Anti-SARS-CoV-2 activities of tanshinone IIA, carnosic acid, rosmarinic acid, salvianolic acid, baicalein, and glycyrrhetinic acid between computational and in vitro insights

Abstract

Six compounds namely, tanshinone IIA (1), carnosic acid (2), rosmarinic acid (3), salvianolic acid B (4), baicalein (5), and glycyrrhetinic acid (6) were screened for their anti-SARS-CoV-2 activities against both the spike (S) and main protease (Mpro) receptors using molecular docking studies. Molecular docking recommended the superior affinities of both salvianolic acid B (4) and glycyrrhetinic acid (6) as the common results from the previously published computational articles. On the other hand, their actual anti-SARS-CoV-2 activities were tested in vitro using plaque reduction assay to calculate their IC₅₀ values after measuring their CC₅₀ values using MTT assay on Vero E6 cells. Surprisingly, tanshinone IIA (1) was the most promising member with IC₅₀ equals 4.08 ng μl^-1. Also, both carnosic acid (2) and rosmarinic acid (3) showed promising IC₅₀ values of 15.37 and 25.47 ng μl^-1, respectively. However, salvianolic acid (4) showed a weak anti-SARS-CoV-2 activity with an IC₅₀ value equals 58.29 ng μl^-1. Furthermore, molecular dynamics simulations for 100 ns were performed for the most active compound from the computational point of view (salvianolic acid 4), besides, the most active one biologically (tanshinone IIA 1) on both the S and Mpro complexes of them (four different molecular dynamics processes) to confirm the docking results and give more insights regarding the stability of both compounds inside the SARS-CoV-2 mentioned receptors, respectively. Also, to understand the mechanism of action for the tested compounds towards SARS-CoV-2 inhibition it was necessary to examine the mode of action for the most two promising compounds, tanshinone IIA (1) and carnosic acid (2). Both compounds (1 and 2) showed very promising virucidal activity with a most prominent inhibitory effect on viral adsorption rather than its replication. This recommended the predicted activity of the two compounds against the S protein of SARS-CoV-2 rather than its Mpro protein. Our results could be very promising to rearrange the previously mentioned compounds based on their actual inhibitory activities towards SARS-CoV-2 and to search for the reasons behind the great differences between their in silico and in vitro results against SARS-CoV-2. Finally, we recommend further advanced preclinical and clinical studies especially for tanshinone IIA (1) to be rapidly applied in COVID-19 management either alone or in combination with carnosic acid (2), rosmarinic acid (3), and/or salvianolic acid (4).

Fig. 1. Chemical structures of the selected natural compounds (tanshinone IIA 1, carnosic acid 2, rosmarinic acid 3, salvianolic acid B 4, baicalein 5, and glycyrrhetinic acid 6).

Fig. 2. The RMSD of complex (left) and the ligands (right) as a function of simulation time.

Fig. 3. The RMSF of the S protein (left) and the Mpro protein (right).

Fig. 4. Histogram describing the binding interactions between the protein and its ligand during the simulation time of 100 ns for (A) Sal–S and (B) Tan–S, (C) Sal–Mpro, (D) Tan–Mpro, and (E) N3–Mpro.

Fig. 5. Heat map representing the total number of protein–ligand contacts during the simulation time of 100 ns for (A) Sal–S and (B) Tan–S, (C) Sal–Mpro, (D) Tan–Mpro, and (E) N3–Mpro.

Fig. 6. Ligand properties study during the simulation time of 100 ns for (A) Sal–S and (B) Tan–S, (C) Sal–Mpro, (D) Tan–Mpro, and (E) N3–Mpro.

Fig. 7. Graph of cytotoxicity concentration 50 (CC₅₀) on Vero E6 cells using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log cell viability versus normalized response (variable slope).

Fig. 8. Graph of inhibitory concentration 50 (IC₅₀): Antiviral activity against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (hCoV-19/Egypt/NRC-03/2020, accession number on GSAID: EPI_ISL_430820) Vero E6 cells using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitory versus normalized response (variable slope).

Fig. 9. Mode of action for tanshinone IIA against SARS-CoV-2. The significant differences are indicated (* = p < 0.05, and non-significant = ns).

Fig. 10. Mode of action for carnosic acid against SARS-CoV-2. The significant differences are indicated (* = p < 0.05, ** = p < 0.01, and non-significant = ns).

Fig. 11. Graphical representation describing the proposed modes of action for both tanshinone IIA and carnosic acid against SARS-CoV-2.