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Drug repurposing is a rapid approach to identify therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drugs and 49 investigational drugs. The anti-SARS-CoV-2 activities of 230 of these confirmed compounds, of which 38 are approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA-approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set is a useful resource for drug repurposing efforts, including design of new drug combinations for clinical trials for SARS-CoV-2.
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health crisis. As of September 17, 2020, the global case report stands at 30 million, with a death toll of 942,989 (
The requirement of biosafety level 3 (BSL-3) containment laboratories for handling SARS-CoV-2 has limited the number of high throughput screening (HTS) laboratories that are capable of carrying out large scale compound screens using live SARS-CoV-2. Despite these challenges, several drug repurposing screens have been carried out using live SARS-CoV-2, showing promising results (
All compound libraries were assembled internally at NCATS. The NCATS pharmaceutical collection (NPC) contains 2,678 compounds, covering drugs approved by US FDA and foreign health agencies in European Union, United Kingdom, Japan, Canada, and Australia, as well as some clinical trialed experimental drugs (
A SARS-CoV-2 CPE assay was conducted in the BSL3 facilities at the contract research organization Southern Research (Birmingham, AL). Briefly, compounds were titrated in DMSO and acoustically dispensed into 384-well assay plates at 60 nL/well at NCATS, and provided to Southern Research. Cell culture media (MEM, 1% Pen/Strep/GlutaMax, 1% HEPES, 2% HI FBS) was dispensed at 5 µL/well into assay plates, and incubated at room temperature to allow for compound dissolution. Vero E6 African green monkey kidney epithelial cells (selected for high ACE2 expression) were inoculated with SARS-CoV-2 (USA_WA1/2020) at a multiplicity of infection (MOI) of 0.002 in media, and quickly dispensed into assay plates as 25 µL/well. The final cell density was 4,000 cells/well. Assay plates were incubated for 72 h at 37°C, 5% CO2, and 90% humidity. CellTiter-Glo (30 µL/well, Promega #G7573) was dispensed into the assay plates. Plates were incubated for 10 min at room temperature. Luminescence signal was measured on Perkin Elmer Envision or BMG CLARIOstar plate readers. An ATP content cytotoxicity counter-assay was conducted using the same protocol as the CPE assay, without the addition of SARS-CoV-2 virus.
Results from the primary screen and confirmation screens were processed at NCATS using a software developed in-house (
Our aims were two-fold in initiating this program. The first was to identify active compounds that may provide opportunities for repurposing, or identify mechanistic targets of interest. The second was to create a complete HTS reference dataset that can be shared openly with the scientific community for study of disease pathology and new therapeutics development. The CPE reduction assay format has been widely employed to screen for antiviral agents due to its ease of scalability for HTS (
A total of 9,952 compounds were tested in the primary screen, but due to the overlapping composition of the libraries, a significant number of compounds were tested multiply. A total of 8,810 unique compounds in six compound libraries were tested in the primary screen including the NCATS Pharmaceutical Collection (NPC), NCATS Mechanism Interrogation Plate (MIPE), NCATS Pharmacologically Active Chemical Toolbox (NPACT), Epigenomic library, Autophagy library, and anti-infective library. These compounds contain 1,345 approved drugs (by the FDA, EMA, DPD), 751 compounds approved outside of those countries, 1,067 investigational drugs (tested in clinical trials), 1,057 pre-clinical compounds (tested in animals), and 4,472 bioactive compounds (tool compounds) (
Compound library description.
The CPE assay performed well in the primary screen, with an average Z’ factor of 0.83 over 133 plates, from three batched runs (
Assay reproducibility.
Top confirmed anti-SARS-CoV-2 compounds.
