyl-(2S)-2-amino-4-phenylbutanoyl-L-phenylalanyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0017
cyclo[3-(2-naphthyl)-D-alanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-D-phenylalanyl-(2S)-2-amino-4-phenylbutanoyl-L-phenylalanyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0015
cyclo[3-(2-naphthyl)-L-alanyl-D-phenylalanyl-D-arginyl-L-arginyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0026
cyclo[3-(2-naphthyl)-L-alanyl-L-seryl-D-phenylalanyl-D-prolyl-L-lysyl-L-arginyl-L-lysyl-L-seryl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0033
cyclo[D-alanyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0151
cyclo[L-alany-(2R)-2-phenylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0222
cyclo[L-alany-(2S)-2-phenylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0162
cyclo[L-alanyl-(2R)-2-amino-4-phenylbutanoylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0256
cyclo[L-alanyl-(2S)-2-amino-4-phenylbutanoylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0076
cyclo[L-alanyl-D-phenylalanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0285
cyclo[L-alanyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0198
cyclo[L-alanyl-L-phenylalanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0387
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2R)-2-amino-4-phenylbutanoyl-L-cysteinyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0184
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-alanyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0092
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-cysteinyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0221
cyclo[L-alanylglycyl-L-lysyl-L-arginyl-L-lysyl-L-seryl-(2S)-2-phenylglycyl-L-cysteinyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.00095
cyclo[L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl-D-prolyl-L-lysyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.052
cyclo[L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanyl-L-prolyl-L-lysyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.005
cyclo[L-arginyl-L-lysyl-L-seryl-(2S)-2-amino-4-phenylbutanoyl-L-seryl-D-phenylalanylglycyl-L-lysyl]
Dengue virus type 2
-
pH and temperature not specified in the publication
0.0012
L-methionyl-N-(4-nitrophenyl)-L-prolinamide
Dengue virus type 2
-
pH and temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
additional information
the flavivirus NS3 protein is associated with the endoplasmic reticulum membrane via its close interaction with the central hydrophilic region of the NS2B integral membrane protein
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
additional information
physiological function
enzyme NS2B-NS3pro has a critical role in polyprotein maturation and viral infectivity
physiological function
-
the complex of NS2B and NS3 (NS2B/NS3) in the serine protease enzyme is essential for viral replication
physiological function
the N-terminal region of NS3 and its cofactor NS2B constitute the protease that cleaves the viral polyprotein. The NS3 C-terminal domain possesses RNA helicase, nucleoside and RNA triphosphatase activities and is involved both in viral RNA replication and virus particle formation. NS2B-NS3 protein plays multiple roles in the virus life cycle. NS2B-NS3 serves as a hub for the assembly of the flavivirus replication complex and also modulates viral pathogenesis and the host immune response
physiological function
-
the two-component viral protease NS2B-NS3 is required for the production of mature viruses and plays a key role in maintaining infectivity
physiological function
the virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins
additional information
enzyme ligand binding structure analysis, three-dimensional enzyme structure analysis
additional information
molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P1 and P1' subsites) of DENV type 2 to compare the specificity of the protein-substrate binding recognition, overview. Although all substrates are in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength is different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggest that among the ten substrate residues (P5-P5') the P1 and P2 subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites shows preferential binding at the active site. Besides, the P3, P1', P2' and P4' subsites show a less contribution in binding interaction. The catalytic water is detected nearby the carbonyl oxygen of the P1 reacting center of the capsid, intNS3, 2A/2B and 4B/5 peptides. The order of absolute binding free energy between these substrates and the NS2B/NS3 protease is capsid, intNS3, 2A/2B, 4B/5, 3/4A, 2B/3 in descending order in a relative correspondence with previous experimentally derived values. Hydrogen bond patterns of substrate binding
