key: cord-0869375-nkl5tgag authors: Kumar, Sugandh; Singh, Bharati; Kumari, Pratima; Kumar, Preethy V.; Agnihotri, Geetanjali; Khan, Shaheerah; Kant Beuria, Tushar; Hussain Syed, Gulam; Dixit, Anshuman title: Identification of multipotent drugs for COVID-19 therapeutics with the evaluation of their SARS-CoV2 inhibitory activity date: 2021-04-07 journal: Comput Struct Biotechnol J DOI: 10.1016/j.csbj.2021.04.014 sha: c4f382a08a54a7161f43fac9977801ad2afa5398 doc_id: 869375 cord_uid: nkl5tgag The SARS-CoV2 is a highly contagious pathogen that causes COVID-19 disease. It has affected millions of people globally with an average lethality of ∼3%. There is an urgent need of drugs for the treatment of COVID-19. In the current studies, we have used bioinformatics techniques to screen the FDA approved drugs against nine SARS-CoV2 proteins to identify drugs for repurposing. Additionally, we analyzed if the identified molecules can also affect the human proteins whose expression in lung changed during SARS-CoV2 infection. Targeting such genes may also be a beneficial strategy to curb disease manifestation. We have identified 74 molecules that can bind to various SARS-CoV2 and human host proteins. We experimentally validated our in-silico predictions using vero E6 cells infected with SARS-CoV2 virus. Interestingly, many of our predicted molecules viz. capreomycin, celecoxib, mefloquine, montelukast, and nebivolol showed good activity (IC50) against SARS-CoV2. We hope that these studies may help in the development of new therapeutic options for the treatment of COVID-19. infection in brain tissues (21) . The experiments on mice have shown that the SARS-CoV2 infections can cause neuronal distruction and death (22) . In COVID-19 also similar effects have been seen (23) . In light of the above, the therapeutic agents with good CNS penetration ability could have additional advantage (24) . Only a few studies have reported targeting more than one viral protein with a single molecule or using combination therapy (25) (26) (27) (28) (29) . In this study, we attempted to identify molecules that can simultaneously bind to multiple proteins of the SARS-CoV2. The strategy to target multiple proteins originates from the fact that individual viral proteins play specific role in multiple aspects of viral lifecycle such as attachment, entry, replication, morphogenesis and egress. Single molecules that can potentially target many viral proteins can perturb viral lifecycle at multiple points and thereby can be highly efficient in curbing SARS-CoV2 infection. Such strategy will also have a higher barrier towards emergence of resistant mutants. In this work, we have used the 3D-structures of the SARS-CoV2 proteins to identify FDA approved drugs that can bind to these proteins using computational methods. The FDA approved drugs were chosen so that they can be quickly repurposed for treating COVID19. Additionally, we also analyzed if the identified molecules can affect the host proteins that get differentially expressed as a result of SARS-CoV2 infection. We have also tested these molecules using an in-vitro SARS-CoV2 infection model in Vero E6 cells. These molecules can be used as modulators of both the SARS-CoV2 and human proteins. The SARS-CoV2 proteins for which there is no crystal structure reported were modelled using Modeller v9.22 (30) (homology modeling) ( Table 1 ). The modelling template for each protein was identified by performing Delta-BLAST against the PDB database. Proteins were modelled using either single or multiple templates based on the query coverage. Further, the model stereochemistry and other structural parameters were assessed using standalone PROCHECK (31) tool. The ensemble docking approach increases the efficiency by allowing virtual screening against multiple conformations (32) . Therefore, the selected protein structures were subjected to 20ns MD run (total 180 ns) using NAMD 2.6 (33) to explore the flexibility of the binding site were prepared by addition of missing atoms, hydrogens, assignment of bond orders and proper protonation states. The structure of each of the protein was minimized by keeping heavy atoms fixed and then the whole structure was minimized until a RMS gradient of 0.3kcal/mol/Å as implied in Schrodinger. The active site of the modelled proteins were identified using either of the following methods 1) the ligand binding pocket, if the co-crystal structure is available or 2) the