key: cord-0924011-2sywgk0w authors: Khattab, Amira R; Teleb, Mohamed; Kamel, Mohamed S title: In silico study of potential anti-SARS cell entry phytoligands from Phlomis aurea: a promising avenue for prophylaxis date: 2021-10-28 journal: Future virology DOI: 10.2217/fvl-2021-0031 sha: f9fdb746f5d23fe0a6ddd374c2504e6f56da47a2 doc_id: 924011 cord_uid: 2sywgk0w Aim: The severity of COVID-19 has raised a great public health concern evoking an urgency for developing multitargeted therapeutics. Phlomis species was ethno-pharmacologically practiced for respiratory ailments. Materials & methods: An array of 15 phytoligands previously isolated from Phlomis aurea were subjected to molecular docking to explore their potential SARS-CoV-Spike-angiotensin-converting enzyme 2 complex inhibition, that is essential for virus entry to host cell. Results: Acteoside (11) showed the most potent in silico inhibition with an additional merit, over hesperidin (16), of not binding to angiotensin-converting enzyme 2 with well proven in vivo pulmonary protective role in acute lung injury, followed by chrysoeriol-7-O-β-glucopyranoside (12) and luteolin-7-O-β-glucopyranoside (14). Conclusion: Phytoligands (11, 12 and 14) were posed as promising candidates with potential prophylactic action against COVID-19. These phytoligands were prioritized for further biological experimentation because of their acceptable predicted ADME and drug-likeness parameters. Moreover, they could aid in developing multitargeted strategy for better management of COVID-19 using phytomedicines. In silico exploration was performed on an array of 15 phytoligands (Figure 1 ) previously isolated from leaves of Phlomis aurea Decne from Saint Katharine mountain (Sinai, Egypt) which belonged to different phytochemical classes viz. iridoids, in other words, 3-epiphlomurin (1), phlomurin (2) , auroside (5) , lamiide (6), 8-epiloganin (7) and ipolamiide (8) , megastigmane glucoside, in other words, phlomuroside (3), benzyl alcohol glycoside, in other words, benzyl alcohol-O-β-xylopyranosyl-(1→2)-β-glucopyranoside (4) , phenolic glycosides, in other words, syringin (9) and acteoside (11) , phenylethanoid glycoside, in other words, 2-phenylethyl-O-β-xylopyranosyl-(1→2)-β-glucopyranoside (10) , flavonoids, in other words, chrysoeriol-7-O-β-glucopyranoside (12) , acacetin-7-O-β-glucopyranoside (13) and luteolin-7-O-β-glucopyranoside (14) and lignin, in other words, liriodendrin (15) [15] . 2D structures of the phytoligands (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) were downloaded as structure-data file from PubChem (https: //pubchem.ncbi.nlm.nih.gov). Docking simulation Molecular operating environment (MOE) software (version 2015.10, Chemical Computing Group, QC, Canada) was employed for docking simulations using the crystal structure of SARS-CoV-2 spike (S) protein C-terminal domain (SARS-CoV-2-CTD) in complex with human ACE2 (hACE2). SARS-CoV-Spike-ACE2 complex reported to reveal the most recently known hACE2-binding mode was retrieved from the protein data bank (PDB ID: 6LZG) [16] . The complex was prepared by eliminating the unwanted residues, solvents and ligands using the default settings of the 'structure preparation' module. The spatial arrangement of some key residues in the SARS-CoV-2-CTD binding interface was located as the receptor site utilizing 'site finder' feature of MOE, where the site number 8 was selected. Structures of the phytoligands (1-15) under investigation were built in silico, then subjected to default energy minimization and geometry optimization. The ligand placement was set to apply triangular matcher algorithm. Top five conformers of the test ligands that possessed nonredundant poses of the lowest binding energy were generated by utilizing alpha HB as the default scoring function. An induced fitting protocol for docking was employed to record the best possible molecular interactions [17] . Phytoligands were then ordered according to their S-scores with a root-mean-square deviation (RMSD) value <2Å in addition to the graphical representations of the ligand interactions. In