(R)-(+)-Rosmarinic Acid as an Inhibitor of Herpes and Dengue Virus Replication: an In Silico Assessment

In the present computational research of antiviral drugs, (R)-(+)-rosmarinic acid was found to have remarkable antiviral abilities against the complications related to gamma herpes virus and NS5 RNA-dependent RNA polymerase domain complexed with 3′-dGTP and nonstructural protein 1 (NS1) of flavivirus. The present research has recognized that (R)-(+)-rosmarinic acid is an effective antiviral drug for numerous viruses like dengue and herpes virus. This statement was confirmed after evaluating the druggability of target proteins in terms of binding affinities to the tested ligand. Based on the present results, the existence of this molecule in plants and the antiviral potential of this molecule for other viruses have been clearly discussed as follows. Although the molecule was first discovered and isolated from Rosmarinus officinalis, a recent review described that (R)-(+)-rosmarinic acid has been widely isolated from many wild and cultivated plants worldwide over the past three decades. To assess the pharmacological potential of this phytochemical, it was tested on different targets in in silico, in vitro, and in vivo models.

Molecular DockingActive Sites of Viral Proteins

This phase is critical in docking as it helps identify the correct putative binding pocket in the target molecules. Although the protein consists of an enormous number of binding sites, only one site could be used to generate a grid box. Therefore, it is chosen after its site volume and values were found to be higher than the other sites detected from this protein. Ultimately, only one site was chosen as a putative ligand binding pocket because of its potential binding metrics for ligand binding (Vijayakumar et al. 2018).

To locate the ligand binding region, the appropriate ligand site in the protein molecules was examined. Although the molecules possess numerous sites, the ligand binding sites were filtered based on their site score and volume. Here, this site analysis revealed the residue consistency of three viral proteins such as 1F5Q, 2J7W, and 4OIG (Table 1). At the radius of 3 Å, the binding site residues of murine gamma herpesvirus cyclin complexed with human cyclin-dependent kinase 2 have been identified as LYS89, ASP86, GLN85, HIE84, LEU83, PHE82, GLU81, PHE80, LEU143, ALA144, ASP145, PHE146, GLY147, GLU51, LYS33, LEU32, ALA31, LEU134, GLN131, VAL64, ILE64, and LEU55 (Table S1). Similarly, the binding site residues of NS5 RNA-dependent RNA polymerase domain complexed with 3′ dGTP have been identified as PRO319, THR317, PRO258, ASN207, ARG257, LYS211, ALA213, ASP276, PHE277, and ASP278 (Table S1). Furthermore, the binding site residues of flavivirus nonstructural protein 1 (NS1) molecule have been identified as ARG481, VAL450, THR571, LYS575, VAL577, VAL579, VAL358, LYS355, PHE354, TYR299, TRP302, ILE592, GLN602, SER600, and GLY599 (Table S1).

Table 1 Docking scores, binding energies, and H bond interaction values of (R)-(+)-rosmarinic acid (1) with the docked viral proteins

In the present in silico approach, the compatibility of (R)-(+)-rosmarinic acid with the viral proteins of dengue and herpes viruses was examined. Eventually, this tested ligand was a possible drug because it contains better docking metrics for the docked dengue protein, including docking score, electrostatic energy, and hydrogen bonding. Particularly, the docking scores revealed that it comprises potential therapeutic effects to combat these viruses. It was additionally proven by the affinities between this protein and the tested molecule such as hydrogen bonds, π–π stacking, electrostatic potential, and π-cation. The contribution and flexibility of the tested ligand were clearly comprehended with the docked viral proteins, as follows:

i)

Murine gamma herpesvirus cyclin complexed to human cyclin-dependent kinase 2. (R)-(+)-rosmarinic acid was found to have a rather impressive docking score of − 10.847 with this viral protein (Table 1). In order to examine its inhibitory constant and its binding affinities with this enzyme, the docked complex was examined. This research found that (R)-(+)-rosmarinic acid established hydrogen bonding with herpes virus amino acid residues (Fig. 2a): PHE146 (2.14 Å), GLU51 (covalent bond, 1.47 and 2.06 Å), LYS33 (1.70 Å), LEU83 (2.13 Å), and HIE84 (2.14 Å), while the PHE80 was found to be forming a π–π stacking contact (Table S2). Figure 2b shows the functional group involved in the hydrogen bonding contacts with (R)-(+)-rosmarinic acid. Except residues PHE80 and LEU83, all the residues bind with the hydroxyl groups (OH). LEU83, on the other hand, was found in contact with oxygen of the carbonyl group of the ester group (C = O).

ii)

NS5 RNA-dependent RNA polymerase domain complexed with 3′ dGTP: a docking score of − 10.033 was found for (R)-(+)-rosmarinic acid with this viral protein (Table 1). (R)-(+)-rosmarinic acid displayed four hydrogen contact with viral protein amino acid residues (Fig. 3a), such as THR571 (2.28 Å), VAL450 (2.10 Å), PHE354 (1.71 Å), and SER600 (2.54 Å) (Table S2). Except the residues SER600, all the residues bind with the hydroxyl group (OH) of (R)-(+)-rosmarinic acid. LEU83, on the other hand, was found in contact with oxygen (O). The contact lines have been shown quite accurately as binding affinities in Fig. 3b.

iii)

Flavivirus nonstructural protein: a docking score of − 7.259 against this viral protein (Table 1, Fig. 4). (R)-(+)-rosmarinic acid displayed five hydrogen bonding with the herpesvirus amino acid residues. Amino acid residues such as THR317 (Covalent binding), PHE277 (2.76 Å), and ASP278 (1.92 Å) and LYS211 and ARG257 were found as cation and salt bridge contacts; LYS211 in particular binds covalently with (R)-(+)-rosmarinic acid as a π-cation contact at one end and as a salt bridge contact at another (Fig. 4a). Figure 4b shows which functional group is involved in hydrogen bonding contacts with (R)-(+)-rosmarinic acid. Except residues LYS211, all the residues bind with the hydroxyl group (OH) of (R)-(+)-rosmarinic acid (Fig. 4b).

