key: cord-0756941-36wioyqr
authors: Bibi, Nousheen; Farid, Ayesha; Gul, Sana; Ali, Johar; Amin, Farhat; Kalthiya, Umesh; Hupp, Ted
title: Drug repositioning against COVID-19: a first line treatment
date: 2021-09-14
journal: Journal of biomolecular structure & dynamics
DOI: 10.1080/07391102.2021.1977698
sha: 26b6dc9f381906461357bde1fd69cbc1caae8a33
doc_id: 756941
cord_uid: 36wioyqr

COVID-19 disease caused by the SARS-CoV-2 virus has shaken our health and wealth foundations. Although COVID-19 vaccines will become available allowing for attenuation of disease progression rates, distribution of vaccines can create other challenges and delays. Hence repurposed drugs against SARS-CoV-2 can be an attractive parallel strategy that can be integrated into routine clinical practice even in poorly-resourced countries. The present study was designed using knowledge of viral pathogenesis and pharmacodynamics of broad-spectrum antiviral agents (BSAAs). We carried out the virtual screening of BSAAs against the SARS-CoV-2 spike glycoprotein, RNA dependent RNA polymerase (RdRp), the main protease (Mpro) and the helicase enzyme of SARS-CoV-2. Imatinib (a tyrosine kinase inhibitor), Suramin (an anti-parasitic), Glycyrrhizin (an anti-inflammatory) and Bromocriptine (a dopamine agonist) showed higher binding affinity to multiple targets. Further through molecular dynamics simulation, critical conformational changes in the target protein molecules were revealed upon drug binding which illustrates the favorable binding conformations of antiviral drugs against SARS-CoV-2 target proteins. The resulting drugs from the present study in combination and in cocktails from the arsenal of existing drugs could reduce the translational distance and could offer substantial clinical benefit to decrease the burden of COVID-19 illness. This also creates a roadmap for subsequent viral diseases that emerge. Communicated by Ramaswamy H. Sarma

The coronavirus pandemic, also known as coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is creating a severe global health crisis which is one of the worst the world has faced since world war II. An unexplained virus, SARS-CoV-2, derived out of Wuhan, China in late 2019 Zhou et al., 2020) and has since spread to every continent of the world with over 72,692,481 confirmed cases and over 1,619,801 confirmed deaths worldwide as of December 14, 2020 (www.WHO.org). Due to the lack of any demonstrable cure against this devastating virus, researchers around the globe are trying to develop or repurpose antiviral drugs through experimental and computational means to alleviate the pain of this pandemic.

SARS-CoV-2 the major pathogen of the COVID-19 illness is closely related (89.1%) to SARS-CoV and it's a positive singlestranded RNA virus (þssRNA) (StatPearls, 2021) . The interaction of SARS-CoV-2 spike protein with the host receptor angiotensin-converting enzyme 2 (ACE2) drives the infection cycle of the virus by releasing the viral genome. The SARS-CoV-2 genome is comprised of variable number of 6-11 ORFs among which encodes the 16 non-structural proteins and other ORFs encode structural proteins including spike (S), envelope (E), membrane (M), and nucleocapsid (N) (Shereen et al., 2020) . ORF1a and ORF1b contain a frameshift in between which produces two polypeptides: pp1a and pp1ab. These polypeptides are processed by virally encoded chymotrypsin-like protease (3CLpro) or main protease (M pro ) and one or two papain-like proteases into 16 NSPs (Mousavizadeh & Ghasemi, 2021) . All NSPs perform their specific functions like NSP1 and NSP2 suppress the expression of host gene, formation of a multidomain complex by NSP3, NSP5 the main protease (M pro ) with critical role in replication form the multidomain complex (Stobart et al., 2013) . Furthermore, the transmembrane proteins NSP4 and NSP6 (Wang et al., 2016) as well as NSP7 and NSP8 are primases (Te Velthuis et al., 2012) . The dimeric form of NSP9 (an RNAbinding protein) is crucial for viral infection (Egloff et al., 2004) . NSP10 acts as a cofactor for the activation of the replicative enzyme (Bouvet et al., 2014) , NSP12 known as RNAdependent RNA polymerase (Jiang et al., 2021) and NSP13 as helicase (Jang et al., 2020) . While NSP14 and NSP15 shows exoribonuclease and endoribonuclease activities, respectively, and NSP16 with methyltransferase activity (Wang et al., 2016) . NSP10-NSP16 complex is crucial to evade from the host immune system (Rosas-Lemus et al., 2020) . Conclusively, all structural and nonstructural proteins critical for the virus entry, replication and transcription process may serve an important role from drug design perspectives.

