Repurposing of Clinically Developed Drugs for Treatment of Middle East Respiratory Syndrome Coronavirus Infection

Abstract

Outbreaks of emerging infections present the unique challenge of trying to select appropriate pharmacologic treatments in the clinic with little time available for drug testing and development. Typically clinicians are left with general supportive care and often untested convalescent plasma as available treatment options. Repurposing of approved pharmaceutical drugs for new indications presents an attractive alternative to clinicians, researchers, public health agencies, drug developers and funding agencies. Given development times and manufacturing requirements for new products, repurposing of existing drugs is likely the only solution for outbreaks due to emerging viruses. In the studies described here, a library of 290 compounds was screened for antiviral activity against Middle Eastern respiratory syndrome-coronavirus (MERS-CoV) and severe acute respiratory syndrome-coronavirus (SARS-CoV). Selection of compounds for inclusion in the library was dependent on current or previous FDA-approval or advanced clinical development. Some drugs were included that had a well-defined cellular pathway as target. In total, 27 compounds with activity against both MERS-CoV and SARS-CoV were identified. The compounds belong to thirteen different classes of pharmaceuticals including; inhibitors of estrogen receptors used for cancer treatment and inhibitors of dopamine receptor used as antipsychotics. The drugs identified in these screens provide new targets for in vivo studies as well as incorporation into ongoing clinical studies.

Full text

Repurposing of Clinically Developed Drugs for Treatment of Middle
East Respiratory Syndrome Coronavirus Infection
Julie Dyall,
a
Christopher M. Coleman,
b
Brit J. Hart,
a
Thiagarajan Venkataraman,
b
Michael R. Holbrook,
a
Jason Kindrachuk,
a
Reed F. Johnson,
c
Gene G. Olinger, Jr.,
a
Peter B. Jahrling,
a,c
Monique Laidlaw,
d
Lisa M. Johansen,
d
Calli M. Lear-Rooney,
e
Pamela J. Glass,
e
Lisa E. Hensley,
a
Matthew B. Frieman
b
Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
a
; Department of Microbiology and
Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
b
; Emerging Viral Pathogens Section, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Frederick, Maryland, USA
c
; Zalicus Inc., Cambridge, Massachusetts, USA
d
; United States Army Medical Research Institute of
Infectious Diseases, Frederick, Maryland, USA
e
Outbreaks of emerging infections present health professionals with the unique challenge of trying to select appropriate pharma-
cologic treatments in the clinic with little time available for drug testing and development. Typically, clinicians are left with gen-
eral supportive care and often untested convalescent-phase plasma as available treatment options. Repurposing of approved
pharmaceutical drugs for new indications presents an attractive alternative to clinicians, researchers, public health agencies,
drug developers, and funding agencies. Given the development times and manufacturing requirements for new products, repur-
posing of existing drugs is likely the only solution for outbreaks due to emerging viruses. In the studies described here, a library
of 290 compounds was screened for antiviral activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and
severe acute respiratory syndrome coronavirus (SARS-CoV). Selection of compounds for inclusion in the library was dependent
on current or previous FDA approval or advanced clinical development. Some drugs that had a well-defined cellular pathway as
target were included. In total, 27 compounds with activity against both MERS-CoV and SARS-CoV were identified. The com-
pounds belong to 13 different classes of pharmaceuticals, including inhibitors of estrogen receptors used for cancer treatment
and inhibitors of dopamine receptor used as antipsychotics. The drugs identified in these screens provide new targets for in vivo
studies as well as incorporation into ongoing clinical studies.
Middle East respiratory syndrome coronavirus (MERS-CoV)
is an emerging virus, and to date no antiviral or therapeutic
has been approved for treating patients. Since September 2012,
206 cases, including 86 deaths, have been attributed to infection
with MERS-CoV. Currently, supportive care remains the only
available treatment option. As the number of cases continues to
rise and the geographic range of the virus increases, there is a
growing urgency for candidate interventions.
Prior to 2002, coronaviruses were not considered to be signif-
icant human pathogens. Other human coronaviruses such as
HCoV-229E and HCoV-OC43 resulted in only mild respiratory
infections in healthy adults. This perception was shattered in 2002,
when severe acute respiratory syndrome coronavirus (SARS-
CoV) emerged in Guangdong Province, China. This virus rapidly
spread to 29 different countries, resulting in 8,273 confirmed cases
and 775 (9%) deaths (1). While SARS-CoV predominantly im-
pacted Southeast Asia, with significant outbreaks throughout
China, Hong Kong, Taiwan, Singapore, and Vietnam, the virus
was carried outside the region. Importation of the virus into Can-
ada resulted in 251 confirmed cases and 44 deaths (1). The imple-
mentation of infection control measures was able to bring the
epidemic to an end in 2003.
