Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses

Abstract

Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention.
Copyright © 2015. Published by Elsevier B.V.

Full text

Inhibitors of cellular kinases with broad-spectrum antiviral activity for
hemorrhagic fever viruses
q
Emma L. Mohr
a,b
, Laura K. McMullan
a
, Michael K. Lo
a
, Jessica R. Spengler
a
, Éric Bergeron
a
,
César G. Albariño
a
, Punya Shrivastava-Ranjan
a
, Cheng-Feng Chiang
a
, Stuart T. Nichol
a
,
Christina F. Spiropoulou
a,
⇑
, Mike Flint
a
a
Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Road, MS G-14, Atlanta, GA 30333, USA
b
Emory University Department of Pediatrics, Emory-Children’s Center, 2015 Uppergate Drive, Atlanta, GA 30322, USA
article info
Article history:
Received 6 April 2015
Revised 7 May 2015
Accepted 11 May 2015
Available online 16 May 2015
Keywords:
Ebola
Lassa
Antiviral
AR-12
BIBX
abstract
Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a
panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses.
OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382
dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola
virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved
virus–host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeu-
tic intervention.
Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creative-
commons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Viral hemorrhagic fevers (VHFs) cause significant morbidity and
mortality globally. The infectious etiologies of VHFs include mem-
bers of several RNA virus families, including the Arenaviridae,
Bunyaviridae,Filoviridae, and Flaviviridae. The leading cause of
VHF worldwide is Lassa virus (LASV), an arenavirus endemic to
West Africa that causes 300,000–500,000 infections annually
(Ogbu et al., 2007). Ebola virus (EBOV), Marburg virus (MARV),
Junin virus (JUNV), Alkhurma hemorrhagic fever virus (AHFV,
called Alkhumra virus in some reports), and Crimean Congo hem-
orrhagic fever virus (CCHFV), as well as the encephalitic Nipah
virus (NiV), cause sporadic outbreaks, often with high
case-fatality rates (Aljofan, 2013; Kortekaas et al., 2010; MacNeil
and Rollin, 2012; Madani, 2005). These highly pathogenic agents
are all classified as biosafety level 4 (BSL-4) pathogens; there are
no approved therapeutics or vaccines, and medical care for patients
is generally only supportive. Despite the challenges inherent in
studying BSL-4 agents, research into therapies for these viruses is
critical because of the potential for large outbreaks with high
case-fatality rates, as demonstrated by the 2013–2015 EBOV out-
break in West Africa.
Host cell kinases have been implicated in the replication of sev-
eral BSL-4 viruses. One signaling pathway, the phosphatidylinositol
3-kinase (PI3K)/Akt pathway, was reported to be essential for the
propagation of LASV and EBOV in cell culture. Inhibition of the
PI3K/Akt pathway by the small molecule BEZ-235 impeded the
budding of LASV virus-like particles (VLPs) (Urata et al., 2012).
Another inhibitor, LY294002, blocked EBOV entry (Saeed et al.,
2008) and an early event in JUNV infection (Linero and Scolaro,
2009). The replication of Andes virus, a bunyavirus, was blocked
by temsirolimus, an inhibitor of mTOR, another kinase in the
PI3K/Akt pathway (McNulty et al., 2013). We therefore hypothe-
sized that a cellular kinase could be essential for the replication
of multiple highly pathogenic viruses. Identifying such a kinase
http://dx.doi.org/10.1016/j.antiviral.2015.05.003
0166-3542/Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Abbreviations: AHFV, Alkhurma hemorrhagic fever virus; BSL-4, biosafety level
4; CC
50
, 50% cytotoxic concentration; CCHFV, Crimean Congo hemorrhagic fever
virus; EBOV, Ebola virus; EC
50
, 50% effective concentration; GFP, green fluorescent
protein; LASV, Lassa virus; MARV, Marburg virus; NiV, Nipah virus; PBS, phosphate
buffered saline; SI, selectivity index; VHF, viral hemorrhagic fever; VLP, virus-like
particle; VSV, vesicular stomatitis virus.
q
The findings and conclusions in this report are those of the authors and do not
necessarily represent the official position of the Centers for Disease Control and
Prevention.