silico prediction of physicochemical properties, ADMET & drug-likeness parameters Physicochemical properties and drug-likeness were computed by SwissADME software [18] . ADME profiling was performed by PreADMET calculator [19] . The workflow of the research methodology is illustrated in Figure 2 . Molecular docking analysis of Phlomis aurea phytoligands Viral infections are generally initiated with the binding of viral particles to specific host-cell surface receptors. Hence, receptor recognition is considered as a critical determinant of the viral entry and cell invasion. Targeting such process is; therefore, considered an inspiring prophylactic approach against viral infection. In SARS-CoV-2, the viral entry is mediated by an envelope-embedded surface-located spike (S) glycoprotein [20] which utilizes hACE2 for cell entry [21] . The S glycoprotein is cleaved, in most cases, by host cell proteases into S1 and S2 subunits for receptor recognition and cell membrane fusion, respectively. S1 is further subdivided into N-terminal domain and C-terminal domain (CTD), both can function as a receptor-binding entity. Most recently, a pioneer study utilized immunostaining and flow cytometry assays to first identify the S1 CTD of SARS-CoV-2 as the key region that interacts with the hACE2 receptor. A 2.5-A • crystal structure of SARS-CoV-2-CTD in complex with a single hACE2 molecule in asymmetric unit (PDB ID: 6LZG) [16] was solved revealing a clear receptor-binding mode. Further analysis of the virus-receptor interaction on structural basis was performed to identify the key aminoacids involved in complex formation. Accordingly, a series of hydrophilic residues forming an H-bonds network and salt bridge interactions were located along the binding interface [16] . In light of the mentioned information we utilized docking simulation to probe the ability of the studied phytoligands to destabilize the virus-receptor complex or even prevent its formation, where possible accommodation/fitting of such phytochemicals at the SARS-CoV-2-CTD-h2ACE interface and their interaction with the complex key amino acids may provide preliminary promising insights for further biological investigation. Several in silico studies displayed the possible inhibitory mechanisms of various phytochemicals on the ACE2-Spike complex of SARS-CoV-2, and highlighted the structural determinants of important interactions [22] [23] [24] [25] [26] . In absence of a cocrystalized inhibitor at the interface of the studied complex, we employed the 'site finder' feature of MOE 2015.10 to locate the most suitable site for docking the studied phytochemicals into the SARS-CoV-2-CTD-2hACE binding interface taking in consideration the key residues involved in the complex formation. Flexible docking of the 15 phytochemicals under investigation was performed several times in the defined site at the binding interface with hesperidin as a reference viral entry inhibitor [27] . Docking simulations results (Table 1) showed that most of the studied phytoligands displayed good binding affinities compared with hesperidin (16) . Acteoside (11) came at the top of the list recording the best binding affinity (-7.75 kcal/mol) among the studied phytoligands, even better than hesperidin (-7.10 kcal/mol). Liriodendrin (15) , acacetin-7-O-β-glucopyranoside (12) , phlomurin (2), 2-phenylethyl-O-β-xylopyranosyl-(1→2)-β-glucopyranoside (10), Syringin (9), benzyl alcohol-O-β-xylopyranosyl-(1→2)-β glucopyranoside (4), phlomuroside (3) and luteolin-7-O-β-glucopyranoside (13) showed slightly less binding affinities ranging from Gln409, Lys417, Ser494 † The best ligand-receptor complex binding free energy at RMSD Ͻ2Å. ‡ The key residues involved in the SARS-CoV-2-CTD-2hACE complex formation are listed in bold. -6.73 to -6.06 kcal/mol. Moderate fitting was observed in case of the three phytoligands viz. chrysoeriol-7-O-β glucopyranoside (15) , lamiide (6) and ipolamiide (8) with binding affinities of -5.97, -5.72 and -5.68 kcal/mol, respectively. The remaining phytoligands showed low binding affinities. Interaction of Phlomis