Transcriptomics Based Drug Repositioning Pipeline Identifies Therapeutic Candidates For COVID 19

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OPEN Transcriptomics‑based drug

repositioning pipeline identifies
therapeutic candidates
for COVID‑19
Brian L. Le1,2,11, Gaia Andreoletti1,2,11, Tomiko Oskotsky1,2,11, Albert Vallejo‑Gracia3,
Romel Rosales4,5, Katharine Yu1,2,6, Idit Kosti1,2, Kristoffer E. Leon3, Daniel G. Bunis1,2,6,
Christine Li1,2,7, G. Renuka Kumar3, Kris M. White4,5, Adolfo García‑Sastre4,5,8,9,
Melanie Ott3,10 & Marina Sirota1,2*
The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied
a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures
derived from publicly available data. We utilized three independent published studies to acquire or
generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples.
Using a rank-based pattern matching strategy based on the Kolmogorov–Smirnov Statistic, the
signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our
top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation
experiments in human cell lines showed that 11 of the 16 compounds tested to date (including
clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These
initial results are encouraging as we continue to work towards a further analysis of these predicted
drugs as potential therapeutics for the treatment of COVID-19.
SARS-CoV-2 has already claimed at least a million lives, has been detected in at least 40 million people, and has
likely infected at least another 200 million. The spectrum of disease caused by the virus can be broad ranging
from silent infection to lethal disease, with an estimated infection-fatality ratio around 1%1. SARS-CoV-2 infec-
tion has been shown to affect many organs of the body in addition to the lungs2. Three epidemiological factors
increase the risk of disease severity: increasing age, decade-by-decade, after the age of 50 years; being male; and
various underlying medical conditions1. However, even taking these factors into account, there is immense inter-
individual clinical variability in each demographic category considered3. Recently, researchers found that more
than 10% of people who develop severe COVID-19 have misguided antibodies―autoantibodies―that attack the
innate immune system. Another 3.5% or more of people who develop severe COVID-19 carry specific genetic
mutations that impact innate immunity. Consequently, both groups lack effective innate immune responses that
depend on type I interferon, demonstrating a crucial role for type I interferon in protecting cells and the body
from COVID-19. Whether the type I interferon has been neutralized by autoantibodies or―because of a faulty
gene―is produced in insufficient amounts or induced an inadequate antiviral response, the absence of type I
IFN-mediated immune response appears to be a commonality among a subgroup of people who suffer from
life-threatening COVID-19 p neumonia3.
While numerous efforts are underway to identify potential therapies targeting various aspects of the disease,
there is a paucity of clinically proven treatments for COVID-19. There have been efforts to therapeutically target
the hyperinflammation associated with severe COVID-194, as well as to utilize previously identified antiviral
1
Department of Pediatrics, UCSF, San Francisco, CA, USA. 2Bakar Computational Health Sciences Institute,
UCSF, San Francisco, CA, USA. 3Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA,
USA. 4Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 5Global Health
and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 6Biomedical
Sciences Graduate Program, UCSF, San Francisco, CA, USA. 7Shanghai American School, Shanghai, China. 8Division
of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 9The
Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 10Department of Medicine,
UCSF, San Francisco, CA, USA. 11These authors contributed equally: Brian L. Le, Gaia Andreoletti and Tomiko
Oskotsky. *email: [email protected]
Scientific Reports | (2021) 11:12310 | https://doi.org/10.1038/s41598-021-91625-1 1
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medications5,6. One of these antivirals, remdesivir, an intravenously administered RNA-dependent RNA poly-
merase inhibitor, showed positive preliminary results in patients with severe COVID-197. In October 2020, the
FDA approved remdesivir for the treatment of COVID-198. Dexamethasone has also been shown to reduce the
mortality rate in cases of severe COVID-199.
Nevertheless, the lack of treatments and the severity of the current health pandemic warrant the explora-
tion of rapid identification methods of preventive and therapeutic strategies from every angle. The traditional
paradigm of drug discovery is generally regarded as protracted and costly, taking approximately 15 years and
over $1 billion to develop and bring a novel drug to m arket10. The repositioning of drugs already approved for
human use mitigates the costs and risks associated with early stages of drug development, and offers shorter
routes to approval for therapeutic indications. Successful examples of drug repositioning include the indication
of thalidomide for severe erythema nodosum leprosum and retinoic acid for acute promyelocytic leukemia11. The
development and availability of large-scale genomic, transcriptomic, and other molecular profiling technologies
and publicly available databases, in combination with the deployment of the network concept of drug targets
and the power of phenotypic screening, provide an unprecedented opportunity to advance rational drug design.
Drug repositioning is being extensively explored for COVID-19. High-throughput screening pipelines have
been implemented in order to quickly test drug candidates as they are identified12–15. In the past, our group
has successfully applied a transcriptomics-based computational drug repositioning pipeline to identify novel
therapeutic uses for existing drugs16. This pipeline leverages transcriptomic data to perform a pattern-matching
search between diseases and drugs. The underlying hypothesis is that for a given disease signature consisting of
a set of up and down-regulated genes, if there is a drug profile where those same sets of genes are instead down-
regulated and up-regulated, respectively, then that drug could be therapeutic for the disease. This method based
on the Kolmogorov–Smirnov (KS) test statistic has shown promising results for a variety of different indications,
including inflammatory bowel d isease17, dermatomyositis18, cancer19–21, and preterm b
irth22.
In existing work from Xing et al.23, this pipeline has been used to identify potential drug hits from multiple
input disease signatures derived from SARS-CoV or MERS-CoV data. The results were aggregated to obtain a
consensus ranking, with 10 drugs selected for in vitro testing against SARS-CoV-2 in Vero E6 cell lines, with four
drugs (bortezomib, dactolisib, alvocidib and methotrexate) showing viral inhibition23. However, this pipeline
has not yet been applied specifically to SARS-CoV-2 infection.
