key: cord-0867251-t2rf7d73
authors: Mslati, Hazem; Gentile, Francesco; Perez, Carl; Cherkasov, Artem
title: Comprehensive Consensus Analysis of SARS-CoV-2 Drug Repurposing Campaigns
date: 2021-07-27
journal: J Chem Inf Model
DOI: 10.1021/acs.jcim.1c00384
sha: 677bc5f296278b0b24c485be5f2288b21bc83bd6
doc_id: 867251
cord_uid: t2rf7d73

[Image: see text] The current COVID-19 pandemic has elicited extensive repurposing efforts (both small and large scale) to rapidly identify COVID-19 treatments among approved drugs. Herein, we provide a literature review of large-scale SARS-CoV-2 antiviral drug repurposing efforts and highlight a marked lack of consistent potency reporting. This variability indicates the importance of standardizing best practices—including the use of relevant cell lines, viral isolates, and validated screening protocols. We further surveyed available biochemical and virtual screening studies against SARS-CoV-2 targets (Spike, ACE2, RdRp, PL(pro), and M(pro)) and discuss repurposing candidates exhibiting consistent activity across diverse, triaging assays and predictive models. Moreover, we examine repurposed drugs and their efficacy against COVID-19 and the outcomes of representative repurposed drugs in clinical trials. Finally, we propose a drug repurposing pipeline to encourage the implementation of standard methods to fast-track the discovery of candidates and to ensure reproducible results.

Coronavirus disease 2019 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded RNA virus that causes many outcomes, including pulmonary infection and respiratory distress. COVID-19 was declared a pandemic by The World Health Organization in March 2020, 1 months after the original Wuhan outbreak occurred in 2019. 2 As of June of 2021, there were more than 170 million confirmed cases and 3.7 million deaths worldwide. 1 This novel betacoronavirus displays genomic and clinical features similar to the earlier severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) and is speculated to be of a zoonotic origin, with very few credible therapeutic options available to date. 3 Traditionally, development of therapeutics requires greater than two billion dollars and may take up to 10−15 years. 4 Thus, the repurposing of FDA-approved drugs remains an attractive, rapid, and economic option to address SARS-CoV-2 infection. Consequently, an explosion in scientific publications and preprints describing a myriad of computational and experimental drug repurposing studies has emerged. Hundreds (if not thousands) of virtual screening campaigns have been reported, describing drugs that could bind to one of the six main therapeutic targets encoded by SARS-CoV-2 or the host's cell, including the viral receptor binding domain (RBD) of the spike glycoprotein (S protein), main protease (M pro ) and papain-like protease (PL pro ) enzymes, and RNA-dependent RNA polymerase (RdRp), as well as the host cell's angiotensin converting enzyme 2 (ACE2)a human receptor serving as the viral entry pointand the transmembrane protease serine 2 (TMPRSS2). 5 Simultaneously, a large number of highthroughput screening (HTS) campaigns have been reported identifying broadly acting (target unspecific) inhibitors for SARS-CoV-2 virus and/or its specific target proteins. These experimental and computational efforts generated valuable drug repurposing informationalthough frequently contradictive, thereby requiring rigorous benchmarking, standardization and postprocessing.

Computer-aided discovery of repurposed drugs helps avoid costly trial-and-error experiments involving cultured cells, biochemical screenings, and live systems. 6 As an example, baricitinib (a rheumatoid arthritis drug) was predicted using artificial intelligence as a repurposed drug 7, 8 and was later granted an FDA-emergency approval for treatment of COVID-19 in combination with redemsivir. 9, 10 However, most of the computational studies in the COVID-19 repositioning landscape were found to be lacking support from experimental results. In this review, we sought to reconcile in vitro, in silico, and biochemical repositioning studies to identify promising drugs. Prior to using empirical evidence to support in silico findings, we assessed the consensus drug activities among cellbased and biochemical screenings. Our strategy is summarized in the flowchart in Figure 1 . We chose to use cell-culture repositioning studies to benchmark in silico and biochemical screening studies, as well as other cell-culture studies, benefiting from the large amount of cell-based experiments in the literature.

Several reviews reported progress on SARS-CoV-2 drug repositioning; 11−17 we present an extensive overview of the drug classes, the mode of action (MOA) in SARS-CoV-2 patients, the screening protocols that were used to identify them (accentuating interlab consensus findings), and the contradictory clinical trial outcomes.

We investigated half-maximal effective concentration (EC 50 ), half-maximal inhibitory concentration (IC 50 ), and half-maximal activity concentrationAC 50 , as well as half-maximal cytotoxic concentration (CC 50 ) values, for the 19 experimental cellbased articles reviewed and compared overlapping hits identified from multiple independent studies (cross-validation). 18−37 Reported hits were reviewed in the National Center for Advancing Translational Sciences (NCATS) database. 38, 39 Notably, the rich NCATS databases were trained in the computational model REDIAL-20a user-friendly web program to retrieve activity against SARS-CoV-2 targets through DrugBank. 40 Cross-validation of hits between different HTS studies and NCATS results derived from the cytopathic or cytopathogenic effect (CPE) screening results are illustrated in Figure 2 . While these studies utilized Vero E6 cells and similar viral MOIs, differing experimental conditions are probable sources of variability.

Active Compounds from Cell-Based HTS Repurposing Campaign Show Weak Consistency. Initially cellbased HTS experiments dominated in vitro COVID-19 drug repositioning research, involving a large number of assay variables, which in turn may have led to the low consistency in reported drug potency ( Figure 2 ). However, unlike measurements of K I , IC 50 data are assay protocol specific. 41 The following subsections explore some of the cell-based experimental variables derived from SARS-CoV-2 screening campaigns.