Currently, there are no specific and appropriate antiviral therapy for the treatment of SARS-CoV-2 induced illness. Every country needs an immediate response and recovery from this crisis in this critical time, drug repurposing is an attractive approach with reduced time and cost to treat COVID-19. Broad spectrum antiviral agents (BSAAs) are smallmolecules which may inhibit different types of human viruses that exploit similar pathways and host factors to replicate inside the cells. Instead of individual drug, combination of drugs could serve as front-line therapeutics for newly emerging viruses (SARS-CoV-2) and remerging viruses (Andersen et al., 2020) .

The current study is focused on a drug repurposing strategy to find potential drug candidates suitable for targeting possible SARS-CoV-2 infection pathways. Targeting virus at entry and replication stage is attractive and credible strategy to treat covid-19. Therefore, spike (entry protein) and RdRp, M pro and helicase (replication proteins) are selected for the present study as drug target. Through structure based virtual screening and molecular dynamics simulations. We screened all the existing BSAAs (those reported in multiclinical trials against COVID-19 and other approved, investigational and experimental) against SARS-CoV-2 druggable targets to iteratively explore the SARS-CoV-2-BSAAs interactions. The most probable binding mode for selected target-drug candidate from docking experiments were subjected to molecular dynamics (MD) simulations to rationalize the flexibility of their binding sites, mechanism of action of BSAAs and target molecules stability. With the aim to impact the COVID-19 outbreak, drug repurposing through virtual screening, molecular modeling and molecular dynamics (MD) simulation can facilitate and accelerate the search for appropriate antiviral drugs for SARS-CoV-2.

The recently resolved three-dimensional crystal structure of SARS-CoV-2 spike protein (PDB ID: 7BWJ) (Ju et al., 2020) , RNA-Directed RNA polymerase (RdRp; PDB ID: 6M71) (Gao et al., 2020) , main protease (M pro ) (PDB ID: 6LU7) (Jin et al., 2020) , were retrieved from Protein Databank (PDB) (www. rcsb.org) and helicase protein (QHD43415_12) was retrieved from zhang lab (zhanglab.ccmb.med.umich.edu/COVID-19/). The structures of four target proteins were subjected to energy minimization (steepest descent steps of 100, with step size of 0.02, and with 10 conjugate gradient steps with step size of 0.02 (Ð) followed by detachment of any ligand attached and nonstandard residues through UCSF Chimera (Pettersen et al., 2004) and Discovery Studio Viewer (Pazel, 1989) . Structural features of selected target molecules have been elucidated along with binding site identification for inhibitor binding (Figure 1 ).

Electrostatic potential and pKa values for spike, RdRp, M pro and helicase protein were calculated using Bluues Method (Walsh et al., 2012) , that compute the electrostatic properties through generalized Born radii comparable to Poisson-Boltzmann equation. Electrostatic potential of the target proteins was estimated in molecular context. As a gauge of protein stability and function, pKa values for four target molecules were also calculated. Following parameters were used for electrostatic potential and pKa calculation: Solvent probe radius (Ð):1.4 Ð, Salt radius (Ð): 2.0 Ð, Inner dielectric constant: 4, outer dielectric constant: 78.5, and ionic strength:0.150 mol/L.

BSAAs are small-molecules which may inhibit different types of human viruses that exploit similar pathways and host factors to replicate inside cells. In the present study a total of 872 BSAAs were retrieved from the DrugVirus.info database (https://drugvirus.info/). 3 D coordinates of all the BSAAs were obtained using Open Babel (https://sourceforge.net/ projects/openbabel/) followed by energy minimization. The dataset of BSAAs were subjected to structure based virtual screening against selected set of receptors, spike protein, RdRp, Mpro and helicase using Autodock vina wizard of PyRx0.8 (Dallakyan & Olson, 2015) . Default parameters in PyRx was used during docking except grid size and spacing. The grid size and spacing were set according to each target's binding cavity (Table S1 ). The virtually screened best compounds were then docked with the target receptors again to ensure the conformation poses and binding affinities. The interactions were visualized using UCSF Chimera (Pettersen et al., 2004) and Discovery Studio (Pazel, 1989) .

