Potential bioactive compounds as SARS-CoV-2 inhibitors from extracts of the marine red alga Halymenia durvillei (Rhodophyta) – A computational study

1 Introduction

In December 2019, a major cluster of atypical pneumonia caused by a novel coronavirus was reported in Wuhan city, China (Wang et al., 2020; Zhou et al., 2020). This coronavirus was initially named 2019 novel coronavirus (2019-nCoV) by the World Health Organization (WHO) and later renamed severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) (Gorbalenya et al., 2020; Meng et al., 2020). Despite efforts to contain the outbreak within the initial area, the dissemination of SARS-CoV-2 has continued to spread widely to other nations. In March 2020, the WHO declared the uncontrollable spread of SARS-CoV-2 as a pandemic (Cucinotta and Vanelli, 2020). Now, almost 2 years later, this unprecedented pandemic remains a global health threat with a total of 205,338,159 confirmed cases worldwide and 4,333,094 fatalities (WHO, 2021a) causing enormous economic upheaval.

Global collaborations are necessary to find effective way to control SARS-CoV-2 (Kaur and Gupta, 2020). The development of potential drugs and vaccines usually takes a sizable amount of time but, considering the high morbidity and mortality of the SARS-CoV-2, the pace of development had to go at a very fast pace (Belete, 2021). At least 294 vaccine candidates are in various stages of clinical development to date (WHO, 2021b) with some already in use by countries for mass vaccinations (Jabal et al., 2021). Vaccines reduce the risks of getting infected but questions remain regarding the duration of protection and protection response in natural infection (Long et al., 2020), as well to reduce the number of symptomatic cases for those infected (Olliaro et al., 2021). However, concerns for the protection provided by vaccines are raised related to the multiple SARS-CoV-2 variants that have been circulating globally (Iacobucci, 2021; Kirby, 2021).

The use of vaccines is for the precautionary use for the healthy, while the sick requires drugs to recover. The current drugs used include those with appropriate pharmacological and therapeutic efficacies such as remdesivir, hydroxychloroquine, chloroquine, lopinavir, umifenovir, favipiravir, and oseltamivir (Ita, 2020; Jean and Hsueh, 2020). To date, no specific therapeutic drugs have been reported to specifically target SARS-CoV-2 (Wu et al., 2020). Therefore, the search for potential therapeutic regimens to treat the sick is still ongoing by considering the available alternatives. Furthermore, the use of synthetic compounds has shown several adverse effects in the patients. On the other hand, the development of new drugs from natural sources was found to be less toxic and involved lower financial cost (Permana et al., 2020). Furthermore, to the best of our knowledge, there has been no report regarding the extreme toxicity of antiviral compounds obtained from natural product. Therefore, it is crucial to develop an alternative treatment of COVID-19 from natural products, including those from the marine environment such as algae-derived compounds (Hans et al., 2021).

Seaweeds have been recognized as rich and valuable sources of biological compounds that may provide potential solutions to the SARS-CoV-2 pandemic (Pereira and Critchley, 2020). Seaweed compounds showed activity against a broad spectrum of viruses (Jha et al., 2020; Hans et al., 2021). A group from Morocco found one seaweed compound that inhibits the Herpes simplex virus type 1 (HSV-1) by cell viability method (Rhimo et al., 2010). Cirne-Santos et al. (2019) also revealed that the brown seaweed Dictyota menstrualis (Hoyt) Schnetter, Hörnig & Weber-Peukert can inhibit the viral replication of the Zika virus (ZIKV). Moreover, the capability of seaweeds could be an alternative in combating coronaviruses through several mechanisms: penetration, replication, or targeting the host‘s cellular targets (Chen et al., 2020; Zaporozhets and Besednova, 2020).