Cell Lines. A number of cell-based HTS studies used the Vero 76 cell line or a lineage thereof such as Vero E6. [18] [19] [20] [21] [22] [22] [23] [24] [25] [26] [27] [28] [29] [31] [32] [33] [34] [35] [36] [37] 42, 43 Vero E6 cells, originally cloned from the Vero 76 cell line, 44 are African green monkey kidney epithelial cells and are highly permissive to SARS-CoV-2 infection. 45 Vero E6 is broadly considered as the "gold standard" cell line for assaying SARS-CoV-2 viral-induced CPE. 27 Similarly, the ACE2 expression level is significantly elevated in Vero E6 cells. 46 Vero E6 cells are also interferon deficient which makes them more susceptible to viral infection. 47 Moreover, the engineered lineage, VeroE6/ TMPRSS2 cells, displays 10-fold greater mRNA expression levels of cellular TMPRSS2, responsible for S protein cleavage, 48, 49 compared to normal human lung tissue and other human cell lines, making them an attractive choice for use in SARS-CoV-2 repositioning screenings. 45 Calu-3 cells (human lung epithelial cells) and Huh 7.5 cells, derived from parental cell line Huh-7 (human liver cells), are both infection-permissive human-derived cells. 20 However, Huh-7 cells were considered less desirable in some studies due to the low level expression of ACE2 and the lack of TMPRSS2 expression. 50, 51 Caco-2 used in other repurposing studies 31,52 is an immortalized cell line derived from human colorectal adenocarcinoma cells, observed to differentiate into a mixture of intestinal-like cells with heterogeneous properties in cultures. 53 Both Caco-2 and Calu-3 cells (at a lower MOI) were shown to be more efficient in propagating SARS-CoV-2 infectionin contrast to Huh-7 cells and other human-derived cell lines. 54 The differences in infection efficiencies and cell lines translate into variable CPE outcomes in drug screenings which in turn result in inconsistent reported drug activities. For instance, Dittmar et al. 20 evaluated a library (3000 drugs and drug-like molecules) via an HTS campaign using Vero E6, Huh-7.5, and Calu-3 cell lines ( Figure 3 ). The authors identified six and 23 active compounds from Vero E6 and Huh-7.5 screens, respectively, with nine out of the Huh-7.5 active compounds displaying Calu-3 activity. Two drugs (Y-320 and salinomycin) demonstrated high potency in the three cell lines. In the same study, known antivirals, remdesivir and hydroxychloroquine (HCQ), displayed Huh-7.5 EC 50 values more than 10-fold lower than those observed in Vero E6 cells. Additionally, HCQ, chloroquine, and structurally related 20 Variability in potency employing different cell lines was reported in the study by Touret et al., 31 who validated their drugs in Caco-2 cells and reported Arbidol to be less potent in the Vero E6 cells. 31 Viral infection efficiency appears to be cell line dependent and may require optimal endosomal acidification for entry into the host cell. 20, 31 Not surprisingly, it was demonstrated that drug potency in Caco-2 cells was similar to other human cell lines (e.g., Calu-3 cells) compared to nonhuman cells (Vero E6 or BHK-21) and in particular for inhibitors associated with blocking virus entry into the host cell. 52 Variations in Experimental Conditions Used to Determine EC 50 Values. The phenotypic outcome associated with CPE measurements varies with culture conditions and with viral MOI. 22, 55 Reported MOI values that were chosen in various SARS-CoV-2 cell-based repurposing studies ranged between 0.001 31 and 5. 22 Increasing MOI was shown to induce stochastic gene expression profiles which may hinder precise and reproducible readouts for EC 50 values. 56 An MOI of 0.02 resulted in approximately 95% cell mortality and 72 h post infection (hpi) in one study, 22 while a MOI of 0.016 resulted in only 75% cell mortality within the same hpi time frame in another study. 29 In contrast, Weston et al. reported insignificant variation in the EC 50 readouts as a function of MOI. 18 Dose−response curves (DRC), from which EC 50 values are extrapolated, are calculated using a variety of different viral indicessuch as reverse transcription polymerase chain r e a c t i o n ( R T -P C R ) t o q u a n t i f y v i r a l R N A (vRNA). 18, 20, 23, 25, 26, 31, 33, 43 In other studies, DRCs were calculated using CPE mixed analysis of cell morphology, fluorescence intensity of viral markers, position properties of cells, and cell confluence followed by fitting data with sigmoidal dose−response models. 21, 22, 32, 52 Other works extrapolated EC 50 values by measuring viral nucleoprotein levels from supernatants of drug-treated cells that were preinfected with SARS-CoV-2. 29 Importantly, EC 50 values are also impacted by duration of viral infection. 57 While infection time was highly variable in drug screening assays, 72 h infection assessment was the most commonly reported.

Choice of Viral Isolate. Cell-based drug screening assays are generally conducted on the beta coronavirus HCoV-HKU1 or viral isolates obtained from COVID-19 patients of different origins. 18, [20] [21] [22] [23] 25, 27, 29, [31] [32] [33] 43, 52 Few exceptions were reported in the literature. Drayman et al. resorted to a different betacoronavirus predecessor, HCoV-OC43, which is a much less potent version and consequently a safer alternative as a viral isolate in screenings experiments. 30 A singular SARS-CoV isolate was used for the repositioning HTS by Fan et al., derived from a dead smuggled pangolin in 2017 (whose spike protein shares 92.2% amino acid identity with the spike protein of SARS-CoV-2). 28 Ultimately, different coronavirus lineages might have resulted in reported EC 50 variable outcomes due to differences in ACE2-dependent cell entry RDBs affinities. 51 Some Active Compounds from Cell-Based HTS Repurposing Campaign Show Promising Similarities. Despite the interlab variability in potency, some compounds were reported as active against SARS-CoV-2 at low concentration by multiple studies and are confirmed by NCATS experiments and are candidates for future COVID-19 treatments. The chemical structures of these candidate drugs, their speculated MOAs from experimental and/or computational studies involving molecular dynamics (MD), and stages of clinical evaluation are summarized in Table 1 .