In 2012, a novel coronavirus, Middle East respiratory syn-
drome coronavirus (MERS-CoV), was detected in a patient with
severe respiratory disease in the kingdom of Saudi Arabia. To date,
636 laboratory-confirmed cases of MERS-CoV infection have
been reported, including 193 deaths, across nine countries (WHO
Global Outbreak Alert & Response Network, 28 May 2014; http:
//www.who.int/csr/outbreaknetwork/en/). The clinical features of
MERS-CoV infection in humans range from asymptomatic to
very severe pneumonia with the potential development of acute
respiratory distress syndrome, septic shock, and multiorgan fail-
ure resulting in death. Since the first case of MERS-CoV infection
was reported in September 2012 and the virus was isolated, signif-
icant progress has been made toward understanding the epidemi-
ology, ecology, and biology of the virus (2). Several assays for the
detection of acute infection with MERS-CoV by real-time reverse
transcription (RT)-PCR have been developed and are now in
widespread use (3). Over 30 whole- or partial-genome sequences
from different MERS-CoV-infected patients have been posted to
GenBank, and phylogenetic trees have been published by several
groups (3). Dipeptidyl peptidase 4 (also known as CD26) has been
identified as the functional cellular receptor for MERS-CoV (4,5).
Ecological studies have suggested that the virus is of animal origin
and is most closely related to coronaviruses found in a number of
species of bats, with MERS-CoV viral sequences now found in
camels in Saudi Arabia (6–9). Interestingly, a subset of MERS-
CoV patients reported close contact with camels. Camels may
Received 10 April 2014 Returned for modification 2 May 2014
Accepted 14 May 2014
Published ahead of print 19 May 2014
Address correspondence to Matthew B. Frieman, MFrieman@som.umaryland.edu.
J.D. and C.M.C. contributed equally to this work.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.03036-14.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.03036-14
August 2014 Volume 58 Number 8 Antimicrobial Agents and Chemotherapy p. 4885– 4893 aac.asm.org 4885

constitute an intermediate animal host, since camel serum sam-
ples collected in 2003 and 2013 had antibodies to MERS-CoV,
indicating that MERS-CoV circulates in camels (10–12). The re-
cent development of an animal model for MERS-CoV with ade-
novirus vectored human DPP4 in mice will now allow for further
pathogenesis studies with various MERS-CoV strains (13).
The emergences of both SARS-CoV and MERS-CoV have
demonstrated the importance of coronaviruses as potential
emerging human pathogens and highlighted the necessity and
value of effective communications within the international sci-
ence community to facilitate rapid responses to emerging infec-
tious diseases. In July 2013, the International Severe Acute Respi-
ratory & Emerging Infection Consortium (ISARIC) compiled a
list of drugs available to clinicians for treatment of MERS-CoV
infection based on recent experience in treating SARS-CoV infec-
tion and pandemic influenza (14). The most promising and clin-
ically available drugs were ribavirin and interferon (IFN), or a
combination of the two, since they demonstrated efficacy in an in
vivo model for MERS-CoV infection (15,16). This combination
has failed to demonstrate benefit in the small number of severely
ill MERS-CoV patients treated (17). Outside ribavirin and IFN,
the ISARIC recommendations had few alternatives for treating
clinicians. It should be noted that these recommendations are
meant to be fluid and based on the best available information at
the time. As new data become available, these recommendations
may change. Recently, we have shown mycophenolic acid (MPA)
and IFN-␤to be highly effective against MERS-CoV infection in
vitro. Interestingly, the activity of MPA was specific to MERS-
CoV, with little activity observed against SARS-CoV infection
(18,19).
In the work described here, we took the approach of screening
a unique panel of both approved drugs and drugs with a well-
defined cellular pathway for in vitro efficacy against MERS-CoV
infection. This subset was identified previously as having antiviral
activity against a series of other viruses (P. J. Glass, G. G. Olinger,
Jr., and L. M. Johansen, unpublished data). A subset of drugs was
also screened against SARS-CoV with the objective to identify
drugs with broad activity against coronaviruses in preparedness
for potential future emerging coronaviruses. We utilized this ap-
proach with the rationale that drugs that have been approved for
use in humans would be more readily accepted as potential ther-
apeutic options for MERS-CoV infection if shown to have antivi-
ral activity. The screening of approved drugs to identify therapeu-
tics for drug repurposing is a valid approach, and several approved
drugs have been identified as having activity against many viral
diseases (20–22). Here we found that 66 of the screened drugs
were effective at inhibiting either MERS-CoV or SARS-CoV infec-
tion in vitro and that 27 of these compounds were effective against
both MERS-CoV and SARS-CoV. These data demonstrate the ef-
ficiency of screening approved or clinically developed drugs for
identification of potential therapeutic options for emerging viral
diseases and also provide an expedited approach for supporting
off-label use of approved therapeutics.
MATERIALS AND METHODS
Cell lines and virus. Vero E6 cell line (ATCC 1568; Manassas, VA) was
maintained at the Integrated Research Facility (IRF, Frederick, MD) in
Dulbecco’s modified Eagle’s medium (DMEM; Corning, Manassas, VA)
plus 10% fetal bovine serum (FBS). The Jordan strain of MERS-CoV
(GenBank accession no. KC776174.1, MERS-CoV-Hu/Jordan-N3/2012
[23]), kindly provided by Kanta Subbarao (National Institutes of Health,
Bethesda, MD) and Gabriel Defang (Naval Medical Research Unit-3,
Cairo, Egypt), was amplified in Vero E6 cells at a multiplicity of infection
(MOI) of 0.01. On day 4 after infection, when the cytopathic effect (CPE)
was visible, virus-containing supernatants were collected and clarified by
centrifugation. The MERS-CoV titers on Vero E6 cells were determined
by plaque assay. All procedures using live MERS-CoV were performed
under biosafety level 3 conditions at the IRF.