Sample ID | Sample name | CPE EC50 (uM) | CPE % efficacy | Cytotox CC50 (uM) | % Cytotox | Previous reports against CoVs | Approval status | MOA |
---|---|---|---|---|---|---|---|---|
|
||||||||
NCGC00686694 | Remdesivir | 10.0 | 133.1 | N/A | <30 | Clinical ( |
FDA | RdRP inhibitor |
|
||||||||
NCGC00387732 | VPS34-IN1 | 0.63 | 103.0 | 10.0 | −76.5 | None | Bioactive | Autophagy modulator |
NCGC00344081 | STF-62247 | 1.1 | 107.1 | 11.2 | −56.6 | None | Preclinical | Autophagy modulator; Renal cell growth inhibition |
NCGC00507892 | VPS34 Inhibitor 1 | 1.4 | 98.3 | N/A | <30 | None | Preclinical | Autophagy modulator |
|
||||||||
NCGC00346896 | MCOPPB | 3.5 | 85.6 | N/A | <30 | None | Preclinical | ORL1 (OP4, NOP) agonists |
NCGC00370950 | GW 803430 | 3.5 | 93.3 | N/A | <30 | None | Bioactive | Melanin-concentrating hormone receptor 1 antagonist |
NCGC00017063 | Amodiaquine dihydrochloride | 4.0 | 87.2 | N/A | <30 |
|
FDA | Histamine receptor antagonist |
NCGC00485045 | N-Methylspiperone hydrochloride | 4.5 | 80.0 | N/A | <30 | None | Clinical trial | Serotonin 2 (5-HT2) receptor antagonist |
NCGC00016710 | Clemastine fumarate | 7.9 | 96.0 | N/A | <30 | Mpro assay ( |
FDA | Histamine receptor antagonist |
NCGC00386477 | GMC 2-29 | 7.9 | 117.2 | N/A | <30 | None | Bioactive | 5-hydroxytryptamine receptor 1D antagonist |
NCGC00378842 | Lu AE58054 hydrochloride | 10.0 | 97.2 | N/A | <30 | None | Clinical trial | Serotonin 6 (5-HT6) receptor antagonist |
NCGC00013683 | Chlorprothixene | 10.0 | 104.4 | N/A | <30 | None | FDA | Dopamine receptor antagonist |
NCGC00014482 | Methdilazine hydrochloride | 10.0 | 86.4 | N/A | <30 | Virtual: AI prediction ( |
FDA | Antihistamine |
NCGC00179370 | Methotrimeprazine maleate | 10.0 | 84.6 | N/A | <30 | None | FDA | Antagonist for adrenergic, dopamine, histamine, cholinergic and serotonin (5-hydroxytryptamine; 5-HT) receptors |
NCGC00016642 | Piperacetazine | 10.0 | 103.7 | N/A | <30 | None | FDA | Dopamine receptor antagonist |
NCGC00181913 | Difeterol | 10.0 | 113.4 | N/A | <30 | None | Approved outside of US | Antihistamine |
NCGC00386484 | (R)-(-)-LY 426965 dihydrochloride | 10.0 | 110.7 | N/A | <30 | None | Bioactive | Serotonin 2b (5-HT2b) receptor modulator |
NCGC00015608 | Loperamide hydrochloride | 10.0 | 98.6 | N/A | <30 |
|
FDA | Opioid receptor agonist |
NCGC00485321 | Naltrindole isothiocyanate hydrochloride | 10.0 | 114.7 | N/A | <30 | None | Bioactive | Delta opioid receptor antagonist |
NCGC00165726 | AM1241 | 10.0 | 97.6 | N/A | <30 | None | Bioactive | Cannabinoid CB2 receptor agonist |
NCGC00386703 | CpdD hydrochloride | 10.0 | 96.9 | N/A | <30 | None | Bioactive | Ghrelin receptor antagonist |
NCGC00386219 | SB 271046 hydrochloride | 10.0 | 107.5 | N/A | <30 | None | Bioactive | Serotonin 6 (5-HT6) receptor antagonist |
NCGC00386479 | GMC 2-113 | 10.0 | 129.7 | N/A | <30 | Virtual: RdRP ( |
Bioactive | 5-hydroxytryptamine receptor 1D antagonist |
|
||||||||
NCGC00386330 | Z-FA-FMK | 0.13 | 104.8 | N/A | <30 | Mpro assay, |
Bioactive | Cathepsin L inhibitor |
NCGC00485951 | VBY-825 | 0.14 | 97.8 | N/A | <30 |
|
Clinical trial | Cathepsin S inhibitor |
NCGC00345807 | CAA-0225 | 0.20 | 99.3 | N/A | <30 | None | Preclinical | Cathepsin L inhibitors |
NCGC00386232 | Cathepsin Inhibitor 1 | 0.25 | 114.4 | N/A | <30 | None | Bioactive | Cathepsin inhibitors |
NCGC00163432 | Calpeptin | 0.50 | 111.7 | N/A | <30 | Mpro assay, |
Preclinical | Calpain inhibitor |
NCGC00485375 | Z-Gly-Leu-Phe-chloromethyl ketone | 1.3 | 87.2 | N/A | <30 | None | Bioactive | Granzyme B Inhibitor |
NCGC00371151 | Balicatib | 2.0 | 100.3 | N/A | <30 | None | Clinical trial | Cruzipain (Trypanosoma cruzi) inhibitor |
