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Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broad-spectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of general population from emerging and re-emerging viral diseases reinforcing the arsenal of available antiviral options. Here, we reviewed discovery and development of BSAAs and summarized the information on 119 safe-in-man agents in freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections.
Content may be subject to copyright.
Review
Discovery
and
development
of
safe-in-man
broad-spectrum
antiviral
agents
Petter
I.
Andersen
a,1
,
Aleksandr
Ianevski
a,1
,
Hilde
Lysvand
a
,
Astra
Vitkauskiene
b
,
Valentyn
Oksenych
a
,
Magnar
Bjørås
a
,
Kaidi
Telling
c
,
Irja
Lutsar
d
,
Uga
Dumpis
e
,
Yasuhiko
Irie
c
,
Tanel
Tenson
c
,
Anu
Kantele
f
,
Denis
E.
Kainov
a,c,g,
*
a
Department
of
Clinical
and
Molecular
Medicine,
Norwegian
University
of
Science
and
Technology
(NTNU),
7028
Trondheim,
Norway
b
Department
of
Laboratory
Medicine,
Lithuanian
University
of
Health
Science,
44307
Kaunas,
Lithuania
c
Institute
of
Technology,
University
of
Tartu,
50090
Tartu,
Estonia
d
Institute
of
Medical
Microbiology,
University
of
Tartu,
Tartu
50411,
Estonia
e
Latvian
Biomedical
Research
and
Study
Centre,
Riga
1067,
Latvia
f
Helsinki
University
Hospital
(HUS)
and
University
of
Helsinki,
Helsinki
00290,
Finland
g
Institute
for
Molecular
Medicine
Finland,
FIMM,
University
of
Helsinki,
00014
Helsinki,
Finland
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
24
January
2020
Received
in
revised
form
7
February
2020
Accepted
11
February
2020
Keywords:
Virus
Antiviral
drug
Drug
discovery
and
development
Broad-spectrum
antiviral
agents
BSAAs
A
B
S
T
R
A
C
T
Viral
diseases
are
one
of
the
leading
causes
of
morbidity
and
mortality
in
the
world.
Virus-specic
vaccines
and
antiviral
drugs
are
the
most
powerful
tools
to
combat
viral
diseases.
However,
broad-
spectrum
antiviral
agents
(BSAAs,
i.e.
compounds
targeting
viruses
belonging
to
two
or
more
viral
families)
could
provide
additional
protection
of
the
general
population
from
emerging
and
re-emerging
viral
diseases,
reinforcing
the
arsenal
of
available
antiviral
options.
Here,
we
review
discovery
and
development
of
BSAAs
and
summarize
the
information
on
120
safe-in-man
agents
in
a
freely
accessible
database
(https://drugvirus.info/).
Future
and
ongoing
pre-clinical
and
clinical
studies
will
increase
the
number
of
BSAAs,
expand
the
spectrum
of
their
indications,
and
identify
drug
combinations
for
treatment
of
emerging
and
re-emerging
viral
infections
as
well
as
co-infections.
©
2020
The
Authors.
Published
by
Elsevier
Ltd
on
behalf
of
International
Society
for
Infectious
Diseases.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/4.0/).
Introduction
Viruses
are
one
of
the
major
causes
of
morbidity
and
mortality
in
the
world
(DALYs
and
Collaborators,
2018;
Disease
et
al.,
2018;
Howard
and
Fletcher,
2012;
WHO,
2015).
Antiviral
drugs
and
vaccines
are
used
to
ght
viral
infections
in
human
(De
Clercq
and
Li,
2016;
Marston
et
al.,
2014).
Previously,
there
has
been
a
focus
on
one
drug,
one
virus
dogma,
which
relied
on
targeting
virus-
specic
factors.
A
counterpoint
to
this
is
the
one
drug,
multiple
viruses
paradigm,
which
came
with
the
discovery
of
broad-
spectrum
antiviral
agents
(BSAAs),
small-molecules
that
inhibit
a
wide
range
of
human
viruses
(Bekerman
and
Einav,
2015;
de
Clercq
and
Montgomery,
1983;
Debing
et
al.,
2015;
Ianevski
et
al.,
2019;
Rada
and
Dragun,
1977;
Sidwell
et
al.,
1972 ).