Compared to cell-based assays, biochemical assays and highthroughput virtual screenings (HTVS) provide more insights into the MOA. In contrast to M pro -enzymatic and cell-based assays, there was little COVID-19 drug repositioning data for five SARS-CoV-2-encoded targets (Spike, ACE2, RdRp, PL pro , and TMPRSS2). Docking studies, similarly, largely favored prioritizing the M pro as the target in their screenings, although many drugs were demonstrated to have inhibitory effects against a different viral protein. Remdesivir, for example, which was cocrystallized with RdRp, 67 appeared in numerous M pro computational docking campaigns as a predicted hit. 68−72 On the other hand, it is possible that identified drugs in a targetspecific virtual repurposing campaign could act synergistically on more than one SARS-CoV-2 targeas speculated for chloroquine inhibiting M pro activity and interfering with the endosomal acidification process associated with SARS-CoV-2 viral entry. 73 The lack of sufficient biochemical data, in turn, may render it challenging to rule out off-target activity.

In the case of M pro , only drugs that were predicted to be active by at least two different computational studies and displayed activity also in cell-based assays were retained (presented in Figure 4 ).

Repositioning Screenings against the Spike/ACE2 Interface. The glycosylated trimeric spike protein, encoded by SARS-CoV-2, is the ligand by which SARS-CoV-2 enters the host cell. 74 The larger binding interface and higher affinity of the S protein to host the ACE2 receptor likely contributes to its greater virulence compared to the S proteins expressed in SARS-CoV. 75 Additionally, the S protein, localized to the plasma membrane, can trigger receptor-dependent syncytia formation, 76 which is a process not addressed during vaccine development. The crystal structure of the S protein receptorbinding domain complexed with the ACE2 receptor (6M0J) 77 has been used in many virtual screening campaigns for identifying repurposed drugs. 78 82−84 During the proteolytic cleavage products of the 16 nonstructured proteins (NSPs), RdRp is generated from NSP 12 which requires the attachment of NSP 7 and two NSP 8 molecules to assemble a minimally functioning complex. 83 RdRp shares significant homology within the RNA virus Coronaviridae family and is a popular target for SARS-CoV-2 repositioning studies due to a given drug's prospect of high selectivity and low probability of associated cytotoxicity. 82, 83 Many nucleoside analogues have been proposed due to low RdRp replication fidelity. 85 Due to the inhibitory potential of remdesivir for the RdRp in Ebola virus, 67,86 several virtual screening campaigns were conducted. [83] [84] [85] 87 Despite the lack of consensus for hits identified among the virtual repurposing campaigns, two of the in silico shortlisted drugs were reported among the hits identified in cell-based repurposing studies, namely, digoxin (a cardiac glycoside; EC 50 = 0.07 μM) 20 and ritonavir (an HIV protease inhibitor; AC 50 = 22.53 μM). 21, 23, 26, 88 Repositioning Screenings against PL pro . During SARS-CoV-2 replication, 16 NSPs are generated from two large overlapping polyproteins (PPs): PP1a and PPab. 89 Two types of cysteine proteases process these PPs, of which the Nterminal termini are proteolytically processed by the papainlike protease (PL pro ). 90 Moreover, SARS-CoV-2 PL pro efficiently inhibits ISGylation of the central interferon (IFN) regulatory factor thereby diminishing IFN-mediated innate viral immunity. 91 Therefore, PL pro inhibition has been the focus of many SARS-CoV-2 drug repositioning studies as it may impede viral replication and help restore innate viral immunity. 91, 92 The anti-inflammatory drug ebselen was reported as an effective PL pro inhibitor 93 and a potent M pro inhibitor. 94 Only two drugs, ribavirin (an antiviral) (EC 50 = 4.09 μM) 36 and oxprenolol (a beta-blocker) (EC 50 = 20.22 μM), 31 were identified in silico as potential PL pro ligands and were effective in cell-based assays. 23, 31, 87 Targeting PL pro may be challenging in translational development since host deubiquitinases and PL pro recognize the same C-terminal human ubiquitin domain. 95, 96 Repositioning Screenings against M pro . The C-terminal termini of PPs are proteolytically cleaved by a chymotrypsinlike cysteine protease [aka M pro ; 3C-like protease (3CL pro )], hydrolyzing the Gln-Ser peptide bond in the Leu-Gln-Ser-Ala-Gly recognition sequence. 97 Only one FDA-approved compound, boceprevir, was found to be active and in consensus among the M pro biochemical repositioning screening studies. 24,96,98−101 Additionally, the large-scale X-ray crystallographic study led by Gunther et al. and its lack of identifying similar compounds in M pro screens suggests significant inconsistencies. 101 The authors conducted SARS-CoV-2 M pro cocrystallization experiments using a 5935drug library and identified 37 M pro ligands binding at different pockets. 101 Of note, the crystallographic finding in one paper 30 documented masitinib (an antineoplastic drug) as an M pro active site ligand and was not reproduced by Gunther et al. 30, 101 This inconsistency might be explained by the inherent technical difference between crystal soaking and cocrystallization experiments. 102 However, drugs have been found to be active in both the M pro target-directed biochemical and cellbased assays ( Table 2) . With in silico drug repositioning against M pro , only a handful of molecules were found to be common in multiple studies. These molecules were active in cell-based repurposing screenings: dolutegravir, elbasvir, nelfinavir, atazanavir, lopinavir, ritonavir, darunavir, saquinavir, indinavir, oseltamivir, c h l o r o q u i n e , a n d r e m d e s i v i r ( F i g u r e 4). 19 Active compounds identified in in silico and cell-based assays were compared with biochemical assayNCATS screening data. 39 Only the drug darunavir (AC 50 = 39.8 μM) satisfied this triplex consensus. 39 Repositioning Screenings against the TMPRSS2 Protease. TMPRSS2 is a type II serine protease and is primarily expressed in respiratory and gastrointestinal epithelial cells. 117 This serine protease cleaves a specific sequence from the S protein upon ACE2 interaction, thereby promoting cellular endocytosis of the virus and potential syncytia formation. 117 Recently, TMPRSS2 knockout mice demonstrated coronavirus-infection resistance, 118 suggesting this cellular host protein is a potential drug target.