NCGC0016166 | Calpain Inhibitor I, ALLN | 2.0 | 111.1 | N/A | <30 | None | Bioactive | Calpain inhibitor |
|
||||||||
NCGC00263093 | Apilimod | 0.023 | 104.4 | N/A | <30 |
|
Clinical trial | IL-12 Production inhibitor; PIKfyve inhibitor |
NCGC00386313 | Berzosertib | 0.71 | 87.9 | 11.2 | -98.5 | None | Clinical trial | ATR Kinase inhibitor |
NCGC00347280 | IKK-2 inhibitor VIII | 7.1 | 91.7 | N/A | <30 | None | Preclinical | IKK-2 (IKK-beta) inhibitor |
NCGC00387166 | NSC 33994 | 8.9 | 107.6 | N/A | <30 | None | Bioactive | Jak2 inhibitor |
NCGC00159456 | Imatinib | 10.0 | 119.0 | N/A | <30 | Clinical ( |
FDA | Bcr-Abl kinase inhibitor; KIT inhibitor; PDGFR tyrosine kinase receptor inhibitor |
|
||||||||
NCGC00178090 | Pristimerin | 0.11 | 87.4 | 1.1 | −93.2 | SARS Mpro assay ( |
Preclinical | Monoacylglycerol lipase (MGL) inhibitor |
NCGC00385252 | alpha- |
2.4 | 104.0 | N/A | <30 | None | Bioactive | Induces Pbad promoter expression in |
NCGC00351072 | ML414 | 3.2 | 79.6 | N/A | <30 | None | Bioactive | Oligosaccharyltransferase inhibitor |
NCGC00379165 | IT1t dihydrochloride | 3.5 | 96.3 | N/A | <30 | None | Bioactive | CXCR4 inhibitor |
NCGC00485648 | S-15176 difumarate salt | 3.8 | 127.4 | N/A | <30 | None | Bioactive | Oxidative stress inhibitor |
NCGC00384450 | JTV519 Hemifumarate | 5.5 | 85.7 | N/A | <30 | None | Clinical trial | Ryanodine receptor (RyR) inhibitor |
NCGC00253604 | Rescimetol | 8.9 | 81.8 | N/A | <30 | None | Approved outside of US | Antihypertensive agent |
NCGC00164559 | Duloxetine hydrochloride | 10.0 | 90.0 | N/A | <30 | Mpro assay ( |
FDA | Norepinephrine reuptake inhibitor; Serotonin-norepinephrine reuptake inhibitor (SNRI) |
NCGC00181168 | Trifluomeprazine 2-butenedioate | 10.0 | 90.2 | N/A | <30 | None | Bioactive | Antipsychotic agents |
NCGC00169804 | Asteriscunolide D | 10.0 | 93.3 | N/A | <30 | None | Bioactive | Natural product |
NCGC00485925 | Genz-123346 (free base) | 10.0 | 99.4 | N/A | <30 |
|
Bioactive | Ceramide glucosyltransferase inhibitor |
NCGC00015708 | Maprotiline hydrochloride | 10.0 | 103.7 | N/A | <30 | Virtual: Mpro docking ( |
FDA | Norepinephrine reputake inhibitor; tricyclic antidepressant |
NCGC00168786 | Deserpidine | 10.0 | 84.7 | N/A | <30 | Virtual: NSP16 docking ( |
FDA | Angiotensin converting enzyme inhibitor |
NCGC00015096 | Amiodarone hydrochloride | 10.0 | 100.5 | N/A | <30 | Clinical ( |
FDA | Potassium channel blocker |
NCGC00181088 | Melitracen hydrochloride | 10.0 | 97.1 | N/A | <30 | None | Approved outside of US | Antidepressive agents, tricyclic |
NCGC00015428 | (+/-) -Fluoxetine | 10.0 | 115.8 | N/A | <30 |
|
FDA | Selective serotonin reuptake inhibitor (SSRI) |
NCGC00018102 | Flunarizine | 10.0 | 94.1 | N/A | <30 | Virtual: Spike docking ( |
Approved outside of US | Calcium channel blocker |
NCGC00183024 | Proglumetacin | 10.0 | 87.6 | N/A | <30 | None | Approved outside of US | Cyclooxygenase inhibitor |
NCGC00378760 | DMP 777 | 10.0 | 92.5 | N/A | <30 | None | Clinical trial | Leukocyte elastase inhibitor |
NCGC00476094 | Dexanabinol | 10.0 | 110.8 | N/A | <30 | None | Clinical trial | NMDA antagonist |
There were 56 top confirmed hits with EC50 values of ≤10 µM and efficacy values of greater than 80% in the CPE assay, and with greater than 10-fold selectivity index (SI) between cytotoxicity and CPE assays (
Compounds concentration-response curves in the CPE assay.
There have been several previous drug repurposing screens reported for SARS-CoV-2 in 2D cell culture infection models (
Reported human pharmacokinetic properties of FDA-approved top hits.