phytoligands with human SARS-CoV-Spike-ACE2 complex Some of the studied phytoligands showed promising in silico results upon inspecting their binding modes with human SARS-CoV-Spike-ACE2 complex. They were able to efficiently accommodate into the interface and interact with the key aminoacids mostly via hydrogen bonding (Figures 3 & 4) . Hence, they can destabilize the virus-receptor complexation which is dominated by polar bonding interactions with key hydrophilic amino acid residues [16] . Acteoside (11), a phenolic glycoside recording the best binding affinity in the current study, exhibited hydrogen bonding interactions with Gly496 of the SARS-CoV-2-CTD engaged in complex formation ( Figure 3K & L). Additionally, it displayed hydrogen bonding with the nearby amino acids, in other words, Glu406, Arg408, Gln493 and Ser494 on the SARS-CoV-2-CTD side of the interface. These interactions were posed by the sugar hydroxyl groups for Glu406 and Arg408; whereas, Gln493 and Ser494 interacted with the phenolic hydroxyl groups. This highlights that sugar hydroxyl groups and the phenolic hydroxyl groups are the important structural features of the phytoligand. Obviously, acteoside (11) did not show any interactions with hACE2 residues of SARS-CoV-Spike-ACE2 complex as compared with the reference hesperidin (16) which displayed π-π and hydrogen bonding interactions with hACE2 His34 and Ala387, respectively, in addition to its binding to SARS-CoV-2-CTD Gln409, Lys417, Ser494 ( Figure 3U & V) . Recently, in silico prediction studies of physicochemical properties, ADMET and drug-likeness parameters are utilized for the identification of the most promising leads. Herein, SwissADME software was employed to compute the physicochemical properties formulating drug-likeness parameters of the hit phytochemicals (Table 2) [18, 28] . As revealed form our results, acteoside (11) did not inhibit ACE2, which is an added value to its promising anti-SARS-CoV2 potential as the ACE2 inhibition is unfavorable to COVID-19 patients with already developed His A34 Tyr B453 Arg B403 Asp B405 Tyr B495 Gln B409 Arg B408 Asp A30 Pro A389 Arg A393 Glu A37 Lys B417 Tyr B505 Asn A33 Glu B406 Arg B403 Tyr B505 Gly B496 Gln B493 Tyr B505 Asp A38 Glu A37 Asn A33 His A34 Gln B406 Tyr B495 Tyr B453 Arg B403 Gln B493 Ser B494 Asn A33 His A34 Tyr B453 Gln B493 Lys A353 Asp A38 Tyr B505 Arg B403 Thr B415 Gln B493 Tyr B453 Tyr B495 Gly B496 Glu A37 Tyr B505 Arg B403 Lys B417 Arg B408 Gly B416 Thr B415 Gln B409 Asp A38 Asn A33 His A34 Arg B408 Gly B416 Lys A353 Gly B496 Tyr B495 Asn B501 Tyr B505 Arg B403 Thr B415 Gly B416 Lys B417 Gln B409 Arg B408 Glu B406 Tyr B495 Gly B416 Glu B406 Arg B403 His A34 Glu A35 Gly B496 Tyr B453 Tyr B495 Thr B415 Tyr B505 Glu A37 Lys A353 Asp A38 Table 2 . In silico predicted physicochemical properties, ADMET and drug-likeness parameters of the promising phytoligands (11, 12 & 14) . symptoms. These symptoms develop as a consequence to the decreased production of angiotensin 1-7, which exhibits antifibrotic, anti-inflammatory, vasodilatory actions via Mas receptor [38] . Furthermore, there are preliminary data proving that patients taking angiotensin-II inhibitors (ACE-I) exhibit severe symptoms with a higher mortality rate as compared with their counterparts not taking these medications [39] . Accordingly, ACE2 plays a protective role in the animal models of acute respiratory distress syndrome and acute lung injury [40] . Acteoside (11) occurs frequently in several botanical families viz. Scrophulariacea, Lamiacea and Verbenacea [41] . It was reported to exhibit a strong in vivo antiviral activity against influenza and antirespiratory syncytial virus which causes the lower respiratory tract infection in infants and young children in addition to other biological actions including antihepatotoxic, anti-inflammatory, antinociceptive and antioxidant effects [42] . Among the studied phytoligands classes, flavonoids showed the second most promising in silico activity among which chrysoeriol-7-O-β glucopyranoside (12) interacted with SARS-CoV-2-CTD Gly496 and Tyr453 through