The inhibitory effect of the natural compounds of seaweeds is related to their ability to inhibit the virus from attaching to the main protease, i.e., 3 chymotrypsin-like protease Main Protease (3CL-Mpro), which is fundamental for the replication of SARS-CoV-2 (Muteeb et al., 2020; Palese, 2020). The 3CL-Mpro is a key enzyme that has an important role in SARS-CoV-2 replication mechanism. It has been identified by researchers as a protein target in anti-coronavirus drugs (Qamar et al., 2020). X-ray crystallography of the 3CL-Mpro structure was obtained from PDB with 6LU7 code. The structure is comprised of three domains, i.e., domain I (residues from 8 to 100), domain II (residues from 101 to 176) and domain III (residues from 1200 to 306) where domain I and domain II are called as N-terminal domains with β-sheets and 13 β-strands, while domain III is a C-terminal domain that consists of 5 α-helices. The N-terminal domain plays a critical role as binding substrate that is located in a cleft between domain I and domain II. Residues number 176 to 199 from N-terminal and C-terminal domains are the loop that joins these domains. The overall molecular architecture of SARS-CoV-2 3CL-Mpro is illustrated in Fig. 1.

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Fig. 1

X-ray structure of 3CL-Mpro of SARS-CoV-2 (6LU7 pdb): domain I (red), domain II (orange) and domain II (blue), and its inhibitors NO3 within the active site (between domain I and II).

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Nowadays, drug discovery using computational design via molecular docking has become more popular and is becoming an important device used by scientists. Traditional drug discovery methods are expensive and time-consuming. Instead, molecular docking has been proven to be a more robust, effective, low-cost and less time-consuming process (Bharti and Shukla, 2021). Therefore, this study investigated an in silico method that evaluates the inhibitory effect of natural compounds from the red marine alga Halymenia durvillei Bory de Saint-Vincent against SARS-CoV-2. As previously explained, the main protease (3CL-Mpro) is the significant protein in the life cycle of SARS-CoV-2. This enzyme was used as one of the main targets of this computational study. Identifying the potential of seaweed against 3CL-Mpro main protease using computational study approaches has been conducted by Gentile et al. (2020) who showed that some natural compounds from the Marine Natural Product Library could be used as inhibitors with pharmacophore and molecular docking properties. Moreover, an in silico analysis of the marine seaweed database identified some red algae metabolites have a high-affinity against 3CL-Mpro of SARS-CoV-2. However, not all algae from the databases have been studied for their potentials against SARS-CoV-2.

Here, we identify the compounds in Halymenia durvillei that can potentially act as an inhibitor to 3CL-Mpro of SARS-CoV-2 by employing computational approaches. These involved virtual screening, pharmacophore study and molecular docking with 37 compounds of H. durvillei. The findings of this study may provide a new perspective for the development of natural-based drugs that are widely available, with high-efficiency, and low-toxicity for the treatment in the current and future pandemics.

2 Material and methods

3 Results and discussion

In this study, the extraction of phytochemicals from Halymenia durvillei was conducted using two different solvents, ethanol and ethyl acetate, in order to get compounds under both polar and semi-polar conditions. According to Deepak et al. (2018), ethyl acetate extraction of the brown seaweed Sargassum wightii Greville revealed all the tested phytochemicals while ethanol extraction has been used to obtain compounds from algae, especially the red alga Kappaphycus alvarezii (Doty) L.M. Liao which yielded phytochemicals which show mostly high polarity and solubility in water (Bhuyar et al., 2020). Therefore, in this present study, we used ethanol and ethyl acetate as extraction solvents.

This current investigation revealed 37 compounds (Table 1) from GC–MS analysis of ethanolic and ethyl acetate extracts of H. durvillei. Furthermore, GC–MS chromatograms are presented in Fig. 2. The details of all compounds are listed in Table 1. Three compounds were obtained from ethanol extraction namely caryophyllene, eucalyptol and 1,4,7,-cycloundecatriene, while 1,5,9,9-tetramethyl-, Z, Z, Z- and the rest of the compounds were obtained from ethyl acetate extraction. From Table 1, it is shown that eucalyptol from ethanolic extracts was the most compound with 97.55 area % (154 MW). By contrast, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol was the least compound from the ethyl acetate extract with 0.23 area % (296 MW).

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Fig. 2

The chromatogram of GC-MS extraction of H. durvillei (a) ethyl acetate and (b) ethanol.

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Fig. 3

The 2-D chemical structure of natural compounds of H. durvillei.

