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Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.

Aggarwal M, Sharma R, Kumar P, Parida M, Tomar S - Sci Rep (2015)

Bottom Line: The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors.Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively.The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.

ABSTRACT
Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

No MeSH data available.


Related in: MedlinePlus

Influence of the glycerol on CVCP activity.(A) The effect of increase in glycerol concentration (0 to 50%) in the reaction buffer on the enzymatic activity of CVCP has been observed and the relative activity was measured by taking the activity at 20% glycerol as 100%. The graph represents the average data obtained from three experiments. (B) CVCP 3D homology model (magenta) with glycerol in the S1 specificity pocket was generated and superimposed on the crystal structure of AVCP with bound P1 residue (green) (PDB ID: 4AGK). The overall structure shows the presence of two subdomains; the active site is present at the interface of these two subdomains (active site residues are shown in sticks). The S1 specificity pocket is present in the vicinity of the active site (shown in circle) to which the P1 residue Trp267 of native AVCP binds. (C) The zoom-in surface view of the S1 pocket shows that glycerol (blue) in the CVCP structure binds incisively at the position where conserved P1 residue Trp (red) binds in AVCP. The active site is shown in blue color.
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f6: Influence of the glycerol on CVCP activity.(A) The effect of increase in glycerol concentration (0 to 50%) in the reaction buffer on the enzymatic activity of CVCP has been observed and the relative activity was measured by taking the activity at 20% glycerol as 100%. The graph represents the average data obtained from three experiments. (B) CVCP 3D homology model (magenta) with glycerol in the S1 specificity pocket was generated and superimposed on the crystal structure of AVCP with bound P1 residue (green) (PDB ID: 4AGK). The overall structure shows the presence of two subdomains; the active site is present at the interface of these two subdomains (active site residues are shown in sticks). The S1 specificity pocket is present in the vicinity of the active site (shown in circle) to which the P1 residue Trp267 of native AVCP binds. (C) The zoom-in surface view of the S1 pocket shows that glycerol (blue) in the CVCP structure binds incisively at the position where conserved P1 residue Trp (red) binds in AVCP. The active site is shown in blue color.

Mentions: The effect of anti-chaotropic agent glycerol on the enzyme activity is also observed and the CVCP is found to be susceptible for the inhibition with increasing glycerol concentration. Different concentrations of the glycerol (0 to 50%) (v/v) were used and the reaction velocity was measured. At 10% glycerol, negligible increase in the activity is found, however slight increase was noticed at 20% concentration of glycerol. However, further increase in the glycerol concentrations resulted in the decrease of enzymatic activity and at 50% glycerol concentration, almost no activity is detected (Fig. 6A). The results indicate that up to 20% glycerol can be used in the reaction buffer; afterwards it inhibits the catalytic activity of the enzyme.


Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.

Aggarwal M, Sharma R, Kumar P, Parida M, Tomar S - Sci Rep (2015)

Influence of the glycerol on CVCP activity.(A) The effect of increase in glycerol concentration (0 to 50%) in the reaction buffer on the enzymatic activity of CVCP has been observed and the relative activity was measured by taking the activity at 20% glycerol as 100%. The graph represents the average data obtained from three experiments. (B) CVCP 3D homology model (magenta) with glycerol in the S1 specificity pocket was generated and superimposed on the crystal structure of AVCP with bound P1 residue (green) (PDB ID: 4AGK). The overall structure shows the presence of two subdomains; the active site is present at the interface of these two subdomains (active site residues are shown in sticks). The S1 specificity pocket is present in the vicinity of the active site (shown in circle) to which the P1 residue Trp267 of native AVCP binds. (C) The zoom-in surface view of the S1 pocket shows that glycerol (blue) in the CVCP structure binds incisively at the position where conserved P1 residue Trp (red) binds in AVCP. The active site is shown in blue color.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593962&req=5

f6: Influence of the glycerol on CVCP activity.(A) The effect of increase in glycerol concentration (0 to 50%) in the reaction buffer on the enzymatic activity of CVCP has been observed and the relative activity was measured by taking the activity at 20% glycerol as 100%. The graph represents the average data obtained from three experiments. (B) CVCP 3D homology model (magenta) with glycerol in the S1 specificity pocket was generated and superimposed on the crystal structure of AVCP with bound P1 residue (green) (PDB ID: 4AGK). The overall structure shows the presence of two subdomains; the active site is present at the interface of these two subdomains (active site residues are shown in sticks). The S1 specificity pocket is present in the vicinity of the active site (shown in circle) to which the P1 residue Trp267 of native AVCP binds. (C) The zoom-in surface view of the S1 pocket shows that glycerol (blue) in the CVCP structure binds incisively at the position where conserved P1 residue Trp (red) binds in AVCP. The active site is shown in blue color.
Mentions: The effect of anti-chaotropic agent glycerol on the enzyme activity is also observed and the CVCP is found to be susceptible for the inhibition with increasing glycerol concentration. Different concentrations of the glycerol (0 to 50%) (v/v) were used and the reaction velocity was measured. At 10% glycerol, negligible increase in the activity is found, however slight increase was noticed at 20% concentration of glycerol. However, further increase in the glycerol concentrations resulted in the decrease of enzymatic activity and at 50% glycerol concentration, almost no activity is detected (Fig. 6A). The results indicate that up to 20% glycerol can be used in the reaction buffer; afterwards it inhibits the catalytic activity of the enzyme.

Bottom Line: The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors.Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively.The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.

ABSTRACT
Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

No MeSH data available.


Related in: MedlinePlus