additional information
-
the enzyme is a complex of NS2B cofactor and NS3 protein. It adopts an open or closed conformation in crystal structures with different NS2B C-terminus (NS2Bc) positioning. In solution, NS2B-NS3p forms a mixture of open, closed and maybe other intermediate conformations. Analysis of the conformational changes using 19F NMR spectroscopy and enzyme mutant H51A/S75C. Low pH or bovine pancreatic trypsin inhibitor (BPTI) binding promote the conformation change from open to closed, showing the importance of charge forces in the interaction between NS2Bc and NS3p. NS3p residue His51 plays a crucial role in the pH-dependent conformational exchange by charge interacting with NS2Bc
additional information
the protease is a two-component system such that a portion of NS2B in addition to the protease portion of NS3 is also required for proteolytic activity. The enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the closed conformation dramatically increases the activity, strongly suggesting that the closed conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an unlinked construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a closed, active conformation. In contrast, in a linked construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the closed and other alternative conformations. 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF) induction of the closed conformation. Structure-function analysis, overview
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterodimer
-
the biologically active viral protease of NS2B/NS3 complex is termed as heterodimeric
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
analysis of enzyme structures, PDB IDs 2FOM, 2M9P, 2M9Q, 2BHR, and 2BMF
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A125C/V162C
site-directed mutagenesis, the mutant shows 32% reduction in activity due to the cysteine substitution. Linked A125C/V162C, in which both mutations are within the protease domain, does not migrate differently without DTT. The unlinked either trap does not form intermolecular disulfide bonds
H51A
-
site-directed mutagenesis, the mutation of NS3 protein impairs the charge interaction and the pH dependence of the conformational changes. It stabilizes the open conformation, while the addition of BPTI still converts NS2B-NS3p from open to closed conformation
I73C/P106C
site-directed mutagenesis, the linked open trap mutant does not form intermolecular disulfide bonds appreciably, as evidenced by the lack of higher-molecular weight bands. Unlinked I73C/P106C forms a small amount of NS2BS-SNS3pro, which shows retarded migration as NS2B and NS3pro electrophorese as a single disulfide-bonded complex
K117R/T122R
-
mutant is analyzed by 15N-HSQC spectra with and without inhibitor 4-guanidino-benzoic acid-4-nitrophenyl ester
P176G
-
mutation in the 11-amino acid linker region (169-179): a 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis are observed
S75C
S75C/K117C
site-directed mutagenesis, linked S75C/K117C is induced to form the NS2BS-SNS3pro closed conformation in the presence of active site inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that the mutation forms a disulfide bond when the enzyme is resting in the active conformation. The S75C/K117C pair is close together in the closed conformation but far apart in the open conformation and should therefore trap this variant in the closed conformation upon intramolecular disulfide bond formation
additional information
additional information
-
16 possible mutants bearing and an exchanged Ile to Val are analyzed by 15N-HSQC spectra with and without inhibitor 4-guanidino-benzoic acid-4-nitrophenyl ester
additional information
-
7 Ser to Ala mutants are analyzed by 15N-HSQC spectra with and without inhibitor 4-guanidino-benzoic acid-4-nitrophenyl ester
additional information
multiple types of protein constructs have been used for NS2B-NS3pro expression. In one type, a Gly4SerGly4 linker joins NS2B to NS3pro, reported to improve expression and purification, while another type requires co-expression of the unlinked NS2B and NS3 regions. The catalytic activity is slightly different between the linked and unlinked protein constructs, with the unlinked form showing activity approximately 0-5fold greater than that of the linked form, likely due to constraints introduced by the synthetic linker
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
various truncations and mutations in the cNS2B sequence show that conformational integrity of the entire 47 amino acids is critical for protease activity. DENV-2 NS3 protease can be pulled-down and is trans-activated by cNS2B cofactors from DENV-1, -3, -4, and West Nile Virus
