- Journal Details
- Format
- Journal
- eISSN
- 2564-615X
- First Published
- 30 Jan 2017
- Publication timeframe
- 4 times per year
- Languages
- English
Since the outbreak of the SARS-CoV-2 virus, from December 2019 until May 2021, more than 157 million people have been infected, and over 3.2 million deaths have been recorded (1). SARS-CoV-2 is a positive-sense single-stranded RNA (+ssRNA) virus which belongs to
Genomes of some typical Coronaviruses (CoVs), just like SARS-CoV-2 itself, have 6-10 open reading frames (ORFs). The first, the largest open reading frame, encodes 16 non-structural proteins nsps (nsp1-nsp16) (4). Nsp-5 encodes the main protease known as Mpro (chymotrypsin-like cysteine protease, 3CLpro), with an important role in virus replication and gene expression (5). Chymotrypsin-like cysteine protease (306 amino acid residues) (6) has three domains: catalytic domain, C-terminal domain and N-terminal finger domain, labeled as domain I, domain II and domain III, respectively. Domain III has enzymatic activity and no role in binding (7). The active site of this enzyme contains four sites, S1', S1, S2, and S4 (8, 9), while the catalytic site of Mpro preserves catalytic diad (His41-Cys145) located between domain I and II (7). Mpro is highly conserved among all of the Coronaviruses (8). Main role of Mpro in viral replication is its proteolytic activity. Proteolytic cleavage is performed by papain-pike protease (PLpro) and the main protease (Mpro), releasing functional polypeptides from the pp1a and pp1ab polyproteins (10, 11). Because of its important role in viral replication and its highly conserved catalytic domains, main protease is a potential drug target (11).
Finding an effective cure or vaccine against SARS-CoV-2 represents a great challenge to humanity. In order to stop the global pandemic, numerous studies and trials of potential drugs against SARS-COV-2 are underway (12).
One such potential antiviral substance is grapefruit seed extract (GSE), a commercial product made from grapefruit seeds and pulp, that is often used as a dietary supplement (13). Grapefruit (
The aim of this study was to analyze potential inhibitory effect of selected natural compounds from grapefruit seed extract (GSE) using SARS-CoV-2 main protease (Mpro) as a target protein.
In this study, the virtual interaction of the SARS-CoV-2 main protease (Mpro) and 6 selected components of grapefruit seed extract (naringin, narirutin, naringenin, limonin, ascorbic acid and citric acid) was examined. As positive controls of binding affinity, we used acetoside, remdesivir and gallic acid, because of their high binding affinity against Mpro in other molecular docking studies (20, 21, 23). Also, acetoside covalently inhibited Mpro in study of Teli
The three-dimensional crystal structure of SARS-CoV-2 main protease Mpro (PDB ID: 6Y84) was retrieved from Protein Data Bank in PDB format (27). The three dimensional structures of selected components of grapefruit seed extract (ligands) were retrieved from the PubChem (28) chemical database as SDF format and then converted to PDB three-dimensional format using online software Online SMILES Translator and Structure File Generator (29). PubChem CID of naringin (442428), naringenin (932), narirutin (442431), limonin (179651), ascorbic acid (54670067), citric acid (311), acetoside (24978601), remdesivir (121304016) and gallic acid (370), were used.
Preparation of target protein Mpro and ligands, was performed using the software AutoDock Tools, precisely MGLTools version 1.5.6. (30), wherein the molecules were converted to pdbqt format. Using Auto Dock Tools we removed all water molecules from protein and added polar hydrogen atoms as well as Kollmans charges.