A variety of different transcriptomic datasets related to SARS-CoV-2 were published in the spring of 2020.
In May 2020, Blanco-Melo et al. studied the transcriptomic signature of SARS-CoV-2 in a variety of different
systems, including human cell lines and a ferret m odel24. By infecting human adenocarcinomic alveolar basal
epithelial cells with SARS-CoV-2 and comparing to controls, the authors generated a list of 120 differentially
expressed genes. They observed two enriched pathways: one composed primarily of type-I interferon-stimu-
lated genes (ISGs) involved in the cellular response to viral infection; and a second composed of chemokines,
cytokines, and complement proteins involved in the humoral response. After infecting the cell lines, Blanco-Melo
et al. did not detect either ACE2 or TMPRSS2, which are the SARS-CoV-2 receptor and SARS-CoV-2 protease,
respectively25. However, supported viral replication was observed, thereby allowing the capture of some of the
biological responses to SARS-CoV-2.
In May 2020, another study by Lamers et al. examined SARS-CoV-2 infection in human small intestinal orga-
noids grown from primary gut epithelial stem cells26. The organoids were exposed to SARS-CoV-2 and grown
in various conditions, including Wnt-high expansion media. Enterocytes were readily infected by the virus, and
RNA sequencing revealed upregulation of cytokines and genes related to type I and III interferon responses.
A limited amount of transcriptomic data from human samples has also been published. One study detailed
the transcriptional signature of bronchoalveolar lavage fluid (of which responding immune cells are often a
primary component) of COVID-19 patients compared to controls27. Despite a limited number of samples, the
results revealed inflammatory cytokine profiles in the COVID-19 cases, along with enrichments in the activation
of apoptosis and the P53 signaling pathways.
On the drug side, data are available in the form of differential gene expression profiles from testing on human
cells. Publicly-available versions include the Connectivity Map (CMap)28, which contains genome-wide testing
on approximately 1300 drugs, wherein the differential profile for a drug was generated by comparing cultured
cells treated with the drug to untreated control cultures.
Here, we applied our existing computational drug repositioning pipeline to identify drug profiles with sig-
nificantly reversed differential gene expression compared to several diverse input signatures for SARS-CoV-2
effects on human cells. By taking into account a broader view of differentially expressed gene sets from both
cell line and organoid disease models and human samples, the predictions are complementary to other drug
discovery approaches. We identified 102 unique drug hits, from which 25 were identified in at least two of the
signatures, several of which have been already investigated in clinical trials. We furthermore explore our find-
ings in the context of other computational drug repurposing efforts for COVID-19. Finally, we tested 16 of our
top predicted hits in live SARS-CoV-2 antiviral assays. Four of the top predicted inhibitors were tested for virus
inhibition in a human lung cell line, Calu-3, infected with SARS-CoV-2 with quantitation of the secreted virus
assessed by RT-qPCR assay. Thirteen predicted inhibitors (including one tested in Calu-3) were incubated with
SARS-CoV-2 infected human embryonic kidney 293T cells overexpressing ACE2 (293T-ACE2) with viral rep-
lication determined using an immunofluorescence-based assay.
Results
In this study, we applied our drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures
derived from publicly available RNA-seq data (Fig. 1). The transcriptomic data were generated from distinct
types of tissues, so rather than aggregating them together, we predicted therapeutics for each signature and then
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Figure 1. COVID-19 transcriptomics-based bioinformatics approach for drug repositioning. We generated

lists of statistically significant differentially expressed genes from the analysis of three published studies of
SARS-CoV-2 and COVID-19. The drug repositioning computational pipeline compares the ranked differential
expression of the COVID-19 disease signature with that of drug profiles from CMap. A reversal score based
on the Kolmogorov–Smirnov statistic is generated for each disease-drug pair. If a drug profile significantly
(FDR < 0.05) reverses the disease signature, then the drug could be therapeutic for the disease. Across all
datasets, a total of 102 drugs have been identified as potentially therapeutic for COVID-19. Twenty-five drugs
were identified in analyses of at least two of the three datasets. We further conducted pathways analyses and
targeted analyses on the results, focusing on the 25 shared hits. Finally, we validated 16 of our top predicted hits
in live SARS-CoV-2 antiviral assays.
combined the results. We utilized three independent gene expression signatures (labelled “ALV”, “EXP”, and
“BALF”), each of which consisted of lists of differentially expressed genes between SARS-CoV-2 samples and
their respective controls. The ALV signature was generated from human adenocarcinomic alveolar basal epithelial
cells by comparing SARS-CoV-2 infection to mock-infection c onditions24. The EXP signature originated from a
study where organoids, grown from human intestinal cells expanded in Wnt-high expansion media, were infected
with SARS-CoV-2 and then compared to c ontrols26. The BALF signature was from a contrast of primary human
BALF samples from two COVID-19 patients versus three controls27. Each of these signatures was contrasted
with drug profiles of differential gene expression from CMap.
For each of the input signatures, we applied a significance threshold false discovery rate (FDR) < 0.05. We
further applied minimum fold change thresholds in order to identify the driving genes. The ALV signature had
only 120 genes, with 109 genes shared with the drug profiles; in order to maintain at least 100 genes for the
pattern-matching algorithm to work with, we applied no fold-change threshold. For the EXP signature, we applied
a |log2FC| > 2 cutoff, resulting in 125 genes for the expansion signature (108 shared with the drug profiles). For
the BALF signature, we processed the raw read count data to calculate differential gene expression values. We
applied a |log2FC| > 4 cutoff, with the BALF data yielding 1349 protein-coding genes for the lavage fluid signature
(941 shared with the drug profiles). As the sample types across the three datasets were very different, we used
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Figure 2. SARS-CoV-2 differential gene expression signatures reversed by drug profiles from CMap. (A)
Enrichment analysis using GSEA reveals common pathways among input signatures. (B) DEG overlap from
input signatures. Only 1 gene, DKK1, was shared by all 3 signatures. (C) Top 15 drug profiles reversing the ALV
signature (109 genes). For each column, the gene expression values were ranked, with rank 1 being the most
up-regulated gene (in red) and the maximum rank (109 for ALV) being the most down-regulated gene (in blue).
Drug names highlighted in green were hits for a second signature, and drug hits highlighted in purple reversed
all three signatures. (D) Top 15 drug profiles reversing the EXP signature (108 genes). (E) Top 15 drug profiles
reversing the BALF signature (941 genes).
different fold change thresholds to identify the appropriate gene signatures to be used for drug repurposing. The
gene lists for each of these signatures can be found in the supplement (Tables S1, S2, S3).
We used GSEA (Gene Set Enrichment Analysis)29,30 to annotate enriched (FDR 0.05) Hallmark pathways
from each of the input signatures (Fig. 2A). A number of pathways common to at least two signatures were
found. Interferon alpha response and interferon beta response were upregulated in the ALV and EXP signatures.
Adipogenesis and cholesterol homeostasis pathways were downregulated in the EXP and BALF signatures.
KRAS signaling, and mTORC1 (mammalian target of rapamycin complex 1) signaling were enriched in all three
signatures, but not in the same direction, showing the diversity of effects SARS-CoV-2 may have on human cells,
and highlighting a need for utilization of diverse profiles as we do in the present study. When we look at the
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Drug hit Description (current uses) ALV reversal score EXP reversal Score BALF reversal score
Bacampicillin Antibiotic 0.789 0.790 0.596
Benzocaine Anesthetic n.s. 0.766 0.546
Ciclopirox Antifungal n.s. 1 0.361
Immunosuppressant (RA, psoriasis,
Ciclosporin 0.756 n.s. 0.409
Crohn’s)
Clofazimine Antimycobacterial (leprosy) 0.946 0.893 0.558
Ergoid mesylate (dementia, Alzheimer’s,
Co-dergocrine mesilate 0.775 n.s. 0.553
stroke)
Dicycloverine Antispasmodic (IBS) 0.847 n.s. 0..461
Fludrocortisone Corticosteroid n.s. 0.782 0.519
Fluticasone Steroid (asthma, COPD) 0.790 n.s. 0.463
Haloperidol Antipsychotic (schizophrenia) 0.937 0.773 0.507
Isoxicam NSAID n.s. 0.873 0.410
Lansoprazole Proton-pump inhibitor (acid reflux) 0.856 n.s. 0.370
Levopropoxyphene Antitussive n.s. 0.835 0.770
Antineoplastic (Hodgkin’s disease, brain
Lomustine 0.748 n.s. 0.338
tumors)
Metixene Anticholinergic (Parkinson’s) 0.759 n.s. 0.344
Myricetin Flavonoid n.s. 0.823 0.603
Niclosamide Anthelmintic (tapeworms) 0.812 n.s. 0.360
Nocodazole Antineoplastic 0.766 n.s. 0.439
Vasodilatory and anti-inflammatory
Pentoxifylline n.s. 0.791 0.552
(claudication)
Sirolimus Immunosuppressive n.s. 0.768 0.729
Thiamazole Antithyroid agent (Graves disease) n.s. 0.796 0.724
Tocainide Antiarrhythmic 0.798 n.s. 0.714
Vitamin A derivative (acne, acute promye-
Tretinoin n.s. 0.854 0.579
locytic leukemia)
Valproic acid Anticonvulsant (seizures, bipolar disorder) 0.917 0.786 0.546
Zuclopenthixol Antipsychotic (schizophrenia) 0.754 n.s. 0.535
Table 1. Therapeutic hits reversing at least 2 of input SARS-CoV-2 signatures. A wide variety of drugs were
identified by the analysis of multiple signatures. Drug reversal scores are normalized for each signature; drug
entries marked “n.s.” were not significant for reversing that signature.
contributing genes within the three signatures (Fig. 2B), we found one overlapping upregulated gene—Dick-
kopf WNT Signaling Pathway Inhibitor 1 (DKK1). We used the publicly available single-cell RNAseq dataset
GSE12803331 composed of 13 patients (4 healthy, 3 presenting with mild COVID-19 symptoms, and 6 presenting
with severe COVID-19 symptoms) to further characterize the expression of DKK1 (Figure S1). Data were re-ana-
lyzed following the standard Seurat pipeline. From the analyses of the single-cell data, DKK1 is highly expressed
in COVID-19 patients compared to controls, specifically in severe patients and it is expressed by epithelial cells.
After analyzing the input SARS-CoV-2 signatures, we utilized our repositioning pipeline to identify drugs
with reversed profiles from CMap (Fig. 1). Significantly reversed drug profiles were identified for each of the
signatures using a permutation approach: 30 hits from the ALV signature (Table S4), 15 hits from the EXP sig-
nature (Table S5), and 86 hits from the BALF signature (Table S6). When visualizing the gene regulation of the
input signatures and their respective top 15 drug hits, the overall reversal pattern can be observed (Fig. 2C–E).
Interestingly, we found several drugs shared across datasets that significantly reversed the disease signature. For
example, haloperidol, highlighted in purple in Fig. 2C–E, was shown to reverse the disease gene signature from
three datasets, whereas levopropoxyphene, shown in green in Fig. 2D,E, was observed to reverse the disease gene
signature from two of the datasets. In total, our analysis identified 102 unique drug hits (Table S7). Twenty-five
drug hits reversed at least two signatures (p = 0.0334, random sampling), and four drug hits reversed three sig-
natures (p = 0.0599, random sampling) (Table 1, Fig. 3A).