This
paradigm
was
based
on
the
observation
that
different
viruses
utilize
similar
pathways
and
host
factors
to
replicate
inside
a
cell
(Bosl
et
al.,
2019).
Although
the
concept
of
BSAAs
has
been
around
for
almost
50
years,
the
eld
received
a
new
impetus
with
recent
outbreaks
of
Ebola,
Zika,
Dengue,
inuenza
and
other
viral
infections,
the
discovery
of
novel
host-directed
agents,
as
well
as
development
of
drug
repositioning
methodology.
Drug
repurposing,
also
called
repositioning,
redirecting,
reproling,
is
a
strategy
for
generating
additional
value
from
an
existing
drug
by
targeting
disease
other
than
that
for
which
it
was
originally
intended
(Nishimura
and
Hara,
2018;
Pushpakom
et
al.,
2019).
This
has
signicant
advantages
over
new
drug
discovery
since
chemical
synthesis
steps,
manufacturing
processes,
reliable
safety,
and
pharmacokinetic
properties
in
pre-clinical
(animal
model)
and
early
clinical
developmental
phases
(phase
0,
I
and
IIa)
are
already
available
(Figure
1).
Therefore,
repositioning
of
launched
or
even
failed
drugs
to
viral
diseases
provides
unique
translational
opportunities,
including
a
substantially
higher
probability
of
success
to
market
as
compared
with
developing
new
virus-specic
drugs
and
vaccines,
and
a
signicantly
reduced
cost
and
timeline
to
clinical
availability
(Ianevski
et
al.,
2019;
Pizzorno
et
al.,
2019;
Zheng
et
al.,
2018).
Here,
we
detail
the
steps
of
BSAA
repurposing,
from
discovery
of
novel
antiviral
activities
in
cell
culture
to
post-market
studies.
*
Corresponding
author
at:
Department
of
Clinical
and
Molecular
Medicine,
Norwegian
University
of
Science
and
Technology
(NTNU),
7028
Trondheim,
Norway.
E-mail
address:
denis.kainov@ntnu.no
(D.E.
Kainov).
1
Contributed
equally.
https://doi.org/10.1016/j.ijid.2020.02.018
1201-9712/©
2020
The
Authors.
Published
by
Elsevier
Ltd
on
behalf
of
International
Society
for
Infectious
Diseases.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/4.0/).
International
Journal
of
Infectious
Diseases
93
(2020)
268276
Contents
lists
available
at
ScienceDirect
International
Journal
of
Infectious
Diseases
journal
homepage:
www.elsevier.com/locate/ijid
Moreover,
we
summarized
currently
available
information
on
BSAAs
in
freely
available
database,
focusing
on
those
antivirals,
which
have
been
already
tested
in
human
as
antivirals,
anti-
bacterials,
antiprotozoals,
anthelmintics,
etc.
Finally,
we
discuss
future
perspectives
of
using
safe-in-man
BSAAs
for
treatment
of
emerging
and
re-emerging
viral
infections
as
well
as
viral
and
bacterial
co-infections.
Discovery
and
development
of
safe-in-man
BSAAs
Discovery
of
novel
BSAA
activities
in
immortalized
cell
cultures
and
co-cultures
The
discovery
of
novel
activities
of
BSAAs
starts
with
exposing
cells
to
the
candidate
antiviral
agent
at
different
concentrations
and
infecting
the
cells
with
a
virus
or
mock.
Immortalized
cancerous
cell
cultures
and
co-cultures,
which
express
appropriate
viral
receptors,
are
most
commonly
used
in
this
rst
step.
The
half-
maximal
cytotoxic
concentrations
(CC
50
)
for
a
compound
are
calculated
based
on
their
dose-response
curves
obtained
on
mock-
infected
cells.
The
half-maximal
effective
concentrations
(EC
50
)
are
calculated
based
on
the
analysis
of
curves
obtained
on
infected
cells.