Fig. 2figure 2

Docking of (R)-(+)-rosmarinic acid (1) to the 1F5Q murine gamma herpesvirus cyclin complexed to human cyclin-dependent kinase 2. a Residues and hydrogen bond contacts with their distance values among the ligand and catalytic pocket amino acids of the 1F5Q protein. b Revealed interactions between the hydroxy and oxygen groups of (R)-(+)-rosmarinic acid with 1F5Q

Fig. 3figure 3

Docking of (R)-(+)-rosmarinic acid (1) to the 2J7W dengue virus NS5 RNA-dependent RNA polymerase domain complexed with 3' dGTP. a Residues and hydrogen bond contacts with their distance values among the ligand and catalytic pocket amino acids of the 2J7W protein. b Revealed interactions between the hydroxy and oxygen groups of (R)-(+)-rosmarinic acid with 2J7W

Fig. 4figure 4

Docking of (R)-(+)-rosmarinic acid (1) to the 4OIG dengue virus dengue virus nonstructural protein NS1. a Residues and hydrogen bond contacts with their distance values among the ligand and catalytic pocket amino acids of the 4OIG protein. b Revealed interactions between the hydroxy and oxygen groups of (R)-(+)-rosmarinic acid with 4OIG

Biomedical Profiles

Since 1997 to date, it has been reported various pharmacological properties for (R)-(+)-rosmarinic acid as a novel candidate for inflammation, antioxidant potential, cancer, diabetic, and antiviral agent for zoonotic and non-zoonotic diseases, neurodegenerative, hypertensive, and antimicrobial, among other (Noor et al. 2022). In addition, it has also been used as folk medicine, cosmetics, and dietary supplements since ancient times (Baba et al. 2004). In 2011, Hsu et al. (2011) found that (R)-(+)-rosmarinic acid markedly reduced IL-1 and TNF-α release, thereby ameliorating collagen-induced arthritis in vivo. These are part of the innate immune system of the human body. The cytokinins such as interferon-1 and TNF-α play a key role in activating the innate immune system. In agreement with the research reported by Hsu et al. (2011), our results demonstrated that by inducing these two cytokinins, (R)-(+)-rosmarinic acid indirectly could activate the innate immune system to protect the human body from harmful materials.

MM-GBSA

In the MM-GBSA validation, the free energy values of (R)-(+)-rosmarinic acid are found to be − 58.65 for 1F5Q, − 52.17 for 2J7W, and − 48.52 for 4OIG (Table 2). The final results of the investigation imply that (R)-(+)-rosmarinic acid has the strong binding with the docked viral proteins.

Table 2 Rat acute toxicity level of (R)-(+)-rosmarinic acid (1) at various dosesPharmacophore

The data set was divided into active, moderately active, and inactive regions by keeping the activity threshold in the 7.2 range. The (R)-(+)-rosmarinic acid binding domain in terms of its five characteristics as shown in Fig. 5 included the generic pharmacophore hypotheses due to its high survival value. Furthermore, the e-pharmacophore shows that the rosmarinic acid consists of four acceptors (A), four donors (D), one negative ionic (H), and two aromatic rings (R) that were obtained (Fig. 5).

Fig. 5figure 5

Pharmacophore hypothesis of (R)-(+)-rosmarinic acid (1). A denotes hydrogen bond acceptor in pink color, D denotes hydrogen bond donor in blue. and R denotes aromatic rings in brown color from docked phytochemicals; A–D showed the active site of docked phytochemicals developed by e-Pharmacophore

Druggability

The Prediction of Activity Spectra for Substances (PASS) server showed that (R)-(+)-rosmarinic acid has broad pharmacological possibilities as notable by the probability active (Pa) score, with a drug probability active score ranging from 0.710 to 0.956; only the most probable pharmacological activities were included in Table S3. On the other hand, the side effects of the prescribed herpesvirus drugs such as aciclovir, famciclovir, penciclovir, and valaciclovir have been shown in the supplementary data (Table S4S7). Accordingly, the present research suggests that these drugs might lead to further complications for human health after administration as a treatment for the herpes virus. The most significant adverse effects of these drugs were shown in Figs. S4S6. Likewise, the adverse effects of (R)-(+)-rosmarinic acid were also shown in Table S8.

Rat Acute Toxicity

The rate acute toxicity of (R)-(+)-rosmarinic acid was measured by LD50 at log10 (mmol/kg) and LD50 mg/kg levels. The applicability domain models were measured in terms of administration, including i.p., i.v., p.o., and s.c. administrations. The predicted values for acute toxicity are shown in Table 2 and Fig. 6.

Fig. 6figure 6

Acute toxicity for (R)-(+)-rosmarinic acid (1) at different routes of administration at LD50 dose (predicted by Gusar). Abbreviations: i.p., intraperitoneal; i.v., intravenous; s.c., subcutaneous administration

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