We further characterized the common hits by examining their interactions with proteins in humans. We used
known drug targets from DrugBank32 and predicted additional targets using the similarity ensemble approach
(SEA)33. We visualized the known interactions from DrugBank in a network. Figure 3B shows the connectivity
across compounds highlighting both single drug genes (such as SIGMAR1 for haloperidol) and genes shared
across drugs, such as ADRA2A and DRD1 for haloperidol and co-dergocrine mesilate. The proteins with the most
known interactions with our list of 25 drugs included adrenergic receptors (particularly α2 adrenoreceptors),
dopamine receptors, and serotonin receptors.
To confirm the validity of our approach, the inhibitory effects of 16 of our drug hits which significantly
reversed multiple SARS-CoV-2 profiles were assessed in live pCC50 antiviral assays. Next, we wanted to test the
potential of the predicted compounds to inhibit viral activity robustly using different human cell lines—Calu-3
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Figure 3. Common therapeutic hits from drug repurposing pipeline applied to SARS-CoV-2 signatures. (A)
Drug profiles from CMap significantly reversed signatures from the ALV, BALF, and EXP signatures. 25 of the
drugs were significant in at least 2 of the signatures. (B) Drug-protein target network. For the 25 drugs that
reversed at least 2 of the signatures, target information was gathered from DrugBank to identify clusters of drugs
from shared targets.
and 293T-ACE2. The respective selection of 16 and 13 compounds for testing was based on side effect profiles
and compound availability.
The inhibitory effects of haloperidol, clofazimine, valproic acid, and fluticasone were evaluated in SARS-
CoV-2 infected Calu-3 cells (human lung epithelial cell line), with remdesivir also tested as a positive control.
From these five, remdesivir and haloperidol inhibited viral replication (Fig. 4A), and the inhibitory effect was
also observed by microscopy (Fig. 4B). Toxicity assessments for haloperidol, clofazimine, valproic acid, and
fluticasone were evaluated using viability assays (Alamarblue) in Calu-3 cells treated with each compound for
72 h (n = 1, with 2 technical replicates). No significant differences between controls and the biological replicates
were detected using a non-parametric test (Kruskal–Wallis) (Figure S6). Fluticasone and bacampicillin showed
some toxicity in a dose-dependent manner at the highest doses tested. Haloperidol, clofazimine and valproic
acid did not show significantly reduced cell viability (Figure S6).
Additionally, 13 drugs (bacampicillin, ciclopirox, ciclosporin, clofazimine, dicycloverine, fludrocortisone,
isoxicam, lansoprazole, metixene, myricetin, pentoxifylline, sirolimus, tretinoin) were independently assessed
in a live SARS-CoV-2 antiviral assay. Remdesivir was again used as a positive control. This testing involved six
serial dilutions of each drug to inhibit the replication of SARS-CoV-2 in 293T-ACE2 cells using an immunoflu-
orescence-based antiviral a ssay34. All antiviral assays were paired with cytotoxicity assays using identical drug
concentrations in uninfected human 293T-ACE2 cells. Positive control remdesivir and 10 of our predicted drugs
(bacampicillin, ciclopirox, ciclosporin, clofazimine, dicycloverine, isoxicam, metixene, pentoxifylline, sirolimus,
and tretinoin) showed antiviral efficacy against SARS-CoV-2, reducing viral infection by at least 50%, that was
distinguishable from their cytotoxicity profile when tested in this cell line (Fig. 5). Several inhibitors showed
micromolar to sub-micromolar antiviral efficacy, including clofazimine, ciclosporin, ciclopirox, and metixene.
These results not only confirm our predictive methods, but have also identified several clinically-approved drugs
with potential for repurposing for the treatment of COVID-19.
Discussion
Here, we used a transcriptomics-based drug repositioning pipeline to predict therapeutic drug hits for three
different input SARS-CoV-2 signatures, each of which came from distinct human cell or tissue origins. We
found significant overlap of the therapeutic predictions for these signatures. From 102 total drug hits, 25 drugs
reversed at least two signatures (p = 0.0334) and 4 drugs reversed three signatures (p = 0.0599). The diversity of
such signatures yet overlap of highlighted drugs underscores the utility of the current pipeline for identification
of drugs which might be therapeutic for the diverse effects of SARS-CoV-2 infection.
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Figure 4. Haloperidol inhibits viral replication of SARS-CoV-2 in the Calu-3 lung cell line. (A) Calu-3
cells were infected with SARS-CoV-2 at an MOI of 0.05 for 72 h. Viral replication levels were determined by
RT-qPCR from supernatant RNA using specific primers for the E gene. Viral RNA levels relative to DMSO
are graphed. Error bars represent 3 or 4 independent experiments. One-way ANOVA analysis was used to
determine significance. (B) Microscopy: Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.05 for
72 h. Cells were fixed with paraformaldehyde and used for immunofluorescence analysis with dsRNA antibody
(SCICONS) and DAPI stain. Images were acquired and analyzed using ImageXpress Micro Confocal High-
Content Imaging System.
Twenty-five of our drug hits reversed at least two of the three input signatures (Fig. 3). Notably, 14 of the 15
hits from the EXP signature were also hits for the BALF signature, despite being generated from different types
of tissue. The EXP signature was generated from intestinal tissue, whereas the BALF signature was generated
from constituents of the respiratory tract. Among the common hits reversing at least two of the signatures were
two immunosuppressants (ciclosporin and sirolimus), an anti-inflammatory medication (isoxicam), and two
steroids (fludrocortisone and fluticasone). Sirolimus (or rapamycin), an immunosuppressant and an mTOR
inhibitor, is currently undergoing investigation in several clinical trials in COVID-19 patients (NCT04371640,
NCT04341675, NCT04461340). Other hits currently in clinical trials for COVID-19 treatment include ciclo-
sporin (NCT04412785, NCT04392531), niclosamide in combination with diltiazem (NCT04558021), and clo-
fazimine in combination with interferon beta-1b (NCT04465695).
Among our four drug hits that reversed all three signatures, three drugs demonstrated in vitro antiviral
efficacy—bacampicillin, clofazimine, and haloperidol with no toxicity effects (Figure S6). Our group found
haloperidol decreased viral growth in SARS-CoV-2 infected Calu-3 cells (Fig. 4B) in a dose-dependent manner
(Fig. 4A). Haloperidol is a psychiatric medication that is indicated for the treatment of psychotic disorders includ-
ing schizophrenia and acute psychosis. By blocking dopamine (mainly D2) receptors in the brain, haloperidol
eliminates dopamine neurotransmission which leads to improvement of psychotic symptoms35. Haloperidol
can also bind to the sigma-1 and sigma-2 receptors, which are implicated in lipid remodeling and cell stress
response12. As reported by Gordon et al.12, the SARS-CoV-2 proteins Nsp6 and ORF9c interact with the sigma-1
receptor and the sigma-2 receptor, respectively. Moreover, they found that haloperidol decreased viral replica-
tion in SARS-CoV-2-infected Vero E6 cells with, based on their reported pIC50 and pCC50 values, a calculated
Selectivity Index (SI) of 53.712. An SI greater than 10 is the generally accepted minimum cut-off for an antiviral
worth pursuing36,37. In another more recent study, Gordon et al. found in their analysis of a national electronic
medical record database that fewer hospitalized COVID-19 patients who were newly prescribed haloperidol and
other sigma-binding typical antipsychotic medications progressed to requiring mechanical ventilation compared
to those who were newly prescribed atypical antipsychotic medications that do not bind to sigma receptors14.
Our testing of clofazimine demonstrated submicromolar antiviral effects of this drug in SARS-Co-V-2
infected 293T-ACE2 and Vero E6 cells (Figs. 4 and S3). Clofazimine is an orally administered antimycobacterial
drug used in the treatment of leprosy. By preferentially binding to mycobacterial DNA, clofazimine disrupts the
acterium38. In addition to being an antimycobacterial agent, clofazimine also
cell cycle and eventually kills the b
possesses anti-inflammatory properties primarily by inhibiting T lymphocyte activation and proliferation39. Yuan
et al. found that clofazimine inhibits SARS-CoV-2 replication by interfering with spike-mediated viral entry and
viral RNA r eplication40. Their work also demonstrated that clofazimine has antiviral efficacy against SARS-CoV-2
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Figure 5. Viral inhibition and cell viability tests of 13 compounds in 293T-ACE2 cell assays. Several drugs
inhibit viral infectivity. Red, viral infectivity (anti-NP); black, cell viability. The lack of a dose response in cell
viability probably reflects cytostatic and not cytotoxic effects. Data are mean ± sd; n = 3 biologically independent
samples for cell viability data. Cells were incubated for 24 h at 37 °C in 5% C O2. Then, 2 h before infection, the
medium was replaced with 100 μl of DMEM (2% FBS) containing the compound of interest at concentrations
50% greater than those indicated, including a DMSO control. 100 PFU (MOI = 0.025) was added in 50 μl of
DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incubated for
48 h at 37 °C. The cells were then immunostained for the viral NP protein.
in human embryonic stem cell-derived cardiomyocytes and in an ex vivo human lung culture system, as well as
antiviral synergy with remdesivir demonstrating the potential of clofazimine as part of a combination treatment
regimen for COVID-1940.
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Our group found bacampicillin to have micromolar antiviral efficacy in SARS-Co-V-2 infected 293T-ACE2
cells. Bacampicillin is an orally administered prodrug of ampicillin typically prescribed for treating bacterial
infections41. As identified by SPOKE42, bacampicillin was found to downregulate the GDF15 gene and upregulate
the NFKB2 (Nuclear Factor Kappa B Subunit 2) gene in studies by Cmap28 and LINCS43. The GDF15 protein
acts as a cytokine and is involved in stress response after cellular injury, and the NFKB2 is a central activator of
genes involved with inflammation and immune function44. Circulating levels of GDF15 have been found to be
significantly higher in COVID-19 patients who die45. Zhou et al.’s work revealed NF-kappa B signaling as one of
the main pathways of coronavirus infections in humans. While the rapid conversion of bacampicillin to ampicillin
in vivo makes this prodrug a less optimal therapeutic candidate for COVID-19, our findings nevertheless provide
insights into the immunologic and inflammatory landscape from SARS-CoV-2 infection.
Overall, in testing of our drug hits across two human cell line assays, 11 of 16 exhibited inhibition of SARS-
CoV-2 infection. In particular, three of our four consensus drug hits demonstrated antiviral efficacy, with halop-
eridol showing reproducible inhibition in Calu-3 cells, and bacampicillin and clofazimine inhibiting viral activity
in 293T-ACE2 cells without cytotoxicity. Many of our tested drugs can be administered orally, and several are
on the WHO Model List of Essential medications, including ciclosporin, clofazimine, and haloperidol46. These
results suggest that our drug repositioning pipeline can rapidly identify readily available potential therapeutics
in antiviral contexts.
There are several limitations of our approach that should be recognized. In general, drug repositioning pipe-
lines are reliant upon data being encoded in a computable format. Considering variability of conditions under
which experimental and clinical data are obtained, this also implies a particular set of limitations and biases to
the ensuing results. The general basis of the pipeline operates under the hypothesis that reversing the set of tran-
scriptomic changes will correlate with antiviral response. While it is hard to say whether reversing expression will
stymie viral replication, we hypothesize these drugs could treat the symptoms and/or assist in withstanding the
infection while the immune response fights off the virus. It is likely that secondary cellular responses influence a
specific signature including pro-viral as well as antiviral response and should be further explored in future studies.
In our work, we applied the KS-based similarity metric on the CMap database. Rank-based methods, such
as the KS statistic, may suffer from high false positive rates, as genes not differentially expressed can be ranked
high and contribute to the similarity measurement for drug and disease signatures47,48. Moreover, rank-based
approaches can also miss many potential drugs as ranking captured just a small part of information in a gene
expression profile. Alternative methods have been proposed, such as E MUDRA48 and X Sum49; however, they have
not been widely adopted by the community. Future work might include evaluating multiple similarity metrics
on larger d atasets48. The studies that we leveraged here are also limited because of their small sample size, which
might explain the small gene overlap across the signatures. While the different sample types were chosen due
to availability and to capture the heterogeneity of the response to viral infection, data generated from cell lines
and organoids (the ALV and EXP signatures, respectively) might not accurately represent the biological changes
and responses in human infection, especially with regards to the most severe respiratory infections caused by
the SARS-CoV-2 virus. Furthermore, the data sources presented here offer no insight into the temporal aspects
of response to viral infection. In vivo experiments are needed to better understand the biology of the disease
and the effect of these drugs on it. Moreover, although the BALF signature was generated from fluid recovered
from lavage of infected human tissues, this primary response data was aggregated from a very limited sample
size (2 cases and 3 controls). As the sample types across the three datasets were very different, we used different
fold change thresholds to identify the signature genes to be used for drug repurposing. Future work leveraging
additional datasets can provide further insights into therapeutic predictions and drug repositioning oppor-
tunities. Gathering samples from a larger number of patients should generate a more robust gene expression
signature and better inform therapeutic predictions. Furthermore, the drug profiles from CMap were generated
from cell line data; drug data generated from more relevant tissue cultures (e.g. lung tissue) may generate more
appropriate comparisons. Finally, our validation approaches focus on in vitro studies, which are limited. While
a reduction of 50–70% in viral RNA levels meets the generally accepted minimum threshold change of 1 log,
next steps would involve confirming the antiviral effects via established virological methods (e.g. plaque assay)
in addition to further in vivo testing of the proposed compounds.
The drug development response for SARS-CoV-2/COVID-19 is rapidly developing. One drug, remdesivir,
recently received FDA approval for the treatment of COVID-19, and numerous other drugs are being actively
explored for possible therapeutic value in COVID-19 cases. Utilizing a diverse set of transcriptomic SARS-
CoV-2 signatures, our drug repositioning pipeline identified 25 therapeutic candidates. Validation experiments
revealed antiviral activity for 11 of 16 drug hits. Further clinical investigation into these drug hits, in vivo assays
as well as potential combination therapies is warranted to further investigate both the antiviral as well as side
effect profile of the drugs.
Materials and methods

Study design. We have previously developed and used a transcriptomics based bioinformatics approach for
drug repositioning in various contexts including inflammatory bowel disease, dermatomyositis, and spontane-
ous preterm birth. For a list of differentially expressed genes, the computational pipeline compares the ranked
differential expression of a disease signature with that of a profile16,19,28. A reversal score based on the Kolmogo-
rov–Smirnov statistic is generated for each disease-drug pair, with the idea that if the drug profile significantly
reverses the disease signature, then the drug could be potentially therapeutic for the disease.
SARS‑CoV‑2 gene expression signatures. Blanco-Melo et al. generated a differential gene expression
signature using RNA-seq on human adenocarcinomic alveolar basal epithelial cells infected with SARS-CoV-2
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propagated from Vero E6 cells (GSE147507, 67 samples)24. Due to the fast-moving nature of the research topic,
we opted to use this cell line data in lieu of waiting for substantial patient-level data. This work identified 120
differentially expressed genes (DEGs)—100 upregulated and 20 downregulated. We used these 120 genes as the
ALV signature for our computational pipeline (Table S1).
Lamers et al. performed RNA-seq on their organoid samples, from which differentially expressed genes
were calculated26. These samples were grown in a medium with a Wnt surrogate supplement and infected with
SARS-CoV-2 propagated from Vero E6 cells (GSE149312, 22 samples). They detected 434 significant DEGs
(FDR < 0.05). We additionally applied a fold-change cutoff (|log2 FC| > 2), resulting in 125 genes used as the
EXP signature (Table S2).
Xiong et al. performed RNA-seq analysis of BALF samples from two COVID-19 patients (two samples per
patient) and three healthy c ontrols27. We processed their raw read counts in order to construct a differential
signature (see below for details). FASTQ files were downloaded from the Genome Sequence Archive50,51 under
accession number CRA002390. Paired-end reads were mapped to the hg19 human reference genome using
Salmon (v.1.2.0) and assigned Ensembl genes. After read quality control, we obtained quantifications for 55,640
genes in all samples. In order to identify genes differentially expressed between cases and controls for the BALF
samples, we quantified gene expression as raw counts. Raw counts were used as inputs to DESeq2 (v.1.24.0 R
package) to call differentially expressed genes (DEGs). After adjusting for the sequencing platform, the default
settings of DESeq2 were used. Principal components were generated using the DESeq2 function (Figure S2),
and heat maps were generated using the Bioconductor package pheatmap (v.1.0.12) using the rlog-transformed
counts (Figure S3). Values shown are rlog-transformed and row-normalized. Volcano plots were generated using
the Bioconductor package EnhancedVolcano (v.1.2.0) (Figure S4). Retaining only protein-coding genes and
applying both a significance threshold and a fold-change cutoff (FDR < 0.05, |log2 FC| > 4), we obtained 1349
genes to be used as the BALF signature (Table S3).
Pathway enrichment analysis. Functional enrichment gene-set analysis for GSEA (Gene Set Enrich-
ment Analysis) was performed using fgsea (v.1.12.0 R package) and the input gene lists were ranked by log2
fold change. The 50 Hallmark Gene Sets used in the GSEA analysis were downloaded from MSigDB Signatures
database29,52. For GO (Gene Ontology) terms, identification of enriched biological themes was performed using
the DAVID d atabase53.
Drug gene expression profiles. Drug gene expression profiles were sourced from Connectivity Map
(CMap), a publicly-available database of drugs tested on cancer cell lines28. CMap contains a set of differential
gene expression profiles generated from treating cultured human cells with a variety of different drugs and
experimental compounds. These profiles were generated using DNA microarrays to assay mRNA expression.
These drug profiles are ranked genome-wide profiles (~ 22,000 genes) of the effects of the drugs on various cell
lines. 6100 gene expression profiles are presented in CMap. A total of 1309 compounds were tested in up to 5
different cell lines. The overlap between the gene lists of CMap and the SARS-CoV-2 signature is 109 genes.
Computational gene expression reversal scoring. To compute reversal scores, we used a non-paramet-
ric rank-based method similar to the Kolmogorov–Smirnov test statistic. This analysis was originally suggested
by the creators of the CMap database and has since been implemented in a variety of different s ettings16–19,22,28.
As also described by others, the drug signature is compared with the gene expression profiles. By splitting the
gene signature into two lists containing only upregulated genes and downregulated genes, a so-called connectiv-
ity score is estimated via several auxiliary variables using a nonparametric rank-ordered Kolmogorov–Smirnov
(KS) test)47. Similar to past works, we applied a pre-filtering step to the CMap profiles to maintain only drug
profiles which were significantly correlated with another profile of the same drug. Drugs were assigned reversal
scores based on their ranked differential gene expression profile relative to the SARS-CoV-2 ranked differential
gene expression signature. A negative reversal score indicated that the drug had a profile which reversed the
SARS-CoV-2 signature; that is, up-regulated genes in the SARS-CoV-2 signature were down-regulated in the
drug profile and vice versa.
Statistical analysis. P-values were adjusted using the false discovery rate (FDR; Benjamini-Hochberg)
procedure. P-values for individual drug hits were obtained by comparing reversal scores to a distribution of
random scores. Negative reversal scores were considered significant if they met the criterion FDR < 0.05. For
drugs tested multiple times (e.g. different cell lines), we used the most reversed profile (lowest negative score).
For significance values of the number of drugs reversing multiple signatures, we constructed distributions of the
common reversal (reversing two of three signatures) and the consensus reversal (reversing three of three signa-
tures) by randomly sampling the same number of drug profiles for each signature from CMap.
Single‑cell data analysis. Quantification files were downloaded from GEO GSE145926. An individual
Seurat object for each sample was generated using Seurat v.3. While the data has been filtered by 10x’s algorithm,
we still needed to ensure the remaining cells are clean and devoid of artifacts. We calculated three confounders
for the dataset: mitochondrial percentage, ribosomal percentage, and cell cycle state information. For each sam-
ple, cells were normalized for genes expressed per cell and per total expression, then multiplied by a scale factor
of 10,000 and log-transformed. Low quality cells were excluded from our analyses—this was achieved by filter-
ing out cells with greater than 5000 and fewer than 300 genes and cells with high percentage of mitochondrial
and ribosomal genes (greater than 10% for mitochondrial genes, and 50% for ribosomal genes). SCTransform
is a relatively new technique that uses "Pearson Residuals" (PR) to normalize the data. PRs are independent of
Vol:.(1234567890)
sequencing depth54. We "regress out" the effects of mitochondrial and ribosomal genes, and the cell cycling state
of each cell, so they do not dominate the downstream signal used for clustering and differential expression. We
then performed a lineage auto-update disabled r dimensional reduction (RunPCA function). Then, each sam-
ple was merged together into one Seurat object. Data were then re-normalized and dimensionality reduction
and significant principal components were used for downstream graph-based, semi-unsupervised clustering
into distinct populations (FindClusters function) and uniform manifold approximation and projection (UMAP)
dimensionality reduction was used. For clustering, the resolution parameter was approximated based on the
number of cells according to Seurat guidelines; a vector of resolution parameters was passed to the FindClusters
function and the optimal resolution of 0.8 that established discernible clusters with distinct marker gene expres-
sion was selected. We obtained a total of 21 clusters representing the major immune and epithelial cell popula-
tions. To identify marker genes driving each cluster, the clusters were compared pairwise for differential gene
expression (FindAllMarkers function) using the Likelihood ratio test assuming an underlying negative binomial
distribution (negbinom). For visualization of gene expression data between different samples a number of Seurat
functions were used: FeaturePlot, VlnPlot and DotPlot.
Cell lines. For studies at the Gladstone Institutes, Calu-3 cells, a human lung epithelial cell line (Ameri-
can Type Culture Collection, ATCC HTB-55), were cultured in advanced MEM supplemented with 2.5% fetal
bovine serum (FBS) (Gibco, Life Technologies), 1% l-GlutaMax (ThermoFisher), and 1% penicillin/streptomy-
cin (Corning) at 37 °C and 5% CO2. SARS-CoV-2 Isolate USA-WA1/2020 was purchased from BEI Resources
and propagated and titered in Vero E6 cells. For studies carried out at Mount Sinai, SARS-CoV-2 was propagated
in Vero E6 cells (ATCC CRL-1586) and 293T-ACE2 cells (ATCC CRL-3216).
Compounds. Selection of compounds for testing was based on side effect profiles and compound avail-
ability. Bacampicillin (B0070000), ciclopirox (SML2011-50MG), ciclosporin (C2163000), clofazimine (1138904-
200MG), dicycloverine (D1060000), fludrocortisone (1273003-200MG), fluticasone (1285873-100MG), halo-
peridol (H1512-5G), isoxicam (I1762-1G), lansoprazole (1356916-150MG), metixene (M1808000), myricetin
(M6760-10MG), pentoxifylline (1508901-200MG), sirolimus (S-015-1ML), tretinoin (1674004-5X30MG), and
valproic acid (1708707-500MG) were purchased from Sigma-Aldrich. Remdesivir (GS-5734) was purchased
from Selleckchem.
