Retraction Note: 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.

[This retracts the article on p. 976 in vol. 12, PMID: 33312423.].

Hesperidin identified from Citrus extracts potently inhibits HCV genotype 3a NS3 protease.

BACKGROUND: Hepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries. METHODS: The full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI-MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease. RESULTS: Fusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC50 values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MSn analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC50 value of 11.34 ± 3.83 µg/mL. CONCLUSIONS: A FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a.

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.

BACKGROUND: Hepatitis C virus genotype 3a (HCV G3a) is highly prevalent in Pakistan. Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV, medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease. Furthermore, from natural products, active compounds against vital HCV proteins like non-structural protein 3 (NS3) protease could be identified to prevent viral proliferation in the host. AIM: To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts. METHODS: Full-length NS3 without co-factor non-structural protein 4A (NS4A) and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli. The expressed protein was purified by metal ion affinity chromatography and gel filtration. Citrus fruit extracts were screened using fluorescence resonance energy transfer (FRET) assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry (MS)/MS technique. Among different polyphenols, highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay. RESULTS: NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein. Furthermore, in enzyme kinetic studies, NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3. So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease. FRET assay was developed and validated by the half maximal inhibitory concentration (IC50) values of commercially available inhibitors. Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91% of protease activity. Among the compounds identified by LCMS analysis, hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of -10.98. CONCLUSION: Fused NS4A-NS3 protease is functionally more active, which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32 µmol/L.

Computer aided screening of Accacia nilotica phytochemicals against HCV NS3/4a.

BACKGROUND: HCV has become a leading cause of liver cirrhosis and hepatocellular carcinoma and is a major health concern worldwide. To date, there is no vaccine available in the market to tackle this disease, therefore there is a strong need to develop antiviral compounds that can target all genotypes of HCV with the same efficiency. Medicinal plants have low cost and are less toxic therefore, extracts of medicinal plants can serve as important antiviral agents against HCV. This study was designed to screen phytochemicals of Accacia nilotica to find a potent drug candidate that can inhibit HCV infection effectively. RESULTS: Docking of NS3/4A protease and Flavonoids of Accacia nilotica revealed that most of the flavonoids bound deeply with the active site of NS3/4A protease. Compound 01 showed a high ranking on docking score. All other compounds also showed reliable docking scores and had interactions with the binding cavity of NS3/4A protease, suggesting them as a potent drug candidate to block HCV replication. CONCLUSION: To recognize binding interactions of Accacia nilotica phytochemicals with NS3/4A protease, molecular docking was performed to find potential inhibitor against NS3/4A protease of HCV. After post docking analysis, important interactions were found between active compounds and active site of NS3/4A protease. It can be concluded from the study that phytochemicals of Accacia nilotica may serve as a potential drug candidate with relatively simple structural changes against HCV NS3/4A protease.

Molecular screening of phytochemicals from Amelanchier Alnifolia against HCV NS3 protease/helicase using computational docking techniques.

Hepatitis C is serious health concern worldwide caused by HCV. It causes liver cirrhosis and hepato-cellular carcinoma. Development of prevention solutions is under progress. Meanwhile, the treatment of the viral disease using compounds isolated from natural medicinal plants is promising. The traditional use of photo-chemicals from medicinal plants like Amelanchier alnifolia for viral treatment is hopeful. Therefore, it is of interest to screen for flavonoids from Amelanchier alnifolia against protein targets of HCV. Hence, we assessed the binding of flavonoids to HCV NS3/4A protease and helicase proteins. Results show that Quercitin 3- galactoside and 3-glucosideshowed good binding score with protease and helicase respectively. Their interaction/binding sites are documented in this report. This data provide insights for the consideration of flavonoids as potential inhibitors of HCV/NS3/4A protease and helicase.