Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Nov 27, 2020; 12(11): 976-992
Published online Nov 27, 2020. doi: 10.4254/wjh.v12.i11.976
Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry
Mahim Khan, Waqar Rauf, Fazal-e- Habib, Moazur Rahman, Mazhar Iqbal
Mahim Khan, Waqar Rauf, Fazal-e- Habib, Moazur Rahman, Mazhar Iqbal, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad 38000, Punjab, Pakistan
Moazur Rahman, School of Biological Sciences, University of the Punjab, Lahore 54810, Punjab, Pakistan
Author contributions: Khan M was involved in this project from the design to execution and writing of this manuscript, optimized the expression and purification of hepatitis C virus non-structural protein 3 protease, extracted antioxidant compounds and tested their inhibition using the in vitro fluorescence resonance energy transfer assay, contributed to the LCMS/MS analysis to identify phenolics/flavonoids in plant extracts and evaluated their interaction using a modeling approach; Rauf W contributed to the design and execution of the experiments and LCMS/MS data analysis; Habib F performed the LCMS/MS analysis; Rahman M provided help with the purification of hepatitis C virus non-structural protein 3 and manuscript writing; Iqbal M conceived the idea, planned all of the experiments, and contributed to the data analysis, and manuscript writing.
Institutional review board statement: It is certified that the protocol of the study, screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry was approved by the institutional review board.
Conflict-of-interest statement: There is no financial, commercial or other conflict of interest with any author.
Data sharing statement: All data generated or analyzed during this study are included in this manuscript and its supplementary material. Any additional information, if required, may be asked from the corresponding author (hamzamgondal@ gmail.com).
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Mazhar Iqbal, PhD, Professor, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Jhang Road, Faisalabad 38000, Punjab, Pakistan. hamzamgondal@gmail.com
Received: July 27, 2020
Peer-review started: July 27, 2020
First decision: August 22, 2020
Revised: September 3, 2020
Accepted: September 16, 2020
Article in press: September 16, 2020
Published online: November 27, 2020
Processing time: 119 Days and 23 Hours
ARTICLE HIGHLIGHTS
Research background

Hepatitis C virus genotype 3a (HCV G3a) is highly prevalent in many countries including Pakistan. FDA-approved drugs have significantly contributed in effective control of the disease but are expensive and not affordable to a large proportion of the infected population.

Research motivation

Medicinal natural products having antiviral potential could be screened for the cost-effective treatment of the disease. Using such products, inhibition assays against vital viral proteins like non-structural protein (NS) 3 protease could be developed to prevent viral proliferation in the host.

Research objectives

This study developed cost-effective HCV G3a NS3 protease inhibitors from citrus fruit extracts.

Research methods

Codon optimized NS3 protease domain fused with NS4A as well as full-length NS3 constructs were cloned in pET11a expression vector. Both constructs were expressed in Escherichia coli BL21 (DE3) cells and purification was performed using Ni-affinity chromatography followed by gel filtration. The fluorescence resonance energy transfer assay was developed and validated using commercial inhibitors. Furthermore, extracts from different citrus species, were screened on the basis of percentage inhibition. The components of the most active extract were identified using electro spray ionization- mass spectrometry/mass spectrometry technique. Docking was performed with Molecular operating environment software to screen out the potent natural product, which was acquired in purified form and evaluated against NS3/4A protease using fluorescence resonance energy transfer assay.

Research results

We successfully overexpressed and purified genotype 3a NS3 protease domain fused with NS4A and the yield was also higher than full-length NS3 protein. Inhibition of NS3 protease fused with NS4A protein was tested against different citrus extracts and grapefruit mesocarp extract showed highest percentage inhibition of protease activity (91%). Hesperidin was identified as the inhibiting compound in the extract having docking S-score value of -10.98.

Research conclusions

NS3 protease fused with co-factor NS4A was found functionally more active. Hesperidin from the grapefruit mesocarp extract showed the inhibition against NS4A-NS3 protease domain with an IC50 value of 23.32 µmol/L.

Research perspectives

Hesperidin flavonoid may be further explored as potential antiviral agent against HCV as an affordable option for infected population.