⇑
Corresponding author. Tel.: +1 404 639 1294.
E-mail address: ccs8@cdc.gov (C.F. Spiropoulou).
Antiviral Research 120 (2015) 40–47
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might shed light on conserved virus–host interactions. In addition,
therapies targeting such kinases could have broad-spectrum
antiviral activity, a desirable property given the difficulty of devel-
oping therapies for individual hemorrhagic fever viruses. Here, we
report the identification of 2 inhibitors of cellular kinases which
impeded the replication of multiple highly pathogenic viruses.
2. Materials and methods
2.1. Biosafety
All work with infectious virus was conducted in a BSL-4 labora-
tory at the Centers for Disease Control and Prevention (CDC,
Atlanta, GA). All laboratorians adhered to international practices
appropriate for this biosafety level. Experiments involving cDNA
encoding viral sequences were approved by the CDC Institutional
Biosafety Committee.
2.2. Cell lines, viruses, and compounds
A549, Vero-E6, HeLa, and HT-1080 cells were from the CDC
Biologics Branch and HEK-293 cells were from ATCC. These cell lines
were maintained in Dulbecco’s modified Eagle’s medium (DMEM;
Life Technologies, Grand Island, NY, USA) supplemented with 10%
(v/v) fetal calf serum (FCS; Hyclone, Thermo Scientific, Waltham,
MA, USA) and penicillin–streptomycin (Life Technologies). Huh7
cells were from Apath, LLC (Brooklyn, NY, USA) and were propa-
gated in DMEM, 10% (v/v) FCS, and 1 non-essential amino acids
(Life Technologies). Viruses were from the CDC Viral Special
Pathogens Branch reference collection: LASV (strain Josiah); EBOV
(strain Mayinga); AHFV (strain 200300001); CCHFV (strain
IbAr10200). The Kinase Inhibitor Toolbox library and BIBX 1382
dihydrochloride were from Tocris Bioscience (Bristol, UK). The
PI3K Signaling Inhibitor Library and OSU-03012 were from Selleck
Chemicals (Houston, TX, USA). Compounds were diluted in
dimethylsulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO, USA) as
indicated.
2.3. Assays for antiviral activity and cell viability
To test for the inhibition of LASV replication, A549 cells were
seeded at a density of 1 10
4
cells/well of a 96-well plate the
day prior to infection. Compounds were added to the cells, and
1 h later, the cells were infected with LASV at an MOI of 0.2.
After 48 h, the monolayers were fixed with 10% (v/v) formalin
(Sigma–Aldrich) and
c
-irradiated with 2 10
6
rads. The cells were
permeabilized with 0.1% (v/v) Triton X-100 in phosphate buffered
saline (PBS) for 10 min at room temperature, and LASV proteins
were detected with monoclonal antibodies directed against the
LASV glycoprotein and nucleoprotein (1:10,000 dilution in PBS
supplemented with 2% w/v bovine serum albumin) and goat
anti-mouse Alexa 488 (1:1000; Life Technologies). Cells were
stained with CellMask Red and NucBlue (Life Technologies) and
immunofluorescence microscopy was performed using the
Operetta Imaging System (PerkinElmer, Waltham, MA).
The assay for the inhibition of AHFV-induced cytopathic effect
in A549 cells was as described previously (Flint et al., 2014). The
recombinant reporter viruses NiV-luc and EBOV and MARV
expressing green fluorescent protein (GFP) reporter (EBOV-GFP
and MARV-GFP) have also been described (Albariño et al., 2013;
Lo et al., 2014; Towner et al., 2005). The assay for the inhibition
of CCHFV-induced cytopathic effects was based on one described
previously (Paragas et al., 2004). Briefly, compounds were added
to SW13 cells in an 80% confluent monolayer in a 96-well plate, fol-
lowed by infection with CCHFV at an MOI of 0.1, 1 h later. Cell
viability, as measured by CellTiter-Glo (Promega, Madison, WI,
USA), was measured 72 h post infection.
Cell viability was determined concurrently with the virus inhi-
bition assays, but on compound-treated and mock-infected cells,
using CellTiter-Glo (Promega) or PrestoBlue (Life Technologies)
according to the manufacturer’s instructions. Viability was also
assessed by nuclei number, as determined by counting the
NucBlue-stained organelles with Harmony image analysis software
(PerkinElmer). For each assay, values were normalized to
vehicle-only DMSO controls.
For compound titrations, GraphPad Prism (GraphPad Software,
La Jolla, CA, USA) was used to fit a 4-parameter equation to semilog
plots of the concentration–response data and to interpolate the
concentration of compound that inhibited 50% of the virus replica-
tion (EC
50
). The 50% cytotoxic concentration (CC
50
) was similarly
derived using viability data from mock-infected cells. The selectiv-
ity index (SI) was calculated by dividing the CC
50
by the EC
50
.
2.4. Viral titer reduction assay
Titer reduction assays for LASV and EBOV were performed in
A549 and Huh7 cells, respectively. Cells were treated with com-
pounds for 1 h prior to infection. Two days later, culture super-
natants were harvested and virus titrations were performed in
Vero-E6 cells. Three days post infection, the cells were fixed, per-
meabilized, and stained to visualize viral proteins. End-point viral
titers were determined, and the 50% tissue culture infectious dose
(TCID
50
) was calculated using the method of Reed and Muench
(Reed and Muench, 1938).
2.5. Quantitative reverse transcription polymerase chain reaction
assay
Cells were seeded and treated with compounds for 1 h before
infection with LASV at an MOI of 0.1. The medium was removed
24 h post-infection, lysis buffer (MagMax Total RNA isolation kit;
Life Technologies) was added, and RNA was extracted using a
MagMax-96 deep-well magnetic particle processor (Life
Technologies). Quantitative reverse transcription polymerase chain
reaction (qRT-PCR) was performed with the Express One-Step
Superscript qRT-PCR kit (Life Technologies) and analyzed on an
Applied Biosciences 7500 real-time PCR machine (Life
Technologies). LASV nucleoprotein RNA was quantitated using for-
ward (5
0
-AATCAGTTCGGGACCATGC-3
0
) and reverse (5
0
-GTGTTGG
GATACTTTGCTGTG-3
0
) primers and a probe oligonucleotide (5
0
-/5
6-FAM/AGTCAACCT/ZEN/GCCCCTGTTTTGTCA/Iowa Black FQ/-3
0
)
from Integrated DNA Technologies (Coralville, IA). Levels of glycer-
aldehyde 3-phosphate dehydrogenase (GAPDH) RNA, or 18S ribo-
somal RNA in Vero-E6 cells, were determined using control
primer-probe sets (Life Technologies). Viral RNA levels were nor-
malized to GAPDH or 18S RNA and expressed relative to infected,
vehicle-treated controls.
2.6. VLP assembly assay
HEK-293 cells were transfected with plasmids encoding
FLAG-tagged LASV Z protein, or the non-budding G2A mutant
(Perez et al., 2004), using Lipofectamine 2000 (Life Technologies).
Following overnight incubation, the transfection media was
removed and compounds were added to a final DMSO concentra-
tion of 0.5%. After 48 h, cell lysates were prepared with radioim-
munoprecipitation assay (RIPA) buffer supplemented with
protease inhibitors (Complete Protease Inhibitor, Roche,
Indianapolis, IN, USA). The culture medium was clarified by cen-
trifugation at 4000gfor 20 min, and VLPs were concentrated from
the supernatants by centrifugation at 300,000gthrough a 20%
E.L. Mohr et al. / Antiviral Research 120 (2015) 40–47 41

sucrose cushion. Cell lysates and pelleted VLPs were analyzed by
Western blotting with anti-FLAG M2-peroxidase antibody
(Sigma–Aldrich) or anti-bactin antibody (Genscript, Piscataway,
NJ, USA).
2.7. Assay for LASV and EBOV glycoprotein-dependent entry
HIV pseudotyped particles bearing the glycoproteins of LASV,
EBOV from the 1976 Zaire (Genbank accession No. U23187.1)
and 2014 West African (KP178538.1) outbreaks, vesicular stomati-
tis virus (VSV) G protein, or no glycoprotein were prepared and
used as previously (Flint et al., 2004). Briefly, LentiX-293T cells
(Clontech, Mountain View, CA, USA) were co-transfected with plas-
mid DNA encoding the HIV genome containing the firefly luciferase
gene (pNL4–3.Luc.R
-
.E
-
) and expression vectors encoding the viral
glycoprotein or empty vector in a 32:1 ratio. Pseudotyped viruses
were quantitated by determining HIV matrix protein (p24) con-
tent. Viral glycoprotein-dependent entry assays were performed
using HT-1080 cells and 6 ng of p24 pseudotyped particles, with
firefly luciferase activity determined using BrightGlo (Promega)
72 h post-transduction. Cell viability was concurrently determined
by CellTiter-Glo (Promega) in compound-treated and
mock-transduced cells.
3. Results
3.1. Identification of kinase inhibitors with activity against
hemorrhagic fever viruses
We hypothesized that a cellular kinase would be essential for
the replication of multiple BSL-4 viruses. Therefore, we initially
tested a total of 163 kinase inhibitors, focusing on the PI3K/Akt
Table 1
Compounds inhibiting LASV replication in A549 cells.
Compound % infected cells
a
% cell viability
a
Reported targets
b
5
l
M 500 nM 50 nM 5
l
M 500 nM 50 nM
BEZ-235 14 ± 3 19 ± 5 43 ± 7 31 ± 4 32 ± 1 48 ± 3 PI3K, mTOR
OSU-03012 0 ± 0 32 ± 9 94 ± 18 60 ± 4 82 ± 1 94 ± 4 PDK-1, PAK
AZD8055 4 ± 3 17 ± 15 18 ± 4 34 ± 4 36 ± 1 55 ± 2 mTOR
TWS119 24 ± 9 41 ± 16 41 ± 3 71 ± 7 91 ± 2 93 ± 1 GSK-3
SU 4312 32 ± 6 80 ± 19 83 ± 29 96 ± 3 100 ± 2 98 ± 3 VEGFR
BIBX 1382 dihydrochloride 8 ± 1 69 ± 5 75 ± 7 70 ± 4 85 ± 1 94 ± 3 ErbB1, ErbB2, ErbB4
Ki 8751 23 ± 1 70 ± 14 90 ± 18 57 ± 5 99 ± 2 100 ± 4 VEGFR
10-DEBC hydrochloride 11 ± 5 85 ± 25 81 ± 7 69 ± 3 97 ± 2 95 ± 1 Akt, PKB
a
Mean % infected cells or % viability ± standard deviation from 3 replicate wells, normalized to the vehicle-only control.
b
PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PAK, p21-activated kinase; mTOR, mammalian target of rapamycin; PDK-1, 3-phosphoinositide-dependent kinase
1; GSK-3, glycogen synthase kinase 3; VEGFR, vascular endothelial growth factor receptor; EGFR, epidermal growth factor receptor; PKB, protein kinase B; n/a, not applicable.
A
CB
1852 617 206 69 23 8
Mock-infected DMSO
OSU-03012 concentr on (nM)
% infected cells
% viability
Fig. 1. OSU-03012 inhibits LASV and EBOV in cell culture. (A) OSU-03012 induced a concentration-dependent reduction in LASV replication. A549 cells were treated for 1 h
with varying concentrations of OSU-03012 before infection with LASV at an MOI of 0.2. Two days post infection, the cells were fixed, permeabilized, and stained. Green, LASV
proteins; blue, cell nuclei; red, cell cytoplasm. (B) A representative concentration–response curve showing the quantitation of LASV-infected cells (%) normalized to the
vehicle-only control; each point is the mean of 9 fields from each of quadruplicate wells, with error bars indicating the standard deviation. Cell viability (%) of compound-
treated, mock-infected cells is also shown; each point is the mean of quadruplicate wells, with error bars indicating standard deviation. (C) A representative concentration–
response curve showing viability of mock-infected cells, or inhibition of GFP expression in Huh 7 cells infected with the EBOV-GFP reporter virus. Points represent mean
values, and error bars indicate standard deviations calculated from 4 replicate wells.
42 E.L. Mohr et al. / Antiviral Research 120 (2015) 40–47

pathway, for their ability to inhibit the replication of LASV. A549
cells were treated with compounds 1 h prior to mock-infection or
infection with LASV at an MOI of 0.2. Two days post infection,
the cells were fixed, permeabilized, and stained to visualize LASV
proteins and the mock-infected cells were tested for viability
(Supplemental Table 1). BEZ-235, a known inhibitor of LASV VLP
formation (Urata et al., 2012), was used as a positive control. In
addition to BEZ-235, 7 compounds had anti-LASV activity with
minimal cytotoxicity (Table 1). These compounds were tested for
their ability to inhibit the replication of EBOV-GFP, MARV-GFP,
and NiV-luc recombinant reporter viruses, as well as AHFV and
CCHFV. Two compounds, OSU-03012 (also known as AR-12) and
BIBX 1382 dihydrochloride, demonstrated inhibition of both
LASV and other viruses and were further characterized.
Table 2
OSU-03012 activity against selected viruses.
Assay type
a
Cell line Multiplicity of infection Duration (h) EC
50b
(
l
M) CC
50
CellTiter-Glo (
l
M) SI CellTiter-Glo
LASV Immunofluorescence A549 0.2 48 0.5 ± 0.1 5.7 ± 2.5 11
AHFV CPE inhibition A549 0.1 72 2.9 ± 0.8 6.5 ± 1.1 2
NiV-Luc Luc reporter 293T 0.2 48 0.4 ± 0.2 8.2 ± 0.9 21
EBOV-GFP GFP reporter Huh7 0.2 48 0.3 ± 0.07 6.4 ± 1.1 23
MARV-GFP GFP reporter Huh7 0.2 48 0.3 ± 0.1 7.1 ± 0.6 20
CCHFV CPE inhibition SW13 0.1 72 No inhibition 6.7 ± 0.8 n/a
a
CPE, cytopathic effect; Luc, luciferase; GFP, green fluorescent protein.
b
Mean EC
50
and CC
50
values ± the standard deviation from at least 3 independent determinations are shown.
Table 3
Activity of BIBX 1382 dihydrochloride against selected viruses.
EC
50a
(
l
M) CC
50
CellTiter-Glo (
l
M) SI CellTiter-Glo CC
50
PrestoBlue (
l
M) SI PrestoBlue CC
50
nuclei number (
l
M) SI nuclei number
LASV 3.2 ± 2.4 15.3 ± 6.7 6 21.0 ± 1.4 7 63.4 ± 16.7 24
EBOV-GFP 1.1 ± 0.6 19.8 ± 0.3 18 ND ND ND ND
MARV-GFP 1.8 ± 0.2 29.1 ± 16.4 19 ND ND ND ND
ND: not done.
a
Mean EC
50
and CC
50
values ± the standard deviation from at least 3 independent determinations are shown.
BA
DC
2831XBIB21030-USO
LASVEBOV
EC₅₀ = 257 nM
CC₅₀ = 5700 nM
SI = 22
EC₅₀ = 3250 nM
CC₅₀ = 15300 nM
SI = 5
EC₅₀ = 172 nM
CC₅₀ = 6400 nM
SI = 37
EC₅₀ = 2012 nM
CC₅₀ = 19800 nM
SI = 10
Fig. 2. OSU-03012 (A and C) and BIBX 1382 (B and D) inhibit LASV and EBOV titers in a concentration-dependent manner. Cells were in treated with varying concentrations of
compound for 1 h. A549 cells were then infected with LASV at an MOI of 0.2 (A and B), and Huh7 cells were infected with EBOV at an MOI of 0.02 (C and D). Two days post
infection, culture supernatants were harvested and viral titers determined in Vero-E6 cells. Mean titers are shown with error bars indicating standard errors calculated from 3
replicate wells.
E.L. Mohr et al. / Antiviral Research 120 (2015) 40–47 43

Fig. 3. OSU-03012 and BIBX 1382 induce a concentration-dependent decrease in LASV RNA in multiple cell lines. (A) A549, (B) Huh7, (C) Vero-E6, and (D) HT-1080 cells were
treated with the indicated compound concentrations for 1 h, and then infected with LASV at an MOI of 0.1. RNA was extracted 24 h post infection, and qRT-PCR for LASV RNA
or endogenous cell RNA was performed (white bars). Mock-infected cell monolayers were processed for viability assays using CellTiter-Glo (black bars). Columns represent
mean values, and error bars indicate standard deviations calculated from 8 replicate wells.
44 E.L. Mohr et al. / Antiviral Research 120 (2015) 40–47

3.2. OSU-03012 and BIBX 1382 inhibit multiple highly pathogenic
viruses in cell culture
OSU-03012 was reported to be an inhibitor of
3-phosphinositide-dependent protein kinase 1 (PDK-1) and an
inducer of apoptosis (Zhu et al., 2004). It reduced LASV replication
in A549 cells in a concentration-dependent manner (Fig. 1A and B),
with an EC
50
of 0.5
l
M(Table 2). We used 3 different methods to
measure cell viability to ensure the CC
50
value was accurately
determined: by determining cellular ATP (CellTiter-Glo) and reduc-
ing activity (PrestoBlue), and by counting nuclei by high content
image analysis. The CC
50
values were comparable for each assay
(5.7 ± 2.5
l
M for CellTiter-Glo, 5.4 ± 1.7
l
M for PrestoBlue,
3.1 ± 0.8
l
M for nuclei number; Table 2). The SI for OSU-03012
inhibition of LASV in A549 cells was 6–11, indicating an antiviral
effect rather than reduced virus growth due to cell death.
OSU-03012 also inhibited NiV-Luc, EBOV-GFP, and MARV-GFP with
EC
50
values around 0.3
l
M(Fig. 1C and Table 2) and SI values of
approximately 20. OSU-03012 had no detectable effect on CCHFV
replication, and any inhibition of AHFV was difficult to distinguish
from cytotoxic effects (SI = 2–3; Table 2).
BIBX 1382 was reported to be an inhibitor of the ErbB kinases,
including ErbB1, the epidermal growth factor receptor (EGFR)
kinase (Egeblad et al., 2001). In our study, it inhibited LASV,
EBOV-GFP, and MARV-GFP, with EC
50
values ranging 1.1–3.2
l
M
and SI values 6–24 (Table 3). BIBX 1382 had no specific antiviral
effects against NiV-Luc, CCHFV, or AHFV (data not shown).
Next, we tested the effect of OSU-03012 and BIBX 1382 on viral
titers. A549 or Huh7 cells were treated with the compounds for 1 h
prior to infection with LASV or wild-type EBOV, respectively. Two
days post infection, the cell supernatants were harvested and viral
titers were determined. Both OSU-03012 and BIBX 1382 reduced
LASV and EBOV titers by 2–3 logs in a concentration-dependent
manner (Fig. 2A–D) at concentrations below their CC
50
values
(compare concentrations used to CC
50
values in Tables 2 and 3).
To further confirm the antiviral effects of OSU-03012 and BIBX
1382, we measured LASV RNA in various infected cell lines. A549,
Vero-E6, HT-1080, and Huh7 cell lines were treated with the com-
pounds for 1 h, and then infected with LASV at an MOI of 0.1. RNA
was extracted from the infected cells 24 h post infection, and LASV
RNA was quantified by qRT-PCR. The viability of compound-treated
and mock-infected cells was determined simultaneously. Both
compounds induced a concentration-dependent reduction in
LASV RNA in each of the cell lines, with minimal effects on cell via-
bility (Fig. 3).
3.3. Mechanism of action studies
To understand the antiviral mechanisms of OSU-03012 and
BIBX 1382, we used assays that recapitulate individual steps of
the viral replication cycle. First, we tested the ability of these com-
pounds to inhibit LASV and EBOV glycoprotein-dependent entry by
using HIV particles pseudotyped with the viral glycoproteins.
Amiodarone, a recently reported inhibitor of EBOV-glycoprotein
dependent entry, was used as a control (Gehring et al., 2014).
OSU-03012 did not affect entry mediated by any of the glycopro-
teins tested (data not shown). In contrast, BIBX 1382 inhibited
EBOV-glycoprotein dependent entry, with similar potency against
the 1976 and 2014 EBOV sequences (Fig. 4 and Table 4). LASV
glycoprotein-dependent entry was also affected (Fig. 4 and
Table 4). OSU-03012, BIBX 1382, and amiodarone did not inhibit
VSV-G-dependent entry (Fig. 4).
When expressed in cell culture, the LASV Z protein assembles
into VLPs that are released into the culture medium (Perez et al.,
2003; Strecker et al., 2003). To determine if OSU-03012 and BIBX
1382 affected VLP formation, HEK-293 cells were transfected with
plasmids encoding LASV Z, then treated with each compound. VLPs
were harvested from the culture medium, centrifuged through
sucrose cushions, and detected by Western blotting. A mutant Z
protein (G2A) lacking the myristoylation site required for VLP
Fig. 4. Effect of (A) BIBX 1382 and (B) amiodarone on LASV and EBOV glycoprotein-
dependent entry. HT-1080 cells were transduced with HIV particles pseudotyped
with EBOV, LASV, or vesicular stomatitis virus (VSV) glycoproteins in cell culture
medium containing inhibitor compounds. Cell culture media containing the
pseudotype particles and compounds were removed 6 h post transduction and
were replaced with cell culture media without compound. Transduction was
measured by firefly luciferase activity 3 days post transduction. Mock-transduced,
compound-treated cell monolayers were processed for viability assays using
CellTiter-Glo. A representative of 3 independent experiments is shown. Points
represent mean values, and error bars indicate standard deviations calculated from
4 replicate wells.
Table 4
Effects of BIBX 1382 dihydrochloride and amiodarone on EBOV, LASV, and VSV
glycoprotein-dependent entry, and on cell viability.
Virus
glycoprotein
BIBX 1382 Amiodarone HCl
EC
50
(
l
M)
a
CC
50
(
l
M)
a
SI EC
50
(
l
M)
a
CC
50
(
l
M)
a
SI
EBOV (1976)
b
1.2 ± 0.2 24.3 ± 3.9 20 1.4 ± 0.4 47.1 ± 18 34
EBOV (2014) 1.6 ± 0.3 15 1.6 ± 0.4 29
LASV 7.5 ± 3.0 3 10.7 ± 1.3 4
VSV 30.9 ± 8.5 <1 35.4 ± 13.7 1
a
Mean EC
50
and CC
50
values ± the standard deviation from at least 3 independent
determinations are shown.
b
EBOV (1976): EBOV strain isolated during the 1976 Zaire outbreak. EBOV
(2014): EBOV strain isolated during the 2014–2015 outbreak in West Africa.
E.L. Mohr et al. / Antiviral Research 120 (2015) 40–47 45

formation (Perez et al., 2004) served as a negative control. Each
transfected cell monolayer expressed similar amounts of
wild-type Z or G2A mutant regardless of treatment (Fig. 5). As
expected, the G2A mutant was not secreted, while the wild-type
Z protein assembled into VLPs. Neither OSU-03012 nor BIBX
1382 inhibited the release of LASV Z VLPs (Fig. 5).
4. Discussion
Host cell kinases have been implicated in the replication of sev-
eral BSL-4 viruses (Linero and Scolaro, 2009; Saeed et al., 2008;
Urata et al., 2012). Here, we identified 2 inhibitors of cellular
kinases that inhibit multiple such viruses in cell culture.
OSU-03012 (AR-12) is a celecoxib derivative that does not inhi-
bit cyclooxygenase-2, but has been reported to inhibit PDK-1 and
p21-activated kinase (Porchia et al., 2007; Zhu et al., 2004). We
tested several other reported inhibitors of PDK-1 for their ability
to inhibit LASV replication, including PHT-427, BX-912, BX-795,
PS 48, and GSK 2334470. None of these had a specific anti-LASV
activity (data not shown), suggesting that the antiviral effect of
OSU-03012 is probably not mediated through PDK-1 inhibition. It
is important to note that kinase inhibitors, especially those that
bind within the highly-conserved ATP binding site, frequently inhi-
bit multiple cellular kinases. OSU-03012 was reported to induce
apoptosis or autophagy (Gao et al., 2008; Johnson et al., 2005;
Lee et al., 2009; Liu et al., 2013; Zhu et al., 2004) and was the sub-
ject of a phase I clinical trial in patients with advanced or recurrent
solid tumors or lymphoma. Recently, OSU-03012 was reported to
reduce expression of the endoplasmic reticulum chaperone
HSPA5 (also called GRP78 or BiP) (Booth et al., 2015). Such a reduc-
tion could inhibit the assembly of enveloped viruses that rely on
HSPA5 activity for glycoprotein folding. HSPA5 is incorporated into
the EBOV virion (Spurgers et al., 2010), and knocking down HSPA5
in mice conferred protection from lethal challenge with EBOV (Reid
et al., 2014). Although OSU-03012 was reported to decrease the
expression of NPC1 and LAMP1 (Booth et al., 2015), molecules
involved in the entry of EBOV and LASV (Carette et al., 2011;
Cote et al., 2011; Jae et al., 2014), it did not inhibit EBOV or LASV
glycoprotein-dependent entry in our HIV pseudotype system
(Fig. 4). We are currently investigating the effect of OSU-03012
on the maturation of the EBOV and LASV glycoproteins.
BIBX 1382 was reported to be an inhibitor of the ErbB kinases,
including EGFR (Egeblad et al., 2001). Further work is required to
understand the mechanism by which BIBX 1382 inhibits LASV
and EBOV glycoprotein-dependent entry. These two viruses appear
to use different routes of entry, with LASV apparently using
clathrin-mediated endocytosis and EBOV using macropinocytosis
(Nanbo et al., 2010; Saeed et al., 2010; Vela et al., 2007). Since
BIBX 1382 appears to inhibit the entry of both viruses, this sug-
gests that some aspect of the entry mechanism is shared between
the two. The development of BIBX 1382 was halted after a phase I
study revealed low bioavailability and a dose-limiting increase of
liver enzymes (Dittrich et al., 2002). It may therefore be difficult
to justify repurposing this compound as a candidate VHF
therapeutic.
In conclusion, we have identified 2 compounds with antiviral
activity against LASV, EBOV and MARV in cell culture. Our data
suggest that BIBX 1382 is an inhibitor of LASV and EBOV entry.
OSU-03012 reduces LASV and EBOV virus titers and RNA levels,
but does not inhibit entry or LASV Z-protein mediated assembly.
We believe that testing OSU-03012 for antiviral efficacy in appro-
priate animal models is warranted. In addition, OSU-03012 and
BIBX 1382 may be useful tools for understanding interactions
between these viruses and their host cells.
Acknowledgements
We thank Tanya Klimova for assistance with editing this manu-
script. The findings and conclusions in this report are those of the
authors and do not necessarily represent the official position of the
Centers for Disease Control and Prevention.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.antiviral.2015.05.
003.
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