title: Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.
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.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.
authors.forename: Megha
authors.initials: M
authors.lastname: Aggarwal
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.
authors.forename: Pravindra
authors.initials: P
authors.lastname: Kumar
authors.affiliation: Division of Virology, Defence Research and Development Establishment, Gwalior 474002, India.
authors.forename: Manmohan
authors.initials: M
authors.lastname: Parida
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.
authors.forename: Shailly
authors.initials: S
authors.lastname: Tomar
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D065632:Chikungunya Fever / Q000378:metabolism / Q000821:virology; D002646:Chikungunya virus / Q000302:isolation & purification / Q000502:physiology*; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D011487:Protein Conformation
chemical_list: D036022:Capsid Proteins; D011480:Protease Inhibitors; D010447:Peptide Hydrolases