Camostat mesylate was the only drug predicted by multiple in silico studies. 119, 120 The drug is a trypsin-like serine protease inhibitor and blocked coronaviral cell entry via TMPRSS2 inhibition. 121 Recent biochemical HTS reported camostat mesylate activities of IC 50 = 6.7 117 and 2.7 nM. 119 Similarly, biochemical HTS identified nafamostat, FOY 251, and gabaxate with low nanomolar potency ranges. 117 

The above-discussed in vitro SARS-CoV-2 active drugs include antivirals, antimalarials, anticancer therapeutics, immunomodulators, antibacterial agents, antipsychotics, and calcium channel blockers. Inevitably, repurposed drugs will act on their ontological targets and potentially exhibit synergistic complications with adverse side effects. 19 In the following Journal of Chemical Information and Modeling pubs.acs.org/jcim Review subsections, the possible direct and indirect actions of a given drug, or its class, and the results of any clinical trials on COVID-19 patients are discussed. Direct-Acting Antivirals. COVID-19 treatment strategies include antiviral drugs that interfere with SARS-CoV-2 replication enzymes, i.e., proteases and RdRp. Remdesivir is proposed to treat COVID-19 by disrupting the SARS-CoV-2 RdRp machinery. 123 Briefly, remdesivir triphosphate competes with adenosine triphosphate for incorporation into the replicating viral RNA chainresulting in early termination. 123, 124 Remdesivir was used either as a reference drug 21, 52 or a positive control 19 for many repositioning studies, yet, it was also reported as a hit by Choy et al. 23 and Touret et al. 31 Clinical effectiveness appeared to be generally inconclusive for repurposed antivirals. For instance, remdesivir, favipiravir, ribavirin, a ritonavir−lopinavir combination, and Arbidol were reported to be effective in some clinical trials 125−128 but ineffective in others. 129−138 Emtricitabine−tenofovir, a prescription medicine for HIV, revealed reduced viral titers in infected ferrets after 8 days post infection (dpi) in comparison to the control; but after 10 dpi, both the control and the treated ferrets exhibited insignificant statistical outcomes. 50 Thus, SARS-CoV-2 efficacy of current antiviral drugs remains to be determined through further clinical investigations.

Antimalarial Drugs. The exact MOA for antimalarials for treating COVID-19 is still ambiguous. Notable antimalarials (like amodiaquine, which has been widely used for prophylaxis and treatment of malaria for over 60 years) were identified as lead hits by Bocci et al. 19 after they evaluated an FDA drug library based on the chemical Morgan Fingerprint similarity to HCQ. These drugs exhibit broad spectrum antiviral activity and inhibit infection by other SARS, influenza, and Ebola viruses. 50, 139 The potency of other antimalarial drugs (e.g., chloroquine and HCQ) were attributed to interfering with the glycosylation pattern of the host's ACE2 extracellular receptorsthereby disrupting the SARS-CoV-2 spike-mediated entry. 59 Furthermore, HCQ increases endosomal/ lysosomal pH, which in turn interferes with the viral replication process. 19 Extensive antimalarial in vivo trials were conducted with variable and indeterminate outcomes; despite this, many antimalarial agents were deemed promising for clinical studies. For instance, amodiaquine was withdrawn from use in the United States due to rare occurrences of agranulocytosis and liver damage after high doses or prolonged treatment. 140 FDA also cautioned against the use of HCQ and chloroquine outside a hospital setting due to risks of arrhythmia. 141 While several studies advocated HCQ's and chloroquine's anti-SARS-CoV-2 potency, 34,142−148 Weston et al. and others reported ineffective outcomes for either drug during in vivo studies. 18,149−151 Immunomodulators, Antineoplastic Therapeutics, and Antibacterial Drugs. Important severe COVID-19 infection manifestations are shared with neoplasia, inflammation, immune dysfunction, and coagulopathy. 152 Regulation of several cytokines is disordered in the peripheral blood of SARS patients evidenced by an increase in the levels of cytokines and chemokines and a decrease in the levels of anti-inflammatory cytokines such as IL-10. 153, 154 Notably, the release of proinflammatory cytokines, especially interferon (IFN)-α and IFN-γ, was observed to correlate with lethal SARS. 155 The cytokines associated with increased levels in fatal SARS patients are IL-6, IL-1β, IFN, and CXCL10which are mainly secreted by dendritic cells and macrophages, indicating that innate immunity may play a pivotal part in lethal SARS. 154 Therefore, anticancer and immunomodulatory drugsNF-κB and STAT3 regulatorshave been used to manage the "cytokine storm" that is often observed in SARS-CoV-2 patients. In addition, PARP-inhibiting cancer agents and IL-1β and Il-6 inhibiting immunomodulators could prevent caspase-8-mediated necroptosis, whose activation is one of the reported hallmarks of SARS-CoV-2 viral infection. 156 Antibacterial azithromycin, identified by Touret et al., 31 was reported to decrease viral entry into cells by upregulating type I and III IFN expressions (especially IFN-β and IFN-λ), as well as upregulating genes involved in virus recognition (e.g., MDA5 and RIG-I). 31, 157 Another antibacterial, salinomycin, reported by Dittmar et al. 20 as an ionophore, may attenuate viral entry by disrupting endosome acidification. 20, 158 Disagreement in clinical studies include immunosuppressive drugs like tocilizumab and dexamethasone which were demonstrated to be effective in one study 138 but were rebuffed in other works. 159, 160 It is still questionable if immunomodulatory corticosteroids are useful since they have been associated with increased mortality and delayed viral clearance in coronavirus infectious diseases. 161 Antipsychotics. Several FDA-approved drugs with antipsychotic-acting ontology were reported as active against SARS-CoV-2. The MOA of chlorpromazine (CPZ), a lead drug identified by Weston et al., 18 entails the inhibition of clathrin coating in cells, thereby disrupting infection by many viruses that require clathrin-mediated endocytosis (e.g., SARS-Cov-2). 162 It is still unclear whether other antipsychotic dopaminergic antagonists like spiperone, reported to exhibit inhibit human pathogenic polyomaviruses, 31,163 have a similar effect in SARS-CoV-2 infection. Indeed, clinical evaluations of the antipsychotic CPZ revealed ineffective outcomes in vivo. 18 Calcium Channel Blockers. Several antiviral candidates belong to the calcium channel blockers (CCBs) drug class. Since CCBs block intracellular calcium influx, any anti-SARS-CoV-2 effect may reduce the intracellular calcium level. 164 Indeed, Vero E6 cells treated with serial concentrations of calcium chelators BAPTA-AM or 2APB3 and then infected with SARS-CoV-2 exhibited significant inhibition of virus replication in a concentration-dependent manner, confirming the dependent role of intracellular Ca 2+ for SARS-CoV-2 replication. 165 Calcium chelator drugs were also shown to reduce SARS-COV-2 viral titers in infected Vero E6 cells in a dose-dependent manner while also reducing cell viability. 166 Multiple CCBs were evaluated by Touret et al. 31 and Ko et al. 167 for efficacy in vitro with Calu-3 cells and exhibited no activity. While some studies report efficacy of CCB in patients with pre-existing hypertension, 165 their efficacy in vivo remains to be clearly assessed.