Compounds were resuspended in DMSO according to manufacturer’s instructions and serially diluted to the
relevant concentrations for treatment of infected cells.
Infection experiments. Work involving live SARS-CoV-2 was performed in the BSL3 facility at the Glad-
stone Institutes with appropriate approvals. Calu-3 cells were seeded in 96-well plates for 24 h, infected with
SARS-CoV-2 at a multiplicity of infection (MOI) of 0.05, and treated with compounds. 72 h post infection,
supernatant was collected for RNA extraction and the RNA was analyzed using RT-qPCR to quantify viral
genomes present in the supernatant. SARS-CoV-2 specific primers targeting the E gene region: 5ʹ-ACAGGT
ACGTTAATAGTTAATAGCGT-3ʹ (Forward) and 5ʹ-ATATTGCAGCAGTACGCACACA-3ʹ (Reverse) were used to
quantify cDNA on the 7500 Fast Real-Time PCR system (Applied Biosystems). Cells were fixed with paraform-
aldehyde and used for immunofluorescence analysis with dsRNA antibody (SCICONS) and DAPI stain. Images
were acquired and analyzed using ImageXpress Micro Confocal High-Content Imaging System.
In vitro microneutralization assay for SARS‑CoV‑2 serology and drug screening. For studies
at Mount Sinai, SARS-CoV-2 was propagated in Vero E6 cells (ATCC CRL-1586) and 293T-ACE2 cells (ATCC
CRL-3216), as previously d escribed12,34. Two thousand cells were seeded into 96-well plates in DMEM (10%
FBS) and incubated for 24 h at 37 °C, 5% CO2. Then, 2 h before infection, the medium was replaced with 100 μl
of DMEM (2% FBS) containing the compound of interest at concentrations 50% greater than those indicated,
including a DMSO control. The Vero E6 cell line used in this study is a kidney cell line; therefore, we cannot
exclude that lung cells yield different results for some inhibitors. Plates were then transferred into the Biosafety
Level 3 (BSL3) facility and 100 PFU (MOI = 0.025) was added in 50 μl of DMEM (2% FBS), bringing the final
compound concentration to those indicated. Plates were then incubated for 48 h at 37 °C. After infection, super-
natants were removed and cells were fixed with 4% formaldehyde for 24 h before being removed from the BSL3
facility. The cells were then immunostained for the viral NP protein (an in-house mAb 1C7, provided by Dr.
Thomas Moran) with a DAPI counterstain. Infected cells (488 nM) and total cells (DAPI) were quantified using
the Celigo (Nexcelcom) imaging cytometer. Infectivity is measured by the accumulation of viral NP protein in
the nucleus of the Vero E6 cells and 293T-ACE2 cells (fluorescence accumulation). Percentage infection was
quantified as ((infected cells/total cells) − background) × 100 and the DMSO control was then set to 100% infec-
tion for analysis. The IC50 and IC90 for each experiment were determined using the Prism (GraphPad) software.
Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer’s instructions.
Cytotoxicity was performed in uninfected VeroE6 cells with same compound dilutions and concurrent with viral
replication assay. All assays were performed in biologically independent triplicates.
Code availability
The data used for the repositioning pipeline are all publicly available. The code for the drug repositioning pipe-
line was adapted from reference 19 and is available at https://github.com/brianlle/sirota_lab_covid_drug_repos
itioning.
Vol.:(0123456789)
Received: 15 March 2021; Accepted: 17 May 2021
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Acknowledgements
This work was funded in part by the Program for Breakthrough Biomedical Research (PBBR) Grant. This research
was also partly funded by the Defense Advanced Research Projects Agency (HR0011-19-2-0020); by CRIP
(Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research
and Surveillance (CEIRS, contract # HHSN272201400008C); by supplements to NIAID grant U19AI135972 and
DoD grant W81XWH-20-1-0270; by the generous support of the JPB Foundation and the Open Philanthropy
Project (research grant 2020-215611 (5384)); and by anonymous donors to A. García-Sastre. M. Ott acknowl-
edges support through a gift from the Roddenberry Foundation and by NIH 5DP1DA038043. We would like to
thank members of the Sirota Lab for useful discussion, Dr. Boris Oskotsky for technical support, Edna Rodas
for administrative support, Randy Albrecht for support with the BSL3 facility and procedures at the ISMMS,
and Richard Cadagan for technical assistance.
Author contributions
B.L., T.O. and M.S. designed and coordinated the study. B.L. led the drug repurposing efforts. G.A., K.Y., I.K.,
and C.L. helped with data analyses. SARS-CoV-2 virus assays were led by A.V.G., G.R.K., K.L., R.R., K.W., A.G.S.,
and M.O. All the authors contributed to making figures, writing and editing the manuscript.
Competing interests
M.S. is on the advisory board of twoXAR. The García-Sastre Laboratory has received research support from
Pfizer, Senhwa Biosciences and 7Hills Pharma. A.G.S. has consulting agreements for the following companies
involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Accurius and
Esperovax. Other authors declare no competing financial interests.
Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-021-91625-1.
Correspondence and requests for materials should be addressed to M.S.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
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Transcriptomics Based Drug Repositioning Pipeline Identifies Therapeutic Candidates For COVID 19

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OPEN Transcriptomics‑based drug

Scientific Reports | (2021) 11:12310 | https://doi.org/10.1038/s41598-021-91625-1 1

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Figure 1. COVID-19 transcriptomics-based bioinformatics approach for drug repositioning. We generated

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Materials and methods

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Received: 15 March 2021; Accepted: 17 May 2021

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© The Author(s) 2021

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