The Vero E6 cell line (ATCC 1568; Manassas, VA) at the University of
Maryland, Baltimore (UMB), was maintained in minimal essential me-
dium (MEM; Corning, Manassas, VA) supplemented with 10% FBS
(SAFC, Bioscience, Lenexa, KS), 1% penicillin-streptomycin (Gemini
Bio-products, West Sacramento, CA), and 1% L-glutamine (Life Technol-
ogies, Grand Island, NY). Mouse adapted SARS-CoV (MA15) has been
described previously (24). SARS-CoV was amplified in Vero E6 cells for 2
days, when the CPE was visible. SARS-CoV-containing supernatants were
collected and clarified by centrifugation. Titers of SARS-CoV on Vero E6
cells were determined by plaque assay. All procedures using live SARS-
CoV were performed under biosafety level 3 conditions at UMB.
Reagents. Chlorpromazine hydrochloride (CAS 69-09-0) was pur-
chased from Sigma-Aldrich, St. Louis, MO. Imatinib mesylate (CAS
220127-57-1), gemcitabine hydrochloride (CAS 122111-03-9), and
toremifene citrate (CAS 89778-27-8) were purchased from Sequoia Re-
search Products, Pangbourne, United Kingdom. Triflupromazine hydro-
chloride (CAS 1098-60-8) was purchased from the U.S. Pharmacopeia,
Rockville, MD. Dasatinib (CAS 302962-49-8) was purchased from To-
ronto Research Chemicals Inc., Toronto, Canada. Dimethyl sulfoxide
(DMSO) was used as a solvent for the high-throughput screening assay
described below.
Drug library and compound plate preparation. A library of approved
drugs, including some drugs with a well-defined cellular target, was as-
sembled and has been previously described (25). A subset of 290 com-
pounds was selected for screening against MERS-CoV and SARS-CoV
based on the antiviral activity observed in screens against other RNA
viruses (21). For the MERS-CoV and SARS-CoV screens, compounds
were added to compound plates using an acoustic compound dispenser
(Echo 555; Labcyte, Sunnyvale, CA). The compounds were shot in nano-
liter volumes directly onto 96-well plates from master stock solutions.
Following addition of compound, 200 ␮l of DMEM was added to plates,
and plates were frozen at ⫺80°C for a minimum of 24 h prior to shipment
to the IRF and UMB investigators. Compound plates were thawed prior to
the addition of compound to the infectivity assays described below at the
IRF and UMB. For the MERS screen, compounds were plated in 200 ␮lof
media at 4 times the final concentrations such that the addition of 50 ␮lto
assay plates resulted in the appropriate final concentration (200-␮l final
assay volume). For the SARS screens, drugs were plated in 200 ␮l of media
at 2 times the final concentrations such that the addition of 50 ␮l resulted
in the appropriate final concentration (100-␮l final assay volume). All
drug plates were blinded to those performing the infectivity assays.
Cell-based ELISA screen for MERS-CoV antiviral agents. For cell-
based enzyme-linked immunosorbent assay (ELISA) screen, Vero E6 cells
were seeded at 40,000 cells in 100 ␮l DMEM plus 10% FBS per well in
black-, opaque-, or clear-bottom 96 well-plates. After 24 h, test drugs were
transferred from compound plates and added to 3 cell plates in 50 ␮l using
a 96-well liquidator (Rainin Instrument LLC, Oakland, CA). The DMSO
concentration was kept at 0.05% or lower. Duplicate Vero E6 seeded
plates were used for detecting inhibition of MERS-CoV, and one plate was
used for determining the cytotoxicity of compounds. For infection, du-
plicate plates were pretreated with drugs for 1 h before the plates were
transferred into the containment laboratory to add MERS-CoV strain
Hu/Jordan-N3/2012 at an MOI of 0.1 in 50 ␮l of DMEM plus 10% FBS.
After 48 h, plates were fixed with 10% neutral buffered formalin and
removed from biocontainment. MERS-CoV infection was detected with a
rabbit polyclonal antibody to the HCoV-EMC/2012 Spike protein (num-
ber 40069-RP02; Sino Biological Inc., Beijing, CN) followed by staining
Dyall et al.
4886 aac.asm.org Antimicrobial Agents and Chemotherapy

with Alexa Fluor 594 goat anti-rabbit IgG (H⫹L) antibody (Life Technol-
ogies, Grand Island, NY). Fluorescence was quantified on a plate reader
(Infinite M1000 Pro; Tecan US, Morrisville, NC) with an excitation wave-
length of 590 nm and emission wavelength of 617 nm. The drugs with
⬎50% inhibition of Spike expression and ⬍30% toxicity were then
screened with SARS-CoV as described below.
To detect cellular toxicity of drugs in the MERS-CoV screen, one of the
three plates that received the test drugs was used to evaluate the cytotox-
icity of drugs and was not infected with virus. At 48 h after drug addition,
cell plates were analyzed using the CellTiter Glo luminescent cell viability
assay kit according to the manufacturer’s directions (Promega, Madison,
WI), and luminescence was read on the Infinite M1000 Pro plate reader.
SARS-CoV cytopathic effect inhibition assay. For the SARS-CoV
screen, 174 of the 290 drugs were screened against SARS-CoV, including
all the hits that blocked MERS-CoV (72 drugs). The assay used to screen
for inhibition of SARS-CoV replication was different from the one used
for MERS-CoV replication due to differences in equipment for analysis at
UMB and IRF/NIAID. For the SARS-CoV inhibitor screen at UMB, du-
plicate Vero E6 cells were seeded into white opaque 96-well plates (Corn-
ing Costar) at 1 ⫻10
4
cells per well and cultured overnight at 37°C. Cells
were treated with the drugs for2hat37°C and then mock infected or
infected with SARS-CoV (MA15) at an MOI of 1. Cells were cultured at
37°C for 48 h and then analyzed for cell survival using the CellTiterGlo
luminescent cell viability assay (Promega, Madison, WI) according to the
manufacturer’s instructions and read on a SpectraMax M5 plate reader
(Molecular Devices, Sunnyvale, CA). A third identical drug compound
plate was used to assess drug toxicity in the absence of SARS-CoV using
the same Cell-Titer Glo assay (Promega) as above, with cells incubated in
the presence of the drug for 48 h before being assayed.
Data analysis. For the MERS-CoV screen, a minimum of four repli-
cates were performed on two separate days. For the SARS-CoV screen, a
minimum of two replicates were performed on two separate days. Outlier
data points were defined as values that were greater than the median plus
3 standard errors (␴) and were excluded from calculations.
For MERS screening, raw phenotype measurements (T) from each
treated well were converted to normalized fractional inhibition, I,bythe
formula I⫽1⫺(T/V), relative to the median, V, of vehicle-treated wells
arranged around the plate. For SARS screening with a CPE endpoint, the
calculation used to measure the antiviral activity of the compounds was
the Percent Normal. The Percent Normal monitors the reduction in cy-
tolysis of cells due to the presence of compound treatment and is deter-
mined as follows: Percent Normal ⫽(T⫺V)/(N⫺V), where Trepresents
the number of cells infected with SARS-CoV and treated with compound,
Vrepresents the number of cells infected with SARS-CoV but vehicle
treated, and Nrepresents the number of the normal control cells that are
neither infected nor treated with compound.
After normalization, average activity values were calculated between
replicate measurements at the same treatment doses along with ␴
1
, the
accompanying standard error estimates. Drug response curves were rep-
resented by a logistic sigmoidal function with a maximal effect level
(A
max
), the concentration at half-maximal activity of the compound
(EC
50
), and a Hill coefficient representing the sigmoidal transition. We
used the fitted curve parameters to calculate the concentration at which
the drug response reached an absolute inhibition of 50% (EC
50
), limited
to the maximum tested concentration for inactive compounds.
Compounds were considered active if the antiviral activity observed
was ⬎50% I(or Percent Normal) with no or low corresponding cytotox-
icity (⬍30% I).
RESULTS
Overview of screening process. A primary screen of 290 com-
pounds containing both approved drugs and developmental
drugs with defined cellular targets was performed with three-
point dose-response curves to identify compounds with activity
against MERS-CoV using a cell-based ELISA (Fig. 1). The analysis
of the raw screening data indicated that 72 compounds were active
against MERS-CoV (⬎50% inhibition) with no or low cytotoxic-
ity (⬍30% toxicity). In the secondary screen, the 72 compounds
were plated at eight doses for confirmation of antiviral activity
against MERS-CoV as well as to determine EC
50
s in the MERS-
CoV ELISA. The 72 compounds were also evaluated for their an-
tiviral activity against SARS-CoV using a cytopathic effect (CPE)
inhibition assay. An independent screen using a subset of 102
compounds against SARS-CoV infection identified 6 unique
compounds with activity against SARS-CoV.
Overview of drugs active against SARS-CoV, MERS-CoV, or
both. Analysis of data from all screening activities resulted in a list
of 66 compounds that were active against SARS-CoV, MERS-
CoV, or both. In summary, we found six drugs that were active
against SARS-CoV only, 33 drugs that were active against MERS-
CoV only, and 27 drugs that were active against both SARS-CoV
and MERS-CoV. These drugs were grouped based upon their rec-
ognized mechanism of action into 13 different therapeutic classes
that were active against SARS-CoV, MERS-CoV, or both (Table
1). The high hit rates of 21% (60 of 290) for MERS-CoV inhibitors
and 19% (33 of 174) for SARS-CoV inhibitors can be explained by
the fact that the library was enriched for compounds that have
shown antiviral activity against other viruses (Glass et al., unpub-
lished).
FIG 1 Flowchart of screening procedure. A library of 290 compounds was
screened at three doses for activity against MERS-CoV. Seventy-two com-
pounds that had activity against MERS-CoV were subsequently screened
against both MERS-CoV and SARS-CoV. Twenty-seven compounds showed
activity (⬎50% inhibition) against both viruses, while 33 compounds were
active against only MERS-CoV. A 102-compound subset was screened against
SARS-CoV, leading to 6 compounds that were active against only SARS-CoV.
Pharmaceuticals with Activity against MERS-CoV
August 2014 Volume 58 Number 8 aac.asm.org 4887

Pharmaceuticals that inhibited both coronaviruses included
neurotransmitter inhibitors, estrogen receptor antagonists, kinase
signaling inhibitors, inhibitors of lipid or sterol metabolism, pro-
tein-processing inhibitors, and inhibitors of DNA synthesis/re-
pair. Antiparasitics or antibacterials were two classes of pharma-
ceuticals in which function was not obviously linked to
coronaviruses, or viruses in general, but showed antiviral activity
against SARS-CoV and MERS-CoV. We also found that a cathep-
sin inhibitor, E-64-D, blocked both SARS-CoV and MERS-CoV,
though this was not surprising since it is known that cathepsins are
important for the fusion step during virus entry of coronaviruses
(26).
Interestingly, classes of drugs that seem to inhibit only SARS-
CoV or MERS-CoV, but not both, were discovered. Though we
identified only a small number of SARS-CoV-only inhibitors, they
are primarily anti-inflammatories, which interfere with cell sig-
naling associated with the immune response to virus infection.
MERS-CoV was specifically blocked by inhibitors of ion trans-
port, the cytoskeleton (specifically tubulin), and apoptosis.
Specific drugs. Twenty-seven specific drugs inhibited both
MERS-CoV and SARS-CoV infection (Table 2; see also Fig. S1 and
S2 in the supplemental material). We present a selection of drugs
in Fig. 2,3, and 4that are particularly interesting because they have
similar structures or similar mechanisms of action or have been
tested against other viruses. Data on antiviral activity and cytotox-
icity for the remaining compounds that inhibit MERS-CoV and
SARS-CoV are provided in the supplemental material.
In total, 16 neurotransmitter antagonists were found to have
activity against one or both of the coronaviruses (Table 1). Eleven
of these antagonists were active against both MERS-CoV and
SARS-CoV, two against only SARS-CoV, and three against only
MERS-CoV. Two of the neurotransmitter inhibitors that inhibit
both MERS-CoV and SARS-CoV are chlorpromazine hydrochlo-
ride and triflupromazine hydrochloride (Table 2). Both of these
drugs inhibit the dopamine receptor, and they have similar chem-
TABLE 1 Compounds with activity against MERS-CoV and/or SARS-
CoV
a
Pharmaceutical class
No. of compounds with activity
against:
Total no.
of drugs
for class
SARS-
CoV
only
MERS-
CoV
only
SARS-
CoV and
MERS-CoV
Antibacterial agents 1 1 2
Antiparasitic agents 2 4 6
Neurotransmitter inhibitors 2 3 11 16
Estrogen receptor inhibitors 3 2 5
DNA inhibitors 3 1 4
Protein-processing inhibitors 1 3 4
Signaling kinase inhibitors 1 2 3
Cytoskeleton inhibitors 8 8
Lipid, sterol metabolism
inhibitors
22 4
Anti-inflammatory agents 3 3
Ion channel inhibitors 9 9
Apoptosis inhibitors 1 1
Cathepsin inhibitors 1 1
Total 6 33 27 66
a
Drugs showed inhibition (⬎50%) against the virus(es) and low cytotoxicity (⬍30%).
TABLE 2 Specific compounds with activity against MERS-CoV and SARS-CoV
Compound Pharmaceutical class MERS-CoV EC
50
SARS-CoV EC
50
Emetine dihydrochloride hydrate Antibacterial agent 0.014 0.051
Chloroquine diphosphate Antiparasitic agent 6.275 6.538
Hydroxychloroquine sulfate Antiparasitic agent 8.279 7.966
Mefloquine Antiparasitic agent 7.416 15.553
Amodiaquine dihydrochloride dihydrate Antiparasitic agent 6.212 1.274
E-64-D Cathepsin inhibitor 1.275 0.760
Gemcitabine hydrochloride DNA metabolism inhibitor 1.216 4.957
Tamoxifen citrate Estrogen receptor inhibitor 10.117 92.886
Toremifene citrate Estrogen receptor inhibitor 12.915 11.969
Terconazole Sterol metabolism inhibitor 12.203 15.327
Triparanol Sterol metabolism inhibitor 5.283
Anisomycin Protein-processing inhibitor 0.003 0.191
Cycloheximide Protein-processing inhibitor 0.189 0.043
Homoharringtonine Protein-processing inhibitor 0.0718
Benztropine mesylate Neurotransmitter inhibitor 16.627 21.611
Fluspirilene Neurotransmitter inhibitor 7.477 5.963
Thiothixene Neurotransmitter inhibitor 9.297 5.316
Fluphenazine hydrochloride Neurotransmitter inhibitor 5.868 21.431
Promethazine hydrochloride Neurotransmitter inhibitor 11.802 7.545
Astemizole Neurotransmitter inhibitor 4.884 5.591
Chlorphenoxamine hydrochloride Neurotransmitter inhibitor 12.646 20.031
Chlorpromazine hydrochloride Neurotransmitter inhibitor 9.514 12.971
Thiethylperazine maleate Neurotransmitter inhibitor 7.865
Triflupromazine hydrochloride Neurotransmitter inhibitor 5.758 6.398
Clomipramine hydrochloride Neurotransmitter inhibitor 9.332 13.238
Imatinib mesylate Kinase signaling inhibitor 17.689 9.823
Dasatinib Kinase signaling inhibitor 5.468 2.100
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4888 aac.asm.org Antimicrobial Agents and Chemotherapy

ical structures (Fig. 2A), sharing the same core structure, with the
only difference being the nature of the halide group: chlorprom-
azine hydrochloride has a single chlorine, while triflupromazine
hydrochloride has three fluorine atoms surrounding a carbon.
Both chlorpromazine hydrochloride and triflupromazine hydro-
chloride strongly inhibit MERS-CoV and SARS-CoV, with micro-
molar EC
50
s (range, 5.76 ␮M to 12.9 ␮M) and low toxicity (Fig. 2B
and C). No significant difference was observed between the effects
FIG 2 Antiviral activity of chlorpromazine hydrochloride and triflupromazine hydrochloride. (A) Chemical structures of the compounds. Vero E6 cells were
infected with MERS-CoV or SARS-CoV at an MOI of 0.1 or 1, respectively, and treated for 48 h with eight doses of chlorpromazine hydrochloride (B) or
triflupromazine hydrochloride (C). Antiviral activity is shown in blue, and cytotoxicity is shown in red. EC
50
s are indicated. Results are representative of one
experiment (means ⫾standard errosr of the means [SEM]; n⫽2).
FIG 3 Antiviral activity of dasatinib and imatinib mesylate. Vero E6 cells were infected with MERS-CoV or SARS-CoV at an MOI of 0.1 or 1, respectively, and
treated for 48 h with eight doses of dasatinib (A) or imatinib mesylate (B). Antiviral activity is shown in blue, and cytotoxicity is shown in red. EC
50
s are indicated.
Results are representative of one experiment (means ⫾SEM; n⫽2).
Pharmaceuticals with Activity against MERS-CoV
August 2014 Volume 58 Number 8 aac.asm.org 4889

of these drugs on MERS-CoV and SARS-CoV; for example, trif-
lupromazine hydrochloride inhibits both MERS-CoV and SARS-
CoV with approximately the same EC
50
(5.76 ␮M and 6.39 ␮M,
respectively [Fig. 2C]). The similarity in the structures of chlor-
promazine hydrochloride and triflupromazine hydrochloride
would suggest that they inhibit MERS-CoV and SARS-CoV using
the same mechanism of action. Chlorpromazine hydrochloride
has been used to study virus entry by clathrin-mediated endocy-
tosis of several viruses, including West Nile virus (WNV) and
influenza virus (27–31). SARS-CoV also utilizes the clathrin-me-
diated endocytosis pathway for entry (32), suggesting that this
drug may act similarly on MERS-CoV and SARS-CoV and have
potential as a broad-spectrum coronavirus inhibitor.
We identified three inhibitors of the kinase signaling pathway,
two (imatinib mesylate and dasatinib) that are active against both
MERS-CoV and SARS-CoV, and one (nilotinib) that inhibits
SARS-CoV only. Imatinib mesylate and dasatinib are known in-
hibitors of the Abelson murine leukemia viral oncogene homolog
1 (ABL1) pathway. The ABL1 pathway is a signaling pathway in-
volved in cell differentiation, cell adhesion, and the cellular stress
response. Overactivation of the ABL1 pathway can lead to chronic
myelogenous leukemia. Both imatinib mesylate and dasatinib
were developed and approved as inhibitors of this pathway for
treating human cancers, including chronic myelogenous leuke-
mia (33,34). Both imatinib mesylate and dasatinib inhibit SARS-
CoV and MERS-CoV with micromolar EC
50
s (range, 2.1 to 17.6
␮M) and low toxicity (Fig. 3A and B). SARS-CoV does appear to
be more sensitive to both ABL1 inhibitors; for example, the EC
50
of dasatinib against SARS-CoV is 2.1 ␮M, whereas for MERS-CoV
the EC
50
is 5.4 ␮M(Fig. 3A). A third ABL1 inhibitor, nilotinib, was
also used in this study. Nilotinib is able to inhibit SARS-CoV with
a micromolar EC
50
and low toxicity (data not shown) but does not
significantly inhibit MERS-CoV, with the maximum inhibition of
MERS-CoV being 39% at the highest dose tested (data not
shown). However, the fact that nilotinib is able to inhibit SARS-
CoV and partially inhibit MERS-CoV further points to the impor-
tance of the ABL1 pathway in coronavirus replication. Imatinib
mesylate has been shown to block egress of Ebola virus and of
poxviruses and entry of coxsackievirus (20,35,36). These data
suggest that the ABL1 pathway may be important for replication
of many different virus families and, therefore, inhibitors of this
pathway have the potential to be broad-spectrum antivirals.
Gemcitabine hydrochloride is a deoxycytidine analog that in-
hibits DNA synthesis and repair. Gemcitabine hydrochloride in-
hibits both MERS-CoV and SARS-CoV with micromolar EC
50
s
(1.2 ␮M and 4.9 ␮M, respectively) and low toxicity (Fig. 4A).
Interestingly, we identified four DNA synthesis inhibitors that
were active against at least one coronavirus (Table 1), suggesting
that these drugs have potential as antivirals for coronaviruses.
These data also demonstrate the importance of screening large
drug sets, rather than targeted screens of suspected inhibitors, as it
may not have been immediately obvious that a DNA synthesis
inhibitor would have any effect on the replication of an RNA virus.
Toremifene citrate is an estrogen receptor 1 antagonist that
inhibits both MERS-CoV and SARS-CoV with micromolar EC
50
s
(12.9 ␮M and 11.97 ␮M, respectively) and low toxicity (Fig. 4B).
Toremifene citrate has been tested against several filoviruses and
was shown to block filovirus entry (21,37). In the screens de-
scribed here, there were five estrogen receptor inhibitors that
blocked at least one coronavirus (Table 1), and two of these
blocked both MERS-CoV and SARS-CoV with micromolar EC
50
s
(Table 2) and low toxicity. While the antiviral mechanism against
MERS-CoV and SARS-CoV is unknown, these results suggest that
estrogen receptor 1 inhibitors have the potential for broad-spec-
trum antiviral activity.
DISCUSSION
In order to prevent the emergence of a novel virus from growing
into a pandemic or established human pathogen, it is critical that
public health officials and clinicians be able to diagnose the infec-
FIG 4 Antiviral activity of gemcitabine hydrochloride and toremifene citrate. Vero E6 cells were infected with MERS-CoV or SARS-CoV at an MOI of 0.1 or 1,
respectively, and treated for 48 h with eight doses of gemcitabine hydrochloride (A) or toremifene citrate (B). Antiviral activity is shown in blue, and cytotoxicity
is shown in red. EC
50
s are indicated. Results are representative of one experiment (means ⫾SEM; n⫽2).
Dyall et al.
4890 aac.asm.org Antimicrobial Agents and Chemotherapy

tion, control its spread, and treat those afflicted. First and fore-
most, we need more countermeasures that can be used for the
early phase of an epidemic to provide an immediate treatment
response while more-appropriate therapies are being developed.
Given the time and costs associated with licensure of novel thera-
peutics, one feasible and rapid response is through repurposing of
existing clinically developed products. Repurposing of approved
drugs has several advantages, including known safety/tolerability
profiles, availability, lower cost, and familiarity of clinicians in
working with these drugs. Supplying the international community
with robust sets of in vitro and in vivo data on potential drugs for
treatment of emerging viral diseases continues to be a high prior-
ity, as it will allow clinicians to make educated decisions on clini-
cally available drugs for testing in intervention trials.
Here we report that screening of a library of 290 drugs either
clinically developed or with a well-defined cellular pathway iden-
tified 27 compounds with activity against MERS-CoV and SARS-
CoV, 33 compounds with activity against MERS-CoV alone, and 6
compounds with activity against SARS-CoV alone. Overall, we
have demonstrated that libraries of approved compounds can be
used to screen for inhibitors of viruses and have identified a num-
ber of potential antivirals with activity against coronaviruses.
The drugs identified here belong to 13 different classes of phar-
maceutical drugs. For two of the classes, kinase signaling inhibi-
tors and estrogen receptor antagonists, previous work with other
viruses has given insight into how these drugs may affect viral
infections. Three tyrosine kinase inhibitors, imatinib mesylate
(Gleevec), nilotinib (Tasigna), and dasatinib, were developed to
treat human cancers and were later shown to have activity against
several viruses, including poxviruses and Ebola virus (20,36).
Mechanism of action studies revealed that Abl1 tyrosine kinase
regulates budding or release of poxviruses and Ebola virus, dem-
onstrating that the c-Abl1 kinase signaling pathways play an im-
portant role in the egress of these viruses. Here we show that
kinase signaling may also be important for replication of two
members of the Coronaviridae family. Imatinib mesylate and da-
satinib inhibit MERS-CoV and SARS-CoV, while nilotinib inhib-
its only SARS-CoV. The step in viral replication in which these
kinases are involved will need to be investigated further. In vivo
studies performed in the mouse model of vaccinia virus infection
showed that imatinib mesylate was more effective than dasatinib
in blocking dissemination of the virus, and this was attributed to
the immunosuppressive effect of dasatinib (36). Nevertheless, da-
satinib may have value for treating coronaviral infections if a dos-
ing regimen that minimizes immunotoxicity while still blocking
viral replication can be defined. Imatinib mesylate (Gleevec) and
nilotinib (Tasigna) are FDA-approved oral cancer medicines and
are considered promising candidates for development into anti-
virals against poxviruses (38).
Estrogen receptor modulators represent another class of FDA-
approved drugs that have potential as antivirals in the clinic.
Toremifene citrate, which we have shown blocks both MERS-CoV
and SARS-CoV, has previously been shown to inhibit filoviruses
(21). Mechanism of action studies showed that the drug acts at a
late step of virus entry and may inhibit trafficking of the virus to
the late endosome or triggering of fusion for filoviruses (21,37).
Interestingly, the estrogen signaling pathway is not involved in the
virus entry step, indicating that these drugs may have off-target
effects or the estrogen signaling pathway plays an as-yet-undiscov-
ered role in filovirus biology. Toremifene citrate also showed ac-
tivity in the mouse model of Ebola virus infection (21).
Our screen also identified antiviral actives in the pharmaceu-
tical class of neurotransmitter receptor antagonists. These antag-
onists have been developed for psychiatric care as antipsychotics,
antiemetics, anticholinergics, and antidepressants and predomi-
nantly act by blocking the dopamine receptor or H
1
receptor (an-
tihistamine). Chlorpromazine was shown to inhibit clathrin-me-
diated endocytosis of several viruses by preventing the formation
of clathrin-coated pits at the plasma membrane (27). This drug is
currently approved by the FDA as an antipsychotic and for the
treatment of nausea (39) and is occasionally used for short-term
use as off-label treatment of severe migraine (40), making it a
promising candidate for testing as a broad-spectrum antiviral.
Astemizole, an antihistamine that was identified in our screen, is a
strong antagonist of the H
1
receptor (see Fig. S1 and S2 in the
supplemental material). Interestingly, it has been reported that
astemizole is an inhibitor of malaria and showed efficacy in two
animal models of malaria with a mechanism of action similar to
that of chloroquine (41). Although astemizole was withdrawn
from the U.S. market in 1999, it may be worthwhile to reexamine
this drug or existing analogs for short-term use in an acute infec-
tion. Previous work on chloroquine in coronavirus infections by
Barnard et al. has found that while the drug inhibits viral replica-
tion in vitro, chloroquine did not show efficacy in reducing SARS-
CoV virus titers in a nonlethal mouse model (42). Protection stud-
ies using a mouse-adapted SARS-CoV will be performed to
identify the in vivo efficacy of targeted drugs from our screen.
While development of drugs with broad activity against a virus
family or even unrelated viruses is advantageous for several rea-
sons such as immediate availability, lower costs, and recycling of
products from the strategic national stockpile, drug classes that
are more selective in their activity and affect either MERS-CoV or
SARS-CoV should also be further investigated. Our screen iden-
tified 33 MERS-CoV actives (Table 1), and the two largest classes
were cytoskeleton inhibitors (8 drugs) and ion channel inhibitors
(11 drugs). Drugs targeting the cytoskeleton specifically interfere
with microtubule polymerization and are antimitotics developed
for treatment of cancer. Some of them, such as nocodazole, have
also been used in cell biology labs to synchronize the cell division
cycle. Nocodazole’s ability to depolymerize microtubules has been
used to investigate the entry pathway of WNV, and results show
that an intact microtubule network is necessary for trafficking of
internalized WNV from early to late endosomes (27). This drug
had high activity against MERS-CoV but had no activity against
SARS-CoV, suggesting that, in addition to the application as
therapeutics, these drugs may also have value in further elucidat-
ing differences in the virus replication cycle of MERS-CoV and
SARS-CoV.
Two of the 9 ion channel inhibitors, monensin and salinomy-
cin sodium, with activity against MERS-CoV, represent polyether
ionophores that are currently well-recognized candidates for an-
ticancer drugs (43,44). Studies on the mechanism of anticancer
activity have shown that these compounds affect cancer cells by
increasing their sensitivity to chemotherapy and reversing multi-
drug resistance (monensin) in human carcinoma. Furthermore,
ionophore antibiotics also inhibit chemoresistant cancer cells by
increasing apoptosis, and salinomycin was specifically shown to
be able to kill human cancer stem cells (45). Interestingly, these
compounds affected MERS-CoV but not SARS-CoV, indicating
Pharmaceuticals with Activity against MERS-CoV
August 2014 Volume 58 Number 8 aac.asm.org 4891

that MERS-CoV is uniquely susceptible to ionophore activities.
Monensin has also been reported to inhibit La Crosse virus and
Uukuniemi virus infection by blocking the formation and egress
of virus particles (46,47). Further studies will reveal if these drugs
act at a similar step during MERS-CoV infection.
Overall, we identified several pharmaceutical classes of drugs
that could be beneficial for treatment of coronaviral infections.
Interestingly, chlorpromazine hydrochloride and chloroquine
diphosphate were also identified in a similar but independent
study described in the accompanying paper by A. H. de Wilde et al.
(48). These drugs appear to target host factors rather than viral
proteins specifically, and treatment of viral infections in patients
aimed at host factors could reconfigure overt manifestations of
viral pathogenesis into a less virulent subclinical infection and
lower adverse disease outcome (38). The targets identified in this
paper provide new candidates for future research studies and clin-
ical intervention protocols.
ACKNOWLEDGMENTS
We thank Laura Pierce, Anatoly Myaskovsky, Kelly DeRoche, and Crag
Markwood at Zalicus Inc. for compound plate preparation and data inte-
gration. We thank Yingyun Cai and Cindy Allan for outstanding assis-
tance in the development of the drug screen protocol. We thank the IRF
Cell Culture staff in preparing the cells used in this study. In addition, we
acknowledge Laura Bollinger and Jiro Wada at the IRF for technical writ-
ing services and figure preparation for the manuscript.
This work was supported by the Division of Intramural Research of the
National Institute of Allergy and Infectious Diseases (NIAID), the Inte-
grated Research Facility (NIAID, Division of Clinical Research), the Bat-
telle Memorial Institute’s prime contract with NIAID (contract number
HHSN2722007000161) and NIH grant R01AI1095569 (to M.B.F.), and a
subcontract (W81XWH-12-2-0064) awarded to L.M.J. from the U.S.
Army Research Institute of Infectious Diseases (USAMRIID).
L.M.J. was employed at Zalicus Inc. during the time the researched was
performed. M.L. is currently employed at Zalicus Inc. No other authors
have conflicts of interest.
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