DRUGS AGAINST M PRO Since many repurposed drug MOAs were speculated to act on M pro as a target, drugs that showed promising consensus between cell-based assays and biochemical or virtual screenings ( Table 2) were evaluated in-house. A method was adapted from the SARS-CoV fluorescence-based cleavage assay previously described by Hamill et al. 168 with the substrate (Abz-AVLQSGFR-Y (3-NO2) G-NH 2 ; PL Laboratories Inc.) used for SARS-CoV-2 screening.

Prior to screening drugs, we validated the robustness of the M pro assay using a two-compound approach 169,170 based on the primary control, GC-376, 171 and the secondary control, 17a 172 analogue (Figure 5a ). The minimum standard ratio (MSR) derived was 2.55, indicating that the assay was robust and reproducible (<7.5). 170 Interestingly, only boceprevir and Z-FA-FMK out of the nine selected drugs (boceprevir, nelfinavir, MG-132, darunavir, elbasvir, indinavir, Z-FA-FMK, saquinavir, and disulfiram) displayed IC 50 values within the low micromolar potent range in this M pro biochemical assay (Figure 5c and d). Due to the minimal activity observed, a few takeaways were discussed. First, the vast majority of repurposed drugs possess undetermined MOAs and therefore are "moonshot candidates" for COVID-19 clinical trials. Second, the MOA remains to be evaluated rigorously via biochemical assays ( Figure 5 ). Third, the in-house IC 50 values for boceprevir and MG-132 were notably different from the values reported in literature, thereby reinforcing the notion of the implementation of standardized evaluation protocols in the research community.

Throughout the landscape of drug repositioning research, a minority of proposed drugs had undergone a thorough triaging validation. There is a considerable need to implement best practices using a consensus rapid approach to exclude false positives before translational evaluation.

All SARS-CoV-2 antiviral entities must be evaluated in live virus cellular assays (2D cellular assays or 3D organoids) and potential animal models (ferrets, huACE-2 transgenic mice). However, screening thousands of compounds adhering to BSL-3/CL3 safety protocols is time consuming and very expensive. Therefore, our workflow ( Figure 6 ) encourages the use of rigorous computational docking analysis as the first step, thereby prioritizing the biochemical assays experiments for a subset of candidate drugs to evaluate their MOA hypothesis. Subsequent cell-based screenings could then be carried out Journal of Chemical Information and Modeling pubs.acs.org/jcim Review using standardized protocols: EC 50 determination using all three Huh-7, Caco-2 and Calu-3 cell lines; CC 50 determination to evaluate cytotoxicity; and calculation of the therapeutic index (ratio CC 50 :EC 50 ) to determine relevance for in vivo studies. Since SARS-CoV-2 was reported to have preferential tropism toward nasal epithelial cells, pneumocytes, and enterocytes in the bowel, 173 we recommend that all three cell linesHuh-7, Caco-2 and Calu-3be used to orthogonally validate drug potencies but not the Vero E6 cell line due to the factors discussed earlier (see Cell-Based High-Throughput Screens). EC 50 value determination may be standardized by (a) determining viral titers by RT-qPCR quantification 18, 20, 23, 25, 26, 31, 33, 43 and (b) standardizing the MOI. The most common MOIs reported among the cell-based repositioning studies were 0.01 and 0.1; however, using MOI of 0.2 was effective in all three human cell lines with viral titers peaking at 48 hpi. 174, 175 Finally, independent orthogonal validation assays such as the viral titers reduction assay may be performed to concretize results from cell culture experiments. 19 Additionally, the SARS-CoV-2 clinical isolate and its genome must also be evaluated for its relevance before cellbased screenings take place. 176 For example, the D614G mutation affecting the S protein of SARS-CoV-2 strains had been experimentally reported to exhibit more cell infectivity than other known strains. 177, 178 The D614G mutation is reportedly the most dominant variant to date 178 and thus might be the most currently relevant SARS-CoV-2 model to test repurposing drugs against. Due to the rapid emergence of the variants of concern (VOCs), 179 it is recommended that the investigator review the WHO and U.S. CDC updates and determine the VOCs most relevant for in vivo experiments and clinical development.

