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Abstract The most common host entry point of human adapted coronaviruses (CoV) including SARS-CoV-2 is through the initial colonization in the nostril and mouth region which is responsible for spread of the infection. Most recent studies suggest that the commercially available oral and nasal rinse products are effective in inhibiting the viral replication. However, the anti-viral mechanism of the active ingredients present in the oral rinses have not been studied. In the present study, we have assessed in vitro enzymatic inhibitory activity of active ingredients in the oral mouth rinse products: aloin A and B, chlorhexidine, eucalyptol, hexetidine, menthol, triclosan, methyl salicylate, sodium fluoride and povidone, against two important proteases of SARS-CoV-2 PLpro and 3CLpro. Our results indicate only aloin A and B effectively inhibited proteolytic activity of PLpro with an IC50 of 13.16 and 16.08 μM. Interestingly, neither of the aloin isoforms inhibited 3CLpro enzymatic activity. Computational structural modelling of aloin A and B interaction with PLpro revealed that, both aloin isoforms form hydrogen bond with Tyr268 of PLpro, which is critical for their proteolytic activity. Furthermore, 100 ns molecular dynamics (MD) simulation studies predicted that both aloin isoforms have strong interaction with Glu167, which is required for PLpro deubiquitination activity. Our results from the in vitro deubiquitinase inhibition assay show that aloin A and B isomers exhibit deubiquitination inhibitory activity with an IC50 value of 15.68 and 17.51 µM, respectively. In conclusion, the isoforms of aloin inhibit both proteolytic and the deubiquitinating activity of SARS-CoV-2 PLpro, suggesting potential in inhibiting the replication of SARS-CoV-2 virus.

Recent studies have shown that RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin-like protease (3CLpro), and papain-like protease (PLpro) are necessary for SARS-CoV-2 replication. Among these three enzymes, PLpro exhibits both proteolytic and deubiquitinase (DUB) activity and is responsible for disrupting the host’s innate immune response against SARS-CoV-2. Because of this unique property of PLpro, we investigated the inhibitory effects of phytochemicals on the SARS-CoV-2 PLpro enzyme. Our data indicates that the phytochemicals such as catechin, epigallocatechin gallate (EGCG), mangiferin, myricetin, rutin, and theaflavin exhibited inhibitory activity with IC50 values of 14.2, 128.4, 95.3, 12.1, and 43.4, and 7.3 μM, respectively, towards PLpro proteolytic activity. However, the IC50 values of quercetin, oleuropein, and γ-mangostin are ambiguous. We observed that EGCG, mangiferin, myricetin, oleuropein, rutin, and theaflavin have also inhibited the DUB activity with IC50 values of 44.7, 104.3, 29.2, 131.5, 61.7, and 13.2 μM, respectively. Mechanistically, the ligand-protein interaction structural modeling suggests that mangiferin, EGCG, theaflavin, and oleuropein shows that these four ligands interact with Glu167, and Tyr268, however mangiferin and oleuropein showed very weak interaction with Glu167 as compared to EGCG, and theaflavin which reflects their low IC50 values for DUB activity. Our data indicate that the phytochemicals mentioned above inhibit the proteolytic and DUB activity of SARS-CoV-2 PLpro, thus preventing viral replication and promoting host innate immune response. However, the therapeutic potential of these phytochemicals needs to be validated by pre-clinical and clinical studies.