the sugar part, but not with hACE2 ( Figure 3M & N) . Luteolin-7-O-β-glucopyranoside (14) interacted with Tyr453, Ser494 and Gly496 at the SARS-CoV-2-CTD via the sugar hydroxyl groups; whereas, it was bound to ACE2 at His34 and Lys353 through the aromatic ring and the sugar hydroxyl group, respectively ( Figure 3Q & R). Liriodendrin (Syringaresinol-di-O-glucoside) (15) exhibited hydrogen bonding with Arg403, Glu406, Arg408, Gln409 and the key Lys417 at the SARS-CoV-2-CTD side without binding to hACE2 residues ( Figure 3S & T) . It is worth noting that acacetin-7-O-β-glucopyranoside (13) has been reported to exhibit anti-inflammatory action [43, 44] with well-proven efficacy against chronic obstructive pulmonary disease via inhibiting neutrophilic lung inflammation in a murine model of chronic obstructive pulmonary disease [45] . Interestingly, the flavonoid, luteolin-7-O-β-glucopyranoside (14) and the lignan, liriodendrin (15) interacted with the key His34 and Lys353 of the hACE2 via π-π and hydrogen bonding interactions, respectively ( Figure 3 O-R). It is worth mentioning that a single Lys353 mutation was reported to be sufficient to abolish the interactions at the interface. Both compounds also displayed hydrogen bonding with Tyr453, Ser494 and Gly496 of SARS-CoV-2-CTD at the interface. Benzyl alcohol-O-β-xylopyranosyl-(1→2)-β glucopyranoside (4) interacted with SARS-CoV-2-CTD Tyr453, Gly496 and Tyr505 as well as hACE2 Glu37 and Lys353 (Figure 3 E & F). Liriodendrin (15) was reported to exhibit protective role in sepsis-induced acute lung injury via diminishing the release of many proinflammatory mediators, viz. TNF-α, IL-1β, MCP-1, and IL-6 and lung myeloperoxidase accumulation in addition to suppressing the VEGF expression and NF-κB activation in the lung [46] . Among the studied iridoids (1, 2, 5-8), only lamiide (6) exhibited similar interactions with hACE2 Glu37 and Lys353 besides engaging SARS-CoV-2-CTD Glu406 and Tyr453 ( Figure 3G & H) . However, phlomurin (2) in addition to the megastigmane glucoside, phlomuroside (3) (Figures 3A-D) and the phenylethanoid glycoside, 2-phenylethyl-O-β-xylopyranosyl-(1→2)-β-glucopyranoside (10) (Figure 3I & J) failed to exhibit interactions with the key aminoacids of the virus-receptor complex however, they could interact with nearby residues. On the other hand, ipolamiide (8) and syringin (9) were able to fit in the binding interface without considerable interactions with its residues. The phytoligands 'chrysoeriol-7-O-β-glucopyranoside (12) and luteolin-7-O-β-glucopyranoside (14)' showed a slight deviation from the ideal Lipinski's [47] and Veber's [37] drug-like bioavailability parameters. According to Lipinski's rule of five, phytoligands (12 and 14) violated the number of hydrogen bond acceptors and donors. Acteoside (11) recorded an extra Lipinski violation due to its high molecular weight. On the other hand, the studied phytochemicals violated the ideal total polar surface area of the drug-like molecule according to Veber's parameters (number of rotatable bonds ≤10 and total polar surface area ≤140). Again, 11 showed one more violation regarding the number of rotor bonds. Pre-ADMET software [19] was employed for prediction of absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the studied natural products. 12 and 14 were predicted to display moderate human intestinal absorption. 12 recorded higher predicted absorption percentage (42%) than 14 (25%); whereas, 11 was predicted to be poorly absorbed recording only 7.6% intestinal absorption. The three phytochemicals recorded good aqueous solubility. 11 was predicted to be the most readily soluble compound among the group (S = 290.5 mg/l), followed by 14 (S = 85.91 mg/l) and 12 (S = 40.22 mg/l), respectively. This refers to the expected feasibility of the studied phytochemicals to be formulated in various pharmaceutical dosage forms. The three phytochemicals displayed comparable low CNS absorption as detected by their predicted blood-brain barrier penetration values (around 0.03). Thus, the compounds are expected to display limited possible CNS side effects. The compounds displayed medium Caco-2 model and low MDCK model permeabilities. 