key: cord-0051193-kg6d5kzg authors: Kanbarkar, Nikita; Mishra, Sanjay title: Matrix metalloproteinase inhibitors identified from Camellia sinensis for COVID-19 prophylaxis: an in silico approach date: 2020-10-06 journal: ADV TRADIT MED (ADTM) DOI: 10.1007/s13596-020-00508-9 sha: ccc2a2264feb5272e60a7d4bca590235605638e4 doc_id: 51193 cord_uid: kg6d5kzg To respond to the public panic, government and private research organizations of every country keep working on the COVID-19 pandemic, even though still there is a lack of more efficacious medicine for the choice of Coronavirus disease treatment. To counteract on this situation several approved drugs including anti-malarial (hydroxychloroquine and chloroquine), and few anti-viral (remdesvir) agents are choice of treatment for COVID-19. However, these agents suffer from certain limitation in their uses and pointed that there is no specific treatment or vaccine available to counter this contagious disease. Hence, there is urgent requirement to find a specific cure for the disease. In this view, there are several ongoing clinical trials of both western and traditional medicines. In present study, phytochemicals from Camellia sinensis were retrieved from the database and identified based on their ability to inhibit matrix metalloproteinase (MMPs) against SARS-CoV-2 main protease. Camellia sinensis entails of a massive number of phytochemicals with a good source of polyphenols such as Catechin, Epicatechin, Epigallocatechin and (–)-Epigallocatechin gallate. Molecular docking was performed using the GLIDE docking module of Schrodinger Suite software. The analysis displayed docking score for the five polyphenols i.e. theaflavin (− 8.701), 1-O-caffeoylquinic acid (− 7.795), Genistein (− 7.168), Epigallocatechin 3-gallate (− 6.282) and Ethyl trans-caffeate (− 5.356). Interestingly, theaflavin and Epigallocatechin 3-gallate have not revealed any side effects. These polyphenolic compounds had a strong binding affinity with hydrogen bonds and a good drug-likeness score. Therefore, Camellia sinensis could be the beneficial option in the prophylaxis of the COVID-19 outbreak. . Social distancing and home quarantine had started psychological changes in the public. To respond to this panic situation, government and private research organizations of every country start working on the COVID-19 epidemic, although there is still lack of more efficacious medicine for the cure of Coronavirus. COVID-19 has been documnted for two types: (1) Severe Acute Respiratory Syndrome Coronavirus (SARS-Cov) and (2) the Middle East Respiratory Syndrome Coronavirus (MERS-Cov) . Both are RNA viruses with glycoprotein spikes on surface, The family Coronaviridae causes severe respiratory tract dysfunction with symptoms such as cold, fever, body ache, and difficulty in breathing. The first outbreak occurred at the Wuhan China sea food market and has now spread globaly (Liu and Wang 2020) . This disaster had compelled the government to take stringent measures to save lives such as national and international lockdown, social distancing, the extension of vacations, patient hospitalization and quarantine, and many other changes to safe guard their countries (Lin et al. 2020) . To counteract this situation anti-malarial drugs are used i.e. Hydroxychloroquine and Chloroquine (Frie and Gbinigie 2020; WHO, April 2020b) and few anti-viral drugs such as Ritonavir, Tipranavir and Lopinavir has been tested in patients with COVID-19 but still, no specific vaccine or anti-corona virus drug/s are available (Nukoolkarn et al. 2008) . Indian Traditional medicine plays an important role in many viral diseases. Camellia sinensis commonly refered as 'TEA' belongs to family 'Theaceae'. It consists of a huge number of phytochemicals, along with a good source of polyphenols i.e. Catechin, Epicatechin, Epigallocatechin and Epigallocatechin gallate. Among them, Epigallocatechin gallate (constitutes 59%) is a major source of polyphenol (Kaur and Saraf 2011) . According to published literature, polyphenols have been previously reported for its strong potency in the treatment of viral diseases. Camellia sinensis has been documented for antioxidant (Chan et al. 2007 ), Chemoprotective (Kaur and Saraf 2011) , Wound healing (Hajiaghaalipour et al. 2013) , Anti-diarrheal (Besra et al. 2003) , antimicrobial (Farooqui et al. 2015) and numerous anti-viral activities (Xu et al. 2017) . Above cited studies suggests that phytochemicals identified from Camellia sinensis could have