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Fig. 3

The 2-D chemical structure of natural compounds of H. durvillei.

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Most of the identified compounds are fatty acids or lipid constituting the most prominent phytochemicals from this seaweed as shown at Table 1 (see Fig. 3). This result is supported by the research of Chirasuwan et al. (2009) that showed lipid extracts to be potential antiviral agents and Schoggins and Randall (2013) reported that the roles of lipids in the antiviral response to infection are in two ways with directly inhibiting viral infection and regulating the adaptive and inflammatory responses. Moreover, according to recent study of SARS-CoV-2, the role of bioactive plasmalogens (vinyl ether glycerophospholipids) has the ability to enrich lung surfactant formulations to stimulate the respiratory process in severely infected individuals. Therefore, the role of lipid likely plasmogens can be suggested as an anti-viral prophylactic, a lipid biomarker in SARS-CoV and SARS-CoV-2 infections, and a potential anti-viral therapeutic component of lung surfactant development for COVID-19 patients (Deng and Angelova, 2021). These previous studies have shown the effectiveness of several lipid compounds as antiviral agent. Therefore, to confirm the potential of these compounds against viral activity, they were investigated to analyze their intermolecular interactions with the enzyme 3CL-Mpro of SARS-CoV-2 as ligand and protein through pharmacophore and molecular docking study.

A pharmacophore study to determine the biological features of inhibitors of the SARS-CoV-2 was applied and found to generate ligand-based pharmacophore models from the well-known drugs of SARS-CoV-2: remdesivir, lopinavir, ritonavir and hydroxychloroquine. Throughout the 10 generated models, the highest score was chosen as the template with 0.7886 scores using Ligand Scout calculation. The template model has at least two hydrogen donor and hydrogen acceptor features which were used in virtual screening approach. As a result, from 37 compounds of H. durvillei, only two passed the virtual screening accession: 1–2 tetradecandiol and E, E, Z-1,3,12-nonadecatrienome-5,14-diol with their pharmacophore fit at 38.95 and 38.99, respectively. The pharmacophore features (Fig. 4) of 1–2 tetradecandiol are two hydrogen donors and one hydrogen acceptor which interact with hydroxyl groups, respectively, while E, E, Z-1,3,12-nonadecatriene-5,14-diol has two hydrogen acceptors and one hydrogen donor. Furthermore, these two compounds as well as other compounds are the subject of continued study for their molecular interactions between protein and its inhibitors (ligands).

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Fig. 4

Pharmacophore features of (a) 1–2 tetradecandiol and (b) E,E,Z-1,3,12-nonadecatriene-5,14-diol.

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Although only two compounds passed the pharmacophore-based virtual screening approach, the docking result of main protease 3CL-Mpro with the natural compounds of H. durvillei showed considerable fitness scores that are shown in Table 2. It appears that all the natural molecules in monomer docking showed good binding energy and have competitive fitness scores ranging from 27,43 to 17,73 in GOLD and binding affinity ranging from 2.9 to 5.0 in AutoDock, except the positive controls. In both calculation of GOLD and AutoDock, Cholest-5-En-3-Ol (3.Beta.)- is the strongest ligand with a fitness score of 27,43 (GOLD) and 5.0 binding affnity (AutoDock), while the hits compound from the screening approach, 1–2 Tetradecanadiol and E, E, Z-1,3,12-Nonadecatriene-5,14-diol have 21,13 and 25,65 fitness scores in GOLD and −3.6 and −3.8 binding affinity in AutoDock, respectively. On the other hand, N3 and remdesivir that are co-crystallized ligands from X-ray crystallography protein structure (PDB: 6LU7) and well-known drugs of SARS-Cov-2have fitness scores of 24,44 and 16,78 in GOLD, then –6.1 and –5.9 in AutoDock, respectively. This gives information that in GOLD calculation, they are below those from other natural compounds while their scores are just over from the others compounds at AutoDock calculation. The ligand efficiency (LE) calculation showed that the Cholest-5-En-3-Ol (3.Beta.)- is −0.18 kcal/mol, while N3 and remdesivir are −0.12 and −0.14, respectively. According to Schultes et al (2010), the LE calculation showed that some compounds are competitive with the positive controls, which means that the compound are prioritized as the hits compound according to their LE with smaller low affinity for further optimization step. Furthermore, to examine the interaction of these potential compounds in monomeric docking with their competitors (N3 and remsidivir) with active site residues, Fig. 5 illustrates different binding modes of protein–ligand complexes and residues type in Table 3.