Statistical
analyses
can
help
to
determine
if
the
differences
between
CC
50
and
EC
50
are
signicant,
given
the
inherent
variability
of
the
experiment
(Meneghini
and
Hamasaki,
1967).
A
relative
effectiveness
of
a
drug
is
dened
as
selectivity
index
(SI
=
CC
50
/EC
50
).
Cell
viability
assays
and
cell
death
assays
are
commonly
used
to
assess
the
cytotoxicity
and
efcacy
of
BSAAs
(Figure
2A).
Cell
viability
assays
include
MTT,
MTS,
resazurin
or
similar
assays,
mitochondrial
membrane
potential-dependent
dyes-based
assays,
esterase
cleaved
dye-based
assays,
ATP-ADP
assays,
and
assays
that
measure
glycolytic
ux
and
oxygen
consumption.
Other
cell
death
assays
include
LDH
enzyme
leakage
assays,
membrane
imperme-
able
dye-based
assays,
and
apoptosis
assays,
such
as
Annexin
V,
TUNEL,
and
caspase
assays
(Shen
et
al.,
2019).
For
example,
the
Cell
Titer
Glo
(CTG)
assay
quanties
ATP,
an
indicator
of
metabolically
active
living
cells,
whereas
Cell
Tox
Green
assay
uses
uorescent
asymmetric
cyanine
dye
that
stains
the
DNA
of
dead
cells
(Bosl
et
al.,
2019;
Bulanova
et
al.,
2017;
Ianevski
et
al.,
2018;
Muller
et
al.,
2014).
Viral
strains
or
cell
lines
expressing
reporter
proteins
are
also
used
to
assess
the
efcacy
of
BSAAs
in
infected
cells.
For
example,
TZM-bl
cells
expressing
rey
luciferase
under
control
of
HIV-1
LTR
promoter
allowed
quantitation
of
BSAA
action
on
HIV-1
infection
(tat-protein
expression
by
integrated
HIV-1
provirus)
using
rey
luciferase
assay
(Sarzotti-Kelsoe
et
al.,
2014;
Xing
et
al.,
2016).
RFP-expressing
RVFV,
nanoLuc-expressing
CHIKV
and
RRV,
as
well
as
GFP-expressing
FLUAV,
HCV
and
HMPV
also
allowed
identication
of
novel
activities
of
several
BSAAs
(Andersen
et
al.,
2019b;
Bosl
et
al.,
2019;
de
Graaf
et
al.,
2007;
Habjan
et
al.,
2008;
Ianevski
et
al.,
2018;
Jupille
et
al.,
2011;
Kittel
et
al.,
2004;
Lee
et
al.,
2017;
Utt
et
al.,
2016).
In
addition,
qPCR/RT-qPCR,
RNA/DNA
sequencing,
RNA/DNA
hybridization,
immuno-
and
plaque
assays
as
well
as
CRISPR-Cas9
systems
could
be
used
for
detection
of
inhibitory
effects
of
BSAAs
(Boonham
et
al.,
2014;
Fischer
et
al.,
2017;
Konig
et
al.,
2019;
Laamiri
et
al.,
2018;
Landry,
1990;
Perez
et
al.,
2013;
Sashital,
2018;
Zhou
et
al.,
2018).
Interestingly,
CRISPR-
Cas9,
siRNA
and
shRNA
approaches
were
used
for
identication
of
BSAA
targets
(Deans
et
al.,
2016;
Puschnik
et
al.,
2017).
Novel
anti-HSV-2
and
anti-EV1
activities
of
emetine
were
discovered
recently
using
CTG/plaque
assays
in
human
non-
malignant
RPE
cells.
Moreover,
novel
antiviral
activities
of
the
drug
were
identied
using
RFP-expressing
RVFV,
and
GFP-expressing
HMPV
or
FLUAV
strains
in
RPE
cells
(Andersen
et
al.,
2019a).