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant DV NS2B-NS3pro, C-terminally tagged with a hexahistidine tag, purified by Co2+ affinity chromatography
-
recombinant His-tagged enzyme complex mutant H51A/S75C from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
-
recombinant His6-tagged protease DENV2 NS2B-NS3pro linked and form from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, recombinant unlinked enzyme is purified by anion exchange chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
plasmid pDV2-NS2B40-G4SG4-ASR-NS3FL expressed in Escherichia coli strain Rosetta2
-
recombinant expression of codon-optimized N-terminally His6-tagged dengue virus serotype 2 protease (DENV2 NS2B-NS3pro)from linked and unlinked constructs in Escherichia coli strain BL21(DE3)
recombinant expression of His-tagged enzyme complex mutant H51A/S75C in Escherichia coli strain BL21(DE3)
-
the dengue 2 NS2B-NS3pro construct contains 255 residues, including a (Gly)4-Ser-(Gly)4 linker between NS2B (47 residues) and NS3pro (185 residues). In addition, it contains a His-tag at the C-terminus and the T7 gene 10 N-terminal peptide MASMTG at the N-terminus followed by a two-residue cloning artifact (LE), resulting in a 27 kDa protein. To suppress slow autocleavage detected by SDS-PAGE, most samples have the mutation Lys15Ala which cause no significant changes in the appearance of the NMR spectra. Protein is expressed in Escherichia coli
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RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
two sets of prediction approaches. Secondary structure prediction performed using available structure prediction servers. A second approach makes use of the information on the secondary structures extracted from structure prediction servers, threading techniques and DSSP database of some of the templates used in the threading techniques. Consensus on the one-dimensional secondary structure of Dengue virus type 2 protease obtained from each approach and evaluated against data from the recently crystallised Dengue virus type 2 NS2B/NS3 obtained from the Protein Data Bank. The second approach shows higher accuracy compared to the use of prediction servers only and thus is applicable to the initial stage of structural studies of proteins with low amino acid sequence homology against other available proteins in the Protein Data Bank
drug development
medicine
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to validate NS2B as a potential target in allosteric inhibitor development, a cNS2B specific human monoclonal antibody (3F10) is used. 3F10 disrupts the interaction between cNS2B and NS3 in vitro and reduces DENV viral replication in HEK293 cells
drug development
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unique cleavage preferences of the Dengue virus protease promise novelty for the design of potent inhibitors that may be selective for the flaviviral proteases over proteases of the host cell and thus provide a solid base for follow-on structural and functional studies
drug development
flavivirus NS2B-NS3 protease-helicase is a target for antiviral drug development
drug development
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the dengue virus NS2B-NS3 protease (NS2B-NS3p) is an important antiviral target for drug development
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Niyomrattanakit, P.; Winoyanuwattikun, P.; Chanprapaph, S.; Angsuthanasombat, C.; Panyim, S.; Katzenmeier, G.
Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation
J. Virol.
78
13708-13716
2004
Dengue virus type 2
Shiryaev, S.A.; Kozlov, I.A.; Ratnikov, B.I.; Smith, J.W.; Lebl, M.; Strongin, A.Y.
Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses
Biochem. J.
401
743-752
2007
West Nile virus, Dengue virus type 2, Dengue virus type 2 16681, West Nile virus NY99
Othman, R.; Wahab, H.A.; Yusof, R.; Rahman, N.A.
Analysis of secondary structure predictions of dengue virus type 2 NS2B/NS3 against crystal structure to evaluate the predictive power of the in silico methods
In Silico Biol.
7
215-224
2007
Dengue virus type 2
Bera, A.K.; Kuhn, R.J.; Smith, J.L.
Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease
J. Biol. Chem.
282
12883-12892
2007
Japanese encephalitis virus, West Nile virus, Yellow fever virus, Dengue virus type 2, Dengue virus type 4, West Nile virus NY99
Niyomrattanakit, P.; Yahorava, S.; Mutule, I.; Mutulis, F.; Petrovska, R.; Prusis, P.; Katzenmeier, G.; Wikberg, J.E.
Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides
Biochem. J.
397
203-211
2006
Dengue virus type 2
Kiat, T.S.; Pippen, R.; Yusof, R.; Ibrahim, H.; Khalid, N.; Rahman, N.A.
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
2006
Dengue virus type 2
Phong, W.Y.; Moreland, N.J.; Lim, S.P.; Wen, D.; Paradkar, P.N.; Vasudevan, S.G.
Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target
Biosci. Rep.
31
399-409
2011
Dengue virus type 2
de la Cruz, L.; Nguyen, T.H.; Ozawa, K.; Shin, J.; Graham, B.; Huber, T.; Otting, G.
Binding of Low Molecular Weight Inhibitors Promotes Large Conformational Changes in the Dengue Virus NS2B-NS3 Protease: Fold Analysis by Pseudocontact Shifts
J. Am. Chem. Soc.
133
19205-19215
2011
Dengue virus type 2
Luo, D.; Wei, N.; Doan, D.N.; Paradkar, P.N.; Chong, Y.; Davidson, A.D.; Kotaka, M.; Lescar, J.; Vasudevan, S.G.
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
2010
Dengue virus type 2, Dengue virus type 4 (Q2YHF0)
Frecer, V.; Miertus, S.
Design, structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of Dengue virus NS2B-NS3 protease
J. Comput. Aided Mol. Des.
24
195-212
2010
Dengue virus type 2
Luo, D.; Vasudevan, S.G.; Lescar, J.
The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development
Antiviral Res.
118
148-158
2015
Yellow fever virus (P03314), Murray Valley encephalitis virus (P05769), West Nile virus (P06935), Kunjin virus (P14335), dengue virus type I (P17763), Japanese encephalitis virus (P27395), Dengue virus type 2 (P29990), Dengue virus type 4 (Q2YHF0), Kokobera virus (Q32ZD5), Dengue virus type 3 (Q6YMS3), Dengue virus type 2 Thailand/16681/1984 (P29990), Dengue virus type 3 Martinique/1243/1999 (Q6YMS3), Yellow fever virus 17D vaccine (P03314), Dengue virus type 4 Thailand/0348/1991 (Q2YHF0), dengue virus type I Nauru/West Pac/1974 (P17763), Japanese encephalitis virus SA-14 (P27395), Kunjin virus MRM61C (P14335), Murray Valley encephalitis virus MVE-1-51 (P05769)
Zhu, L.; Yang, J.; Li, H.; Sun, H.; Liu, J.; Wang, J.
Conformational change study of dengue virus NS2B-NS3 protease using 19F NMR spectroscopy
Biochem. Biophys. Res. Commun.
461
677-680
2015
Dengue virus type 2
Balajee, R.; Srinivasadesikan, V.; Sakthivadivel, M.; Gunasekaran, P.
In silico screening, alanine mutation, and DFT approaches for identification of NS2B/NS3 protease inhibitors
Biochem. Res. Int.
2016
7264080
2016
Dengue virus type 2
Hill, M.E.; Yildiz, M.; Hardy, J.A.
Cysteine disulfide traps reveal distinct conformational ensembles in dengue virus NS2B-NS3 protease
Biochemistry
58
776-787
2019
Dengue virus type 2 (P29990), Dengue virus type 2 Thailand/16681/1984 (P29990)
Takagi, Y.; Matsui, K.; Nobori, H.; Maeda, H.; Sato, A.; Kurosu, T.; Orba, Y.; Sawa, H.; Hattori, K.; Higashino, K.; Numata, Y.; Yoshida, Y.
Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity
Bioorg. Med. Chem. Lett.
27
3586-3590
2017
Dengue virus type 2
Weng, Z.; Shao, X.; Graf, D.; Wang, C.; Klein, C.D.; Wang, J.; Zhou, G.C.
Identification of fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors
Eur. J. Med. Chem.
125
751-759
2017
Dengue virus type 2
Yotmanee, P.; Rungrotmongkol, T.; Wichapong, K.; Choi, S.B.; Wahab, H.A.; Kungwan, N.; Hannongbua, S.
Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2 A molecular dynamics study
J. Mol. Graph. Model.
60
24-33
2015
Dengue virus type 2 (Q91H74), Dengue virus type 2 Thailand/NGS-C/1944 (Q91H74)
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