Using relevant primary tissue organoid or explant models to clarify pharmacological properties (kinetics, absorption, cytotoxicity, and dosage) was seen as a necessary integration in the workflow proposed by Si et al. 50 The authors suggested to test anti-SARS-CoV-2 compounds in an airway chip that contains highly differentiated human lung epithelium cells expressing high levels of serine proteases involved in viral entry. 50 The human organ chip model was claimed to have successfully predicted the inability of chloroquine, HCQ, and Arbidol to work in animals and human patients, thus validating recent reports. 180−182 The penultimate "rung" in the flowchart is evaluating the repurposed drug in an appropriate COVID-19 animal model. 183 The K18-hACE2 mouse, in which transgenic human ACE2 expression is driven by the human K18 promoter in mouse epithelial cells, is a powerful model for SARS-COV-2 virus nasal administration. Post infection, the mice experiences severe respiratory illness and succumbs in 4 days. This model has been used to evaluate the antiviral efficacy of an inhibitor to TMPRSS2. 184 

The route of identification of antiviral candidates through drug repositioning is convenient, saves preclinical development time, and has been deemed safe by regulatory agencies following clinical trials. However, few repositioning efforts have borne fruit since the concept was deployed. 185 Excluding serendipitous findings or drugs retailored based on rational MOA, the challenge remains to reposition drugs to the new target tissue. For COVID-19 treatment, the plethora of autonomous protocols to assess antiviral activity, such as sparse independent triaging, internal orthogonal validation and external data validation data, makes drug repurposing unreliable Therefore, the COVID-19 research community should implement a collective effort to standardize and coordinate screening protocols and deploy the proposed pipeline to identify drug candidates for clinical translation. 

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An OpenData Portal to Share COVID-19 Drug Repurposing Data in Real Time

A Machine Learning Platform to Estimate Anti-SARS-CoV-2 Activities

Comparability of Mixed IC50 Data − A Statistical Analysis

Screening of FDA-Approved Drugs Using a MERS-CoV Clinical Isolate from South Korea Identifies Potential Therapeutic Options for COVID-19. bioRxiv preprint

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2. bioRxiv preprint

The Genome Landscape of the African Green Monkey Kidney-Derived Vero Cell Line

Enhanced Isolation of SARS-CoV-2 by TMPRSS2-Expressing Cells

Analysis of ACE2 in Polarized Epithelial Cells: Surface Expression and Function as Receptor for Severe Acute Respiratory Syndrome-Associated Coronavirus

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

SARS-CoV-2 Entry Factors Are Highly Expressed in Nasal Epithelial Cells Together with Innate Immune Genes

Human Organ Chip-Enabled Pipeline to Rapidly Repurpose Therapeutics during Viral Pandemics

Functional Assessment of Cell Entry and Receptor Usage for SARS-CoV-2 and Other Lineage B Betacoronaviruses

Identification of Inhibitors of SARS-CoV-2 in-Vitro Cellular Toxicity in Human (Caco-2) Cells Using a Large Scale Drug Repurposing Collection. preprint

Caco-2 Cell Line

Replication Kinetics, and Cell Damage Profiling of SARS-CoV-2 and SARS-CoV with Implications for Clinical Manifestations, Transmissibility, and Laboratory Studies of COVID-19: An Observational Study

Patient-Derived Mutations Impact Pathogenicity of SARS-CoV-2; medRxiv preprint

Effect of Multiplicity of Infection on Transcription in Escherichia Coli Cells Infected by Bacteriophage Lambda

Testing Therapeutics in Cell-Based Assays: Factors That Influence the Apparent Potency of Drugs

Fast Identification of Possible Drug Treatment of Coronavirus Disease-19 (COVID-19) through Computational Drug Repurposing Study

Hydroxychloroquine in COVID-19: Potential Mechanism of Action Against SARS-CoV-2

Molecular Structure Analyses Suggest Strategies to Therapeutically Target SARS-CoV-2

Clofazimine Broadly Inhibits Coronaviruses Including SARS-CoV-2

Ontological Modeling and Analysis of Experimentally or Clinically Verified Drugs against Coronavirus Infection

Raloxifene as a Treatment Option for Viral Infections

Antiviral Activity of Digoxin and Ouabain against SARS-CoV-2 Infection and Its Implication for COVID-19. preprint

Pulmonary Delivery of Nanostructured Lipid Carriers for Effective Repurposing of Salinomycin as an Antiviral Agent

Structural Basis for Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

Computational Screening of Repurposed Drugs and Natural Products against SARS-Cov-2 Main Protease (Mpro) as Potential COVID-19 Therapies

A Search for Medications to Treat COVID-19 via in Silico Molecular Docking Models of the SARS-CoV-2 Spike Glycoprotein and 3CL Protease

Silico Potential of Approved Antimalarial Drugs for Repurposing Against COVID-19

Identify Potent SARS-CoV-2 Main Protease Inhibitors via Accelerated Free Energy Perturbation-Based Virtual Screening of Existing Drugs

Veesler, D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Identifying and Repurposing Antiviral Drugs against Severe Acute Respiratory Syndrome Coronavirus 2 with in Silico and in Vitro Approaches