Given
that
emetine
also
inhibits
ZIKV,
EBOV,
RABV,
CMV,
HCoV-OC43
and
HIV-1
infections
(Chaves
Valadao
et
al.,
2015;
MacGibeny
et
al.,
2018;
Mukhopadhyay
et
al.,
2016;
Shen
et
al.,
2019;
Yang
et
al.,
2018),
and
that
it
is
an
FDA-approved
anti-protozoal
drug,
it
may
represent
a
promising
safe-in-man
BSAA
candidate.
Evaluation
of
BSAAs
in
human
primary
cell
culture
and
co-cultures
Immortalized
cell
cultures/co-cultures
and
reporter
viral
strains
represent
excellent
model
systems
for
the
discovery
of
novel
activities
of
safe-in-man
BSAAs.
However,
these
genetically
modied
systems
have
certain
limitations
(attenuated
or
incom-
plete
virus
replication
cycle,
accumulation
of
mutations
during
repeated
cell
and
virus
passaging,
defective
innate
immune
responses
and
viral
counter-responses,
etc.)
(Carter
and
Shieh,
2015).
Thereby,
novel
antiviral
activities
of
BSAAs
should
be
further
validated
in
primary
human
cells
using
different
viral
strains
(including
wild-type
viruses),
different
viral
loads,
different
times
of
compound
addition,
different
endpoint
measurements
and
compound
concentration
range.
Primary
cell
cultures
give
more
accurate
images
of
drug
responses
(Alves
et
al.,
2018;
Denisova
et
al.,
2012;
Koban
et
al.,
2018;
Postnikova
et
al.,
2018).
They
have
a
low
population
doubling
level
and
therefore
more
closely
recapitulate
the
physiological
conditions
observed
in
vivo.
Primary
cells
are
cells
isolated
directly
from
tissues
or
blood
using
enzymatic
or
mechanical
methods.
The
cells
are
character-
ized
by
their
high
degrees
of
specialization,
are
often
fully
differentiated
and
thus
require
dened
culture
conditions
(se-
rum-free
media)
in
order
to
preserve
their
original
phenotype.
Peripheral
blood
mononuclear
(PBMC),
placental,
amniotic
and
fetal
primary
cultures
as
well
as
vaginal/cervical
epithelial
and
male
germ
cells
have
been
used
intensively
to
validate
BSAA
activity
(Barrows
et
al.,
2016;
Denisova
et
al.,
2012;
Fink
et
al.,
2018;
Rausch
et
al.,
2017;
Robinson
et
al.,
2018).
Although
primary
cell
cultures
are
relevant
systems
for
validation
of
BSAAs,
there
are
technical
difculties
limiting
their
use,
such
as
ethical
issues,
purity
of
population
of
primary
cells,
and
limited
shelf
life
of
the
cells.
In
addition,
age,
race,
sex
and
other
genetic
and
epigenetic
factors
of
donor
cells
should
be
considered
to
determine
common
biological
effect
across
a
signicant
number
of
donors
thereby
avoiding
minor
variants
(Lee
et
al.,
2014;
Zhang
et
al.,
2013).
The
obstacles
associated
with
use
of
human
primary
cell
cultures
can
be
bypassed
using
human
embryonic
stem
cells
(ESCs)
and
human
induced
pluripotent
stem
cells
(iPSCs).
ESCs
are
isolated
from
surplus
human
embryos,
whereas
iPSCs
are
obtained
Figure
1.
Discovery
of
novel
activities
and
follow-up
development
of
broad-spectrum
antiviral
agents
(BSAAs).
Yellow
shading
indicates
a
process
of
discovery
and
development
of
safe-in-man
BSAAs,
for
which
pharmacokinetic
(PK)
properties
in
pre-clinical
(animal
model)
and
early
clinical
developmental
phases
(phase
0-IIa
trials)
are
already
available.
Abbreviations:
ESCs,
human
embryonic
stem
cells;
iPSCs,
human
induced
pluripotent
stem
cells
(iPSCs).
P.I.
Andersen
et
al.
/
International
Journal
of
Infectious
Diseases
93
(2020)
268276
269
by
reprogramming
somatic
cells.
These
cells
proliferate
extensively
and
retain
multi-lineage
activity,
which
allows
them
to
generate
virtually
any
cell
type
of
the
body.