Statistical analysis of the screening results was performed in Origin 2018. Box plot for four different groups with normal distribution was constructed. Histogram was constructed and linear curve fitting was performed to define the best fit model. Normal probability plot was constructed to assess the approximate normal distribution of four datasets. Cystoscope 3.8.2 (https://cytoscape.org/) was used to construct the drugreceptor network of top ten selected drugs for each target molecule. Network analyzer was used with following statistical parameters: clustering coefficient of 0.072, network diameter of 4 and radius of 1, network centrality of 0.286 and characteristic path length of 2.862.

Molecular dynamics (MD) simulations were performed for best ranked protein-ligand complexes for all studied targets(drug-protein) using GROMACS package version 5.0.5 (Van Der Spoel et al., 2005) . PRODRG server (Sch€ uttelkopf & van Aalten, 2004) was used to pick up the topologies of top binding affinity BSAAs. The parameters of all the target and drugs molecules were optimized by using the GROMOS96 54a7 force field (Schmid et al., 2011) having better propensities and free energy of hydration. Periodic cubic box equipped with solvent molecules was used for all the systems. For non-bonded interactions periodic boundary condition was used and similarly for strong electrostatic interactions particle mesh Ewald method was used (Essmann et al., 1995) . Systems were neutralized with Na þ Cl counter ions. The prepared systems in the periodic box containing protein, ligand, ions and solvent were minimized using a steepest-descent algorithm for 50,000 steps. NVT (number of atoms, volume, temperature) ensemble was used to add constraint to protein-ligand complex for 100ps during heating. NPT (constant pressure, constant temperature) ensemble was used at constant pressure (1 bar) and temperature (300 K) to simulate the systems for 100 ns. To the end of simulations all the systems were analyzed for stability and fluctuations using VMD (Humphrey et al., 1996) , PyMol (http://www.pymol.org) and GROMACS tools.

In biomolecular simulations binding free energy calculation is a state function based on predicted affinities of the receptor-ligand complex. Using Molecular mechanics Poisson-Boltzmann Surface Area (MM-PBSA) (Iqbal & Iqbal, 2014; Wrapp et al., 2020) , binding free energy calculations were performed to measure the affinities of shortlisted drugs with respective target molecules. Overall, 100 frames from the trajectories were processed to estimate the net energy of the systems through the following equation,

All above mentioned terms involves the computation of several energy components: electrostatic energy, van der Waals energy and internal energy summed from molecular mechanics and polar contribution towards solvation energy. The analysis also takes into account the contribution from non-polar term towards solvation energy and inhibitor entropy. In the calculations of the MM-PBSA, the external dielectric constant was set to 80.0, the internal dielectric constant was 1.0 and the reciprocal grid spacing of 1.0 Å was used.

Electrostatic potential helps to understand the contribution of electrostatic potential on structural aspects of the protein and protein-ligands interactions through analysis of positive and negative potential. pKa values of the protein determine its pH characteristics which is measure of protein flexibility and structural feature to accept or release the ligand.

The spike protein of SARS-CoV-2 biding site showed more positive potential. His 69 , Lys 458 , Arg 664 , Lys 786 and Phe 855 are the highly solvent accessible residues of spike protein ( Figure  2A and B). Thr 874 is the most buried residue, H (hydrogen atom) is the most positive potential atom of protein belonging to Leu 977 and the most positive pKa atom is CZ (carbon atom) belong to Arg 457 (Table1). The electrostatic potential of binding site of RdRp enzyme of SARS-CoV-2, showed positive and negative potential. Gln 117 , Arg 118 , Lys 267 , Arg 365 and Glu 919 are the highly solvent accessible residues of RdRp ( Figure 2C and D). HD12 is the most buried atom belong to Leu 663 , HB3 (hydrogen atom) is the most positive potential atom of RdRp belonging to Asp 304 and the most positive pKa atom is CZ (carbon atom) belong to Arg 457 (Table1). The electrostatic potential of M pro binding site showed more negative potential. Tyr 154 , Arg 222 , Thr 4 , Arg 60 and Asn 142 are the most solvent accessible residues of the M pro ( Figure 2E and F) HE1 (hydrogen atom) is the most buried atom of Met 162 , HZ3 is the most positive potential atom of Lys 336 and OH is the most positive pKa atom of Tyr 161 (Table 1) . Helicase enzyme binding cavity showed both positive and negative potential. Whereas, Asn 596 , Met 68 , Lys 189 , Arg 392 and Arg 579 are the highly solvent accessible residues ( Figure 1G and H). CE2 is the most buried atom belong to residue Val 372 . NZ is the most positive potential atom belong to Lys 347 . CZ is the most positive pKa atom belong to Arg 579 (Table 1) .