11 recorded the highest Caco-2 model permeability value (almost two-fold) compared with 12 and 14. On the other hand, 14 was predicted to be two-fold more absorbable by MDCK than 12. 11 displayed the least MDCK permeability value among the group. Interestingly, all compounds were considered poorly bound to plasma proteins (PPB ranges from 62 to 73%) indicating that much of the unbound compound will be available for transport across various membranes to display its pharmacological activities. They were predicted to be devoid of cytochromes P450 2D6 (CYP2D6) inhibition activities but not CYP3A4, addressing the possibility of limited predicted drug interactions. Finally, the median lethal dose (LD 50 ; mg/kg) of the studied compounds in rodents was predicted employing ProTox [35] , the toxicity predictor program, to be 5000 mg/kg; thus, classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) as class V concerning acute oral toxicity. In the current study, we conducted docking simulation to predict the in silico inhibitory potential of a set of 15 phytoligands previously isolated Phlomis aurea, a wild Sinai peninsula plant, against the functional activity of SARS-CoV-Spike-ACE2 complex. Among the studied phytoligands, the phenolic glycoside 'acteoside (11) ' showed the most potent in silico inhibitory action with an additional merit, over the reference hesperidin (16), of not binding to ACE2 which was reported recently to possess a pulmonary protective role in acute lung injury and acute respiratory distress syndrome in vivo. The second most active phytoligands were flavonoids viz. acacetin-7-O-βglucopyranoside (13), chrysoeriol-7-O-β-glucopyranoside (12), followed by the lignan 'liriodendrin (15) ' and the iridoid 'lamiide (6)'. Our results provide promising leads from Phlomis aurea plant for designing and developing drug candidates with phytoprophylactic potential against COVID-19. Besides, pulmonary inflammation and fibrosis are recognized now as the first death causes of COVID-19 patients [48] . The role of anti-inflammatory agents in the effective management of symptoms during COVID-19 has been suggested by clinical practitioners [2] . Interestingly, three of the most active phytoligands in our study have well reported anti-inflammatory actions such as acteoside (11) , acacetin-7-O-β-glucopyranoside (13), liriodendrin (15) , with the former compound possessed antifibrotic action additionally. Accordingly, these phytoligands could aid in developing multitargeted strategy for better management and reducing the likelihood of COVID-19 using phytomedicines. Moreover, preparation of quality controlled P. aurea leaf extract aiming to possess highest levels of these phytoligands is warranted. However, in vitro and in vivo experimentation now follows in order to validate the predicted antiviral potential of the most promising phytoligands in P. aurea and/or its leaf extract standardized to the therapeutic levels of these phytoligands. The predicted ADME and drug-likeness parameters were computed for the three most promising phytochemicals (11, 12 & 14) . Results showed that chrysoeriol-7-O-β-glucopyranoside (12) and luteolin-7-O-β-glucopyranoside (14) recorded relatively better predicted drug-like criteria compared with acteoside (11) . Thus, these phytochemicals deserve to be further subjected to in vivo pharmacokinetic studies on experimental animals. • In silico analysis of 15 Phlomis aurea phytoligands against SARS-CoV-Spike-ACE2 complex. • Acteoside possessed the most potent in silico inhibition without binding to ACE2. • The study posed some phytoligands with prophylactic potential against COVID-19. 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Material preparation and data collection were performed by AR Khattab and MS Kamel. Methodology, software, formal analysis, data curation, writing -original draft was performed by M Teleb. The first draft of the manuscript was written by AR Khattab and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.