Molecular docking was performed using AutoDock Vina 1.1.2. following standard procedures (31). We selected eight amino acid residues within chain A of the main protease (Mpro) as the catalytic binding site, namely His41, Phe140, Gly143, Ser144, Cys145, His163, His164 and Glu166, following the relevant literature (10,12). Cys145 – His41 dyad is a highly conserved region that forms an active site that has a catalytic function (7). Cys145, Gly143 and Ser144 are S1 residues which makes the oxyanion hole. His163 is yet another S1 residue, while Glu166 is a part of S2. According to Kumar
AutoDock Vina produced nine binding modes for every tested ligand with particular docking score (binding energy). The most stable binding mode for ligand is the mode with the least binding energy. The best binding modes and corresponding affinities of selected compounds of grapefruit seed extract (narirutin, naringin, naringenin, limonin, ascorbic acid abd citric acid) and binding affinities of positive controls (acetoside, remdesivir and gallic acid) against main protease of SARS-CoV-2 are presented in Table 1. These positive controls of binding affinities were chosen because of their high binding affinity against Mpro in other molecular docking studies.
Best docking scores (lowest binding energy) of selected compounds of grapefruit seed extract (GSE) as well as of the positive controls and their distance from best mode
| Distance from best mode | |||
|---|---|---|---|
| Ligands | Affinity (kcal/mol§) | rmsd l.b.† | rmsd u.b‡ |
| Narirutin | -10.5 | 0.000 | 0.000 |
| Naringin | -10.1 | 0.000 | 0.000 |
| Naringenin | -8.2 | 0.000 | 0.000 |
| Limonin | -9.9 | 0.000 | 0.000 |
| Ascorbic acid | -6.7 | 0.000 | 0.000 |
| Citric acid | -6.4 | 0.000 | 0.000 |
| Acetoside | -10.0 | 0.000 | 0.000 |
| Remdesivir | -9.6 | 0.000 | 0.000 |
| Gallic acid | -6.4 | 0.000 | 0.000 |
Legend: † root mean square deviation lower bound; ‡ root mean square deviation upper bound; §- kilocalorie per mole;• compounds of GSE; • positive controls
The highest binding affinity was obtained for flavonoid narirutin (-10.5, rmsd l.b. 0.000; rmsd u.b. 0.000) followed by a very similar affinity of naringin (-10.1, rmsd l.b. 0.000; rmsd u.b. 0.000) and slightly lower of limonin (-9.9, rmsd l.b. 0.000; rmsd u.b. 0.000). These binding affinities were similar to values of binding affinites for positive controls acetoside (-10.0, rmsd l.b. 0.000; rmsd u.b. 0.000) and remdesivir (-9.6, rmsd l.b. 0.000; rmsd u.b. 0.000). The binding affinities acquired for citric acid (-6.4, rmsd l.b. 0.000; rmsd u.b. 0.000) and positive control gallic acid (-6.4, rmsd l.b. 0.000; rmsd u.b. 0.000).
Visualized interactions showed similarity in narirutin, naringin, limonin binding modes to Mpro close to position of Lys5 (Figure 1B; Figure 2B; Figure 3B.)
Figure 1
A-B. Visualisation of the SARS-CoV-2 main protease (A) (PDB ID: 6Y84) with ligand narirutin and their best mode binding interactions (B) via PyMOL 2.4.
Figure 2
A-B. Visualisation of the SARS-CoV-2 main protease (A) (PDB ID: 6Y84) with ligand naringin and their best mode binding interactions (B) via PyMOL 2.4.
Figure 3
A-B. Visualisation of the SARS-CoV-2 main protease (A) (PDB ID:6Y84) with ligand limonin and their best mode binding interactions (B) via PyMOL 2.4.
Also, the naringin and naringenin showed similarity for binding modes to main protease close to position of Ser284 (Fig 2B; Fig 4B), just like ascorbic acid and citric acid for binding modes close to position of Glu290 and Glu288 (Figures: 5B, 6B.)
Figure 4
A-B. Visualisation of the SARS-CoV-2 main protease (a) (PDB ID:6Y84) with ligand naringenin and their best mode binding interactions (b) via PyMOL 2.4
Figure 5
A-B. Visualisation of the SARS-CoV-2 main protease (a) (PDB ID:6Y84) with ligand ascorbic acid and their best mode binding interactions (b) via PyMOL 2.4.
Figure 6
A-B. Visualisation of the SARS-CoV-2 main protease (a) (PDB ID:6Y84) with ligand citric acid and their best mode binding interactions (b) via PyMOL 2.4.
Except for these similar interactions, narirutin showed interaction close to position Gly138 and naringenin close to position Arg4 (Fig 1B; Fig 4B.)
Visualisation of the SARS-CoV-2 main protease with positive controls (acetoside, remdesivir and gallic acid) via PyMOL 2.4. showed that all three had interactions close to positions of Lys137 and Gln127 (Fig 7B; Fig 8B; Fig 9B) and also limonin interacted near position Lys137 (Fig 3B.)
Figure 7
A-B. Visualisation of the SARS-CoV-2 main protease (a) (PDB ID:6Y84) with ligand acetoside and their best mode binding interactions (b) via PyMOL 2.4.
Figure 8
A-B. Visualisation of the SARS-CoV-2 main protease (a) (PDB ID:6Y84) with ligand remdesivir and their best mode binding interactions (b) via PyMOL 2.4.
Figure 9
A-B. Visualisation of the SARS-CoV-2 main protease (a) (PDB ID:6Y84) with ligand gallic acid and their best mode binding interactions (b) via PyMOL 2.4.
Numerous studies and clinical trials about vaccines as well as a potential antiviral drugs are underway to hopefully put an end to the SARS-CoV-2 pandemic (35, 36).
There are five approaches of viral inhibition at different levels: 1.virus binding to the host receptor; 2. viral endocytosis; 3. neutralizing the virus particle; 4. targeting the viral structural proteins like the membrane, envelope and nucleocapsid protein and 5. restoration of host’s innate immunity (38).
Some of the potential drug targets of SARS-CoV-2 are spike protein (S), small envelope protein (E), matrix protein (M) and nucleocapsid protein (N). They all have a role in virus survival and propagation, and that is why their inhibition can lead to virus decay, which makes them ideal for drug targeting (35, 37). However, the best key targets in developing the antiviral drugs are the two proteases, 3CLpro (chymotrypsin-like cysteine protease, also known as Mpro) and PLpro (papain-like protease) (39). These proteases have a critical role in viral replication, whereby their proteolytic activities lead to the formation of functional non-structural proteins (11).
In this study, we analyzed virtual interaction between selected compounds from grapefruit seed extract (narirutin, naringin, naringenin, limonin, ascorbic acid, citric acid) as a potential inhibitors against main protease Mpro of SARS-CoV-2 using molecular docking method. As positive controls we used acetoside, remdesivir and gallic acid, because of their high binding affinity in other molecular docking studies and potential inhibitory effect against Mpro (20, 22, 23). The binding affinity of selected compounds ranged, from the highest for narirutin (-10.5 kcal/mol) to the lowest for citric acid (-6.4 kcal/ mol). The binding affinities for narirutin (-10.5 kcal/mol) and naringin (-10.1 kcal/mol) were slightly higher than binding affinity of positive control acetoside (-10.0 kcal/mol). According to Teli
Some of the studies observed the antiviral effect of grapefruit seed extract (17). The research by Go
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Best docking scores (lowest binding energy) of selected compounds of grapefruit seed extract (GSE) as well as of the positive controls and their distance from best mode
| Distance from best mode | |||
|---|---|---|---|
| Ligands | Affinity (kcal/mol§) | rmsd l.b.† | rmsd u.b‡ |
| Narirutin | -10.5 | 0.000 | 0.000 |
| Naringin | -10.1 | 0.000 | 0.000 |
| Naringenin | -8.2 | 0.000 | 0.000 |
| Limonin | -9.9 | 0.000 | 0.000 |
| Ascorbic acid | -6.7 | 0.000 | 0.000 |
| Citric acid | -6.4 | 0.000 | 0.000 |
| Acetoside | -10.0 | 0.000 | 0.000 |
| Remdesivir | -9.6 | 0.000 | 0.000 |
| Gallic acid | -6.4 | 0.000 | 0.000 |
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