Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus

Scientific Reports
Amrita SahaPaban Kumar Dash

Abstract

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z' value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for ...Continue Reading

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Citations

Jan 9, 2020·Expert Opinion on Therapeutic Targets·Vitor Won-Held RabeloPaula Alvarez Abreu
Jun 11, 2019·Expert Opinion on Drug Discovery·María-Jesús Pérez-PérezEva-María Priego
Feb 11, 2020·Frontiers in Cellular and Infection Microbiology·Shweta SaraswatDeepak Sehgal
Mar 20, 2019·Expert Review of Anti-infective Therapy·Shree Madhu BhatGovindakarnavar Arunkumar
Jul 1, 2020·ACS Synthetic Biology·Peng ZhangYuan Lu

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Datasets Mentioned

BETA
EU372006

Methods Mentioned

BETA
protease assay
FRET
flow cytometry
protein folding

Software Mentioned

Bio
Office excel
GraphPad Prism
Flex Analysis
MetSite
Biocommand Bioprocessing
MASCOT
CellQuest Pro
Gen5

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