ligand bound cocrystal structure of a close homolog or 3) the active site was predicted using SiteMap algorithm in Schrodinger v9.3 molecular modelling software (34) . The proteins with active site pocket volume of <150 Å 3 were removed as smaller pockets may not be amenable to docking. The pockets were further selected by sequence comparisons and available literature. Finally, 9 proteins were selected for docking. The molecular docking was performed using the Glide module of Schrodinger molecular modelling software (www.schrodinger.com/glide). The docking was performed using default settings except that the formation of intramolecular hydrogen bonds was rewarded and the enhancement of planarity of conjugated π groups was checked on. Strain correction terms were applied, thus, if a docking pose has high internal strain, the docking score will be penalized and the pose may get removed from final results. A maximum of 10 poses were generated for each of the molecules. The final ranking of the molecules was obtained by calculating the average glide score in the five snapshots of a viral protein generated by molecular dynamics simulation to include the effect of binding site dynamics. The molecules showing a docking score of -8.5 (11) or better were selected for further analysis. The obtained hits were subjected to MM-GBSA analysis as implied in Glide module of Schrodinger modeling software for further selection of better hits. The receptor residues within 5Å of the ligands were considered flexible for the MM-GBSA procedure with other default settings. Since the MM-GBSA binding energies reflect approximate free energies of binding, a more negative value indicates stronger binding. Similar to average glide score, average MMGBSA score was also calculated for each of the ligand for each viral protein. The differentially expressed genes were obtained from the data reported by Blanco-Melo et al (35) . The drug-gene interaction database (DGIdb) that contains information about the drugs and their target genes was employed to identify the drugs that can modulate the differentially expressed genes in COVID-19. The drug gene interaction was obtained from various online resources such as Drugbank, and BindingDB. It was utilized to identify the drugs that can target both the viral as well as human proteins. Further, it was analysed whether a drug is agonist or antagonist for a given human protein for optimum therapeutic effect. The calculations were performed on a high performance Linux cluster. The flowchart of the methodology is presented in Fig. 1 . The FDA approved drug library was docked in the generated snapshots using Glide. The MMGBSA score was also calculated for each of the ligand in individual snapshots. (B) Transcriptomics data from SARS-CoV2 infected and normal human samples identified significantly differentially expressed genes (|log 2 FC>1|, p-value<0.01) as a result of infection. A protein-protein-interaction network was created using these genes. They were also analyzed for their involvement in biological pathways using Ingenuity Pathways Analysis (IPA). The drug gene interactions were also analysed and molecules were tested using SARS-CoV2 infection model in Vero E6 cells for antiviral activity. As stated earlier nine viral proteins (Table 1) were selected for molecular docking. The computational analysis of ligands binding to various proteins is a powerful method to quickly identify potential molecules for further studies. These methods have been successfully used in various studies (38-40). In the first stage, the molecules were docked into the SARS-CoV2 protein snapshots obtained by molecular dynamics using Glide module of Schrodinger in standard precision (SP) mode. The molecules were then ranked using average Glide score. The MM-GBSA was then performed to ensure the appropriate selection of top hits. All hits were visually inspected for interactions with receptor residues. The following strategy was adopted for the current study to identify candidate drugs: 1. drugs that can inhibit viral entry into host cell by perturbing the function of surface glycoproteins like the spike. 2. blocking the functions of viral enzymes that plays a vital role in replication such as 2'-O-ribose methyl transferase, RNA-dependent RNA polymerase, endoRNAse, helicase, 3'-to-5' exonuclease, 3C-like main protease and papain-like protease. 3. drugs that can also affect differentially expressed host proteins in COVID-19 along with the viral proteins. The hallmark feature of coronaviruses is their transmembrane spike (S) glycoprotein as this protein is the reason for its name "Corona" in Latin meaning, "Crown". SARS-CoV-2 uses its spike (S) protein to attach to host cells. The spike protein exists as homo-trimers. Each monomer is about 180kDa and has two distinct subunits S1 and S2. While the receptor binding is mediated by S1 subunit with the help of receptor binding domain (RBD), the fusion between the viral envelope and the host cellular membranes is facilitated by the S2 subunits upon the cleavage of S1-S2 junction by host proteases (41) . The S1 subunit of spike protein in SARS-CoV2 has four distinct domains: NTD, CTD1, CTD2 and CTD3, of these the "up" conformation of CTD1 is responsible for binding with ACE2 receptor (42 (43) . Recently, some of the SARS-CoV2 strains containing mutants of the Sprotein (D614G) with high infectivity have been reported. This mutant does not change the structure of S-protein but increases its binding with human TMPRSS2 protein (44) . This mutation is away from the identified binding site. Our molecular docking analysis suggest that capreomycin, posaconazole, mefloquine, nebivolol, angiotensin II, celecoxib and trimethoprim bind to spike protein with appreciable affinity (Supplementary Table 1 ) (Fig. 2 ). Other groups have also predicted the binding of posaconazole to spike protein which further substantiates our analysis (45) . Posaconazole is an antifungal agent used in the prevention of invasive fungal infections and is also shown to inhibit the entry of Chikungunya virus (46) and replication of Zika and Dengue viruses by binding to oxysterol-binding protein (sterol transporter) (47) . Mefloquine is an antimalarial drug used in chloroquine resistant malaria. Nebivolol is an antihypertensive molecule with a very good safety profile in subjects with obstructive respiratory comorbidities (48) and can be an important drug to consider in SARS like diseases. Capreomycin is a polypeptide (isolated from Streptomyces capreolus) used in the treatment of multidrug resistant tuberculosis. Its mechanism is similar to aminoglycosides and used in the inhalation therapy of pulmonary tuberculosis by spray-drying technology (49, 50) . It can be a promising prophylactic agent against SARS-CoV2 using similar application strategy (Fig. 2 ). (51) . Previous studies with SARS-CoV1 suggest that N protein inhibits TGF-beta, AP-1, NF-kB signaling and type 1 interferon production but induces apoptosis. The sera of COVID-19 patients shows the presence of IgG, IgA, and IgM antibodies against N protein suggesting its role in eliciting humoral immune response (52, 53) . In the current study the crystal structure of N-terminal dimerization domain of nucleocapsid phosphoprotein with a ligand (PDB ID: 6WKP) is used. The ligand binds at the dimer interface and has interactions with residues of both of the chains. The active site was defined as residues lying within 5Å of the cocrystallized ligand. Our study predicts that nelarabine, paclitaxel, regadenoson, quinaprilat and bromfenac are among top molecules binding to N protein (Supplementary Table 1 2′-O-Methyl Transferase (Nsp16) of SARS-CoV2 belongs to the S-adenosylmethioninedependent methyl transferase family and is activated upon binding to Nsp10. Nsp10 binds with a conserved four amino acid sequence 'KDKE' of Nsp16 in its catalytic pocket and activates its methyltransferase activity. Capping of viral mRNA at 5′-end is one of the viral strategy for protecting viral transcripts from host 5′ exoribonucleases and escaping the host innate immune response by mimicking as host mRNAs, thus Nsp16 is the potential target for antiviral therapeutics (54) . (Fig. 4 ). Methotrexate acts as an antimetabolite and thus used as an antineoplastic drug. It is also used in treatment of inflammatory diseases like rheumatoid arthritis. It decreases the de novo synthesis of purines and pyrimidines and forms dimers with thymidylate synthase (TS), hence also has anti-parasitic effect (55) . Methotrexate is also shown to effectively inhibit Zika and Dengue virus replication (56) . Zidovudine is used in HIV1 treatment (21) , histrelin and triptorelin are gonadotropin-releasing hormone analogs used in the treatment of central precocious puberty and endometriosis (57) . Lanreotide is a long-acting analog of somatostatin and is used for the management of acromegaly, a condition caused by excess secretion of growth hormone. Octreotide is also a somatostatin analog currently used for the treatment of There are many small molecules, peptides and peptidomimetics that have been developed against these proteases (58) (59) (60) . The 3CL pro is a cysteine protease having three domains: β-barrel Domain I (residues and II (residues 102-184) and α-helix domain III (residues 201-306) similar in structure to chymotrypsin (61) . The functional protease is a dimer that cleaves polyprotein 1ab in 11 regions at its specific cleavage site (P1) of Leu-Gln↓(Ser, Ala, Gly). The sequences of SARS-CoV2 and SARS-CoV main protease are highly similar (96% identity) and so their 3D structures, barring some surface residues. However, enigmatically the inhibitors of SARS-CoV 3CL pro lopinavir and ritonavir that were also recommended for use against SARS-CoV2 have not shown expected results in the clinical trials for COVID-19 (62 Previous studies report α-ketoamides, lopinavir and ritonavir as inhibitor of 3CL pro (63, 64) . Ruprintrivir inhibits human rhinovirus (HRV) 3C protease and has shown broad-spectrum anti-HRV activity (65) . Others have also indicated it to be useful against SARS-CoV2 main protease (66) . Indinavir is shown to inhibit HIV protease by blocking its active site and leads to immature virus particle formation, however high doses have been linked to lipodystrophy syndrome (67) . Naldemedine, is a μ-opioid receptor antagonist used for the treatment of opioidinduced constipation (68) . PL pro is a domain within nsp3 of pp1a/pp1ab with proteolytic activity. It cleaves three sites at Table 1 ). Galidesivir has broad-spectrum antiviral activity (in vitro) against many RNA viruses in nine different families, including the coronaviruses (71) . The binding of galidesivir with PL pro is shown in Fig. 6 . This drug has been under clinical trials for COVID- Helicase enzyme (Nsp13) of SARS-CoV2 is motor protein essential for unwinding of both dsDNA and dsRNA and has metal binding (Zn2+) N-terminal and helicase domain (Hel). It is involved in formation of RTC of SARS-CoV2 along with RdRp, which is known to enhance its activity (75) . The SARS-CoV2 helicase has 99.8% sequence similarity with that of SARS-CoV. Since it is one of the most conserved proteins in Nidoviruses and is essential for viral RNA synthesis, it is an attractive target for antiviral drug development. A recent review summarizes its importance as a drug target in COVID-19 (76) . In the current studies the cryo electron microscope structure of helicase-RdRp (PDB: 6XEZ) was used. The residues within 5Å of the ADP bound to helicase enzyme were defined as the active site. Our analysis shows that eratapenem, methotrexate, clofarabine, trimethoprim, ascorbic acid, cefixime, and pibrentasvir bind to the helicase with high affinity (Fig. 7) . Clofarabine is a potent HIV-1 inhibitor (77) . Pibrentasvir, is a HCV NS5A inhibitor effective against all HCV genotypes (78). In the current studies, we have used the structure of RdRp complexed with remdesivir (PDB: 7BV2). The residues falling withing 5Å of the remdesivir were defined as active site. Our analysis shows that fludarabine, cobicistat, capreomycin, regadenoson, doxazocin, pibrentasvir, elbasvir, indinavir and remdesivir among others that can bind with RdRp (Fig. 8 ). Fludarabine is used for the treatment of hematological malignancies. It inhibits various critical enzymes and results in the inhibition of DNA synthesis. It has been predicted to be ative against SARS-CoV2 RdRp by other groups as well (74, 83) . Ribavirin is broad spectrum antiviral used for treatment of RSV infection, hepatitis C and viral hemorrhagic fevers (84) . It is a well known RdRp inhibitor. Cobicistat is known to inhibit the cytochrome-mediated metabolism of HIV protease and was approved in 2012 by FDA as pharmacoenhancer for HIV treatment (85). Other groups have also predicted that cobicistat and capreomycin can inhibit SARS-CoV2 protease (86) (87) . Pibrentasvir and elbasvir are HCV NS5A inhibitors and indinavir is potent HIV protease inhibitor (88) . Another molecule monteleukast, a leukotrine inhibitor used as antihistaminic was also showing good affinity towards RdRp (docking score -9.42). The molecules we identified to bind to RdRp can serve as potential alternatives to remdesivir. The Nsp15 is EndoRNase with endoribonuclease activity. It cleaves the 5′ and 3′ of uridylate residues in RNA by forming 2′-3′cyclic phosphodiester. Its mechanism is similar to that of RNase A, RNAse T1 and XendoU (89) . Its NendoU activity can interfere with the host's innate immune response and masks the exposure of viral dsRNA to host dsRNA sensors (90) . The crystal structure of SARS-CoV2 Nsp15 cocrystallized with U5P (PDB: 6WLC) was used in the current studies. The active site was defined by the residues falling with 5Å of the cocrystallized ligand. The active site is situated near the N-terminal and is surrounded by beta sheets and a helix. In our analysis, drugs such as quinapril, octreotide, folic acid, and macimorelin were found to bind to Nsp15 with appreciable affinity (Fig. 9 ). Quinapril is an angiotensin converting enzyme (ACE) inhibitor and the ACE inhibitors have been suggested to be beneficial for COVID-19 patients (91) . Folic acid is essential for DNA and protein synthesis and in the adaptive immune response (92) . The dose dependent effect of folic acid on rotavirus infected mice has been reported indicting its antiviral activity (93) . Additionally, the role of folic acid in the prevention of cellular entry of SARS-CoV2 has been reported (94). Macimorelin is used for the diagnosis of adult growth hormone deficiency (95) . Interestingly other groups have also predicted it to be active against SARS-CoV2 (96, 97). Nsp14 is the 3'-5'exonuclease that plays a role in proofreading mechanism (98 Our molecular docking predicted that cangrelor, venetoclax, pimozide, nilotinib, droperidol, nebivolol, indacaterol, ezetimibe, simeprevir, siponimod, lapatinib, elagolix bind to Nsp14 ( Supplementary Fig. 1 ). Moreover, it binds to the envelope protein of HCV and inhibits infection with many HCV genotypes (101) . Droperidol is also predicted by other groups to be effective against SARS-CoV2 infection (102) . Ezetimibe is shown to inhibit formation of capsid-associated relaxed circular DNA of Hepatitis B Virus (HBV) (103) and is also shown to inhibit Dengue infection by interfering in formation of replication complex (104) . Indacaterol is the β2-adrenoceptor agonist and used in the treatment of chronic obstructive pulmonary disease (COPD) since it induces bronchodilation (105) . It is a promising candidate for therapeutics against SARS-CoV2 due to its ability to regulate genes involved in suppressing proinflammatory cytokine production and attenuation of airway hyper-responsiveness (106) . However, dose and treatment schedule needs to be evaluated due to its counter effect on the expression of RNase L which is vital for antiviral response. Since one of our major objectives was modeling of the intrinsic flexibility of the SARS-CoV2 proteins by molecular dynamics simulation and finding drugs that can adjust with the site flexibility. We provide a summary of the top drugs for individual proteins and their docking scores in the frames generated by molecular dynamics along with the average MMGBSA score Table 2 . The drugs with consistently good docking scores will have a better average. This approach is novel and is not reported anywhere before for screening of drugs against SARS-CoV2 as per the best of our knowledge. A heatmap (Fig. 10) was generated using the docking scores to summarize the binding of important drugs to multiple proteins. Individually or in combinations these drugs can serve as potential therapeutics with the capacity to modulate both the viral as well as human proteins. The identification of molecules targeting multiple viral proteins simultaneously will effect the virus life cycle at multiple stages and will also have a higher barrier toward evolution/emergence drug resistant mutants, a common problem with many direct acting antivirals (DAA) against RNA viruses. Using a combination of drugs that target various different viral proteins to achieve synergistic effect against viral dissemination. The detailed list of drugs and their docking scores is given in Supplementary table 1. As stated earlier, the differentially expressed genes (DEGs) were obtained from the data reported by Blanco-Melo et al (35) . The DEGs were selected based on the following criteria: Table 2 ). This criteria was chosen to select genes showing the most significant variation. The gene ontology (GO) enrichment analysis on these DEGs indicates immune system processes, such as type II interferon signaling (IFNG), innate defence response, cytokine and chemokine siganling, RAGE receptor binding, secretary granuale are among the most enriched ontologies (Supplementary Table 3 ). It is important to note that these are among the processes usually activated in infection-associated inflammation. Protein-protein-interaction network analysis was done using Cytoscape to identify the network Table 4 ). It is worth to mention that among the human proteins many of them (e.g. indinavir, chloroquine, sequinavir, and ritonavir, that are common between our study and the said study further corroborating our findings. It is interesting to find many drugs with multitargeting ability against hub proteins as well as SARS-CoV2 proteins. Such drugs can have a significant therapeutic utility for COVID-19 (Fig. 11 ). We assessed the activity of few of the compounds that bind to more than one SARS-CoV2 proteins in an in-vitro cell culture model of SARS-CoV2 in VeroE6 cells. We used the SARS-CoV2 virus (ILS-01)) isolated from oropharyngeal swab sample of confirmed COVID19 positive patient. Cell-free Viral RNA Cytotoxicity vitro, were used as positive controls in this study that further validated the activity and potency of the molecules tested in this study. Among the 5 drugs tested mefloquine has the lowest IC 50 value at 0.37 uM and montelukast, which was also predicted by others to possess anti-SARS-CoV2 potential has a higher IC 50 value of 18.82 uM (Fig 12, Table 3 ). To further validate the anti-SARS-CoV2 potency of the tested drugs we performed an immunofluorescence assay by staining for SAR-CoV2 nucleocpasid protein in SARS-CoV2 infected Vero-E6 cells subjected to treatment with the vehicle control or drugs at their IC50 concentrations. We observed marked reduction (around 50% or higher) in the percentage of infected cells subjected to treatment with the drugs in comparison to the vehicle control (Fig 13 A and B) . We also observed that the drug treatment at the indicated IC50 concentrations had very minimal effect on the total cell number in comparison to the vehicle control (Fig 13 C) . Overall these observations suggest that the treatment with drugs resulted in marked inhibition of viral gene expression with minimal effect on cellular viability. Overall these observations strongly validate our in-silico finding, however further screening is required in physiologically relavant cell lines and in-vivo animal models to fully establish the anti-SARS-CoV2 potential of the identified leads. The computational drug repurposing studies came into forefront bacause of the speed and memory of the modern computers supplemented by the availability of the algorithms and data from studies in the past. Moreover, the crystal structures of many SARS-CoV2 proteins were reported during 2020 that made the structure based screening studies feasible. Various libraries (e.g. FDA approved) were screened against targets like RdRp, main protease (3CLpro), spike protein, membrane protein, and non-structural proteins (Nsps) using various strategies (74, (108) (109) (110) (111) (112) . For example, the main protease (Mpro) was screened by three docking algorithms. The authors selected the molecules that are commonly predicted by all algorithms (108) . Similarly, structure based docking followed by molecular dynamics studies have been used by Capreomycin is a promising candidate with potential to inhibit SARS-CoV2 at mutltiple stages of viral lifecycle, as it can bind with high affinity to spike protein and the viral proteases and methyl transferase, which play a crucial role in viral entry, replication and transcription. In our in-vitro asays it has shown good inhibitory activity. Mefloquine, an antimalarial drug, has shown good affinity towards spike protein in our insilico and in-vitro studies. It is pertinent to note that recently mefloquine has been shown to prevent the entry of SARS-CoV-2 into host cells. It has shown potent inhibitory activity against SARS-CoV2 in multiple cell lines (117) . Many other studies reported similar observations corroborating our findings (109, 112, 118) . Our analysis further indicated that some drugs that bind viral proteins also target some of host proteins that are differentially expressed in lung tissue during SARS-CoV2 infection. The predicted drug candidates that interact with the viral protein(s), in parallel can also specifically target the host signalling pathways vital to control viral infection or disease manifestation. For instance, nebivolol a β-adrenoreceptor blocker, which stimulates nitric oxide production by endothelial nitric oxide synthase (48) is found to bind to PL pro and exonuclease of SARS-CoV2. Nitric oxide is used to reverse pulmonary hypertension and shown to improve severe hypoxia in SARS-CoV1 (119) and SARS-CoV2 patients. Hence, nebivolol can be a promising therapeutic strategy with dual benefit; i) to curb SARS-CoV2 infection and ii) reversal of severe hypoxia manifestation in critical Covid-19 patients via its direct effect on nitric oxide synthase. It is interesting to note that recently nebivolol is found to inhibit SARS-CoV2 in submicromoar range similar to our study (120) . A major contributor of COVID-19 pathology is hyper-inflammation and cytokine hyperactivity. Strategies to reduce the inflammation and cytokine hyperactivity has shown promising results. Celecoxib, a selective cyclooxygenease-2 (COX-2) inhibitor, which lowers the effect of proinflammatory cytokines IL-6 and IL-1β (121) interface and by inhibiting the main protease. Apart from the anti-asthmatic effect it is also reported to cross BBB and reduce neuroinflammation (128) . A recent paper reviewed its antiviral, anti-inflammatory, anti-allergic and anti-fibrotic activities. It has also been suggested that montelukast should be tried as therapeutic option in COVID-19 (129) . Interestingly, the drugs montelukast, celecoxib and nebivolol can cross blood brain barrier (128, 130, 131) , which gives additional advantage to counter neurological manifestations of COVID-19. It is again pertinent to note that these drugs have shown appreciable inhibitory activity (IC 50 ) against SARS-CoV2 in our study. Lapatinib binds to Nsp14, a viral protein crucial for viral RNA synthesis (132) . Lapatinib is a HER2 inhibitor, which can also trigger TBK1 activation that plays a crucial role in anti-viral signalling. Computational studies have predicted lapatinib to be able to bind many SARS-CoV2 proteins including the main protease (110, 133) . Thereby, lapatinib has the dual advantage of inhibiting SARS-CoV2 replication as well as upregulating anti-viral signaling. (134) (135) (136) . Saralasin belongs to a class of drugs called angiotensin receptor blockers (ARBs). It is worth mentioning some other ARBs (e.g. losartan) are also in clinical trials as therapeutics for COVID-19 (https://clinicaltrials.gov, IDs: NCT04287686, NCT04312009, NCT04311177). Moreover, it is reported to bind to many other SARS-CoV2 targets (137, 138) . Therefore, it is also a good candidate worth consideration. Similarly, bronchodialator indacaterol, which targets the exonuclease is also a promising agent due to its ability to regulate genes involved in suppressing pro-inflammatory cytokine production and attenuation of immune response (139) (140) (141) . Another study has reported indacaterol to be able to bind spike protein of SARS-CoV2 (142) . The transcription complex activator protein 1 (AP1) is composed of homo/hetero dimers of Fos, Jun, CREB and other activated transcription factors (ATFs). The studies on the SARS-CoV1 infection in the Vero and Huh7 cell shows that nucleocapsid protein is the potent activator of (AP-1) (143) . Interestingly, asthmatic patients show higher expression of c-Fos in their epithelial cells. It is also observed that TNF-α induced ROS and intracellular glutathione depletion in the airway epithelial cells induces the production of AP-1 and leads to the pulmonary fibrosis (144, 145) . Our analysis suggests that paclitaxel and bromocriptine, which dock with nucleocapsid and Nsp4 proteins can also effectively bind to c-Fos and thereby would be beneficial in inhibiting the SARS-CoV2 as well as in alleviating lung injury observed in COVID19. Interestingly bromocriptine is reported by other studies also to be able to bind to main protease (146) or Nsp14 (147) . The transcriptome analysis revealed that S100/calgranulin is upregulated during SARS-CoV2 infection. This protein is also found in higher quantity in the Bronchoalveloar Lavage Fluid (BALF) and sputum of patients with inflamed lungs, COPD, and ARDS (148) . Calgranulin is polypeptide released by the activated inflammatory cells such as leukocytes, PBMC phagocytes and lymphocytes and is accumulated at the sites of chronic inflammation. It is the ligand for RAGE receptors and is the major initiator of cascading events that amplify inflammation (149) . Our analysis suggests that the anti-inflammatory agent methotrexate which has high affinity to the Nsp16 protein of SARS-CoV2 also shows appreciable binding to calgranulin and can thereby be useful to curtail systemic inflammation observed in lungs during COVID19 in addition to its inhibitory effect on SARS-CoV2. Interestingly, methotrexate is recently reported to inhibit the replication of SARS-CoV2 (150) . Another paper suggested to use methotrexate with leucovorin rescue for the treatment of severe COVID-19 (151) . Methotrexate is also in clinical trial currently for the treatment of mild COVID-19 (https://clinicaltrials.gov/ct2/show/NCT04610567). The expression of endogenous prolactin is also upregulated during SARS-CoV2, which leads to prolactin induced STAT5 activation and its pathways. Prolactin has a dual role in human physiology functioning as a hormone (secreted from anterior pituitary gland) and cytokine (secreted by immune cells). It causes anti-apoptotic effect and induces proliferation in immune cells in response to antigens leading to increased production of immunoglobulin, cytokines, and autoantibodies (152) . We envisage that prolactin may be one of the significant player in trigger of cytokine storm implicated in COVID19. Interestingly, our study suggests that zidovudine which target the O'-methyl transferase (Nsp16) can also bind to prolactin and can be of high significance in management of COVID19 due to dual ability to affect Nsp16 and prolactin. The COVID-19 creates an inflammatory state involving proinflammatory cytokines e.g. IL-6, TNF-α etc. IL-6 stimulate ferritin and hepcidin synthesis (153) . The hyper-ferritinemia is associated with generation of ROS and RNS leading to enhanced systemic inflammation. As a result a devastating cycle is propogated where increased ferritin leads to higher inflammation (increased IL6) resulting in further increase in ferritin levels (154) . Hyper-ferritinemia has been linked with poor prognosis in COVID-19 patients, evidenced by high levels of ferritin in nonsurvivors as compared to survivors (155, 156) . In this milieu, iron chelators can be extremely helpful by reducing the hyper-ferritinemia and systemic inflammation. Deferoxamine is an iron chelator that also increases degradation of ferritin by lysosomes leading to reduction of free radicals and subsequently inflammation. It also limits the chances of ARDS and tissue fibrosis. Our analysis indicates that deferoxamine binds to RdRp and PL pro protein of SARS-CoV2 with good affinity. Therefore it can be a good candidate for the therapeutics of COVID-19. Currently, desferaox is under clinicl trials for the treatment of COVID-19. We could test a few molecules that showed potent anti-SARS-CoV2 activity in in-vitro models. The identified molecules are commonly used drugs and hence can be quickly repurposed. Their combinations can also have synergistic effcts against SARS-CoV2. We hope these molecules will prove to be useful in our fight against COVID-19. Apparently, the cytotoxicity of some molecules is high. As the in-vitro assays were performed in Vero-E6 cells, a monkey kidney epithelial cell line, we expect that the cytotoxicty may be lower in human cell lines. However, they are FDA approved drugs already being used for treating various clinical conditions at the recommended dosage and may be benefical for SARS-CoV2 therapeutics. Also the treatment for SARS-CoV2 will likely last only for a short duration, therefore weighing the potential benefits vs toxicity they could be very useful in curbing SARS-CoV2 infection. Currently, there are no approved coronavirus treatments and therefore there is a pressing need for drugs that can be effective therapeutics for COVID-19. Our study predicted promising drug candidates with high binding affinity towards many of SARS-CoV2 proteins. These drugs are expected to be more effective than drugs that target single viral proteins due to their ability to affect multiple aspects of viral lifecycle and enhance the barrier towards the evolution of drugresistant mutants, a usual phenomenon observed in RNA viruses. Overall our study predicts promising agents with potential to inhibit crucial viral processes, upregulate anti-viral host response and alleviate severe lung disease condition thereby providing attractive avenues for design of potential and multipronged therapeutic strategies against COVID 19. The work is designed and conceptualized by SK, AD, GA and GHS. Data generation and work performed by SK, PVK, PK, BS and AD. 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