The
ESCs-
and
iPSC-derived
cells
have
been
used
successfully
to
investigate
the
efcacy
of
several
BSAAs
against
HBV,
ZIKV,
CHIKV
and
HSV-1
infections
(Table
S1)
(Ferreira
et
al.,
2019;
Iwasawa
et
al.,
2019;
Lanko
et
al.,
2017;
Simonin
et
al.,
2019;
Xia
et
al.,
2017;
Zhou
et
al.,
2017).
iPSCs,
ESCs
and
primary
tissue
cells
can
be
used
to
generate
complex
cultures
termed
organoids.
Organoids
are
miniature
and
simplied
version
of
organs.
Establishing
human
airway,
gut,
skin,
cerebral,
liver,
kidney,
breast,
retina
and
brain
organoids
allowed
researchers
to
study
toxicity
and
efcacy
of
several
safe-in-man
BSAAs
against
coronaviruses,
inuenza,
enteroviruses,
rotaviruses
and
aviviruses
(Li
et
al.,
2017;
Sacramento
et
al.,
2017;
Watanabe
et
al.,
2017;
Xu
et
al.,
2016;
Yin
et
al.,
2015;
Yin
et
al.,
2018;
Yin
et
al.,
2016;
Zhou
et
al.,
2017).
However,
iPSCs,
ESCs
and
iPSCs/ESCs-
derived
organoids
have
the
same
disadvantages
as
human
primary
cells
(genetic
differences,
line-to-line
and
organoid
batch-to-batch
variability).
On
the
other
hand,
these
models
allow
researchers
to
predict
the
behavior
of
viruses
in
vivo
and,
therefore,
to
reduce
animal
use
and
in
cases
where
animal
models
are
unavailable
to
initiate
clinical
trials.
For
example,
novel
anti-ZIKV
activities
of
enoxacin,
amodia-
quine
and
niclosamide
were
discovered
recently
using
human
neural
progenitor
cells,
human
pluripotent
stem
cell-derived
cortical
neural
progenitor
cells,
and
human
induced
neural
stem
cells,
respectively
(Cairns
et
al.,
2018;
Xu
et
al.,
2019;
Zhou
et
al.,
2017).
Enoxacin
is
an
oral
broad-spectrum
uoroquinolone
antibiotic,
which
also
possesses
anti-HCV
and
anti-HIV-1
activities
in
immortalized
cell
cultures
(Kashiwase
et
al.,
1999;
Young
et
al.,
2010).
Amodiaquine
is
an
anti-malaria
drug,
which
also
possesses
antiviral
activities
against
DENV,
HCV,
RRV,
SINV,
WNV,
EFV,
EBOV,
LASV,
RABV,
VZV,
and
HSV-1
in
immortalized
cell
cultures
(Boonyasuppayakorn
et
al.,
2014;
Hulseberg
et
al.,
2019;
Mazzon
et
al.,
2019).
Niclosamide
is
an
orally
bioavailable
anthelmintic
drug
which
inhibits
the
broadest
range
of
viruses
in
vitro
and,
in
Figure
2.
ABC
of
BSAA
development
process.
(A)
Testing
BSAA
toxicity
(left
panel)
and
efcacy
(right
panel)
in
immortalized
cell
cultures
and
co-cultures.
(B)
Testing
BSAA
toxicity
(left
panel)
and
efcacy
(right
panel)
in
animal
models.
If
BSAA
is
repositioned
from
another
disease
(i.e.
its
PK/PD
and
toxicity
proles
are
available
for
the
animal
model)
it
could
bypass
the
safety
studies.
(C)
Clinical
trials
of
BSAAs.
(Left
panel)
Pharmacokinetics
(PK)
and
safety
studies.
(Right
panel)
Efcacy
studies.
If
the
drug
is
repositioned
from
another
disease
(i.e.
its
safety
prole
in
man
is
available)
it
could
bypass
the
PK
and
safety
studies
in
man.
270
P.I.
Andersen
et
al.