ability to inhibit MMPs (enzyme belongs to family Proteases), which is associated with chemokine activation and contributes significantly in the degradation of myelin proteins and generation of auto-antigens. MMPs and their inhibitors are involved in remodeling of the extracellular matrix (ECM) during normal physiological conditions (Marten and Zhou 2005; Gupta 2016 ). Therefore, this study was planned to analyse one of very prominent, economical, and popular beverage source, Camellia sinensis for the prophylaxis of COVID-19. Camellia sinensis has been reported for more than hundred phytochemicals and can be a potential candidate for the COVID-19 prophylaxis. Thus, with the aid of molecular doking, this study aimed to analyse Camellia sinensis for its possible therapeutic efficacy as per available phytochemicals in data base. The phytochemicals of Camellia sinensis were retrieved from ChEBI online tool (https ://www.ebi.ac.uk/chebi /), and molecular weight, molecular formula, PubChem CID and Canonical SMILE of phytochemicals were recorded (Kanbarkar et al. 2020 ). All the identified phytochemicals were predicted for their Matrix Metalloproteinase inhibition activity by submitting the Canonical SMILE with the help of an online tool-Swiss Target Prediction http://www.swiss targe tpred ictio n.ch/ (Gfeller et al. 2014 ). The drug-likeness properties of the identified phytochemicals were determined from Molsoft online tool https ://molso ft.com/mprop / (Khanal et al. 2019) . As per Lipinski's rule of five, molecular weight; lipophilicity (MolLogP); number of hydrogen bond acceptor; number of hydrogen bond donor; and drug-likeness score were noted. The side effects of the identified phytochemicals were calculated using the ADVERPred online tool http://www.way2d rug.com/adver pred/ (Ivanov et al. 2008) . It predicted probable activity and inactivity values for each compound along with their side effects. The gene set data obtained for each compound from DIGEPpred. http://www.way2d rug.com/GE/ (Lagunin et al. 2013) were submitted to STRING online tool https ://strin g-db.org/ (Szklarczyk et al. 2017 ) and the KEGG pathway was downloaded. The pathway predicts possible mechanism of action which could be followed by the identified phytochemicals. All the selected polyphenolic compounds of Camellia sinensis were downloaded from PubChem and prepared using the LigPrep version 4.8 (Schrodinger LCC) (Adnan et al. 2020) . LigPrep generates energy minimized structure with multiple tautomer and stereoisomer's, which was further used as input to molecular docking. The phytochemicals were subjected to molecular docking to explore its conformational space and orientation of substituents in the binding pocket of the target proteins. The crystal structure of SARS-CoV-2 main protease (PDB ID: 6LU7) in complex with a peptide for the present study was downloaded from the RCSB protein data bank (Berman et al. 2002) database. For protein preparation, the standard protocol of protein preparation wizard (Schrodinger, LLC) was followed and minimized the protein structure until the RMS gradient for heavy atom reached 0.3 A°. The crystal bound ligand was selected to enumerate a binding site grid with a scaling factor of 1.0 and partial charge cutoff of 0.25 for the Van Der Waals radius. Molecular docking simulations were performed using the GLIDE docking module of Schrodinger Suite software (Adnan et al. 2020) . The glide approximates a complete systemic search of the conformational, orientational and positional space of the ligand in the protein binding pocket. The glide docking produces different poses for each input ligand, and each pose was scored and ranked by the glide docking scores (kcal/mol). By using keyword 'Camellia sinensis' in data base, total 122 phytochemicals were retrieved and their canonical SMILES were recorded for generating data in the further steps. All the retrieved phytochemicals predicted for MMPs property and out of 122 phytochemicals, twelve phytochemicals were identified based on their inhibition potential of MMPs (Table 1) . Theaflavin was predicted to inhibit eight MMPs, being the highest inhibitor in the listed polyphenolic compounds. The drug-like properties or physicochemical properties such as molecular weight, lipophilicity (MolLogP), number of hydrogen bond acceptor (NHBA), number of hydrogen bond donors (NHBD) and drug-likeness score (DLS) were calculated for the tweleve identified compounds ( Table 2 ). The ranking order of DLS was perceived as follows: 2-(4-hydroxybenzyl) quinazolin-4(3H)-one > Genistein > 1-O-caffeoylquinic acid > Theaflavin > Epigallocatechin 3-gallate > Cordysinin A > Vanillic acid > Ethyl trans-caffeate > Gedunin > Inflatin E > Inflatin D > Inflatin f. All these compounds follows the Lipinski's rule, except epigallocatechin 3-gallate and 1-O-caffeoylquinic acid. (-)-Epigallocatechin 3-gallate MMPs-2, 9, 12, 13, 14 3. Cordysinin A MMPs-1, 2 4. 1-O-caffeoylquinic acid MMPs-2, 12 5. Genistein MMPs-2, 9, 12 6. Inflatin E MMPs-1, 2, 3, 7, 8, 9, 13 7. Inflatin F MMPs-1, 2, 3, 7, 8, 9, 13 8. Inflatin D MMPs-1, 2, 3, 7, 8, 9, 13 9. 2-(4-hydroxybenzyl) quinazolin-4(3H)-one MMPs-1, 9 10. Vanillic acid MMPs-2, 8, 9, 12 11. Ethyl trans-caffeate MMPs-1, 2, 9 12. Gedunin MMPs-1, 3, 9 The possible side effects of selected phytochemicals are listed in Table 3 . The Theaflavin, Epigallocatechin 3-gallate, and Gedunin displayed no side effects, whereas vanillic acid indicated four major side effects such as hepatotoxicity, nephrotoxicity, arrhythmia, and cardiac failure with their probable activity and in-activity. The molecular docking study was performed for twelve identified phytoconstituents that have been predicted for Matrix Metalloproteinase enzyme inhibition potential against SARS-CoV-2 main protease. We observed that Theaflavin had maximum and Gedunin had minimum docking score (− 8.401 to − 3.169 kcal/mol respectively). Based on docking score, the ranking of all compounds were: Theaflavin > 1-O-caffeoylquinic acid > Genistein > Epigallocatechin 3-gallate > Ethyl trans-caffeate > 2-(4-hydroxybenzyl) quinazolin-4(3H)-one > Cordysinin A > Inflatin D > Inflatin E > Vanillic acid > Inflatin F > Gedunin (Table 4 ). The binding interactions of each compound with SARS-CoV-2 main protease protein were represented in the form of several hydrogen bonds and interaction residue analysis revealed that most of the compounds formed interaction with conserved catalytic dyad (Cys145 and His41) amino acid residue (Characteristic features from SARS-CoV-2 main protease). The analysis of docked poses of Theaflavin revealed that this compound occupies the catalytic site of SARS-CoV-2 main protease and interacts with catalytic His41 and Cys145 amino acid residues. This compound also interacted with His41, Leu141, Glu166, Met165 side chain amino acid residues, and formed several H-bond interactions (Fig. 1a) . Gedunin a steroid compound, in the binding pocket obtained a U-shaped conformation forming H-bond with His41, Asn142 has the lowest docking score (Fig. 1b) . The gene set data has described the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway followed by the compounds to display the possible molecular mechanism (Table 5 ). It was detected that all compounds followed more than one pathway to hit the target, but all compounds displayed one common pathway that is cancer pathway. It could be believed that naturally occurrring compounds may follow single and multiple pathways may have an anti-viral mechanism at the end. Table 5 displayed the list of pathways, gene counts, and modulated proteins. COVID-19 is a viral infection and emerging with increasing prevalence. It has cought the attention of researchers, doctors, and health care professionals worldwide. Owing to countless deaths and a limitations in available drugs, it has caused the terrible human condition to eradicate the virus. Therefore, potential and cost-effective medicine/s is warrented for best prophylactic (pre and post COVID-19) effect. The present study aimed to investigate the possible effect of Camellia sinensis using in silico approach in the management of COVID-19. Numerous research projects are ongoing over the globe and several has been conducted to identify the potential medicine candidate for the COVID-19 crisis. In this life-threatening situation, traditional medicine/s might be effective for treatment of COVID-19. Warm water decoction made by using various species in Ayurveda called 'Kashay' is generally in practice in the prevention of fever, headache, common cold, and inflammation (Tillu et al. 2020) . Similarly, the warm decoction of dry or fresh leaves of Camellia sinensis with or without sugar is also used in daily life style to overcome stress and has been documented for anti-inflammatory, analgesic (De Lima MotaI et al. 2015) and CNS stimulant activity (Rubab et al. 2020 ). In the present study with the help of in silico approach, we predicted potential phytochemicals of Camellia sinensis for the inhibition