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Fig. 5

The binding modes of selected potential compounds within the active site of X-ray structure main protease (6LU7 pdb). (a) 1–2 tetradecandiol, (b) E,E,Z-1,3,12-nonadecatriene-5,14-diol, (c) Cholest-5-En-3-Ol (3.Beta.)-, (d) remdesivir , (e) NO3 and (f) superimposed image of the selected potential compounds.

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Fig. 6

Interactions between complexes comprised of ligands and dimeric state of the 3CL-Mpro. The co-crystalized ligand N3 bound to subunit 1 (a) and 2 (d), remdesivir bound to subunit 1 (b) and 2 (e), and Cholest-5-En-3-Ol (3.Beta.)- bound to subunit 1 (c) and 2 (f) of dimer state of the 3CL-Mpro (6LU7 PDB). The active site residues of protein are shown in the green sticks visalization, the ligands are shown with grey stick representations and the interacting residues of hydrogen bonds within 3 Å are shown in yellow dashes. The figures were constructed with PyMOL 2.5.

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In addition to analyzing the importance of the residues in the 3CL-Mpro Table 3 shows the classification of the residues in the active site from the different ligands. The importance of residues describes their contribution to the strength of the ligand in the protein cavity where they are significantly matched. Moreover, this list of active residues from candidate compounds and the competitors confirmed that they are also in the same cleft with similar active residues within 5 Å. The residues of the 3CL-Mpro are from number 24 to 192: THR24, THR25, THR26, LEU27, HIS41, MET49, GLY143, SER144, CYS145, HIS163, HIS164, MET165, GLU166, LEU167, PRO168, GLN189, THR190, ALA191 and GLN192 (see Fig. 5).

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Fig. 7

Allosteric site in monomer state.

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Since the monomeric docking showed the potential result for natural compounds, this study also had done to calculate the data set of H. durvillei compounds in biological conformation of the 3CL-Mpro in dimer state. This study was purposed to identifying the binding affinity and molecular interaction of each compound bound to each subunit of 3CL-Mpro. In the Table 2, dimer docking calculation was done by AutoDock which was divided into subunit 1 and subunit 2. Docking studies between compounds, positive controls (N3 and remdesivir) and 3CL-Mpro (dimer state) showed that all ligands bound precisely into the active site into each subunit. The highest affinity score between natural compounds is Cholest-5-En-3-Ol (3.Beta.)- with −7.3 for each subunit. These scores compared with the positive controls of N3 and remdesivir are significantly competitive due to the scores of N3 in subunit 1 and subunit 2 are −7.7 and –7.6, while remdesivir showed the higher scores than other ligand with –8.4 and –7.9 at each subunit. These calculations were depicted in the Fig. 6 to analyze the molecular interaction between ligands and the protein.

Most of compounds are bound precisely into the active site, however, there are some compounds which found its site with low energy affinity score near the active site. In subunit 1, 1,4,7,-Cycloundecatriene, 1,5,9,9-Tetramethyl-, Z,Z,Z- and Benzene Ethenyl have another active site which 1,5,9,9-Tetramethyl-, Z,Z,Z- attach into the cleft of α3 and α4, while Benzene Ethenyl has the allosteric site near to the active site that is located between L14 and L15. Meanwhile, Phenol, 4-(2,2,3,3-Tetramethylbutyl)- and Benzene,1,2-Dimethyl- in subunit 2 were bound between domain II of subunit 2 and domain III of subunit 1. The states of both compounds are quite far from the active site with ± 14 Å distance.

