TITLE

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

AUTHOR(S)
Aggarwal, Megha; Sharma, Rajesh; Kumar, Pravindra; Parida, Manmohan; Tomar, Shailly
PUB. DATE
October 2015
SOURCE
Scientific Reports;10/9/2015, p1
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
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 x 10³ 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.
ACCESSION #
110327900

 

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