of Matrix Metalloprotease (MMPs). MMPs play an important role in immunity, inflammation, cell growth, organ morphogenesis, wound healing, angiogenesis, apoptosis, and embryonic development. Overexpression of MMPs was also observed in various pathological conditions such as cancer, corneal endogens, skin ulceration, neurological diseases, arthritis, and fibrotic lung diseases, etc. (Gupta 2016) . The twelve identified phytochemicals from Camellia sinensis were predicted as MMPs inhibitors in Swiss Target Prediction data base, namely: Theaflavin, (-)-epigallocatechin 3-gallate, cordysinin A, 1-O-caffeoylquinic acid, genistein, inflatin E, inflatin F, inflatin D, 2-(4-hydroxybenzyl) quinazolin-4(3H)-one, vanillic acid, ethyl trans-caffeate, and gedunin with MMPs: − 1, − 2, − 3, − 7, − 8, − 9, − 12, − 13 and − 14 ( Table 1) . Overexpression of these MMPs initiates various diseases. MMP-1 originate breast cancer growth, metastasis, cardiac hypertrophy and heart attack (Marten and Zhou 2005) , MMP-2: involved in chronic lung diseases (Kong et al. 2009 ), MMP-3: participates in rheumatoid arthritis and ankylosing spondylitis (Sun et al. 2014 ), MMP-7: degraded natural immunity of lung and intestine (Burke 2004 ), MMP-8: activates Interleukin and (Dabo et al. 2015) , MMP-12: participates in the aneurysm, atherosclerosis, and emphysema (Chen 2004 ), MMP-13: osteoarthritis and rheumatoid arthritis (Takaishi et al. 2008 ) and MMP-14: promote hepatocellular carcinoma and metastasis (Chen et al. 2011; Murugan et al. 2009 (Jin et al. 2005 ) and genistein (Kousidou et al. 2005) were identified as inhibitors of MMPs involved in various disease conditions such as cancer and lung diseases. Furthermore, by using the STRING online tool, study predicted probable pathways that could be followed by the compounds to identify the possible mechanism. All phytochemicals were observed to for their common pathway (Cancer pathways). Table 5 (Fig. 2) . Considering the above mentioned anti-viral research on the tea polyphenols and its easy availability, this study was planned to perform the in silico molecular docking of predicted phytochemicals against SARA-CoV-2 main protease. The main reason behind the compounds selection having MMPs inhibition activity for the targeting SARS-CoV-2 main protease was that both, drug and target belong to the same family 'protease'. The proteases have been Fig. 2 Anti-viral potential of polyphenols from Camellia sinensis along with their mechanism earlier documented for their role in several biological pathways. The dysfunction of this enzyme may result in an exhaustive range of diseases. Recently used anti-viral drugs for COVID-19 managemnt: Ritonavir, Tipranavir, and Lopinavir are belongs to the class of 'protease inhibitors'. There are mainly five classes of protease namely: Metalloproteases, Aspartic acid protease, Serine protease, Cysteine protease, and Threonine protease. Metalloproteases and aspartic acid protease act through a 'peptide bond hydrolysis' mechanism whereas Serine, Cysteine, and Threonine protease act through 'peptide bond cleavage' mechanism (Drag and Salvesen 2015) . The polyphenols such as epicatechin, epicatechingallate, epigallocatechin, and epigallocatechingallate are previously reported in the tea and inhibits MMPs-2, and -9. Further, also directed to have a chemoprotective effect against various cancers. In addition, Genistein inhibits a wide variety of cancer cells by inhibiting MMPs -2 and -9. Polyphenols such as Theaflavin, 1-O-caffeoylquinic acid, genistein, (-)-epigallocatechin 3-gallate, and ethyl transcaffeate displayed the higher docking score against SARS-CoV-2 main protease enzyme. Therefore, in silico molecular docking investigations suggests that Camellia sinensis could target SARS-Cov-2 main protease in the management of COVID-19. In the view of previous reported anti-viral activities of Camellia sinensis and in silico study data in present study supports the beneficial effect of traditional Ayurvedic/ herbal medicine in the management of COVID-19 crisis by targeting SARA-CoV-2 main protease. Significantly the anti-viral potential is evident from the predicted docking score of polyphenols such as theaflavin, (-)-epigallocatechin 3-gallate, Genistein, 1-O-caffeoylquinic acid, and Ethyl trans-caffeate. Drug likeness characteristics and no or less side effects of theaflavin, (-)-epigallocatechin 3-gallate directs the future scope of these polyphenols. 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