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Computational Drug Repositioning and Elucidation of Mechanism of Action of Compounds against SARS-CoV-2

The COVID-19 crisis called for rapid reaction from all the fields of biomedical research. Traditional drug development involves time consuming pipelines that conflict with the urgence of identifying effective therapies during a health and economic emergency. Drug repositioning, that is the discovery of new clinical applications for drugs already approved for different therapeutic contexts, could provide an effective shortcut to bring COVID-19 treatments to the bedside in a timely manner. Moreover, computational approaches can help accelerate the process even further. Here we present the application of computational drug repositioning tools based on transcriptomics data to identify drugs that are potentially able to counteract SARS-CoV-2 infection, and also to provide insights on their mode of action. We believe that mucolytics and HDAC inhibitors warrant further investigation. In addition, we found that the DNA Mismatch repair pathway is strongly modulated by drugs with experimental in vitro activity against SARS-CoV-2 infection. Both full results and methods are publicly available.


Introduction
Drug repositioning or drug repurposing aims to find a new clinical application for a drug already in use but for a different purpose. Usually, in drug repurposing, a known and potentially therapeutic target is selected and an experimental search for existing compounds able to modulate the target activity is performed. Computational drug repurposing offers a complementary approach to prioritise compounds for experimental validation. Several different methods have been proposed in the literature [1][2][3] and some have already been applied to SARS-CoV-2 4 . These can be broadly classified into methods searching for small molecules able to bind an active pocket starting from three-dimensional structure of the target protein 5 and those searching for compounds able to modulate the expression of the target mRNA 6, 7 . While most of the existing drug repositioning pipelines focus on the former approach, here we used the latter to identify FDA approved drugs able to modulate the expression of genes expressed in the airway epithelium and that are known to interact with SARS-CoV-2 proteins. Next, we attempted a completely agnostic approach to identify drugs reversing the gene expression profile induced by SARS-CoV-2 infection. Finally, we investigated potential effective mechanisms of action by identifying molecular pathways that are consistently dysregulated by a set of 24 drugs recently proposed as effective to reduce SARS-CoV-2 infection in treated Vero cells. By application of the proper computational tools, we were thus able to identify a set of drugs that should be further experimentally validated and that may have a potential beneficial role in COVID19 treatment, together with insights about mechanisms of action that could help to identify the most effective targets against the infection.
The Gene2drug 6 and Drug Set Enrichment Analysis (DSEA) 9 are two bioinformatic tools for drug-target prioritization and drug mode of action elucidation, respectively (see next Sections). They both use a pathway-based version of the CMap. A Pathway-based Expression Profiles (PEP) is obtained from a Gene Expression Profiles (GEP) by iteratively applying Gene Set Enrichment Analysis (GSEA 10 ) to the GEP for each pathway in a database such as the Gene Ontology or KEGG. In particular, both tools use all the pathway collections included in the MSigDB v6.1 10 , as previously published 9 .
The publicly available online implementation of Gene2drug (http://gene2drug.tigem.it) was used to identify drugs downregulating ACE2 and TMPRSS2. The tool automatically identifies all the gene sets involving the input genes across all of the pathway databases. Similarly, for the DSEA analysis the online tool was used (http://dsea.tigem.it).
Since the Gene2drug analysis applied to the SARS-CoV-2 human molecular interactors required customization, it was performed offline using the gep2pep Bioconductor package 11 . In particular, all the human interactors of each for each of the 27 SARS-CoV-2 proteins 12 were defined as a gene-set. The 27 obtained gene sets were then added to each of the pathway collections included in the MSigDB v7 10 . Finally, the CMap GEPs were converted to PEPs based on these newly created gene set collections. In order to prioritize drugs according to their PEPs, Gene2drug analysis was performed for the 27 SARS-CoV-2 related gene sets, using all the MSigDB gene sets as statistical background. Since the statistical background was different for each gene set collection, the average scores were computed to obtain the final prioritization. The dataset of PEPs is publicly available 13 .
Drugs predicted to reverse the SARS-CoV-2 induced signature were identified using the publicly avaliable MANTRA tool (http://mantra.tigem.it). RNA-seq raw counts for the identification of differentially expressed genes (DEGs) in response to SARS-CoV-2 were downloaded from GEO database (accession number GSE147507). Raw counts were first normalized using the edgeR tool while DEGs identified by using the limma package with its voom method in the R statistical environment 3.6. The obtained GEP was added to the MANTRA network using the "reverse node" feature. In this way, the GEP is reversed (most up-regulated genes appear as the most down-regulated and vice-versa) and the closest nodes in the network correspond to drugs inducing the opposite GEP as compared to that of SARS-CoV-2 infection.
Two-dimensional structure images and annotations for the compounds reported in the tables were obtained from the PubChem database 14 .

Identification of drugs reducing the expression ACE2 and TMPRSS2
It has been recently shown tha SARS-CoV-2 entry in host cells is mediated by the ACE2 receptor and requires priming by the TMPRSS2 protease 12 . We thus sought to identify drugs that could potentially reduce the expression of both genes, although the benefit of such an approach is being debated 15 . To this end, we applied a computational drug repositioning approach named Gene2Drug (http://gene2drug.tigem.it) 6 . This tool computes an Enrichment Score (ES) and the corresponding P-value for each of 1309 small molecules of the Connectivity Map dataset (Broad Institute), including FDA approved drugs, based on how much they tend to down-regulate the genes of interest (ACE2 and TMPRSS2), as well as other genes involved in the same pathways. Results from the analysis identified 10 drugs shown in Table 1 (p < 0.05). Most of these drugs are potentially relevant: carbenoxolone has shown antiviral activity against the Dengue virus 16 ; indomethacin is a non-steroidal anti-inflammatory drug (NSAID) inhibiting Prostaglandin E2 synthase (PTGES2) with a demonstrated efficacy against SARS-CoV 17 . Interestingly, in a recent study, SARS-CoV-2 viral NSP7 was found to interact with PTGES2 12 and thus the authors suggested indomethacin as potentially useful in treating patients. Here, however, indomethacin was selected by Gene2Drug because it appears to lower expression of genes in the ACE2 pathway, which does not include PTGES2 itself. Therefore indomethacin could be a high priority molecule to be tested as it could have a double effect. Another NSAID is present among the drugs identified in Table  1, nimesulide which could have a similar effect as indomethacin. Gene2Drug also identified nicarpidine, an angiotensin inhibitor, an obvious candidate but found using a purely data-driven approach.

Identification of drugs reducing expression of SARS-CoV-2 interactors
Another potentially effective approach to the identification of therapeutic agents counteracting SARS-CoV-2 infection is to target the host-virus interaction. In order to investigate cellular proteins interacting with viral proteins, we analyzed a set of 332 protein interactions between 26 SARS-CoV-2 proteins (plus 1 mutant) and human proteins obtained by performed an affinity purification-mass spectrometry analysis 12 . Since each of these interactions could be key to hijack the host during the course of infection, we sought to prioritize existing drugs potentially interfering with them. In particular, we applied once more Gene2drug 6 , this time exploiting its gene-set wise analysis capability. Specifically, for each viral protein, we generated a corresponding gene-set containing all the host protein interactors, thus obtaining 27 gene-sets. We then applied Gene2Drug to identify those compounds that are able to downregulate the expression of most of the genes across the 27 gene-sets at the same time, which appears a particularly effective strategy given the currently limited understanding of the specific infection mechanisms. The top 20 hits are reported in Table 2. Interestingly, one of the hits is niclosamide, an antihelminthic drug, that 2/11 was found to have antiviral efficacy against SARS-CoV-2 in a recent screening using VERO cells 18 . Other notable candidates found by our analysis are: fenoterol, which was also identified as potentially effective in a recent study on computational drug repositioning 19 ; alexidine, an antibiotic and a selective inhibitor of the mitochondrial phosphatase Ptpmt1, that was found to inhibit replication of the CytoMegaloVirus (CMV) infection 20 ; oxytetracycline, another antibiotic that has shown antiviral activity against Dengue virus 21 and the ability to reduce HIV-1 RNA transcripts within extracellular vescicles 22 ; clofibrate, a peroxisome-proliferator activated receptor-alpha (PPAR-) agonist previously used as a cholesterol-lowering agent, which was found to have antiviral activity against MDV, an alphaherpesvirus that infects chickens and causes a deadly lymphoma 23 ; flupentixol, a neuroleptic agent that was found to inhibit hepatitis C virus entry 24 and coxsackievirus replication 25 ; gossypol, a natural phenol derived from the cotton plant known to inhibit spermatogenesis, that was shown to have also antiviral activity 26 ; ms-275 and trichostatin A, two histone deacetylases inhibitors (HDACi), the latter found to inhibit expression of herpes simplex virus genes 27 ; interestingly valproic acid, another HDACi, was proposed for repositioning against SARS-CoV-2, as the viral protein NSP5 was found to interact with HDAC2 12 .

Identification of drugs reversing the transcriptional signature induced by SARS-CoV-2 infection
A data-driven approach to drug repositioning is to identify compounds that are able to revert a disease-related signature 3 . We developed the MANTRA tool exploiting the cMap dataset (Broad Institute) to generate and explore drug networks for drug repositioning 28,29 . In drug networks, nodes represent drug-induced transcriptional profiles, and edges represent similarities between them. We sought to use this approach to prioritize drugs reverting SARS-CoV-2 induced gene expression at the wholegenome level without assuming any prior knowledge of molecular mechanisms involved in the disease progression. Recently, gene expression profiles of primary human bronchial epithelial (NHBE) infected with SARS-CoV-2 have been made publicly available 30 (GSE147507 from the GEO database). We obtained the corresponding raw data and computed the differential expression of 15,330 genes between infected and non infected cells. Out of these, only 37 genes were significantly differentially expressed (adjusted p < 0.1), most of which were over-expressed in infected cells and included interleukin 6 (IL6) and 8 (IL8), chemokines and interferon alpha inducible proteins. The MANTRA approach is based on Gene Set Enrichment Analysis and therefore it requires in input a list of all the measured genes ranked according to their differential expression, including both significant and non-significant genes. We thus sorted all the 15,330 genes according to their differential expression, but with genes most down-regulated following infection at the top of the list and those most up-regulated at the bottom. We then queried the MANTRA drug network with this profile and looked for drugs inducing a similar transcriptional profile, which therefore may potentially induce a profile opposite to that of the infection. We thus found 11 significant drugs (transcriptional distance < 0.8) listed in Table 3. One of the 11 drugs is sirolimus, also known as rapamycin, an inhibitor of mechanistic Target Of Rapamycin Complex 1 (mTORC1), and used clinically as an immunosuppressive agent. This is an obvious candidate for this kind of drug repositioning, where the aim is to find a drug inducing an opposite repsonse to that of the virus, as most of the genes overexpressed during viral infection are immune-related. Of course, this does not mean that it is clinically relevant as suppressing the immune response could be detrimental to the cells. Nevertheless, Rapamycin was also suggested in the SARS-CoV-2 interactors' study based on the nuclecapsid interaction with the mTOR translational repressors LARP1 12 and on rapamycin reported in vitro activity against MERS 31 . Other interesting candidates found by MANTRA are: ambroxol (a metabolite of bromhexine), a mucolytic agent with reported antiviral activity against influenza-virus among others 32, 33 and with inhibition activity against TMPRSS2 34 ; corticosterone, a glucocorticoid; idoxuridine, a nucleoside analogue used to inhibit replication of DNA viruses; naltrexone, a mu-opiod receptor antagonist used for opioid and alcohol dependence, with reported antiinflammatory effect 35 ; nordihydroguaiaretic acid an antioxidant compound found in the creosote bush (Larrea tridentata) with reported antiviral activity against West Nile and Zika viruses 36 .

Mechanism of action of compounds with reported in vitro antiviral action against SARS-CoV-2 infec-
tion. Understanding the biological mechanisms underlying a pathological condition at the molecular level can greatly help the identification of potentially effective drugs. One way of obtaining such knowledge is the analysis of existing small molecules that have been observed to elicit varying levels of therapeutic efficacy through drug screening. Although the mode of action of positive hits from large-scale drug screening can be difficult to identify, it is likely that such hidden mechanism is shared among most of the hits. In order to investigate and exploit this assumption, we previously developed the Drug Set Enrichment Analysis (DSEA 9 , http://dsea.tigem.it). DSEA is able to highlight molecular pathways that are consistently and specifically dysregulated by most drugs in a set by analyzing drug-induced expression profiles in the CMap dataset.
In order to identify compounds that are able to counteract SARS-CoV-2 infection, Jeon et al 18 recently screened a collection of 35 small molecules previously observed as potentially effective against SARS-CoV infection, plus 15 drugs suggested by infectious diseases specialists. Among the 50 screened drugs, 24 showed reduction of the SARS-CoV-2 N protein levels in infected VERO cells 18 . Of the 24 drugs, 10 were present in the cMap dataset used by DSEA (amodiaquine, ciclosporin, desoxycortone, digoxin, loperamide, mefloquine, niclosamide, ouabain, proscillaridin, tetrandrine). We thus sought to analyze 3/11 these 10 drugs through the DSEA tool in order to understand the common mechanism of action underlying their antiviral effect. These drugs were found by DSEA to modulate targets of the transcription factors (TFs) shown in Table 4, including: NF-kB, a master regulator of stress response; ATF6, a TF involved in the integrated stress response and with a known role in enhancing herpesvirus gene expression 37 ; STAT5B, a member of the STAT family activated in response to cytokines and growth factor. DSEA also highlighted several biological processes as modulated by these drugs (Table 4), among which those related to DNA mistmatch repair pathway (MMR). Interestingly, DNA MMR pathway activation has been recently reported to be required for the replication of the coronavirus infection bronchitis virus (IBV) 38 .

Conclusions
Computational methods for drug repositioning could narrow down the search for effective drugs to counteract novel epidemics.
Here we showed how a number of existing tools can be used towards this aim, covering different approaches such as targeting of host proteins interacting with viral proteins, reversion of disease-induced transcriptional profiles, and mode of action investigation for drugs with demonstrated in vitro activity against SARC-CoV-2 infection. Overall, we found 39 compounds that could be tested experimentally among which NSAIDs, antihistamines, antibiotics and antihelmintics, antipsychotics, corticosteroids, mucolytics and HDAC inhibitors (HDACi). Bromhexine, of which ambroxol is a metabolite, has been recently proposed as a prophylactic treatment for SARS-COV-2 patients 39 based on its reported activity against TMPRSS2 and its over-the-counter availability and safety levels. In our study, we find that ambroxol also induces a transcriptional profile opposite to that induced by SARS-CoV-2 infection in vitro, adding to the evidence of a possible role of this drug in counteracting the virus. Of interest, HDAC2 was found to interact with SARS-CoV-2 proteins 12 and the screening on inhibitors of infection on VERO cells identified several members of the cardiac glycosides as effective 18 . Cardiac glycosides have been recently found to have strong HDACi activity 40,41 . It will be therefore interesting to verify experimentally whether HDACi have beneficial role in reducing viral replication in host cells. Finally, by interrogating the possible mode of action of positive hits of a drug screening in VERO cells 18 , we identified the DNA MMR pathway as possibly involved in the mode of action of these drugs, and therefore hinting that this pathway could be involved in the host response to the viral infection. Interestingly, HDAC proteins are involved in the modulation of the DNA MMR pathway, thus hinting that HDAC inhibtors, if proven to be effective, may act through this pathway.

Data Availability
All the cited methods, the full lists of prioritized drugs and pathways, and all the necessary data to reproduce the results, are publicly available 13 .

Funding
The

Acknowledgements
We thank Diego Carrella of the TIGEM Bioinformatics Core for support with the MANTRA webtool. Non-steroidal anti-inflammatory drug most commonly used in rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, acute shoulder pains, and acute gouty arthritis.     Table 4. Drug Set Enrichment Analysis results for 24 small molecules showed to lower the level of the N protein in SARS-CoV-2. The reported pathways are consistently dysregulated by most drugs in the set as found by the DSEA based on the analysis of their induced transcriptomes. Top 10 pathways are reported for the Transcription Factor Targets collection and for the three Gene Ontology collections. Enrichment Scores (ES) and the corresponding p-value (PV) are reported for each gene set.