Revealing SARS-CoV-2 Functional Druggability Through Multi-Target Cadd Screening of Repurposable Drugs

: The emergence of SARS/MERS drug resistant COVID-19 with high transmission and mortality has recently been declared a deadly pandemic causing economic chaos and significant health problems. Like all coronaviruses, SARS-CoV-2 is a large virus that has many druggable components within its proteome. In this study, we focused on repurposing approved and investigational drugs by identifying potential drugs that are predicted to effectively inhibit critical enzymes within SARS-CoV-2. We shortlisted seven target proteins with enzymatic activities known to be essential at different stages of the virus life cycle. For virtual screening, the energy minimization of a crystal structure or modeled protein was carried out using Protein Preparation Wizard (Schrödinger LLC, 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs (n=5903) that are already approved by worldwide regulatory bodies including the FDA, using the ZINC database. Screening was performed against viral targets using three sequential docking modes (i.e. HTVS, SP and XP). Our in-silico virtual screening identified ~290 potential drugs based on the criteria of energy, docking parameters, ligand and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. A top lead from each target group was further subjected to molecular dynamics simulation (MDS) using the Desmond module to validate the efficacy of the screening pipeline. All of the simulated hit-target complexes were predicted to strongly interact and with highly stable binding. Thus, we have identified a number of approved and investigational drugs with high likelihood of inhibiting a variety of key SARS-CoV-2 proteins. Follow-up studies will continue to identify inhibitors suitable for combination therapy based on drug-drug synergy to thwart resistance. In addition, the screening hits that we have identified provide excellent probes for understanding the binding properties of the active sites of all seven targets, further enabling us to derive consensus molecules through computer-aided drug design (CADD). While infections are expanding at a rampant pace, it must be recognized that resistance will grow commensurately through either genetic shift and/or genetic drift to all small molecule drugs identified. Vaccines should provide a more permanent solution through prevention, but resistivity is still a possible scenario. Nevertheless, a persistent multi-target drug development program is essential to curb this ongoing pandemic and to keep reemergence in check. both 2+ Mn 2+ conserved the for the of various Other that doses of some and hepatic and restenosis . NADH dianion is a that arises the is that Guanine- N 7 methyltransferase (ExoN) Hits. Diosmin is a flavone glycoside that is mostly found in citrus fruits. It is a non-prescribed dietary supplement that is primarily used for venous diseases, though there is limited clinical data to support its efficacy. Common adverse effects include mild gastrointestinal disturbances and cardiac arrhythmias 115 . Interestingly other studies have reported diosmin to be a coronavirus main protease inhibitor. Hidrosmin is a synthetic bioflavonoid that is used to treat chronic venous insufficiency of the lower limbs 116 . A crude preparation of Diosmin and Hidrosmin has been determined to be highly effective against Covid-19 117 . N -Desmethyl-4-hydroxy tamoxifen beta-D-glucuronide (E/Z Mixture) has also been identified a strongly binding ligand. Tamoxifen has also been proposed as an anti-COVID19 drug as it can induce autophagy associated with the unfolded protein to kill infected cells and thus the its binding activity established viral helicase as an important target in COVID-19. We found several small molecule compounds including inosine, 5-F-UMP, fenoterol, didanosine and doxifluridine as potential helicase inhibitors. While Inosine, 5-F-UMP and Fenoterol have high affinity towards ATP-Nucleic acid interface, our top target against Helicase is inosine

Currently, there is no approved treatment for COVID-19 and there is a critical need to identify effective agents against it. The discovery and development of novel compounds that specifically target SARS-CoV-2 will require an extended period of preclinical testing before they can enter clinical trials. Due to limited time, there is an urgent need for faster treatment options. One approach is through screening already approved drugs in libraries that could be repurposed for SARS-CoV-2. In this study, we have used a multipronged drug discovery approach through advanced screening that is in line with the World Health Organization's (WHO) guidance to repurpose approved drugs with demonstrated acceptable safety profiles. As potential new treatments are identified, mechanistic and clinical studies can be immediately performed to determine the efficacy of such repurposed drugs.

RATIONAL STUDY DESIGN.
Enzymes generally have binding sites that recognize small molecules and thus, they are comparatively more druggable than non-enzymatic proteins. We have selected seven essential coronavirus enzymes as targets, namely 3CLPro, PlPro, RdRP, Helicase, NandoU, ExoN and 2O-MT, and subjected them to virtual screenings. Details of these targets are described in the next section below. The 3D structures were accessed from among the available PDB X-ray crystal structures as well as from predicted structures available from the I-Tasser server (https://zhanglab.ccmb.med.umich.edu/COVID-19/). The energy minimization and accompanying relaxation of crystal structures or of modeled proteins was carried out using the Protein Preparation Wizard followed by a short 20ns MD simulation (Schrödinger LLC, 2020-1). Following active site selection based on data mining and COACH predictions, we performed high-throughput virtual screening (HTVS) of compounds (n=5903) approved by worldwide any regulatory bodies including the FDA secured from the Zinc database (zinc.docking.org). The screening was performed using the Virtual Screening Wizard (Schrödinger, 2020-1) consisting of three sequential docking modes ( HTVS, SP, and XP). Preliminary in-silico virtual screening identified ~290 potential drugs based on criteria including energy, docking parameters, ligand and binding site strain energies, and fit score. Compounds specific to each target protein were further analyzed for binding free energy perturbation by the molecular mechanics' method using Prime MM-GBSA, followed by refining the hits to the best 32 drugs. A topscoring lead from each target group was further subjected to a molecular dynamic simulation (MDS) using the Desmond module (Schrödinger LLC, 2020-1) to validate the screening pipeline; Overall Study Design(Fig1). Table 1. illustrates drugs targeting SARS-CoV-2 specific structures, and Table 2 lists drugs targeting the host proteins.  Table 2. Repurposable drugs from global approved drug libraries that have known interactions with host proteins/pathways. These drugs were selected from literature showing applications against the coronavirus family. Baricitinib JAK kinase Non-receptor tyrosine kinase that transduces cytokine-mediated signals 39 Chloroquine Endosome/Angiotensin -Converting Enzyme-2 (ACE2) Viral receptor protein on host cells which binds to viral spike protein 40 Umifenovir (Arbidol) ACE2 Viral receptor protein on host cells which binds to viral spike protein 37 Camostat mesylate TMPRSS2 (transmembrane protease, serine 2) Protease produced by host cells that facilitates the binding of the spike protein to ACE2 41 L-163491 AT2 (angiotensin AT2 receptor) Effector involved in blood pressure and volume regulation of the cardiovascular system C-21 (CAS 477775-14-7) AT2 Effector involved in blood pressure and volume regulation of the cardiovascular system 42 CGP-42112A AT2 Effector involved in blood pressure and volume regulation of the cardiovascular system 42 Drug Repurposing for COVID-19: While drugs have initially been produced for use against a specific target and disease, drug repurposing offers a new and faster approach to initiate research-based methodologies. The utility of protein modeling and molecular docking has shown that approved drugs specified for certain uses can have a significant impact on other diseases 43 . For example, loperamide is an approved drug for controlling acute and chronic diarrhea that has exhibited inhibition of the MERS-CoV replication cycle. A number of studies are currently in progress exploring the use of antiviral drugs that were approved for influenza, hepatitis C virus, and human immunodeficiency virus (HIV) 1 against COVID-19, though with limited efficacy as well as at least in some cases added morbidity due to serious side effects. When a drug is repurposed with efficacy and safety demonstrated for other diseases, the timeline to availability to the patient population is reduced, the cost of production is lower, and the distribution channels are already in place. So far, no drugs have been approved specifically for COVID-19, although several studies are underway with the goal of repurposing drugs with other indications as summarized herein. On March 18, 2020, the WHO launched a multinational clinical trial investigating a number of drugs in clinical trials to evaluate their efficacy against COVID-19, but these trials remain inconclusive 44 . One example of repurposing is the use of hydroxychloroquine (H-CQ), an antimalarial repurposed for COVID-19, that is showing mixed results, with the FDA recently (April 24 th ) issuing warning after drug-induced deaths. Other drugs or combinations of drugs that are or will be tested include remdesivir, combinations of lopinavir and ritonavir, and lopinavir, ritonavir and interferon beta, and chloroquine or hydroxychloroquine. These treatments regimens will be evaluated relative to appropriate controls, which in this case, refers to standard care including respiratory support as required.
Recent reports have suggested that Remdesivir may be efficacious in treating COVID-19, including a study that showed that this antiviral drug successfully inhibited MERS-CoV replication in monkey models 45 .
Remdesivir is a mono-phosphoramidate adenosine nucleotide analog, which is metabolized to its active form, GS-441524. This active form interferes with the functioning of viral RNA polymerase and evades proofreading by viral exoribonuclease leading to decrease in viral RNA production 26 . Remdesivir was first showcased as a promising antiviral against Ebola when it showed successful activity against Ebola variants in cell based assays and in a rhesus monkey model 31 . Remdesivir was then taken into various clinical trials with little to no success in comparison to other candidates that proved to be more effective 46 . This raises the question whether it will again prove to be ineffective in the clinical setting, after demonstrated success in the laboratory settings, but initial results are promising. Lopinavir-Ritonavir combinations sold as Kaletra or Aluvia by AbbVie are also under evaluation for the treatment of COVID-19. Lopinavir is an HIV-1 aspartate protease inhibitor. Ritonavir is a potent inhibitor of cytochrome P450 3A4 and is combined with lopinavir to increase lopinavir's plasma half-life through diminishing its metabolism 47 . On March 18, 2020, a study published in the New England Journal of Medicine detailed a clinical trial using lopinavir-ritonavir in adult patients with severe COVID-19 47 . The study concluded that the lopinavir-ritonavir combination did not improve survival or recovery time, and instead showed severe adverse effects in patients. However, the authors noted that more severe cases of COVID-19 were enrolled in this study than in some other clinical trials. Therefore, it is prudent to wait for definitive clinical results evaluating the efficacy and safety of lopinavir-ritonavir versus COVID-19. Lastly, chloroquine is a medication primarily used in the treatment of malaria that belongs to the 4aminoquinoline drug class. In vitro antiviral activity of chloroquine has been known since 1969, although through the mechanism for this activity remains undetermined 48 . More recent studies suggest that chloroquine may block viral infection by increasing the endosomal pH required for viral-host cell fusion, further, this drug has been shown to interfere with the glycosylation of cellular receptors 40 . Chloroquine has been widely used in human patients for a variety of other indications with a fairly safe track record. The question remains whether this treatment will prove to be efficacious and safe for COVID-19 patients in a clinically robust trial, although cardiac liabilities may prove to be limiting. Beyond the drugs just mentioned, there are various other candidates in development or being tested with unknown mechanisms of action in SARS-CoV-2 specifically. Identifying SARS-CoV-2 essential proteins as targets for repurposable drugs: Out of many proteins (~29) known to be produced by the virus, there are several critical non-structural proteins in SARS-CoV-2 that may be valuable targets for antiviral drugs. In the coronaviridae family, a replicase is used to translate most of the viral genomic RNA to synthesize two replicase polyproteins, pp1a and pp1ab. These two polyproteins are processed by two proteases, (1) Papain-Like Protease (PLpro) and (2), coronavirus 3-Chymotrypsin-Like Protease (3CLpro), generating 16 nonstructural proteins [49][50][51] . This proteolytic processing is essential for generating functional replication complexes 52 . As such, both PLpro and 3CLpro are promising antiviral targets and have already shown promise against COVID-19 in the drug combination lopinavir-ritonavir. PLpro has a core catalytic domain containing 316 amino acids. This protease cleaves the N-terminal region of the polyprotein to generate three different nonstructural proteins (1/2/3). It is also suggested that PLpro may have de-ubiquitinating activity, due to its structural similarities with cellular deubiquitinating enzymes 53 . The enzyme 3CLpro contains a cysteine-histidine dimer within its active site that directs proteolytic activity. This protease has the ability to cleave 11 different sites of the replicase polyprotein to produce a mature protein that anchors replication/transcription complexes and releases mature NSPs. Structural analyses and computational screening with 3CLpro as a target have shown promising results for drug candidates against SARS-CoV 35,36 .
(3) Viral non-structural protein (nsp) 14: Nsp14 has been implicated in SARS-CoV-2 as possessing two different activities: an exoribonuclease (ExoN) activity acting on both ssRNA and dsRNA in a 3' to 5' direction, and an N7-guanine methyltransferase activity (N7-MTase) 54 60 . Although there are no reports as of yet revealing drugs that successfully target guanine-N7 methyltransferase (ExoN) for treatment of SARS-CoV or MERS-CoV, given that ExoN is important in coronaviruses for viral RNA synthesis and replication fidelity as well as for avoiding recognition of CoV RNA by the host, we feel it is a promising drug target.
(4) Nonstructural uridylate-specific endoribonuclease (NendoU) Nsp-15 activity is found in the Nterminal domain. This active site has been shown in MERS-CoV and SARS-CoV and is suggested to be a genetic marker common to coronaviruses. MERS-CoV Nsp16 appears to display unique features compared to its homologs. Nsp7/Nsp8 display higher binding affinity for Nsp15, also affecting enzymatic activity. Nsp15 from SARS-CoV appears to be an inhibitor of mitochondrial antiviral signaling adaptor, inducing apoptosis. Nsp15 activity is stimulated by manganese ions (Mn2+), and the enzymes generate 2'-3' cyclic phosphate ends 61 . Nsp15 functions as a homohexamer, although the enzyme has some activity as a monomer 62 . The structures of MERS-Nsp15 and SARS-Nsp15 have been superimposed showing high homology 63 . Previous studies established that Nsp15 from both SARS-CoV and MHV can be stimulated by Mn 2+ 64 . The activity of MERS-Nsp15 increased with the addition of Mn 2+ , suggested by improved RNA binding affinity. The spatial arrangements revealed that residues S290 and Y339 in MERS-Nsp15 correspond to residues S293 and Y342 in SARS-Nsp15, which are postulated to interact with the substrate and confer uridylate specificity 64 , suggesting that there is conserved recognition for uridylate. 63 76 . RdRp is an established drug target for the treatment of SARS-CoV due to its role in viral RNA replication and its importance for SARs-CoV pathogenesis. We are particularly interested in targeting RdRp since it has been suggested to be targeted by hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasir) for inhibition of coronaviruses.

Target Proteins(Receptors) preparation. Protein preparation
was performed using the Protein Preparation Wizard in Maestro (Schrödinger, L. L. C. "Schrödinger Release 2020-1." (2020).). All co-crystallized atoms including calcium and chlorine were deleted. Crystal ligands were not deleted because they were used for grid generation. Each of the seven models developed were optimized and then minimized using the OPLS3e force field (Schrödinger-LLC 2020-1." (2020). Further the models were subjected to a 20 ns MD simulation (MDS). The target complex with trans-isomers was subjected to MDS and trajectory analysis was conducted using the Desmond software (Schrödinger-LLC 2020-1). The full system was prepared by using Maestro's Protein Preparation Wizard. The system was solvated in TIP3P water models with 0.15 M NaCl and placed in physiological solution. A simulation box covering the entire enzyme system was introduced with a 10 Å buffer space. The simulation was run for 20 ns at 300 K and standard pressure (1.01325 bar). OPLS-AA 2005 force field parameters were elected for utilization during this preparation and all later simulations.

Target library preparation.
A virtual library of n=5903 drug compounds was downloaded from the Zinc database (http://zinc.docking.org/substances) under the 'world' subset (approved drugs in major jurisdictions, including the FDA, i.e DrugBank approved). The database was then checked for redundancy, and duplicates were removed. Ligand preparation was performed using LigPrep, which generated variations of the ligands, eliminated reactive species and optimized the ligands. Optimization was performed under the OPLS3e force field. EPIK minimization was performed on possible states at pH 7.0 ± 2.0. Tautomers were generated for each ligand retaining specific chirality combinations with a maximum of 32 structures per ligand (Schrödinger, L. L. C. 2020-1).
Receptor grid generation. COACH analysis was performed to determine the location and size of the active site 78 . The shape and properties of the receptor are represented on a grid to ensure that possible active compounds are not missed. The centroid of the COACH predicted binding pocket residues and was used to generate grids using default values of protein atom scaling (1.0 Å) within a cubic box. The force field employed for grid generation was OPLS3e 79 .

High-Throughput Virtual Screening (HTVS).
The screening was performed with default parameters including ionization states, Epik state penalties within the Glide (Grid-based ligand docking from energetics) module of Schrödinger suite 80 . The scaling factor was maintained at a default of 0.8 and a partial charge cut-off was limited to 0.15. The OPLS3e force field was used during the docking process. The HTVS ligand docking was the first to be performed, followed by SP and XP docking on the top 10% of scoring hits from each previous step. The XP docking aids in removing false positives, and the scoring function is much stricter than the HTVS. The greater the XP Glide score, the better calculated affinity of the hit in binding to the protein target. Further, the estimation of free binding energies for the best hitdocked complexes using MM force fields and implicit solvation was performed using the molecular mechanics/generalized Born surface area (MM-GBSA) method within virtual screening workflow of Schrödinger suite 2020-1. The binding energy was calculated based on the following equation.
The leads were ranked on the basis of binding free energy calculation for their respective protein-ligand complexes.

Molecular Dynamics Simulation (MDS).
The top hit for each target was subjected to a 20 ns MD simulation (100 ns for XAV-939 + 3CLPro) as described above in the Target/receptor Preparation section to validate the interaction and thereby the HTVS pipeline performance.

RESULTS AND DISCUSSION
Following in-silico HTVS, molecular modeling, MDS, and utilizing resources from the literature, we identified a series of potential leads molecules from the repurposable drug libraries against specific proteins that are critical for the ability of SARS-CoV-2 to infect and reproduce within the host cell. Additionally, all of the simulated hit-target complexes were found to be strongly interacting and exhibited highly stable binding indicating the potential of being used for treating COVID-19. Outputs from these analyses are presented in tables and graphically, and are discussed in the following sections.

2'-O-Methyltransferase (2'-O-MT) hits.
Maltotetraose is an oligosaccharide of four units of alpha-Dglucopyranose linked by alpha-(1-4) bonds. This is a sugar variant of a substance that is found to inhibit the growth and reproduction of bacteria 81,82 . Crocin/Crocetin are the major bioactive ingredients of saffron.
There have been several studies that have shown the various efficacies of these drugs, including as neurotropic and chemotherapeutic agents 83,84 . Crocin/Crocetin are popular agents to be tested in clinical settings due to their anti-oxidative properties 85 . Paclitaxel is a diterpene alkaloid natural product and belongs to a family of drugs that target tubulin leading to an abnormality of the mitotic spindle assembly, chromosome segregation, and consequently defects of cell division 86 . Paclitaxel is one of the most widely used anticancer drugs for the treatment of various cancers 87 . Other studies have shown that low doses of paclitaxel show promise in treating some non-cancer diseases including renal and hepatic fibrosis and artery restenosis [88][89][90] . NADH dianion is a species of NADH that arises from the deprotonation of the two diphosphate OH groups. Iohexol is a compound most well-known as a nonionic, water-soluble radiographic contrast medium used especially for renal disease determination. This compound is absorbed from cerebrospinal fluid into the bloodstream and is eliminated by renal excretion. Heparin is a very interesting hit as it has been reported in multiple studies to increase the liklihood of survival of terminal COVID-19 patients 91,92 . By mechanism that is not well understood but involves heparin's activity of reducing hypoxia as well via inhibiting the cytokine storm 93 . There is a minor report suggesting that hepcidin hormone mimics the spike protein of Covid-19, 94 and heparin is known to interfere with hepcidin 95 . This suggests that heparin's anti-COVID activity may present a multifaceted therapy option. MMGBSA re-ranking brought this molecule to the bottom despite a high glide energy score due to penalties exacted due to extra-active site exposure of the bulky compound.  97 . Interestingly, the highest scoring among the aminoglycoside hits is Paromomycin which is an antiparasitic used to treat amoebiasis, visceral leishmaniasis and cryptosporidiosis in immunocompromised patients. With a potential to treat pulmonary tuberculosis as well, the range of indications of this drug is indeed very wide and if found effective against COVID-19, it could prove to be a valuable addition to the arsenal of combination therapies.
Lactulose is a synthetic disaccharide of galactose and fructose which can be produced by the isomerization of lactose. This compound has been used for treating bacterial infections, constipation, and cancer. An important note on lactulose is that it is not hydrolyzed by mammalian enzymes, therefore, ingested lactulose passes through the stomach and small intestine without degradation 98 . Framycetin is an aminoglycoside antibiotic isolated from Streptomyces lavendulae that shows broad-spectrum antibacterial activity. This drug has been used as a therapeutic against a variety of cancers 99,100 . Amikacin is an aminoglycoside antibiotic that is on the WHO list of essential medicines. It is a prokaryotic translation inhibitor that binds to the 16S ribosomal subunit. Bekanamycin is another aminoglycoside that inhibits prokaryotic translation by binding to the highly conserved A site of 16S rRNA in the 30S ribosomal subunit. This compound has the lowest antibacterial activity of the aminoglycosides in clinical use and manifests a moderate level of toxicity, therefore, it is no longer used as a first line antibiotic 101 . Lividomycin A is an aminoglycoside that shows antibiotic activity against several Gram +/-bacteria by inhibiting protein synthesis 102 . Lapatinib ditosylate is the salt of Lapatinib, a synthetic quinazoline that blocks the phosphorylation of various epidermal growth factor receptors and inhibits cyclin D protein levels in human tumor xenografts and cell lines 103,104 . Uridylate-specific endoribonuclease (NendoU) Hits. Daidzin is an isoflavone natural product found in several Legumimosae such as the Japanese Kudzu root and is the 7-O-glucoside of the isoflavone daidzein.
It is shown to have anticancer and antiallergenic activities 105 . Diadzin and Haloperidol have been identified in multiple virtual screenings 106,107 . SCHEMBL24383 (Active principle of Strychnos potatorum Linn. Seed extract) has been extensively used to combat respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and bronchitis. Interestingly, given its predicted anti-Covid-19 activity, it may result in a dual therapeutic potential drug. Metrizamide is a non-ionic iodine-based radiocontrast agent that is widely used in lumbar myelography. It is only found in individuals that have taken this compound 108 .
Haloperidol glucuronide is a metabolite of the commonly prescribed antipsychotic drug on the WHO's list of Essential Medicines. Haloperidol is used in the treatment of schizophrenia, mania in bipolar, delirium, and other neurological diseases 109,110 and is on the WHO's List of Essential Medicines. 4-Hydroxy phenytoin glucuronide is a metabolite of the widely used antiepileptic phenytoin. The adverse effects of phenytoin can range from moderate diseases like gingival hyperplasia to severe more effects including toxic epidermal necrolysis and teratogenic effects 111 . Similarly, acetaminophen O-β-D-glucose iduronate is a metabolite of acetaminophen generated in the liver by UDP-glucuronyltransferase. It is highly water soluble and is excreted through the kidneys. Acetaminophen was recommended by WHO in the case of COVID-19 due to a concern over ibuprofen being an ACE2 inhibitor that might increase viral entry 112 . The advisory was subsequently revised to state that both medications are appropriate 113 . A metabolite of acetaminophen could prove to be an added advantage of already being used as a drug. p-Aminophenyl-alpha-Dgalactopyranoside is an experimental phenolic glycoside that competitively binds to heat-labile enterotoxin B pentamers by mimicking host cell receptors (intracellular adenylyl cyclase) 114 . Guanine-N7 methyltransferase (ExoN) Hits. Diosmin is a flavone glycoside that is mostly found in citrus fruits. It is a non-prescribed dietary supplement that is primarily used for venous diseases, though there is limited clinical data to support its efficacy. Common adverse effects include mild gastrointestinal disturbances and cardiac arrhythmias 115 . Interestingly other studies have reported diosmin to be a coronavirus main protease inhibitor. Hidrosmin is a synthetic bioflavonoid that is used to treat chronic venous insufficiency of the lower limbs 116 . A crude preparation of Diosmin and Hidrosmin has been determined to be highly effective against Covid-19 117 . N-Desmethyl-4-hydroxy tamoxifen beta-Dglucuronide (E/Z Mixture) has also been identified a strongly binding ligand. Tamoxifen has also been proposed as an anti-COVID19 drug as it can induce autophagy associated with the unfolded protein response to kill infected cells and thus contain the virus 118 . Octane-1,3,5,7-tetracarboxylic acid B belongs to the class of organic compounds known as tetracarboxylic acids and derivatives classified under zinc ion binding compounds that target Carboxypeptidase A1. Bempedoic acid is a prodrug that is converted to its active form in the liver. It is an FDA approved treatment for hyper cholesterolemia and has few adverse effects. This compound inhibits adenosine triphosphate citrate lyase, an enzyme within the cholesterol biosynthesis pathway 119 . Helicase (hel) Hits. The FDA approved drug ivermectin recently showed significant anti-COVID-19 antiviral activity, suggesting its therapeutic potential 120 . While the target of ivermectin in COVID-19 is yet to be confirmed, it has been reported that the its antiviral activity in the case of flaviviruses, particularly Australian Kunjin virus (West nile virus variant) 121 is based on targeting viral helicases. While our screening did not show ivermectin as a top helicase inhibitor, its binding activity established viral helicase as an important target in COVID-19. We found several small molecule compounds including inosine, 5-F-UMP, fenoterol, didanosine and doxifluridine as potential helicase inhibitors. While Inosine, 5-F-UMP and Fenoterol have high affinity towards ATP-Nucleic acid interface, our top target against Helicase is inosine. Inosine is nucleoside commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. It has been tried in various clinical settings, most notably in multiple sclerosis 122 . 5-F-UMP is a pyrimidine ribonucleoside 5'-monophosphate having 5-fluorouracil as the pyrimidine component. It shows antineoplastic activity by being inorporated in RNA and it inhibits RNA processing, thereby inhibiting cell growth. Fenoterol is a β adrenoreceptor agonist that is used as an inhaled bronchodilator asthma medication 123 . Didanosine is particularly interesting as it is a reverse transcriptase (HIV) inhibitor 124 . Also, Didanosine has been profiled as a beneficial drug in the case of the COVID-19 type of lung fibrosis by matching single cell RNA sequencing data 125 . Inhibition of inosine-5'monophosphate dehydrogenase (IMPDH) has been shown to control COVID-19 replication 126 , which could be due to inosine accumulation. Didanosine is also a medication used to slow the progress of HIV/AIDS and is a nucleoside analogue of adenosine. There are several common adverse effects associated with this medication including diarrhea, vomiting, and peripheral neuropathy 127 . Fenoterol has already been recommended to reduce severe pulmonary symptoms of COVID-19 patients 128 . Doxifluridine has been suggested as a possible COVID-19 inhibitor based on similarities with active antivirals already tested by artificial intelligence 129 . Doxifluridine is a nucleoside analog prodrug that interferes with RNA transcription by competing with uridine triphosphate for incorporation into the RNA strand. It is used as a cytostatic agent in chemotherapy in several countries in Asia 130 .

Papain-like proteinase (PlPro)Hits.
Troxerutin is a naturally occurring flavonoid that has been reported to show promise as a vasoprotective agent by improving hepatic homeostasis 131 . As there have been many in silico drug screening efforts with the coronavirus Main Protease that include troxerutin as a potential inhibitor of 3CLPro 132 . Also, the famous 'Chai-Ling decoction' a Chinese medicine herbal formulation with reported protection from SARS, MERS and now COVID-19 has troxerutin as a major component 133 135 . Both of these compounds belong to the class of aminocyclitol glycosides similar to streptomycin. This class of compounds have been used previously to target RNA metabolism of viruses 136 . Hyaluronic acid is a non-sulphated glycosaminoglycan (GAG) and is composed of repeating polymeric disaccharides of D-glucoronic acid and N-acetyl-D-glucosamine linked by a glucuonidic β (1→3) bond 137 . It is a key molecule involved in skin moisture that has the capability to retain water. Monoxerutin is another flavonoid which has been patented to be used against Hepetitis C 138 .
Anastrozole is an aromatase inhibitor approved by the FDA to treat postmenopausal women diagnosed with  Main proteinase (3ClPro) Hits. XAV-939 is a beta-catenin signaling inhibitor that has been shown to have promise in treating prostate cancer 141 . XAV-939 is also an effective inhibitor of PARP and Wnt pathway. PARPs enhance IFNγ production and can halt viral infections 142,143 . Thus, a dual mode of action is expected of XAV-939 if it is found to inhibit the main protease as well. Crocin is carotenoid diester that is responsible for the color of saffron, and is popularly used in India as a treatment for arthritis and psychological disorders 144 . Loperamide is used to treat gastrointestinal symptoms including diarrhea with few side effects and is on the WHO's List of Essential Medicines. Isoquercetin is a flavonoid natural product that can be isolated from various plant species and has shown promise as an anti-cancer agent 145 . Danoprevir is a 15membered ring macrocyclic peptidomimetic inhibitor of the hepatitis C protease NS3/4A 146 . Other virtual drug screenings have reported danoprevir as a possible coronavirus main protease inhibitor 147 . In clinical trials, danoprevir had a positive effect on recovery and faster discharge of COVID-19 patients 148 .
Cefoperazone is a cephalosporin antibiotic. It is one of the few cephalosporin antibiotics effective in treating Pseudomonas bacterial infections 149 . In China most of the treatment regimens reported using cefoperazone to prevent secondary infections in COVID-19 patients 150,151 . Nevirapine is a non-nucleoside reversetranscriptase inhibitor 152 . This compound is FDA approved for use in adult patients infected with HIV-1. Nevirapine has also been revealed in various in-silico drug screening with the main protease 153,154 .
Pentostatin is a purine analog that is widely used as a treatment for hairy cell leukemia 155 . Similar to pentostatin, cladribine is also a therapy for hairy cell leukemia. And also like nevirapine, cladribine has also been predicted to block main protease 3CLPro in many reports 154,156 .
Approved drugs encompass major jurisdictions including the FDA approved drugs (i.e. DrugBank). Virtual screening was performed to investigate which compounds are predicted to have a strong binding affinity to key COVID-19 proteins. With proteins identified as potential targets for the World approved drugs/compound library, our study demostrated that, in principle that multiple repurposable drugs should be used to treat COVID-19 as targeted therapies. The identified drug molecules have been further summarized to better represent their properties where possible including dose, toxicity, pharmacodynamic data and combinatorial compatibility. These properties will assist clinicians in making a decision on whether they are suitable to test in treating COVID-19 patients and also help to determine if they are suitable for multi-drug therapy (MDT) regimen. Known Mechanism of Action of drugs from approved libraries. Understanding the mechanism of action for each repurposable drug is important in predicting if the compound will be capable of combating COVID-19. To enable identification of drugs suitable for combination therapies, we have assembled mechanisms of action for each drug from current indications and from the Drug Bank. This information in addition to our current findings will provide key guidance regarding which compounds are likely to show effectiveness in blocking target proteins and suppressing this new coronavirus. Table 10 presents the mechanism of action for the following repurposable drug compounds that were screened from the ZINC database.

Known drug-drug interactions of leads from approved libraries.
While understanding the mechanism of action and pharmacodynamics of each drug compound is important, combination therapy requires an understanding of potential drug-drug interactions. Listed in Table 10 is a compilation of the drugs screened from the ZINC library and their respective interactions with the other compounds. Each interaction listed exhibits the effect that the host drug has in terms of metabolism or other side effects that could take place. For the drug compounds that do not have other compounds listed, data was unavailable. The data was gathered from DrugBank 157 .
For the drugs mentioned in Table 10, the possibility of combination therapy offers benefits and as well has highlighting which drugs could have a negative effect when used with others. For hydroxychloroquine, a drug that has received attention as a potential therapeutic to COVID-19, we have gathered data of its effect in combination therapy with other compounds that have been screened. When used in combination with Paclitaxel and Doxifluridine, the risks associated with each compound can increase, potentially leading to complications or even death. The drugs in Table X show which compounds should benefit from combination therapy and which likely will not. Understanding the dynamics of combination therapy and the compounds listed will better help combat COVID-19 and to understand the drug compounds in use.

CONCLUSION
The rapid spread of SARS-CoV-2 continues to create havoc in health systems and economies, affected every nation in the world. As this is a novel coronavirus, there are no vaccines currently available, though they are being developed at an unprecedented pace and we must resort to small molecule therapeutics against COVID-19. There has been an international focus on the potential efficacy of repurposable drug candidates including remdesivir and hydroxychloroquine, among others. With each controlled clinical study that comes out, it becomes more evident that nations were too quick to push certain compounds, consequently reducing the supply to patients who needed them to treat other already indicated diseases. There is a possibility that these previously mentioned compounds may still have a place in the clinical realm as a treatment for COVID-19, but not as stand-alone therapies as currently utilized. It is clear that until a SARS-CoV-2 specific compound is developed and clinically approved, the best way is to find treatment through a multifaceted approach. This philosophy is at the forefront of our work, first by screening approved compounds for repurposing potential, and by identifying the best possible combinations providing a multifaceted attack on SARS-CoV-2. In this context, we commenced a CAAD through HTVS approach using large pool of world approved drug libraries to identify potential drugs for immediate deployment. Our insilico studies have identified a series of repurposable drugs that can be utilized in clinical trials. Our study also outlined the primary mechanism of action of these drugs which can further aid clinical trial designs to invistigate combinations of compounds that have individually failed to treat COVID-19. It is important to note that caution must be taken with some of these drugs, especially the anti-oncolytic and anti-psychotics that can have severe adverse effects in the context of their utility as antivirals. Also, special notice must be taken with compounds such as crocin, haloperidol glucuronide, doxifluridine, ivermectin, anastrozole, XAV-939, isoquercetin, and phenytoin, that these drugs would be expected to be administered in lower doses than what is normally administered for their original indications. We predict that these drugs will not suffice as candidates that may have stand-alone efficacy in treating COVID-19. Rather, these compounds should be tested in combinatorial studies as an additive to a drug with lower toxicity to the host as a potential treatment with synergistic efficacies. Application of our multi-pronged drug discovery approach is consistent with the WHO's desire to repurpose approved drugs that have acceptable safety profiles. Our study further highlights that target-based drug repurposing is a promising strategy, specifically in helping predict the best combination therapy to avoid the limitations of suboptimal performance by individual drugs. Furthermore, implementation of this approach should be an effective method to pre-select approved drug(s) for clinical testing and evaluation for clinical use. The advantage of using this approach relative to developing new drugs is a potentially vast savings of both cost and time. If any of these approved compounds are safe and effective in patients, then this may significantly alter the degree of infectivity of SARS-CoV-2 and/or the duration of COVID-19 illness in the coming months and years.   Fig 1. Schematic road map of the overall study design. The protein models from various protein bank and other sources were optimized and relaxed by MD simulations. The relaxed structures were then mapped for active site and used to generate GLIDE Grid for HT-virtual screen with world approved drug libraries.

Fig 2. Graphical illustration of SARS-CoV-2 life cycle along with target proteins used in HTVS. The infection cycle starts when SARS-CoV-2 Spike protein binds to the Human ACE2
receptor. An S1-induced post-stable S2 conformation allows either viral-host cell fusion (1a) or endocytosis (1b). Fusion directly allows the viral RNA to enter the host cell (2), but endocytosis require lysosomal degradation of coat and envelop for release of viral nucleocapsid in cytoplasm. The large viral script is known to encode 29 viral proteins (3), including the 7 essential nonstructural proteins that are selected as targets in our paper. A replicase is used to translate most of the viral genomic RNA to synthesize two replicase polyproteins, pp1a and pp1ab, and many small ORFs (4). The two major polyproteins are processed by two proteases, PLpro and 3CLpro (5), generating 16 nonstructural proteins. ExoN posesses a viral exoribonuclease activity that acts on both ssRNA and dsRNA in a 3' to 5' direction (9). Viral Helicase plays a critical role in viral replication by expediting appropriate folding (7). The enzyme 2'-O-MT methylates the viral 2' end which is important for the virion to avoid host recognition of their RNA (8). RdRp is involved in viral-host cell replication through catalyzing template synthesis of polynucleotides in the 5' to 3' direction (7). NandoU is a Mn 2+ dependent hexamer (dimer of trimer) enzyme with sparse functional information. The most prominent theory regarding NandoU is that the activity of this protein is responsible for protein interference with the innate immune system. For viral assembly of S, E and M proteins in the endoplasmic reticulum, along with the N protein are combined with the (+) gRNA to become a helical nucleoprotein complex. They assemble to form a virus particle in the endoplasmic reticulum-Golgi apparatus compartment, this particle is then excreted from cell through exocytosis within the smooth vesicles.     (Table 6) within the Guanine-N7 methyltransferase (ExoN) binding pocket (Schrödinger LLC_2020-1) depicting amino acid side chain residues colored according to their biochemical nature i.e. red = acidic, violet = basic, green = hydrophilic, and light blue = hydrophobic. The bold lines depict peptide backbone and red arrows depict molecular interactions.