Structure based drug design strategy was used to screen the dataset of BSAAs (Table S1 ) selected against the promising druggable targets (spike, RdRp, M pro and helicase) of SARS-CoV-2. High throughput screening using molecular docking approach resulted in broad range of BSAAs showing broad range of binding affinities towards four target molecules ( Figure 3A -C), ranging from À1 to À13.0 kcal/mol. Among all BSAAs Imatinib showed strong binding affinity towards multiple targets molecules. Imatinib a cancer growth blocker showed high binding affinity towards RdRp, M pro and spike protein of SARS-CoV-2. Suramin, a 100-year-old multifunctional drug with broad array of action against parasites to virus and cancer, showed the high binding affinity towards spike and helicase. Glycyrrhizin an anti-inflammatory drug and Bromocriptine showed affinity towards RdRp, M pro and helicase ( Figure 3D ). Drugs targeting various stages of SARS-CoV-2 life cycle are shown in Figure 3E and Table 2 . These finding from the present study can reduce the translational distance to repurpose the BSAAs against SARS-CoV-2 induced disease.

Potential drugs against spike protein

The forefront of SARS-CoV-2 infection is spike protein with important receptor binding domain (RBD) vital for host attachment (Assaad & Assaad-Khalil, 2020) . As an ideal drug target for COVID-19 to stop the viral entry into host cell, we identified some BSAAs as potential drugs against spike-RBD to interfere the binding of virus with host receptor. Screening of BSAAs library resulted in broad range of inhibitors with binding energy ranging from À2.7 to À9.7 kcal/ mol. Among all the BSAAs, Suramin showed binding affinity with binding free energy of À9.7 kcal/mol. Asn 440 , Asn 450 , Glu 484 and Gly 485 showed conventional hydrogen bond with the Suramin and Tyr 451 make pi-alkyl interaction along with number of hydrophobic interactions by Asp 442 , Ile 468 , Thr 470 , Glu 471 and Ile 472 ( Figure 4A and B) . Imatinib also showed good binding affinity with binding free energy of À9.1 kcal/ mol. Imatinib is an anticancer drug (Chen et al., 1997) used for the treatment of covid-19 (Assaad & Assaad-Khalil, 2020). Binding interaction analysis revealed that Asn 440 and Leu 441 made conventional hydrogen binding while two pialkyl interactions by Arg 436 and one pi-sigma interaction by Tyr 451 was observed ( Figure 4C and Table 2 ). The best ten BSAAs for spike protein on the basis of binding energy and RMSD score are given in the table S2A along with mode of action and drug status. 

RNA-dependent RNA polymerase (RdRP) is crucial for viral life cycle, and as catalytic site of the RdRp is the most conserved and accessible, so could be used as an attractive drug candidate to inhibit viral replication. Virtual screening of BSAAs against RdRp of SARS-CoV-2 showed high binding affinity of Glycyrrhizin with binding free energy of À12 kcal/mol. Imatinib also showed good binding affinity towards catalytic cavity of RdRp enzyme. Glycyrrhizin drug interaction with RdRp revealed binding within the palm domain conserved motif ( Figure 4D Figure 4E and F and Table2). The best screened antiviral drug along with status and therapeutic potential is given in supplementary Table S2B .

Main protease (M pro ) of SARS-CoV-2 is a promising drug target as it plays critical role in viral replication and transcription. M pro showed high binding with Imatinib with binding free energy of À9.0 kcal/mol. Verdinexor, which is an investigational drug and has shown inhibition of influenza virus replication was bound with M pro of SARS-CoV-2 with binding free energy of À8.5 kcal/mol. The detailed interaction analysis of high affinity drugs showed that Imatinib formed three conventional hydrogen bonds with the Cys 145 , Gly Table 2 ). The final screened best ten BSAAs with virtual screening high binding affinity score for M pro have been provided in data file S2C.