N3 in subunit 1 was bound by residues in the active site and formed hydrogen bonds in 3 Å with backbone Thr190 and Glu166. On subunit 2, N3 has non-polar interaction with residues at the active site and polar interactions with the side chains Phe449, Asn451 and His473. Remdesivir has polar interaction with backbone atoms with Phe140 in subunit 1 and Phe449 in subunit 2. Moreover, for the highest score among other natural compounds, Cholest-5-En-3-Ol (3.Beta.)- bound to the backbone of Thr190 with hydrogen bond in subunit 1 and this compound formed two hydrogen bond atoms with backbone atoms O and N of Thr499.

On the other hand, to identify candidate inhibitors of 3CL-Mpro activity, this study performed a virtual screen by docking of natural compound from H. durvillei targetting the putative allosteric site of the protein in monomer state. The data set of natural compounds and other positive controls (N3 and remdesivir) were docked to the protein receptor. Eventually, the docking calculations of AutoDock present seven allosteric sites, including the active site. In Fig. 7, it is shown that most of ligands in the first conformation or low energy bound to the allosteric site A and D, while other sites B, C, E, F (active site) and G only bound to the ligands in others conformations (see Table 5).

The residues of each allosteric sites of 3CL-Mpro are shown in Table 4. The identification was done by UCSF Chimera calculation of amino acids within 5 Å. Allosteric site A and B located at the domain III of receptor, allosteric site C and D located between domain II and III, and allosteric site E, F and G are between domain I and II. Allosteric site A bound with ligands in a cleft that is surrounded with α-helices 6 (residue 201–214) , 7 (residue 227–237), and 9 (261–274) with loop number L17 (residue 176–200), L19 (238–243) and L21 (275–292). Allosteric site B consists of L18 and α9. Allosteric site C is located between N-terminal loop (residue 1–10), L21 and β-sheets 9 (residue 121–131) and α6. Allosteric site D consists of L1, L11, L21, β7, β8, β9, β11. β12 and α10. Allosteric site E consists of α1, β1- β4, L8 and L10. Allosteric site F was included by α1, β1, β2, β4, β5, β7, β11, β12, L3, L10 and L17. The last allosteric site G consists L14, L16, L17 and β12- β13 (see Figure 7)

The allosteric site docking revealed that all ligands bound to the several sites of the receptors. However, most of the compounds showed that the other lowest energy was found at the allosteric site D rather than the other site. This phenomenon also has been shown by N3 that has the allosteric site in the D point and remdesivir was in the A point. Even though, most of compounds has the allosteric site to bound, the comparison of the lowest energy between allosteric sites and initial active site are competitive.

Regarding the ADME properties (see Table 6), the Lipsinki’s rule of five showed that most of natural compound of H. durvillei do not fulfill the Lipsinki properties. Even though molecular weight of each compound is not more than 500 kDa, number of HBA, HBD and PSA has fulfilled the standard of Lispinki’s rule of five. LogP and Rbond of some of compounds have higher value than the standard of Lipsinki’s rule of five which are not acceptable into the Lipsinki’s rule. This also happens to the Cholest-5-En-3-Ol (3.Beta.)- which is the most potential inhibitor of 3CL-Mpro with LogP which is little higher than the standard of LogP with more than 5.

Based on pharmacokinetics prediction result of the natural compound of H.durvillei in Table 7, some of the compounds have high ability in intestinal absorption. In gastrointestinal (GI) and blood–brain-barrier penetration properties only E,E,Z-1,3,12-Nonadecatriene-5,14-Diol, 2-Pentadecanone, 6,10,14-Trimethyl-, 15-Isopropenyloxacyclopentadecan-2-One and Z-(13,14-Epoxy)Tetradec-11-En-1-Ol Acetate have high absorption in digestion tract and penetration into the central nervous systems among the ten potential compounds, which would be delivered to the organ target blood barrier (blood-barrier of lung). Moreover, E,E,Z-1,3,12-Nonadecatriene-5,14-Diol, Neophytadiene (2,6,10-Trimethyl,14-Ethylene-14-Pentadecne), 3,7,11,15-Tetramethyl2-Hexadecen-1-Ol, and 2-Pentadecanone, 6,10,14-Trimethyl- have shown that these compounds has interaction with P-glycoprotein substrate which can cause drug-drug interactions and limiting celullar uptake of drugs from blood into brain and from digestion tract (intestinal lumen) to epithelial cells (Lin and Yamazaki, 2003). Meanwhile, most of compounds had no potential to inhibit P450 protein, however, there are still some compounds from the top ten candidate compounds that could inhibit each inhibitor of P450 protein. According to Bibi (2008), some of the prominent pharmacokinetic interactions between drugs are due to the P450 protein. The inhibitors of these types of protein can reduce metabolism, especially in drug-drug interactions.