Syncytia Formation by SARS-CoV-2-infected Cells

Structure of the SARS-CoV-2 Spike Receptor-Binding Domain Bound to the ACE2 Receptor

Drug Repurposing for Coronavirus (COVID-19): In Silico Screening of Known Drugs Against the SARS-CoV-2 Spike Protein Bound to Angiotensin Converting Enzyme 2 (ACE2) (6M0J). medRxiv preprint

Identification of SARS-CoV-2 Cell Entry Inhibitors by Drug Repurposing Using in Silico Structure-Based Virtual Screening Approach

Identifying SARS-CoV-2 Entry Inhibitors through Drug Repurposing Screens of SARS-S and MERS-S Pseudotyped Particles

Antiviral Activity of Nucleoside Analogues against SARS-Coronavirus (SARS-CoV)

SARS-CoV-2 RNA Dependent RNA Polymerase (RdRp) − A Drug Repurposing Study

Targeting SARS-CoV-2 RNA-Dependent RNA Polymerase: An in Silico Drug Repurposing for COVID-19

RNA-Dependent RNA Polymerase as a Target for

Mechanism of Inhibition of the Reproduction of SARS-CoV-2 and Ebola Viruses by Remdesivir

Analysis of Therapeutic Targets for SARS-CoV-2 and Discovery of Potential Drugs by Computational Methods

Repurposing FDA-Approved Phytomedicines, Natural Products, Antivirals and Cell Protectives against SARS-CoV-2 (COVID-19) RNA-Dependent RNA Polymerase

The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme

Drug for Corona Virus: A Systematic Review

Dikic, I. Papain-like Protease Regulates SARS-CoV-2 Viral Spread and Innate Immunity

Ebselen as a Highly Active Inhibitor of PL Pro CoV2. bioRxiv preprint

Tideglusib, and Shikonin Are Nonspecific Promiscuous SARS-CoV

The SARS-CoV-2 Main Protease as Drug Target

Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors

Role of Proteolytic Enzymes in the COVID-19 Infection and Promising Therapeutic Approaches

GC-376, and Calpain Inhibitors II, XII Inhibit SARS-CoV-2 Viral Replication by Targeting the Viral Main Protease

Both Boceprevir and GC376 Efficaciously Inhibit SARS-CoV-2 by Targeting Its Main Protease

Soaking Suggests "Alternative Facts": Only Co-Crystallization Discloses Major Ligand-Induced Interface Rearrangements of a Homodimeric TRNA-Binding Protein Indicating a Novel Mode-of-Inhibition

Predicting Commercially Available Antiviral Drugs That May Act on the Novel Coronavirus (SARS-CoV-2) through a Drug-Target Interaction Deep Learning Model

Silico Drug Repurposing for SARS-CoV-2 Main Proteinase and Spike Proteins

Quinolines-Based SARS-CoV-2 3CLpro and RdRp Inhibitors and Spike-RBD-ACE2 Inhibitor for Drug-Repurposing Against COVID-19: An in Silico Analysis

Peptide-like and Small-Molecule Inhibitors against Covid-19

An Investigation into the Identification of Potential Inhibitors of SARS-CoV-2 Main Protease Using Molecular Docking Study

In Silico Prediction of Potential Inhibitors for the Main Protease of SARS-CoV-2 Using Molecular Docking and Dynamics Simulation Based Drug-Repurposing

Drug Repurposing for Candidate SARS-CoV-2 Main Protease Inhibitors by a Novel In Silico Method

Silico Identification of Clinically Approved Medicines against the Main Protease of SARS-CoV-2, Causative Agent of Covid-19

In Silico Drug Repurposing for COVID-19: Targeting SARS-CoV-2 Proteins through Docking and Consensus Ranking

Drug Repurposing and Polypharmacology to Fight SARS-CoV-2 through the Inhibition of the Main Protease

Identification of Atovaquone, Ouabain and Mebendazole as FDA Approved Drugs Tar-Geting SARS-CoV-2 (Version 4). ChemRxiv preprint

Silico Drug Repurposing for Targeting SARS-CoV-2 Mpro

Identification of Potential Molecules against COVID-19 Main Protease through Structure-Guided Virtual Screening Approach

Silico Exploration of the Molecular Mechanism of Clinically Oriented Drugs for Possibly Inhibiting SARS-CoV-2's Main Protease

An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

Camostat Mesylate Inhibits SARS-CoV-2 Activation by TMPRSS2-Related Proteases and Its Metabolite GBPA Exerts Antiviral Activity

TMPRSS2 Inhibitor Discovery Facilitated through an in Silico and Biochemical Screening Platform. bioRxiv preprint

Computational Screening of FDA Approved Drugs of Fungal Origin That May Interfere with SARS-CoV-2 Spike Protein Activation, Viral RNA Replication, and Post-translational Modification: A Multiple Target Approach

Simultaneous Treatment of Human Bronchial Epithelial Cells with Serine and Cysteine Protease Inhibitors Prevents Severe Acute Respiratory Syndrome Coronavirus Entry

An Integrated Drug Repurposing Strategy for the Rapid Identification of Potential SARS-CoV-2 Viral Inhibitors

COVID-19 Drug Repurposing: A Review of Computational Screening Methods, Clinical Trials, and Protein Interaction Assays

Probable Molecular Mechanism of Remdesivir for the Treatment of COVID-19: Need to Know More

Experimental Treatment with Favipiravir for COVID-19: An Open-Label Control Study

Arbidol Combined with LPV/r versus LPV/r Alone against Corona Virus Disease 2019: A Retrospective Cohort Study

Compassionate Remdesivir Treatment of Severe Covid-19 Pneumonia in Intensive Care Unit (ICU) and Non-ICU Patients: Clinical Outcome and Differences in Post-Treatment Hospitalisation Status

