Limits...
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

Z’ factor analysis.The scatter plot showing the positive control (triangle shaped) and negative control (square shaped) data for the Z’ factor calculation. 24 samples were used for computing the means and standard deviations. The experiment was performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4593962&req=5

f4: Z’ factor analysis.The scatter plot showing the positive control (triangle shaped) and negative control (square shaped) data for the Z’ factor calculation. 24 samples were used for computing the means and standard deviations. The experiment was performed in triplicate.

Mentions: The assay was validated statistically by determining the Z’ factor and the coefficient of variation (CV). The Z’ factor is used for the assessment of the efficiency of HTS assay. It can be defined as the degree of separation between the mean values for the positive control and the mean value of the background signal. The Z’ factor with value greater than 0.5 has been considered as an indicator of an authentic and veritable screening assay. The CV of less than 10% represents the less variation within the groups of positive and negative controls. The Z’ factor for the CVCP was calculated as 0.64 using the FRET peptide substrate, which confirms the high sensitivity and efficiency of the assay. The scatter plot distribution of the group reads for both the positive and negative controls is shown in Fig. 4. The value of Z’ factor suggests high quality of the assay with high signal to noise ratio. The degree of variability is 8.68% in the positive control group. The Z’ factor and the CV reveal the reliability of the present FRET based assay in HTS of inhibitors against CVCP.


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)

Z’ factor analysis.The scatter plot showing the positive control (triangle shaped) and negative control (square shaped) data for the Z’ factor calculation. 24 samples were used for computing the means and standard deviations. The experiment was performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Z’ factor analysis.The scatter plot showing the positive control (triangle shaped) and negative control (square shaped) data for the Z’ factor calculation. 24 samples were used for computing the means and standard deviations. The experiment was performed in triplicate.
Mentions: The assay was validated statistically by determining the Z’ factor and the coefficient of variation (CV). The Z’ factor is used for the assessment of the efficiency of HTS assay. It can be defined as the degree of separation between the mean values for the positive control and the mean value of the background signal. The Z’ factor with value greater than 0.5 has been considered as an indicator of an authentic and veritable screening assay. The CV of less than 10% represents the less variation within the groups of positive and negative controls. The Z’ factor for the CVCP was calculated as 0.64 using the FRET peptide substrate, which confirms the high sensitivity and efficiency of the assay. The scatter plot distribution of the group reads for both the positive and negative controls is shown in Fig. 4. The value of Z’ factor suggests high quality of the assay with high signal to noise ratio. The degree of variability is 8.68% in the positive control group. The Z’ factor and the CV reveal the reliability of the present FRET based assay in HTS of inhibitors against CVCP.

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