Prior work has shown multiple advantages and health benefits of human consumption of red algae (Rhodophyta) such as cardiovascular, cancer, diabetes, anti-bacterial and anti-viral effects (Gamero-Vega et al., 2020) while another study also showed that marine red algae species have crucial anti-viral roles such as in HHV-1 viral DNA synthesis (Montanha et al., 2009). However, there are still lots of red algae with unknown potentials. This study has been conducted to examine one of the most common and largest tropical seaweed, H. durvillei by in silico approaches.

The study found from GC–MS results that ethanolic and ethyl acetate extraction yielded significantly different 3 and 34 compounds, respectively. Belkacami et al. (2020) showed different extraction results using methanol (polar) with 32,6 DC (dielectric constant) and chloroform (semi-polar) with 4,81 DC in the marine green alga Caulerpa racemosa (Forsskål) J. Agardh with methanolic extracts fewer than chloroform extracts. The same differences in polarity and DC were also observed in the current study which indicates that the phytochemicals extracted using semi-polar solvents like ethyl acetate (6 DC) and chloroform were higher than those obtained with polar solvent such as methanol and ethanol with 24,6 DC (Joshi and Adhikari, 2019).

In addition, the pharmacophore study revealed that inhibitor pharmacophore features should have a hydrogen bond donor as well as a hydrogen bond acceptor. Consequently, the virtual screening result of this study established two compounds: 1–2 tetradecandiol and E, E, Z-1,3,12-nonadecatriene-5,14-diol. Most notably, this study is the first to investigate the potential of Indonesian H. durvillei against SARS-CoV-2 through computational study. Our results provide a molecular docking approach in monomer and dimer state of 3CL-Mprotease by analyzing the compounds' molecular interactions. Throughout the result, our study revealed that H. durvillei compounds have a more remarkable fitness score than NO3 and remdesivir whether in monomeric or the biological state (dimeric) state of protein. The allosteric docking study identified that the protein has seven allosteric binding sites. Moreover, the fact that the highest abending affinity scores are from lipid has supported a previous study that lipid could be the potential drug against viruses, especially SARS-CoV-2. Based on the study of Han and Mallampalli (2015) lipid could act as lung surfactant by contributing 70% of its molecular weight. It has important role to stimulate the local pulmonary host defence mechanisms by playing as a barrier against adhesion of microorganisms and could improve the phagocytosis process by alveolar macrophages (inflammation).

Based on ADMET and Pharmacokinetic study, some compounds have not fulfilled the Lipinski's rule of five, while in pharmacokinetics identification, most of compounds are acceptable for absorption in digestion tract. Although our hypothesis is supported numerically, the number of compound types from this alga still needs to be identified and analyzed with more advanced molecular approaches. Future work should therefore include extraction using another solvent, such as non-polar ones and follow-up work in molecular dynamics simulation to assessing molecular behaviors between inhibitors and 3CL-Mpro protein. Moreover, following these promising results, some studies under in vitro and in vivo conditions must now be carried out to further investigate the effectiveness of active compounds in H. durvillei in the treatment of COVID-19. Importantly, the molecular dynamics study and MMGBSA or MMPBSA calculations should be performed for further advanced investigation in in silico study.

4 Conclusions

As noted earlier, the 37 phytochemicals derived from Halymenia durvillei that target the 3CL-Mpro of SARS-CoV-2 are able to interact with binding sites of the target protein effectively as well as exhibit competitive fitness scores. Therefore, the outcomes from computational-based drug discovery could be made a reference for the molecular interaction of the main protease with its ligand inhibitors against SARS-CoV-2. These initial promising results should be assessed further and validated using in vitro and in vivo approaches to better define their treatment mechanism of COVID-19.