Inhibitory effect of phytochemicals towards SARS-CoV-2 papain like protease (PLpro) proteolytic and deubiquitinase activity

Abstract

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.

Keywords: PLpro; SARS-CoV-2; natural compounds; phytochemicals; replication.

Inhibition of proteolytic and deubiquitinase activity of SARS-CoV-2 PLpro enzyme by phytochemicals.

FIGURE 1

Inhibition of proteolytic activity of SARS-CoV-2 PLpro enzyme by selected alkaloids (A), xanthones (B), terpenoids (C) and organosulfur molecules (D). Selected alkaloids, xanthones, terpenoids, and organosulfur compounds were screened for their inhibitory activity against the SARS-CoV-2 PLpro enzyme as described under Materials and Methods. The fluorescence intensity was used to calculate the percent proteolytic activity considering DMSO treated control as 100% activity. Blank values were subtracted before calculating the percent activity. Representatives of four individual experiments (n = 4) with triplicate values were presented graphically and analyzed using GraphPad Prism 8. p <.05 considered as statistically significant compared to the DMSO control.

FIGURE 2

Inhibition of proteolytic activity of SARS-CoV-2 PLpro enzyme by selected flavanols (A), isoflavones (B) and flavanones (C). Selected flavanols, isoflavones and flavanones were screened for their inhibitory activity against the SARS-CoV-2 PLpro enzyme as described under Materials and Methods. The fluorescence intensity was used to calculate the percent proteolytic activity considering DMSO treated control as 100% activity. Blank values were subtracted before calculating the percent activity. Representatives of four individual experiments (n = 4) with triplicate values were presented graphically and analyzed using GraphPad Prism 8. p-values <.05 considered as statistically significant compared to the DMSO control.

FIGURE 3

Inhibition of proteolytic activity of SARS-CoV-2 PLpro enzyme by selected miscellaneous phytochemicals. Miscellaneous phytochemicals were selected and screened for their inhibitory activity against the SARS-CoV-2 PLpro enzyme as described under Materials and Methods. The fluorescence intensity was used to calculate the percent proteolytic activity considering DMSO treated control as 100% activity. Blank values were subtracted before calculating the percent activity. Representatives of four individual experiments (n = 4) with triplicate values were presented graphically and analyzed using GraphPad Prism 8. p-values <.05 considered as statistically significant compared to the DMSO control.

FIGURE 4

Selected phytochemicals Flavanols (A), Flavonones (B), Miscelleneous Drugs (C), and Xanthones (D) that exhibited inhibitory effect against deubiquitinase (DUB) activity of SARS-CoV-2 PLpro enzyme. Phytochemicals that inhibited the proteolytic activity of SARS-CoV-2 PLpro enzyme were selected and screened for their inhibitory effect towards DUB activity as described under Materials and Methods. The fluorescence intensity was used to calculate the percent DUB activity considering DMSO treated control as 100% activity. Blank values were subtracted before calculating the percent activity. Representatives of four individual experiments (n = 4) with triplicate values were presented graphically and analyzed using GraphPad Prism 8. p-values <.05 considered as statistically significant compared to the DMSO control.

FIGURE 5

Dose-dependent inhibition of SARS-CoV-2 PLpro proteolytic activity by EGCG, catechin, myricetin, mangiferin, and γ-mangostin, oleuropein, quercetin, rutin, and theaflavin: The phytochemicals that exhibited at least 50% inhibition of proteolytic activity of PLpro enzyme were selected and screened for their dose-dependent inhibitory activity as described under Materials and Methods. The fluorescence intensity was used to calculate the percent enzymatic activity considering DMSO treated control as 100% activity. Blank values were subtracted before calculating the percent activity. Representatives of four individual experiments (n = 4) with triplicate values were analyzed using GraphPad Prism8 and presented graphically. IC50 values were calculated using non-linear regression (curve fit) with four variable dose vs. inhibition by GraphPad Prism8.

FIGURE 6

Dose-dependent inhibition of SARS-CoV-2 PLpro DUB activity by EGCG, mangiferin, myricetin, oleuropein, rutin, and theaflavin: The phytochemicals that exhibited at least 50% inhibition of DUB activity of PLpro enzyme were selected and screened for their dose-dependent inhibitory activity as described under Martials and Methods. The fluorescence intensity was used to calculate the percent enzymatic activity considering DMSO treated control as 100% activity. Blank values were subtracted before calculating the percent activity. Representatives of four individual experiments (n = 4) with triplicate values were analyzed using GraphPad Prism8 and presented graphically. IC50 values were calculated using non-linear regression (curve fit) with four variable dose vs. inhibition by GraphPad Prism8.

FIGURE 7

Interaction of selected phytochemicals with SARS-CoV-2 PLpro. (A) Interaction of EGCG with Glu¹⁶⁷, Cys¹¹¹ and Tyr²⁶⁸ of PLpro through hydrogen bonding. (B) Interaction of mangiferin with Tyr²⁶⁸ and Lys¹⁵⁷ of PLpro through hydrogen bonding. (C) Interaction of oleuropein with Tyr²⁸⁶ of PLpro through hydrogen bonding. (D) Interaction of Theaflavin with Try²⁶⁸ and Glu¹⁶⁷ of PLpro through hydrogen bonding. Site view and ligand interaction maps were presented to illustrate the hydrogen bonding.

FIGURE 8

Fingerprinting population map of SARS-CoV-2 PLpro-ligand interaction. (A) EGCG shows a stronger interaction strength with Glu¹⁶⁷ than Tyr²⁶⁸. (B) Mangiferin demonstrates a stronger interaction strength with Tyr²⁶⁸. (C) Oleuropein shows a weak interaction with Tyr²⁶⁸. (D) Theaflavin shows a weaker interaction with Tyr²⁶⁸ than Glu¹⁶⁷ of PLpro but this interaction is stronger than EGCG, Mangiferin, and Oleuropein. The interaction of the molecules with specific amino acids of PLpro enzyme are represented as black bar.