Inhibition of NS2B-NS3 protease from all four serotypes of dengue virus by punicalagin, punicalin and ellagic acid identified from <i>Punica granatum</i>

Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC50 values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC50 of 0.91 ± 0.10, 0.75 ± 0.05, 0.42 ± 0.03, 1.80 ± 0.16 µM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV. Communicated by Ramaswamy H. Sarma</p

Effect of detergents on the activity of the HRV 3C protease activity.

<p>Effect of detergents on the activity of the HRV 3C protease activity.</p

Expression conditions used for proteins expression.

<p>Expression conditions used for proteins expression.</p

List of oligonucleotides used for cloning.

<p>List of oligonucleotides used for cloning.</p

SDS-PAGE showing the HRV 3C protease activity in the presence various buffers, increasing concentrations of variety of salts, additives and detergents commonly used during extraction and purification of proteins.

<p>‘‘C” represents the cleavable fusion protein treated with protease in the absence of any additive.</p

Analysis of activities of the HRV 3C and TEV proteases at 25°C towards their target proteins (His8-MBP-HRV 3C-MTD or His8-MBP-TEV-MTD, His₈-MBP-HRV 3C-betaC1 or His₈-MBP-TEV-beta C1 and His₈-HRV 3C-100K or His₈-TEV-100K).

<p>Respective cleavable fusion proteins were incubated with HRV 3C protease or TEV protease at ratio of 50:1 for 1 hour and analysed by SDS-PAGE. ‘‘U” and ‘‘T” represent the cleavable fusion protein untreated and treated, respectively, with the HRV 3C or TEV protease, Note: in case of cleavage of 100K fusion protein, 2kDa band was not observed because of low %age of polyacrylamide used in SDS-PAGE.</p