Sample name | Cmax (ng/ml) | MW (g/mol) | Cmax (µM) | Elimination T1/2 | Dosing regimen | References |
---|---|---|---|---|---|---|
(+/-) -Fluoxetine | 15–55 | 309.33 | 0.05–0.18 | 1–3 days | Single dose 40 mg PO |
|
Amiodarone hydrochloride | 5,000–41,000 | 681.78 | 7.33–60.14 | 9–36 days | Single dose 5 mg/kg IV |
|
Amodiaquine dihydrochloride | 32 ± 3 | 464.8 | 0.069 | 5.2 ± 1.7 h | Single dose 600 mg PO |
|
Chlorprothixene | 430 ± 81 | 315.9 | 1.36 | 25.8 ± 13.6 h | Single dose 100 mg IV |
|
Clemastine fumarate | 0.577 ± 0.252 | 460 | 0.0013 | 21.3 ± 11.6 h | Single dose 1.34 mg PO |
|
Deserpidine | 0.172 | 578.66 | 0.0003 | 42.9 ± 17.8 h | Single dose 0.25 mg PO |
|
Duloxetine hydrochloride | 110 | 333.88 | 0.33 | 6.96–14.9 h | 60 mg BID PO |
|
Imatinib | 3,395 ± 2,409 | 493.6 | 6.88 | 10–18.9 h | Single dose 600 mg PO |
|
Loperamide hydrochloride | 2 | 477 | 0.0042 | 9.1–14.4 h | Single dose 2 mg PO |
|
Maprotiline hydrochloride | 25 | 313.87 | 0.080 | 45 h | Single dose 75 mg PO |
|
|
332.9 | |||||
Methotrimeprazine maleate | 3.44 | 444.6 | 0.0077 | 10.8 h | Single dose 25 mg PO |
|
|
410.6 | |||||
Remdesivir | 4,420 | 602.58 | 7.34 | 1.05 h | Single dose 225 mg IV |
|
Discontinued drugs. No PK data available.
Cmax: maximum serum/plasma concentration; MW: molecular weight; Elimination T1/2: elimination half life; PO: per os (oral dosing); IV: intravenous.
Four drugs approved outside of the US were also identified as novel compounds with anti-SARS-CoV-2 effects: difeterol, rescimetol, melitracen HCl, and proglumetacin. Furthermore, we identified 7 novel clinical trial drugs with anti-SARS-CoV-2 activities: N-methylspiperone HCl, Lu AE58054 HCl, balicatib, berzosertib, JTV519 hemifumarate, DMP 777, and dexanabinol. In addition to the above novel hits, four drugs, approved by the FDA and elsewhere, methdilazine, maprotiline HCl, deserpidine, and flunarizine, were previously reported in virtual screens against SARS-CoV-2 targets without supporting biological data. Here, we report their activities against SARS-CoV-2 infection. In addition, we have confirmed 53 approved drugs with anti-SARS-CoV-2 effects that were reported previously (
In contrast to the other reported drug repurposing screens for SARS-CoV-2 using a single drug concentration in the primary screens (
We identified 319 compounds with activity against SARS-CoV-2 CPE from a qHTS of 8,810 unique compounds. Among the top 56 hits identified with <10 µM EC50 values and >80% efficacies, the anti-SARS-CoV-2 activity of 37 of them has not been reported elsewhere. Of these novel top hits, three were FDA approved drugs with novel anti-SARS-CoV-2 activity. Chlorprothixene is a dopamine receptor antagonist, a classic antipsychotic agent approved for treatment of schizophrenia (
We also confirmed the anti-SARS-CoV-2 activity of five compounds that were reported as virtual screening hits but had yet to be confirmed experimentally, including methdilazine by an AI prediction algorithm (
For
It is worth briefly reflecting on the limitations of the drug repurposing assay approach. A number of small molecules of interest for treating COVID-19 that are currently in clinical trials were not hits in our assay. For example, the TMPRSS2 inhibitors camostat and nafamstat are protease inhibitors approved in Japan for treating pancreatitis, and known to inhibit TMPRSS2 (
Importantly, the comprehensive primary screen datasets of this study for approved and investigational drugs, and mechanism-based bioactive compounds have been made publicly available in real-time on the NCATS OpenData Portal (
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession numbers can be found in the article/
PS, ZI, and RTE prepared the assay ready plates. RB and LR conducted the CPE and cytotoxicity assays. PS, CK‐T, KMW, and SGM curated the compound libraries. CZC and MDH designed the experiments. CZC, BMB, and WZ wrote the manuscript. RH, MS, XH, HG, and TZ performed data analysis and data uploads. All authors provided critical reading of the manuscript.
This work was supported by the Intramural Research Program of National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, United States.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: