Bisindolylmaleimide IX: A novel anti-SARS-CoV2 agent targeting viral main protease 3CLpro demonstrated by virtual screening and in vitro assays

The emergence of SARS/MERS drug-resistant SARS-CoV2 comes with higher rates of transmission and mortality. Like all coronaviruses, SARS-CoV-2 is a relatively large virus consisting of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs by identifying potential drugs that are predicted to effectively inhibit critical enzymes. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral multiplication cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2’-O-MT. For virtual screening, the energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard(Schrodinger 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 approved by worldwide regulatory bodies. The screening was performed against viral targets using three sequential docking modes (i.e. HTVS, SP, and XP). Our in-silico virtual screening identied ~290 potential drugs based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specic 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. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. Herein we report the evaluation of in-vitro ecacy of selected hit drug molecules on SARS-CoV-2 inhibition. Among eight molecules included in our evaluation, we found inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), as the potent inhibitor of SARS-CoV-2 in-vitro. Further, in-silico predicted target validation through enzymatic assays conrmed 3CLpro to be the target. Therefore, our data support advancing BIM IX for clinical evaluation as a potential treatment for COVID-19. This is the rst study that has showcased the possibility of using bisindolylmaleimide IX to treat COVID-19 through this pipeline. (A) an N-terminal Zn(II) binding domain and (B) a hinge domain and (C) a helicase domain 35 . is known of the viral non-structural proteins (nsp’s) 12 activity, but activity occurs on two domains, on the N-terminal subunit NiRAN, and bacteria by inhibiting protein synthesis 64 . 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 65,66 . glycogen BIM potent inhibition of PKC α, PKC βI, PKC βII, PKC γ, and PKC for 123 BIM IX in-silico series of repurposable drugs that can clinical trials. From our nine selected approved compounds that underwent in-vitro studies against SARS-CoV-2, found two compounds with high inhibitory activity. One of these was ivermectin, which showed signicant viral inhibition, conrming the results of a previous study 82 . Like previous reports, our in-vitro studies found that ivermectin has nonselective toxicity to the ATCC E6 Vero cells at ≤ 50 μM and 16.67 μM based on the number of nuclei counted. This shows that high doses (>16.67 μM) of ivermectin have high cytotoxicity as reported by other groups 129,130 , and this must be considered when preparing to use this drug in COVID-19 clinical trials as its currently approved dosage is very low (25mg) 131 . The second compound with signicant inhibitory activity against in-vitro SARS-CoV-2 was bisindolylmaleimide IX (BIM IX). Further, BIM IX spececaly blocked 3CLpro in vitro enzymatic assay our in-silico predictions. The most successful drugs against SARS and MERS have targeted this enzyme including repurposed Lopinavir and Ritonavir 132 Furthermore, PKC inhibition, anti-bronchitis, and anti-inammatory effects may with a multifaceted therapy option, while not validated ExoN inhibition 534, 554 & 560 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 137 . 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 eld was used during the docking process. The HTVS ligand docking was the rst 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 the calculated anity of the hit in binding to the protein target. Further, the estimation of free binding energies for the best hit-docked complexes using MM force elds and implicit solvation was performed using the molecular mechanics/generalized Born surface area (MM-GBSA) method within the virtual screening workow of Schrödinger suite 9 . The binding energy was calculated based on the following equation. and ExoN (Exonuclease) active site. Ivermectin was included as a positive control. Other shortlisted molecules included RdRP hits (paromomycin, amikacin, and lactulose), 3CLpro hits (iopromide & troxerutin), and one NendoU hit (haloperidol). In vitro studies on SARS-CoV-2 were performed at the United States Army Medical Research Institute for Infectious Diseases (USAMRIID). Both entry and spread assays were performed on ATCC Vero E6 cells infected with the SARS-CoV-2 virus. Entry Assay: Vero E6 cells, in 96 well plates, were pre-treated with 14 different test compounds starting at a concentration of 50 uM with 3 fold dilutions down to 0.76 nM for approximately 1hr at 37°C. SARS-CoV-2/MT020880 was then added to the test compound treated cells for 1 hr. at 37°C at an MOI of 0.4. After 1 hr., the cells were washed with PBS before adding additional compounds back in fresh culture media to the cells for 24 hr. at 37°C. Cell media was removed and washed with PBS and plates were submerged in the formal xing solution for 24 hrs., then permeabilized with 0.2% Triton-X for 10 min at RT and treated with blocking solution (3% BSA/PBS). Infected cells were detected using a primary detection antibody recognizing the SARS-CoV-2 nucleocapsid protein (Sino Biological, 40143-R001) and Alexa Fluor 488 conjugated antibody (goat α rabbit), then counterstained with DAPI for visualization of the cell nuclei. Infected cells were enumerated using Operetta high content imaging instrument and data analysis was performed using the Harmony software (Perkin Elmer). Spread Assay: A similar protocol was utilized to the entry assay described above with the following modications. The virus was used at an MOI of 0.02 and the assay was incubated for 48 hrs. rather than 24 hr. after infection of the cells.


Introduction
A novel coronavirus was rst reported in Wuhan, the capital city of the Hubei province in China in December of 2019 1 . This pathogen has been named SARS-CoV-2 2 and the disease caused by it is named, COVID-19 (Coronavirus Disease-2019).
The genome of coronaviruses consists of a single-stranded, positive-sense RNA, causing respiratory and enteric disease in mammals including humans. This family of viruses consists of a large genome, ranging from 28 to 32 kilobases 3 . Coronavirus family members are organized into three subsets based on antigenic and genetic facets: α-CoVs, β-CoVs, and γ-CoVs 4,5 . MERS-CoV, SARS-CoV, and SARS-CoV-2 are all β-coronaviruses, where both SARS-CoV and CoV-2 are derived from the lineage B and MERS-CoV is derived from the lineage C β-coronavirus 6 . Interestingly, these viruses have similar genomic structures with functional proteins encoded at the 5' end, and structural proteins encoded at the 3' end of the genome 7 . β-Coronaviruses have accessory proteins dispersed throughout their structural genes with both SARS-CoV and SARS-CoV-2 having seven different accessory proteins, while MERS-CoV has ve different accessory proteins 3,7 .
The discovery and development of novel compounds that speci cally 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 that could be repurposed for SARS-CoV-2. In this study, we have used a multi-pronged 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 pro les.

Rational Study Design
Enzymes generally have binding sites that recognize small molecules and thus, are comparatively more druggable than nonenzymatic proteins. We have selected seven essential coronavirus enzymes as potential targets, namely 3CLpro, PLpro, RdRP, Helicase, NendoU, ExoN, and 2O-MT, and subjected them to virtual screenings (Fig 2). Details of these targets are described in the following sections 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 8 . The energy minimizations and accompanying relaxations of 3D structure models (crystal structures/modeled proteins) were carried out using the Protein Preparation Wizard followed by a short 20ns MD simulation 9 . 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 regulatory bodies including the FDA secured from the Zinc database or were advanced investigational molecules 10 . The screening was performed using the Virtual Screening Wizard 9 consisting of three sequential docking modes (HTVS, SP, and XP). Preliminary in-silico virtual screening identi ed ~290 potential drugs based on criteria including energy, docking parameters, ligand and binding site strain energies, and t score. Compounds speci c to each target protein were further analyzed for binding free energy perturbation by the molecular mechanics' method using Prime MM-GBSA 9 , followed by re ning the hits to the best 32 lead candidates. A top-scoring lead from each target group was further subjected to a molecular dynamic simulation (MDS) using the Desmond module 9 to validate the screening pipeline (Overall Study Design; Fig1).
Drug Repurposing for COVID-19: While drugs have initially been produced for use against a speci c 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 speci ed for certain indication can have a signi cant impact on other diseases 11 . For example, loperamide is an approved drug for controlling acute and chronic diarrhea that has exhibited inhibition of the MERS-CoV replication cycle 12 . Several studies are currently in progress exploring the use of antiviral drugs that were approved for in uenza, hepatitis C virus, and human immunode ciency virus (HIV) 1 against COVID-19, though with limited e cacy as well as at least in some cases added morbidity due to serious side effects. When a drug is repurposed with e cacy 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.
Identifying SARS-CoV-2 proteins as essential 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 are valuable targets for antiviral drugs as illustrated below.
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 [13][14][15] . This proteolytic processing is essential for generating functional replication complexes 16 . As such, both PLpro and 3CLpro are promising antiviral targets and have already shown promise against COVID-19 in the drug combination therapy using 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). PLpro is also suggested to have de-ubiquitinating activity, due to its structural similarities with cellular deubiquitinating enzymes 17 (Fig 2). The enzyme 3CLpro contains a cysteine-histidine dimer within its active site that directs proteolytic activity. This protease can 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 18,19 .
(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) 20,21 . The activity of N7-MTase adds the N7-methyl guanosine cap during mRNA cap synthesis that is necessary for nsp16, activated by nsp10, to facilitate 2'-O-ribose methylation of the viral mRNA cap 22,23 26 . Although there are no reports as of yet revealing drugs that target guanine-N7 methyltransferase (ExoN) of SARS-CoV or MERS-CoV, given that ExoN is important in coronaviruses for viral RNA synthesis and replication delity as well as for avoiding recognition of CoV RNA by the host, we assert it is to be a promising drug target (Fig 2).
(4) Nonstructural uridylate-speci c endoribonuclease (NendoU) Nsp-15 activity is found in the N-terminal 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 a higher binding a nity 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 (Mn 2+) and the enzymes generate 2'-3' cyclic phosphate ends 27 . Nsp15 functions as a homohexamer, although the enzyme has some activity as a monomer 28 . The structures of MERS-Nsp15 and SARS-Nsp15 have been superimposed showing high homology 29 . Previous studies established that Nsp15 from both SARS-CoV and MHV can be stimulated by Mn 2+ 30 . 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 speci city 30 , suggesting that there is conserved recognition for uridylate. 29,31,32 (5)2'-O-Methyltransferase (2'-O-MT). Following the addition of the N7-methyl guanosine cap, nsp16, activated by nsp10, mediates mRNA cap 2'-O-ribose methylation to the 5'-cap structure of viral mRNAs. This has been shown in SARS-CoV and MERS-CoV and shown to be universal to coronaviruses 33,34 . 2'-O-Methylation is important for the host immune system to discern self RNA from nonself RNA. Thus, through 2'-O-methylation of viral RNA, coronaviruses can subvert host innate immune responses by avoiding host recognition of their RNA 34  (6) Viral helicase is essential to viral genome replication and is therefore a potential target for antiviral drug development. Virusencoded RNA helicases have important roles during viral life cycles for folding and replication of viral RNA 35 . As such, Nsp13 possesses NTPase and RNA helicases to facilitate hydrolysis of NTPs and unwind RNA (Fig 2). In one study, it was demonstrated that myricetin and scutellarein are strong inhibitors of SARS-CoV helicase protein by affecting its ATPase activity 36 . Helicase is a multi-functional protein with a zinc-binding domain in the N-terminus displaying RNA and DNA duplex-unwinding activities with 5' to 3' polarity. The activity of helicase is dependent on magnesium (Mg 2+ ). As such, bismuth salts have been shown to inhibit NTPase and RNA helicase activities of SARS-CoV-2 nsp13 8,37 . Sequence annotation by Ivanov et al. 27 has shown that SARS-CoV nsp13 is divided into three domains: (A) an N-terminal Zn(II) binding domain and (B) a hinge domain and (C) a helicase domain 35 . Little is known of the viral non-structural proteins (nsp's) 12 activity, but activity occurs on two domains, on the N-terminal subunit NiRAN, and motif BN on the C-terminus. These sites have been shown in SARS-CoV and are suggested to apply to coronaviruses in general. 38,39 (7) RNA-directed RNA polymerase (RdRP) plays a critical RNA replication in RNA viruses due to its involvement in catalyzing template synthesis of polynucleotides in the 5'-3' direction. Further, RdRP is essential for the initiation of RNA replication in the host cell, a key step in the RNA viruses infection cycle 40,41 . Jingyue Ju et al. (2020) have demonstrated the importance of RdRP activity for SARS-CoV pathogenesis [Cite Ref].They showed that without RdRP, there is a complete disruption of SARS-CoV -RNA replication and viral growth halted (Fig 2). Additionally, these authors also suggested that the hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) would target the active site of RdRP to inhibit coronaviruses 42 . 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 RdRP is of a particular interest as a drug target.

Results And Discussion
Following in-silico HTVS, molecular modeling, MDS, and utilizing resources from the literature, we identi ed a series of potential lead molecules from the repurposable drug libraries against proteins that are critical for the ability of SARS-CoV-2 to infect and reproduce within the host cell (Tables 1-7). Additionally, all of the simulated hit-target complexes were found to be strongly interacting and exhibited highly stable binding indicating the potential of being potent inhibitors of key SARS-CoV-2 enzymes and therefore promising candidates or leads for further development. Outputs from these analyses are presented in tables (Tables 1-7) and graphically (Top three interaction maps Supplementary Figs 1 ,3,5,7,9,11&13), and are discussed in the following sections.  45,46 . Crocin/Crocetin are popular agents to be tested in clinical settings due to their anti-oxidative properties 47 . 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 48 . Paclitaxel is one of the most widely used anticancer drugs for the treatment of various cancers 49 . Other studies have shown that low doses of paclitaxel show promise in treating some non-cancer diseases including renal and hepatic brosis and artery restenosis [50][51][52] . NADH dianion is a species of NADH that arises from the deprotonation of the two diphosphates 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 the cerebrospinal uid 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 likelihood of survival of terminal COVID-19 patients 53,54 by a mechanism that is not well understood but likely involves heparin's activity of reducing hypoxia as well via inhibiting the cytokine storm 55 . There is a minor report suggesting that hepcidin hormone mimics the spike protein of SARS-CoV2, 56 and heparin is known to interfere with hepcidin 57 . 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.  59 . 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 the 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. The Paromomycin RdRP complex was simulated for 20ns and the interaction was highly stable throughout the simulation validating the pipeline for RdRP (Supplementary g.4).
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 60 . Framycetin is an aminoglycoside antibiotic isolated from Streptomyces lavendula that shows broad-spectrum antibacterial activity. This drug has been used as a therapeutic against a variety of cancers 61,62 . 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 rst-line antibiotic 63 . Lividomycin A is an aminoglycoside that shows antibiotic activity against several of the Gram+/-bacteria by inhibiting protein synthesis 64 . 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 65,66 . Uridylate-speci c endoribonuclease (NendoU) Hits (Table 3). Ligand interaction maps of the top three hits are shown in 'Supplementary g. 5.' Daidzin is an iso avone natural product found in several Legumimosae such as the Japanese Kudzu root and is the 7-O-glucoside of the iso avone daidzein. It is shown to have anticancer and antiallergenic activities 67 . Daidzein and Haloperidol have been identi ed in multiple virtual screenings 68,69 . MD Simulation of the Daidzin-NendoU complex for 20ns revealed a stable and energetically favorable interaction and validated the pipeline for the NendoU virtual screening (Supplementary g.6). SCHEMBL24383 (Active principle of Strychnos potatorum Linn. seed extracts) has been extensively used to combat respiratory diseases including 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 70 . 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 71,72 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 including gingival hyperplasia to severe more effects including toxic epidermal necrolysis and teratogenic effects 73 . Similarly, acetaminophen O-β-Dglucose 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 74 . The advisory was subsequently revised to state that both medications are appropriate 75 . A metabolite of acetaminophen could prove to be an added advantage of already being used as a drug. p-Aminophenyl-alpha-D-galactopyranoside is an experimental phenolic glycoside that competitively binds to heat-labile enterotoxin B pentamers by mimicking host cell receptors (intracellular adenylyl cyclase) 76 .  78 . A crude preparation of Diosmin and Hidrosmin has been determined to be effective against COVID-19 79 . N-Desmethyl-4-hydroxy tamoxifen beta-D-glucuronide (E/Z Mixture) has also been identi ed as a strong binding ligand. Tamoxifen has also been proposed as an anti-COVID-19 drug as it can induce autophagy associated with the unfolded protein response to kill infected cells and thus contain the virus 80 . Octane-1,3,5,7-tetracarboxylic acid B belongs to the class of 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 hypercholesterolemia and has few adverse effects. This compound inhibits adenosine triphosphate citrate lyase, an enzyme within the cholesterol biosynthesis pathway 81 . Bisindolylmaleimide IX (BIM IX) a was strongly bound to ExoN active site for a 100 ns simulation (Fig 4& Fig   5(B)) and the stable complex had a tight binding with the whole molecule involved in interactions with 3CLpro active site side-chain amino acid residues (Fig 4(B)& Fig 5(B)). Helicase (HEL) Hits (Table 5) inhibiting cell growth. Fenoterol is a β adrenoreceptor agonist that is used as an inhaled bronchodilator asthma medication 85 .
Didanosine is particularly interesting as it is a reverse transcriptase (HIV) inhibitor 86 . Also, Didanosine has been pro led as a bene cial drug in the case of the COVID-19 type of lung brosis by matching single-cell RNA sequencing data 87 . Inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) has been shown to control SARS-CoV-2 replication 88 , which could be due to inosine accumulation. Didanosine is also a medication used to slow the progress of HIV/AIDS and is a nucleoside analog of adenosine.
There are several common adverse effects associated with this medication including diarrhea, vomiting, and peripheral neuropathy 89 .
Fenoterol has already been recommended to reduce severe pulmonary symptoms of COVID-19 patients 90 . Doxi uridine has been suggested as a possible SARS-CoV-2 inhibitor based on similarities with active antivirals already tested by arti cial intelligence 91 . Doxi uridine 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 92 .  Papain-like proteinase (PLpro) Hits ( Table 6). The ligand interaction maps of the top 3 hits are shown in 'Supplementary g. 11.' Troxerutin is a naturally occurring avonoid that has been reported to show promise as a vasoprotective agent by improving hepatic homeostasis 93    Main proteinase (3CLpro) Hits (Table 7). Ligand interaction maps of the top 3 hits are shown in 'Supplementary g. 13.' XAV-939 is a beta-catenin signaling inhibitor that has been shown to have promise in treating prostate cancer 103 . XAV-939 is also an effective inhibitor of PARP and Wnt pathway. PARPs enhance IFNγ production and can halt viral infections 104,105 . Thus, a dual-mode of action is expected of XAV-939 if it is found to inhibit the main protease as well. MD Simulation of XAV-939-3CLpro docked complex for 100ns revealed a stable and energetically favorable interaction and validated the pipeline for the 3CLpro virtual screening (Supplementary g.14). Crocin is a carotenoid diester that is responsible for the color of saffron and is popularly used in India as a treatment for arthritis and psychological disorders 106 . 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 avonoid natural product that can be isolated from various plant species and has shown promise as an anti-cancer agent 107 . Danoprevir is a 15-membered ring macrocyclic peptidomimetic inhibitor of the hepatitis C protease NS3/4A 108 . Other virtual drug screenings have reported danoprevir as a possible coronavirus main protease inhibitor 109 . In clinical trials, danoprevir had a positive effect on recovery and faster discharge of COVID-19 patients 110 . Cefoperazone is a cephalosporin antibiotic. It is one of the few cephalosporin antibiotics effective in treating Pseudomonas bacterial infections 111 . In China, most of the treatment regimens reported using cefoperazone to prevent secondary infections in COVID-19 patients 112,113 . Nevirapine is a non-nucleoside reverse-transcriptase inhibitor 114 . 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 115,116 . Pentostatin is a purine analog that is widely used as a treatment for hairy cell leukemia 117 . Similar to pentostatin, cladribine is also a therapy for hairy cell leukemia. And also like nevirapine, cladribine has been predicted to block main protease 3CLpro in many reports 116,118 . Bisindolylmaleimide IX a PKC inhibitor, strongly bound to 3CLpro for a 100 ns simulation (Fig   3 & Fig 5(A)) and the stable complex exhibited a tight binding with the whole molecule involved in interactions with 3CLpro active site side-chain amino acid residues (Fig 3(B) & Fig 5(A)).
In-vitro anti-viral testing. All compounds unless stated otherwise were procured from Selleck Chemicals LLC (Houston, TX). As per screening results, Troxerutin (3CLpro & PLpro) and BIM IX (3CLpro & Exon) had possible dual targets, while other targets were shortlisted based on novelty and scores. Mild anti-SARS-CoV-2 activity at higher concentrations; at 25µM Amikacin (7.2%), Troxerutin (2.8%), Paromomycin (2.3%%) & Haloperidol (9.8%%) had non-translatable activity (Figs 6 and 7). Lactulose & Iopromide had no observed inhibitory activity, which could be due to low intracellular accumulation as Lactulose is anti-diarrhea and Iopromide is an MRI contrast agent. This supports the predicted interaction and MOA and can be used to improve or screen more members from the same chemotypes and similar pharmacophore. Lactulose (Anti-Diarrhea) and Iopromide (MRI contrast) both showed no activity at the concentration range tested. This could be due to a lack of cell permeability as both are not used for intracellular functions. An inhibitory concentration (IC50) was estimated by curve tting over linear regression of Log 10 drug concentrations vs. normalized data (percentage, Table 8). The IC50 was plotted on the variable slope of the drug responses (Figs 6 and 7). Bisindolylmaleimide derivatives are widely used as inhibitors of protein kinase C (PKC) isoforms. Bisindolylmaleimide IX (BIM IX), also known as Ro 31-8220, is an advanced investigational molecule and one of the most commonly used PKC inhibitor standards 119 . BIM IX is an imidothio carbamic ester, as well as a bis-indole maleimide. Several reported pharmacological effects of BIM IX; stimulation of c-Jun-N-terminal kinase (JNK) expression, an activator of p38 MAPK through phosphorylation; activation of glycogen synthase activity in adipocytes, among other effects [120][121][122] . BIM IX shows potent inhibition of PKC α, PKC βI, PKC βII, PKC γ, and PKC ε 123 . BIM IX also inhibits MSK1, MAPKAPK1, RSK, GSK3β, and S6K1 with a potency similar to that for PKC 123 . BIM IX signi cantly decreases apoE secretion from primary human macrophages by inhibiting vesicular transport of apoE to the plasma membrane without signi cantly affecting apoE mRNA or apoE protein levels 123 . Bim IX has been extensively studied in several animals and human tissue types showing a safe pro le [124][125][126] . In the context of SARS CoV2, the anti-in ammatory and anti-bronchitis pro le of this molecule may prove to be an additional therapeutic advantage 125 . While the effect of PKC inhibition on this particular virus is not known, but the SARS-CoV pathogenesis pro le is known to be greatly dependent on PKC 127,128 .
3CLpro enzyme inhibition assay. To characterize the mechanistic aspects of BIM IX activity, biochemical studies of the 3CLpro enzyme-inhibitor complex were performed. We used a peptide substrate with a C-terminal 7-amino-4-methylcoumarin (AMC) with a uorogenic reporter group to perform kinetic assays of 3CLpro inhibition. BIM IX and Haloperidol were tested at four concentrations and the rate of substrate cleavage was recorded at different time points (Fig.8). Haloperidol had a very mild activity, while BIM IX had signi cant activity. Finally, BIM IX concentrations were varied relative to a xed concentration of the peptide substrate and monitored over time. Inhibition was observed with an IC 50 value of 113.7 ± 5.2 µM (Fig.9).

Conclusion
The rapid spread of SARS-CoV-2 continues to create havoc in health systems and economies, affected every nation around the world. As this is a novel coronavirus, vaccines are being developed at an unprecedented pace and we must also resort to the rapid development of therapeutics for COVID-19. There has been an international focus on the potential e cacy 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 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. Until a SARS-CoV-2 speci c compound is developed and clinically approved, the best way is to nd treatment through a multifaceted approach of repurposing as well as developing new drugs. This philosophy is at the forefront of our work, rst by screening approved compounds for repurposing potential, and by identifying the best possible combinations providing a multi-faceted attack on SARS-CoV-2. In this context, we commenced a CADD through the HTVS approach using a large pool of world approved drug libraries to identify potential drugs for immediate deployment (Fig.1). Our in-silico studies have shortlisted a series of repurposable drugs that can be utilized in clinical trials. From our nine selected approved compounds that underwent in-vitro studies against SARS-CoV-2, we found two compounds with high inhibitory activity. One of these was ivermectin, which showed signi cant viral inhibition, con rming the results of a previous study 82 . Like previous reports, our in-vitro studies found that ivermectin has nonselective toxicity to the ATCC E6 Vero cells at ≤50 μM and 16.67 μM based on the number of nuclei counted. This shows that high doses (>16.67 μM) of ivermectin have high cytotoxicity as reported by other groups 129,130 , and this must be considered when preparing to use this drug in COVID-19 clinical trials as its currently approved dosage is very low (25mg) 131 . The second compound with signi cant inhibitory activity against invitro SARS-CoV-2 was bisindolylmaleimide IX (BIM IX). Further, BIM IX spece caly blocked 3CLpro in vitro enzymatic assay con rming our in-silico predictions. The most successful drugs against SARS and MERS have targeted this enzyme including repurposed Lopinavir and Ritonavir 132 . Furthermore, PKC inhibition, anti-bronchitis, and anti-in ammatory effects may align with a multifaceted therapy option, while not validated ExoN inhibition can have a negative effect on viral tness for transmission by increasing defective genomes packed. Overall, this is the rst study that showcases the possibility of using bisindolylmaleimide IX spece c derivatives to further evaluate their suitability to treat COVID-19. More studies are needed to assess their effectiveness. Target Proteins (Receptors) preparation. Protein preparation was performed using the Protein Preparation Wizard in Maestro 9 . All co-crystallized atoms including calcium and chlorine were deleted. Co-crystalized ligands within active site were not deleted because they were used for grid generation. All water with less than 3 hydrogen bonds (Sample water orientation) with 'non waters' i.e. receptor protein or ligand were deleted. Each of the seven models developed was subjected to preparation by the Wizard in Maestro 9 . Each of the complexes was optimized with minimized hydrogens of altered species and then minimized using the OPLS3e force eld 9,135 . Further, the models were subjected to MD simulation (MDS) after solvation in water and 0.15M NaCl (physiological solution). A simulation box covering the entire enzyme system was introduced with a 10 Å buffer space. The simulation was run for 100 ns at 300 K and standard pressure (1.01325 bar). The target complex trajectory analysis for interacting residues and was conducted using the Desmond software 9 .

Methods
Target library preparation. A virtual library of n=5903 drug compounds was downloaded from the latest Zinc database 10 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 eld. EPIK minimization was performed on possible states at pH 7.0 ±2.0. Tautomers were generated for each ligand retaining speci c chirality combinations with a maximum of 32 structures per ligand 9 .
Receptor grid generation. COACH analysis was performed to determine the location and size of the active site 136 137 . 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 eld was used during the docking process. The HTVS ligand docking was the rst 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 the calculated a nity of the hit in binding to the protein target. Further, the estimation of free binding energies for the best hit-docked complexes using MM force elds and implicit solvation was performed using the molecular mechanics/generalized Born surface area (MM-GBSA) method within the virtual screening work ow of Schrödinger suite 9 . The binding energy was calculated based on the following equation.

Molecular Dynamics Simulation (MDS).
To determine the stability of the ligand receptor complex especially during exiblity of the binding site an to determine the energetic strain on the docked ligand that could result in immediate yoff (<10ns). The top hit for each target was subjected to a 20 ns MD simulation (100 ns for XAV-939 + 3CLpro, BIM IX + 3CLpro, & BIM IX + ExoN) as described above in the Target/receptor Preparation section to validate the interaction and thereby the HTVS pipeline and MM-GBSA performance. If the top hit ew off imediately due to major conformation change in the receptor site than the receptor was simulated alone for 20ns ro reach a stable state and screnning was repeated with the resulting minimized structure.
In-vitromicroneutralization assay.BIM IX was shortlisted for its predicted a nity for both 3CLpro (Main protease) and , then permeabilized with 0.2% Triton-X for 10 min at RT and treated with blocking solution (3% BSA/PBS). Infected cells were detected using a primary detection antibody recognizing the SARS-CoV-2 nucleocapsid protein (Sino Biological, 40143-R001) and Alexa Fluor 488 conjugated antibody (goat α rabbit), then counterstained with DAPI for visualization of the cell nuclei. Infected cells were enumerated using Operetta high content imaging instrument and data analysis was performed using the Harmony software (Perkin Elmer). Spread Assay: A similar protocol was utilized to the entry assay described above with the following modi cations. The virus was used at an MOI of 0.02 and the assay was incubated for 48 hrs. rather than 24 hr. after infection of the cells.
Compound screening to identify 3CLpro inhibitors.
Fluorescence-based biochemical assay for inhibitors of 3CLpro. Inhibition assays were performed in 96-well black plate in triplicate at 25 °C. Reactions containing varying concentrations of inhibitor (0-1000 µM) and 3CLpro enzyme (0.4 µM) in Tris-HCl pH 7.3, 1 mM EDTA were incubated for approximately ve minutes. Reactions were then initiated with TVLQ-AMC probe substrate (40 µM), shaken linearly for 5 s, and then measured continuously for uorescence emission intensity (excitation λ: 364 nm; emission λ: 440 nm). Data were t using nonlinear regression (dose-response inhibition, variable slope) analysis in GraphPad Prism 7.0. Figure 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 HTvirtual screen with world approved drug libraries. The top 10% of these compounds were subjected to high accuracy docking (SP/XP) which were then further re ned to the top 10%. This was followed by a secondary rescoring (GBSA). Top leads were subjected to MD simulations of the top compounds for each viral target tested as a methodological validation. 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). NendoU is a Mn2+ dependent hexamer (dimer of trimer) enzyme with sparse functional information. The most prominent theory regarding NendoU 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 budding mediated by fusion of smooth walled vesicles to plasma membrane (11)(12)   Results of a 100 ns MD simulation (A) Root mean square deviations difference between guanine-N7 methyltransferase (ExoN) and bound ligand BIM IX (<4 Å). Graph obtained for RMSF value of ligand (purple line) from the protein back bone (green line). The ligand was tightly bound to the active site throughout the simulation. The complex progressed towards a more stable state during the simulation. This suggests two binding conformers of same ligand within the binding site. (B) Schematic 2D representation of bound ligand interactions of BIM IX throughout the simulation. (C) Root mean square uctuation between the binding site of target protein and interacting ligand. (D) Critical protein ligand contacts of amino acid side chain residues with the interaction properties.  SARS CoV2 anti-viral entry assay; Synchronized infections were conducted for viral entry as described in Materials and Methods. Each curve shows a dose-response to the indicated 8 drug compounds (Color coded; key in set). The results are presented as the PFU formed in the presence of drug as a percentage of the PFU formed and each plotted value is the mean with + standard deviations of experiment performed in triplicate.

Figure 7
SARS-CoV2 Anti-viral spread assay; Synchronized infections were conducted for viral spread as described in Materials and Methods. Each curve shows a dose-response to the indicated 8 drug compounds (Color coded; key in set). The results are presented as the PFU formed in the presence of drug as a percentage of the PFU formed and each plotted value is the mean with + standard deviations of experiment performed in triplicate.

Figure 8
Preliminary compound screening for 3CLpro inhibitors. Fluorescence-based biochemical activity assay for (A) BIM IX (B) Heloperidol and (C) Disul ram using TVLQ-AMC probe substrate. Each point is the mean of compounds screened in duplicate (1-50 µM) or a DMSO control (0 µM) for n = 2 replicate screens. Emission is normalized to 0 µM at 3 h to identify compounds with inhibitory activity toward 3CLpro.