Favipiravir versus Arbidol for COVID-19: A Randomized Clinical Trial. medRxiv preprint

The Correlation between Viral Clearance and Biochemical Outcomes of 94 COVID-19 Infected Discharged Patients

Factors Associated with Prolonged Viral Shedding and Impact of Lopinavir/Ritonavir Treatment in Hospitalised Non-Critically Ill Patients with SARS-CoV-2 Infection

Real-World Efficacy and Safety of Lopinavir/Ritonavir and Arbidol in Treating with COVID-19: An Observational Cohort Study

Clinical Efficacy of Lopinavir/Ritonavir in the Treatment of Coronavirus Disease

Arbidol Monotherapy Is Superior to Lopinavir/Ritonavir in Treating COVID-19

Arbidol/IFN-A2b Therapy for Patients with Corona Virus Disease 2019: A Retrospective Multicenter Cohort Study. Microbes Infect

A Screen of Approved Drugs and Molecular Probes Identifies Therapeutics with Anti−Ebola Virus Activity

International Notes Agranulocytosis Associated with the Use of Amodiaquine for Malaria Prophylaxis

FDA Cautions against Use of Hydroxychloroquine or Chloroquine for COVID-19 Outside of the Hospital Setting or a Clinical Trial Due to Risk of Heart Rhythm Problems

Rethinking the Role of Hydroxychloroquine in the Treatment of COVID-19

Hydroxychloroquine, a Less Toxic Derivative of Chloroquine, Is Effective in Inhibiting SARS-CoV-2 Infection in Vitro

In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Breakthrough: Chloroquine Phosphate Has Shown Apparent Efficacy in Treatment

Associated Pneumonia in Clinical Studies

Treating COVID-19 with

Efficacy of Hydroxychloroquine in Patients with COVID-19: Results of a Randomized Clinical Trial. medRxiv preprint

Clinical and Microbiological Effect of a Combination of Hydroxychloroquine and Azithromycin in 80 COVID-19 Patients with at Least a Six-Day Follow up: A Pilot Observational Study

No Evidence of Rapid Antiviral Clearance or Clinical Benefit with the Combination of Hydroxychloroquine and Azithromycin in Patients with Severe COVID-19 Infection

Hydroxychloroquine in Patients with Mainly Mild to Moderate Coronavirus Disease 2019: Open Label

Pneumonia Who Require Oxygen: Observational Comparative Study Using Routine Care Data

Repurposing Anticancer Drugs for COVID-19-Induced Inflammation, Immune Dysfunction, and Coagulopathy

The Immunobiology of SARS

Cytokine Storm in COVID-19: The Current Evidence and Treatment Strategies

Interferon-Mediated Immunopathological Events Are Associated with Atypical Innate and Adaptive Immune Responses in Patients with Severe Acute Respiratory Syndrome

Azithromycin for COVID-19: More Than Just an Antimicrobial?

Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function

Tocilizumab Treatment in COVID-19: A Single Center Experience

The Use of Corticosteroid as Treatment in SARS Was Associated with Adverse Outcomes: A Retrospective Cohort Study

Mis-Assembly of Clathrin Lattices on Endosomes Reveals a Regulatory Switch for Coated Pit Formation

High-Throughput Cell-Based Screen for Chemicals That Inhibit Infection by Simian Virus 40 and Human Polyomaviruses

2-Aminoethoxydiphenyl Borate Affects the Inositol 1,4,5-Trisphosphate Receptor, the Intracellular Ca2+pump and the Non-Specific Ca2+leak from the Non-Mitochondrial Ca2+stores in Permeabilized A7r5 Cells

Calcium Channel Blocker Amlodipine Besylate Is Associated with Reduced Case Fatality Rate of COVID-19 Patients with Hypertension

FDA Approved Calcium Channel Blockers Inhibit SARS-CoV-2 Infectivity in Epithelial Lung Cells

Comparative Analysis of Antiviral Efficacy of FDA-Approved Drugs against SARS-CoV-2 in Human Lung Cells: Nafamostat Is the Most Potent Antiviral Drug Candidate. bioRxiv preprint

Development of a Red-Shifted Fluorescence-Based Assay for SARS-Coronavirus 3CL Protease: Identification of a Novel Class of Anti-SARS Agents from the Tropical Marine Sponge Axinella Corrugata

Minimum Significant Ratio − A Statistic to Assess Assay Variability

Assay Operations for SAR Support

Broad-Spectrum Antivirals against 3C or 3C-Like Proteases of Picornaviruses, Noroviruses, and Coronaviruses

Discovery of N-(Benzo[1,2,3]Triazol-1-Yl)-N-(Benzyl)-Acetamido)Phenyl) Carboxamides as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Inhibitors: Identification of ML300 and Noncovalent Nanomolar Inhibitors with an Induced-Fit Binding

A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19

Differential Immune Activation Profile of SARS-CoV-2 and SARS-CoV Infection in Human Lung and Intestinal Cells: Implications for Treatment with IFN-β and IFN Inducer

Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19

The Global Landscape of SARS-CoV-2 Genomes, Variants, and Haplotypes in 2019nCoVR

Emergence of a Highly Fit SARS-CoV-2 Variant

COVID-19)

Efficacy and Safety of Lopinavir/Ritonavir or Arbidol in Adult Patients with Mild/ Moderate COVID-19: An Exploratory Randomized Controlled Trial

A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19

Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Randomized Clinical Trial

Animal Models of Mechanisms of SARS-COV-2 Infection and COVID-19 Pathology

A Novel Highly Potent Inhibitor of TMPRSS2-like Proteases Blocks SARS-CoV-2 Variants of Concern and Is Broadly Protective against Infection and Mortality in Mice

What Are the Odds of Finding a COVID-19 Drug from a Lab Repurposing Screen?