Nsp13 (helicase), is a multi-functional protein with metal binding N-terminal domain and C-terminal helicase domain. Due to essential role helicase in replication, transcription, and translation of SARS-COV-2 it is promising druggable target. In the virtual screening analysis, Suramin showed high binding affinity towards helicase with the binding free energy of 11.6 kcal/mol. Asn 179 , Asn 516 , Asp 534 and Arg 560 made conventional hydrogen bond with the Suramin. Apart from this Tyr 515 and Arg 560 formed pi-interactions along with number of hydrophobic interactions by Asn 177 , Arg 178 , Thr 410 , Leu 417 , Thr 532 , Gln 537 , His 554 and Asn 557 . Apart from Suramin, Glycyrrhizin also returned good binding affinity with binding energy of À10.7 kcal/mol. Asn 179 , Asn 516 , Arg 560 made conventional hydrogen bond along with number of hydrophobic interactions by Asn 177 , Arg 178 , Pro 406 , Pro 408 , Thr 532 , Asp 534 and Asn 557 ( Figure 4J -L and Table 2 ). The final screened best ten BSAAs with virtual screening high binding affinity score for helicase have been provided in data file S2D Therapeutics implications of top screening drugs are also shown in the supplementary table.

To validate the conformational behavior of the best screened BSAAs bound with respective target protein molecule, GROMACS package (Pazel, 1989) was used for all atom MD simulations for 100 ns. We have taken following protein-drug conjugates; spike-Suramin, RdRp-glycyrrhizin, M pro -Imatinib and helicase-Suramin along with their apo state to measure the conformational changes through comparative analysis. 

The stability of protein is the description of all the net forces to determine whether the protein will remain in folded state or assume non-native congregating structures. Therefore, the stability of protein is important to study the function of protein as alteration in protein stability would lead to misfolding or degradation of protein. Root Mean Square Deviation (RMSD) is the measure of conformational difference of the protein backbone from start to end state or comparison between the two poses of same molecule. In spike protein apo protein RMSD staring from $0nm stabilize between $0.2-$0.4 nm till 40 ns with little increase in stability to $0.35 and showed stability between $0.35 to$0.4 for the rest of simulation experiment. In spike-Suramin complex an increased RMSD was observed from $0nm to $0.4 in the first 40 ns of the simulations and stabilize between $0.35 to $0.4 for the remaining simulations experiments ( Figure 5A ). In RdRp apo and bound state both the systems showed RMSD variation in the first 20 ns of simulations and get stabilize between $0.2 to $0.3 throughout the simulations ( Figure 5B ). RMSD measures of Mpro apo and bound state (Mpro-Imatinib) showed stability in the RMSD value from start of simulations till 40 ns. The system showed increase in RMSD value from $0.2-$0.35 nm between 45 ns-55ns and for rest of simulations system get stabilized ( Figure 5C ). RMSD measure of helicase apo and bound state showed increased RMSD value in the start of simulation and different behavior throughout experiment. Helicase apo starting from $0nm stabilize between $0.2-$0.25 nm till 20 ns and showed increased RMSD of $0.25 nm between 30-40ns and get stabilize rest of the time. In helicase bound state (helicase-Suramin complex) the value of RMSD increased in the beginning form $0nm to $0.3 nm and get stabilize between $0.2-$0.25 nm for 50 ns of simulations and increased stability was monitored between 0.25 to 0.4 nm for rest of the simulations ( Figure 5D ). Binding pocket RMSD of each target molecule Spike, RdRp, Mpro and helicase is calculated as shown in Table  S3 . Root mean square fluctuation (RMSF) plot of spike, RdRp, M pro and helicase showed overall stability in the region encompassing the binding cavity ( Figure 6A-D) .

Conformational adjustments of spike, RdRp, m pro and helicase upon drug binding

Structural changes were captured for the spike protein at different time intervals upon binding of Suramin compound. A loop region in apo state (residue range: Ser 366 -Tyr 369 ) was converted to b-sheet in bound state of spike protein RBD region. Gly 431 -Asn 437 a b-sheet in apo form was converted to loop in bound sate. Tyr 508 and Arg 509 the part of b-sheet in apo form was changed to loop in bound sate of spike protein. Leu 546 -Val 551 was extended b-sheet observed in bound sate and Ser 325 -Arg 328 , b-sheets shortening was monitored in bound sate in comparison to apo state of spike protein. The Suramin bound spike protein showed significant fluctuation to aid in binding (Figure 7 ). Significant conformational adjustments were observed in and around the RBD of spike protein at secondary structure and residue level that are main determinant of interactions. The analysis of spike protein conformational adjustments upon binding of small molecule inhibitor suggests that it might reduce chances of binding with host receptor and will not trigger the signaling cascade for viral infection.

The dynamic trajectories of apo and drug bound state of RdRp enzyme revealed significant conformational changes. In unbound state there are 31 alpha helices of RdRp and loss of two helices were observed at Asp 269 -Leu 271 and Thr 739 -Ala 747 in bound form of RdRp. Apart from these alterations in helix length was also observed throughout the protein. Overall, the topology of b-sheet showed minor fluctuation with increase in sheet length at few places (residue range: Phe 753 -Leu 758 , Asp 761 , Phe 766 ) (Figure 8 ). Most of the conformational changes were observed in the palm, thumb, and finger motifs that are main contributor of RdRp active site for drug binding. So, these predictions conclude that binding of BSAAs in the active site will not allow the binding of RNA and will stop RNA synthesis. These results are consistent with Weisberg et al. (2020) and Al-Kamel and Grundmann (2021) . Moreover, the docking analysis of shortlisted BSAAs and RNA with RdRp revealed strong binding of drugs with binding free energy of -12kcal/mol and À8.8 kcal/mol as compared to À7.8 kcal/mol binding free energy of RMA molecule ( Figure S1 ).

Comparative analysis of the trajectories generated at different ns (M pro -Imatinib) with apo state of M pro showed important structural twists. A short b-sheet in apo state was extended four residues long sheet from Thr 111 -Tyr 118 . Another short b-sheet in apo state is extended by five residues long sheet from Ser 121 to Ala 129 in bound state. A loop region in the apo state is converted to b-sheet comprising of residues ranging from Val 157 -Glu 166 . A loop region in unbound state encompassing Arg 40 -Ile 43 was converted to an extended helix in the bound sate of M pro . Asp 187 -Gln 189 a loop region at the cavity mouth was moved upward to widen the cavity opening. His 41 , Tyr 54 , Asn 142 and Cys 145 a loop region was tilted towards the binding cavity to make covalent interactions with the Imatinib (Figure 9 ).

Comparative analysis of the trajectories generated at different ns with helicase apo and helicase bound (helicase- Suramin) state showed significant structural turns. Some secondary structure elements were built and while the few loss of regular secondary structure was observed in bound state in comparison to unbound form. From Val 60 -Leu 65 a loop region a loop region in apo state was converted to short b-sheet and up to nine residues long helix was created form Ala 117 -Thr 125 as compared to loop region in the apo state. Two short helices with inter loop region of seven residues was converted to one long helix from Asp 260 -Gly 273 in bound state. Helix6 was extended form His 290 to Gly 294 in bound state as compared to apo state. A loop at the cavity opening encompassing Asp204-Asp207 was extended to open the mouth of cavity. Asn 179 , Asn 516 and Arg 560 showed interesting conformational switches towards the cavity to aid in hydrogen bonding with drug molecule (Figure 10 ).

After molecular dynamics simulations trajectories for all the protein ligand complexes from last 10 ns were extracted to calculate the binding free energy MM-PBSA. While the Suramin bound with spike protein depicted the binding energy of À12.63 kcal/mol, similarly the MM-PBSA energy calculation of Glycyrrhizin in complex with RdRp enzyme revealed relatively higher binding free energy of À13.54 kcal/ mol. Imatinib in complex with M pro showed binding energy of À11.25 kcal/mol, while Suramin bound to helicase showed binding free energy of À9.28 kcal/mol (Table 3) .

The COVID-19 disease caused by the SARS-CoV-2 virus has disrupted modern global infrastructure. The development of rapid and effective antiviral agents is remarkably challenging due to evident cost and time-consuming nature. In this era of COVID-19 mostly public discussion is centered on vaccine, but vaccine use may not defeat the virus completely especially as mutant forms emerge which are not responsive to the vaccines (https://www.ft.com/content/c2aa5ea4-66b9-4f64-9e74-7c89c12f9461), as well as vaccine distribution challenges. Drug repurposing is an alternate approach to expedite the identification of potential drugs for rapid management of emerging and remerging infections and offer an immediate integration into routine clinical practice. The present study is designed for expeditious screening and identification of BSAAs against spike, RdRp, M pro and helicase of the SARS-CoV-2 to combat the diseases at various stages. Initial screening showed that Imatinib, Suramine, Glycyrrhizin and Bromocriptine have affinity towards multiple targets. Remdesivir and Azithromycin already approved by FDA and NIH for COVID-19 treatment also came up among the top ten high binding affinity drugs for RdRp that further strengthen our strategy. Suramin is approved antiprotozoal drug with broad spectrum antiviral activity against Zika virus, Ebola virus, Hepatitis C virus and Human influenza virus. Suramin also inhibits the binding of dengue virus to host cells through a direct effect on the viral envelope protein (Chen et al., 1997) . Inhibition of host cell attachment was also found for herpes simplex (Aguilar et al., 1999) and hepatitis C (Garson et al., 1999) viruses, which explained the previously reported protective effects of Suramin against in vitro herpes simplex virus infections (Alarc on et al., 1984) and in vivo infections of ducks with duck hepatitis B virus (Offensperger et al., 1993) . Imatinib is an approved anticancer drug also known as ABL1-Tyrosine kinase inhibitor of Abelson murine leukemia virus oncogene 1 (ABL1) pathway which is critical for many viral replications. In vitro studies confirmed the role of Imatinib activity against MERS-CoV and SARS-CoV by inhibiting the kinase signaling pathway which is important for replication (Dyall et al., 2014) . Imatinib have the potential to become broad-spectrum antivirals for the treatment of SARS-CoV-2 as a number of clinical trials demonstrated that Imatinib will interfere with release or replication of SARS-CoV-2 and would have significant impact on COVID-19 patients in intensive care units (https://clinicaltrials. gov/ct2/show/NCT04394416). Imatinib showed affinity towards all targets (Spike, RdRp, M pro and Helicase), and most preferable residues were Asn, Asp, Arg and Thr. Glycyrrhizin was reported as the most active drug inhibiting replication of the SARS-CoV (Numazaki, 2003) and can be repurposed to stop SARS-CoV-2 replication. Bromocriptine is dopamine receptor agonist and can be used for the treatment of diabetes (DeFronzo, 2011) . Fumihiro Kato in 2016 and Chan JF et al in 2017 reported the antiviral activity of Bromocriptine against dengue virus and zika virus, respectively (Chan et al., 2017; Kato et al., 2016) . Instead of individual drug, combination of drugs against viruses could serve as front line therapeutics for newly emerging viruses (SARS-CoV-2) and remerging viruses (Bibi et al., 2020; Shyr et al., 2020) . Therefore, the cocktail of antiviral drugs from the present study with explicit antiviral effect can be used for various stages of SARS-CoV-2 infection. Due to the lack of experimental support, there are limitations in validating these in silico proposed work. However, the data provide support for experimental validation. Combination of drugs that can attack the virus differently through different mechanism can be an optimum approach.

In conclusion BSAAs revealed to have potential against SARAS-CoV-2 as antiviral agents. Particularly, Imatinib, Glycyrrhizin Bromocriptine, Suramin revealed good affinity towards multiple targets. MD simulations of shortlisted high biding affinity drugs against Spike, RdRp, Helicase and M pro revealed a formation of stable complex throughout the simulations. RMSD and RMSF analysis revealed that the interaction of shortlisted BSAAs with respective target were very stabilizing. Therefore, BSAAs could offer significant clinical benefit to decrease the burden of COVID-19 illness. These molecules can either be used as main drug or combination as multitargeted therapy against SARS-CoV-2.

Due to the speedy outburst of the SARS-CoV-2 and high mortality rate, there is burning needs to control this highly transmissible disease either by novel therapeutic development or repurposing the existing antivirals to inhibit this virus are essential. Throughout the world several clinical trials are running by targeting structural and/or nonstructural proteins of SARS-CoV-2 to find efficient drug. The future goal of this study is to complete clinical trial of shortlisted BSAAs and find the most effective antiviral drugs for SARS-CoV-2 infection.

The schematic figure was created in BioRender.com.

NB: conceived and designed the experiments, performed experiments and manuscript writing, AF and SG: made a significant contribution by performing virtual screening experiments. FA, JA, UK and TH: made a substantial contribution in revising the manuscript for intellectual content.

No potential conflict of interest was reported by the authors. 

The data supporting the findings of the article are available in the Protein Data Bank (PDB) at http://www.rcsb.org. The data needed to evaluate the article is present in the article and supplementary material.

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The deadly coronaviruses: The 2003 SARS pandemic and the 2020 novel coronavirus epidemic in China

Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin