key: cord-0968180-tx8e6i45 authors: Chikhale, Rupesh V.; Sinha, Saurabh K.; Khanal, Pukar; Gurav, Nilambari S.; Ayyanar, Muniappan; Prasad, Satyendra K.; Wanjari, Manish M.; Patil, Rajesh B.; Gurav, Shailendra S. title: Computational and network pharmacology studies of Phyllanthus emblica to tackle SARS-CoV-2 date: 2021-07-13 journal: Phytomedicine Plus : International journal of phytotherapy and phytopharmacology DOI: 10.1016/j.phyplu.2021.100095 sha: f25b44d36356cbc5f5b0e6faccb050fce260613c doc_id: 968180 cord_uid: tx8e6i45 Background Since December 2019, SARS-CoV-2 had been a significant threat globally, which has accounted for about two million deaths. Several types of research are undergoing and have reported the significant role of repurposing existing drugs and natural lead in the treatment of COVID-19. The plant Phyllanthus emblica (Synonym-Emblica officinalis) (Euphorbiaceae) is a rich source of vitamin C, and its use as an antiviral agent has been well established. Purpose The present study was undertaken to investigate the potency of the several components of Phyllanthus emblica against three protein targets of 2019-nCoV viz. NSP15 endoribonuclease, main protease, and receptor binding domain of prefusion spike protein using molecular docking and dynamics studies. Methods The docking simulation studies were carried out using Schrödinger maestro 2018-1 MM share version, while dynamics studies were conducted to understand the binding mechanism and the complexes' stability studies. Results Out of sixty-six tested compounds, Chlorogenic acid, Quercitrin, and Myricetin were most effective in showing the highest binding energy against selected protein targets of SARS-CoV-2. The network pharmacology analysis study confirmed these compounds' role in modulating the immune response, inflammatory cascade, and cytokine storm through different signaling pathways. Conclusion Current pharmacoinformatic approach shows possible role of Phyllanthus emblica in the treatment and management of COVID-19. In December 2019, a unique and unprecedented worldwide pandemic arose from the Wuhan city of China, known as the "Severe Acute Respiratory Syndrome Coronavirus-2" (SARS-CoV-2) and had become a significant peril to the world to high mortality and morbidity rates. The coronaviruses are single-stranded RNA (genome size-26-32-kilobases), which plays a dangerous role in the initial RNA synthesis of the infectious cycle and acts as a substrate packaging into the progeny virus . In coronaviruses, two-thirds of the genome encodes a replicase polyprotein processed by viral protease, which cleaves into 16 non-structural proteins (NSPs) and is involved in transcription and replication (Chikhale et al., 2020a) . The non-structural protein 15 of SARS-CoV-2 (Nsp15) is a nidoviral RNA uridylate-specific endoribonuclease (NendoU), and the exact functional relevance of Nsp15 remains unknown. Recent studies claimed that the NendoU activity of NSP15 is mainly attributed to protein interference with innate immune response and is considered an essential segment for the natural progression of coronaviruses . Another NSP that helps enter 2019-nCoV into the host cells is spike (S) protein composed of two subunits S1 and S2. This S protein plays a vital role in receptor recognition and the host cell membrane's fusion process. During viral infection in the host cells, the target cell proteases activate the S protein by cleaving it into two subunits, which are essential to activate the membrane fusion domain after viral entry into target cells (Hoffmann et al., 2020) . The current focus is the development of novel therapeutics including natural antivirals and specific vaccines. In-silico tools have employed a diverse set of computational approaches to understand the relative performance of the predictions for SARS-CoV-2 by repurposing existing drugs and the alleged role of natural lead in treating COVID-19 (Chikhale et al., 2020a (Chikhale et al., , 2020b (Chikhale et al., , 2021 Patil et al., 2020; Khanal et al., 2020 Khanal et al., , 2021a Khanal et al., , 2021b . However, it may take months or years to develop such effective treatments, and hence the quest for prompt treatment is the utmost need of the hour. During the 2003 severe acute respiratory syndrome (SARS) pandemic, traditional medicinal plants' efficacy has been established (Li et al., 2005) . Consequently, based on historical and traditional evidence, researchers have started investigations on medicinal plants for SARS-CoV-2 (Chikhale et al., 2020a (Chikhale et al., , 2020b (Chikhale et al., , 2021 . The medicinal plants, with their therapeutic prominence are a gift to humankind to attain a healthy life. Phyllanthus emblica L. (syn. Emblica officinalis Gaertn.) (Euphorbiaceae), commonly-known as Indian Gooseberry or Amla, is a remarkable tropical south-east Asian shrub, cultivated throughout India. Moreover, it is reported for its antiinflammatory, antipyretic, antioxidant, anticancer, anti-hyperlipidemic, adaptogenic, antidiabetic, nootropic, antimicrobial and immunomodulatory, anti-bacterial potential (Variya et al., 2016; ) . Antiviral efficacy of P. emblica in the treatment of HIV (El-Mekkawy et al., 1995) , Coxsackie VB3 (Liu et al., 2009 ), hepatitis B virus (Xiang et al., 2010 , and herpes simplex virus (Xiang et al., 2011) has evidenced with scientific reports. Out of various parts of E. officinalis, the fruits have a significant place in Rasayana to treat several infectious and non-infectious diseases (Variya et al., 2016) . The present research investigation highlights phytoconstituents from the Indian gooseberry using an in-silico approach targeting three different SARS-CoV-2 proteins viz. NSP15 endoribonuclease, main protease, and receptor-binding domain (RBD) of the prefusion spike protein. Schrödinger Glide SP module was used for Molecular docking studies of a total of sixty-six phytoconstituents of P. emblica with the experimentally solved crystal structures of NSP15 endoribonuclease (PDB: 6W01), SARS-CoV-2 spike RBD (PDB: 6M0J), and SARS-CoV-2 main protease (PDB: 6WNP) having the resolution of 1.9, 2.45 and 1.45Å respectively (Supplementary File) (Chikhale et al., 2020a) . The AMBER18 software package was used for MD simulations, and ligands were parameterized with ANTECHAMBER. The prepared protein-ligand complexes were subjected to 100 ns MD simulations on Nvidia V100-SXM2-16GB Graphic Processing Unit using the PMEMD.CUDA module. Further, the 100 ns trajectories were subjected to MM-GBSA analysis using Amber18 and Amber18 tools on all the 10,000 frames (Supplementary File). The down-regulated and up-regulated protein-based targets of phytoconstituents were retrieved from DIGEP-Pred at the pharmacological activity (Pa)>0.5. The complete proteins were then queried in the STRING database to visualize the protein-protein interaction and GO analysis (Gene Ontology Consortium, 2004) for cellular and molecular functions and the biological spectrum. Similarly, the probably regulated pathways were identified concerning pathways was constructed using Cytoscape (Shannon, 2003) . The network was treated as directed and evaluated by setting map node size from "low values to small sizes" and map node color from "low values to bright colors" based on edge count for both. To assess the possible potential and understand the possible mechanism of sixty-six phytoconstituents molecular docking simulations were carried out on three proteins i.e. Table S1 ), only the best five tested ligands and the reference drug with respect to different proteins have been discussed in the present investigation. The second docking study was done on the crystal structure of the SARS-CoV-2 spike receptor-binding domain bound with ACE2. SARS-CoV-2 uses a homotrimeric glycoprotein spike to get entry into the host cells ACE-2 receptor. Such a binding relationship is enhanced by attaching the subunit S1 to the host cell receptor and converting the subunit S2 to a highly stable postfusion conformation. Besides, the S1 subunit receptor-binding domain (RBD) consists of 5 twisted β sheets β1, β2, β3, β4, and β7 that are anti-parallel to each other. There was an extended inclusion of β4 to β7 containing some α loops called receptor binding motif (RBM). This RBM includes most of the residues necessary to link n-COVID-19 to ACE-2. S2g ). The hydroxyl group of the terminal amino chain of quinoline exhibited H-bonding with Asn 450. The quinoline ring's quaternary nitrogen showed H-bonding with Glu 484 amino acids with a docking score-4.6 kcal/mol ( Supplementary Fig. S2h ). The third docking study was performed on the X-ray structure of SARS-CoV-2 main protease bound to Boceprevir. The SARS-CoV-2 main protease (3CL pro ) comprises approximately 306 amino acids, responsible for coronavirus replication and polypeptide processing into functional proteins. Each 3CL pro consists of three domains, with domains I (residues 8-101) and II (residues 102-184) having an anti-parallel β-barrel structure. Domain III (201-303 residues) has five α-helices, arranged in a mostly anti-parallel globular cluster and further connected to Domain II through an extended loop region (residues 185-200). Table S2 ). Similarly, the interaction of phytoconstituents, regulated proteins, and associated pathways are represented in Figure 2 . The gene ontology of regulated proteins identified the regulation of the multiple pathways that are concerned with infectious and non-infectious diseases. Some pathways involved with viral infections, like human papillomavirus, herpesvirus, and Epstein-Barr virus infection are also modulated. The subjects with lower immunity are more prone to the COVID-19 infection. In the present study, the majority of the regulated pathways like HIF-1, p53, IL-17, PI3K-Akt, FoxO, Wnt, NF-kappa B, TNF, MAPK, Rap1, and AMPK signaling pathways are directly or indirectly concerned with immunity manipulation and regulating inflammation (Johnson and Chen, 2012; Li et al., 2019; Muñoz-Fontela et al., 2016; Palazon et al., 2014; Šedý et al., 2014; Zenobia and Hajishengallis, 2015) . Further, the secondary phytoconstituents from P. emblica may also be involved in managing the cytokine storm as the multiple pathways related to chemokines are also regulated (Supplementary Table S1 ). During the bioactive-target-pathway network analysis, the apigenin was identified as (Lv et al., 2014) , it may not be directly involved in the inhibition of the coronavirus infection. However, it could play an essential role in immune regulation and possible anti-inflammatory activity in COVID-19 infection as reported for both properties (Hosseinzade et al., 2019) . Though Chlorogenic acid, Quercitrin, and Myricetin reflected the comparatively less modulatory effect of multiple proteins in the network, they may not be directly involved in regulating multiple pathways as apigenin does. It suggests the apigenin from E. officinalis in COVID-19 infection could majorly contribute to the immune regulation and possess antiinflammatory action in the infective tissue, whereas, Chlorogenic acid, Quercitrin, and Myricetin could directly act over the coronavirus to amplify its anti-viral activity. The molecular dynamics studies (MDS) of target protein-ligand complexes were performed to understand the binding mechanism and the complexes' stability over the period, explaining and modeling its in-vitro or in-vivo efficacy. The top-scoring ligands in complex proteins from the molecular docking experiments were selected for the MDS. Remdesivir, a drug approved by the US-FDA for emergency use in the COVID-19 pandemic, was also studied as the control for these simulation studies. The NSP15 endoribonuclease bound to Chlorogenic acid and Remdesivir were simulated for 100 ns in the explicit solvent model at physiological salt concentration. The NSP15 endoribonuclease protein RMSD converged with lower fluctuations between 1.5 to 2 Å for the first 40 ns, which later fluctuated with a steep fall and rose to 3 Å and remained between 2-3 Å for the remaining simulations period (Figure 3.1a) . The RMSF for individual residues show a higher fluctuation for amino acids (aa) between 35 to 50 of 3.5 Å, but the rest of aa had lower changes (Figure 3.1b) . The bound ligand, chlorogenic acid, had a low RMSD of 0.5 Å for the first 50 ns, which gradually rose for 20 ns to reach and stabilize at 3 Å (Figure 3.1c) . It suggests the protein-ligand complex's stability. The gradual rise in Ligand RMSD means a smooth positional change from its original binding site to a more stabilized position (Figure 3.1d and 1e ). This shifting of chlorogenic acid was stabilized by forming hydrogen bonds between the hydroxy groups and the residue Leu347 (Figure 3.1e) . In the MDS of the NSP15 endoribonuclease-Remdesivir complex, the protein RMSD was more stable with fluctuations between 1-3 Å (Figure 3 .2a) compared to the NSP15 endoribonuclease-chlorogenic acid complex. The RMSF for aa was very similar for the NSP15 endoribonuclease-Remdesivir and NSP15 endoribonuclease-chlorogenic acid complexes (Figure 3.2b) . The Ligand RMSD for Remdesivir converged between 4 to 6 Å from 5 ns to the end of the simulation with fewer fluctuations between 25 to 50 ns ( Figure 3 .2c), which could be attributed to the conformational changes in the Remdesivir ligand ( Figure 3.2d and 3 .2e). The SARS-CoV-2 main protease-Quercitrin and SARS-CoV-2 main protease-Remdesivir complexes were simulated for 100ns, and their binding modes were analyzed. The protease in complex with Quercitrin converged slowly to 3 Å during the initial 20 ns and remained stable throughout the simulation with few fluctuations around 30 and 60 ns ( Figure 4 .1a). The RMSF lower than 2.5 Å reflects the high stability of the aa (Figure 4.1b) . The ligand RMSD for Quercitrin converged and stabilized between 2 to 3.5 Å with occasional fluctuations (Figure 4.1c) . The visual inspection of the trajectory shows the breaking of the hydrogen bond with Arg188 around 20 ns of the simulation, which is also reflected from the ligand RMSD. The formation of a hydrogen bond with Glu166 stabilizes the complex ( The SARS-CoV-2 receptor-binding domain (RBD) was docked with the compounds under investigation, and remdesivir was included as a standard drug for comparing the binding on the natural products. Remdesivir is a prodrug that acts by interfering with the RNA-dependent RNA polymerase function. However, recently the alternate mechanism of action reported the concentration-dependent effect of Remdesivir. These reports provide for further investigation in the possible alternate binding sites and mechanism of action for Remdesivir. The top-scoring compound, Myricetin bound to RBD, and Remdesivir docked on the RBD were simulated for 100 ns, and the binding modes were analyzed. The RBD domains RMSD was between 1.5 to 2.5 Å, and the RMSD for its aa was below 2.5 Å, which is highly acceptable for a domain region of a protein ( Figure 5.1a and 5.1b) . The RBD bound ligand Myricetin had RMSD below 2 Å for the initial 25 ns of the simulation, which later fluctuated between 2 to 5 Å for the rest of the simulation. The visualization of the trajectory and different frames from the trajectory shows the intact hydrogen bond between Ile78 and Myricetin throughout the period. This complex gets more stabilized towards the end of the simulation by forming another hydrogen bond with Gly72 ( Figure 5 .1c, 5.1d and 5.1e). The SARS-CoV-2 RBD bound Remdesivir showed initial stability in its RMSD but has several very high fluctuations of about 5-10 Å. The aa residues showed higher RMSF of 0.5 to 3.5 Å over the simulation period suggesting lowered stability or no effect of Remdesivir binding on the RBD (Figure 5.2a and 5.2b) . The ligand had very high RMSD due to structural conformation changes and the absence of initial stage interactions. However, towards the end of the simulation, it forms hydrogen bond interaction with Glu74 and Arg71, and possibly these interactions keep the ligand attached to the receptor (5.2c, 5.2d, and 5.2e). The Molecular Mechanics/Generalized Born Surface Area calculations were performed to calculate the binding free energy of the complexes. The complete trajectories for 100 ns were used for the study, and the results are tabulated in Table 1 In the present research investigation, a total of sixty-six phytoconstituents of X-ray structure of SARS-CoV-2 main protease bound to boceprivir at 1.45 Å. 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