/
International
Journal
of
Infectious
Diseases
93
(2020)
268276
some
cases,
in
vivo
(Cairns
et
al.,
2018;
Fang
et
al.,
2013;
Huang
et
al.,
2017;
Hulseberg
et
al.,
2019;
Jurgeit
et
al.,
2012;
Kao
et
al.,
2018;
Mazzon
et
al.,
2019;
Stachulski
et
al.,
2011;
Wang
et
al.,
2016;
Wu
et
al.,
2004).
These
safe-in-man
BSAAs
represent
promising
drug
candidates.
Evaluation
of
BSAAs
in
animal
models
In
vitro
and
ex
vivo
models
do
not
fully
reect
the
complexity
and
physiology
of
living
organisms.
Therefore,
several
in
vivo
models
have
been
developed
to
test
novel
antiviral
activities
of
BSAAs.
These
include
immunocompetent
and
genetically
or
chemically
immunocompromised
mice,
guinea
pigs,
hamsters,
ferrets,
pigs,
macaques
and
other
animals
(Figure
2B)
(Alves
et
al.,
2018;
Haese
et
al.,
2016;
Louz
et
al.,
2013;
Morrison
and
Diamond,
2017;
Taylor,
2017;
Thangavel
and
Bouvier,
2014).
PK/PD
studies
determine
drug
absorption,
dosage
and
half-life
of
BSAAs.
Toxicological
studies
determine
if
the
drugs
have
any
adverse
effects
on
the
tissues
and
organs
of
the
animals
and
dene
the
dosage
of
adverse
effects
(Alabaster
and
In
Vivo
Pharmacology
Training
Group,
2002;
Parasuraman,
2011;
Rizk
et
al.,
2017).
Studying
the
efcacy
of
BSAAs
is
generally
done
by
treating
the
animal
with
the
drug
or
vehicle
and
infecting
it
with
a
virus
of
interest.
Endpoints
are
usually
body
weight/
mortality
(depending
on
the
virus),
histopathology,
virus
titers
in
organs,
presence
of
clinical
signs
and
development
of
immunity
(Oh
and
Hurt,
2016;
Smee
and
Barnard,
2013).
Although
animal
models
can
give
the
initial
characterization
of
BSAA,
it
is
important
to
keep
in
mind
that
they
differ
signicantly
from
humans,
with
respect
to
symptoms,
disease
manifestation,
susceptibility,
immune
responses,
patho-
genesis,
and
pharmacokinetics
(Barré-Sinoussi
and
Montagutelli,
2015;
Shanks
et
al.,
2009).
Often
animals
require
higher
concentration
of
an
experimental
antiviral
as
compared
to
effective
in
vitro
concentrations.
Moreover,
it
is
relatively
difcult
to
achieve
micromolar
EC50
in
vivo.
For
example,
aminoglycoside
antibiotics,
kasugamycin
and
neomycin
were
successfully
tested
against
ZIKV,
FLUAV,
and
HSV-2
infections
in
mice
(Gopinath
et
al.,
2018).
Polyether
antibiotic
salinomycin
also
showed
anti-FLUAV
effect
in
mice
(Jang
et
al.,
2018).
In
addition,
investigational
anticancer
agent,
avopiridol,
was
effective
against
FLUAV
in
mice
(Soderholm
et
al.,
2016).
These
ndings
support
further
development
of
these
and
other
BSAAs.
Clinical
trials
and
post-clinical
studies
of
BSAAs
Clinical
trials
are
the
most
critical
and
time-consuming
step
of
a
drug
candidates
journey
to
being
approved
(Figure
2C).
However,
safe-in-man
BSAAs
make
this
journey
relatively
short,
because
they
have
been
already
at
phase
0,
I
and,
sometime,
at
IIa
of
clinical
trials
as
antibacterial,
antiprotozoal,
anticancer,
etc.
agent;
i.e.
they
have
been
administered
at
sub-therapeutic
doses
to
healthy
volunteers
to
ensure
the
drugs
are
not
harmful
to
the
participants.
Thus,
safe-in-man
BSAAs
enter
phase
II
and
III,
which
assess
the
efcacy,
effectiveness,
safety
and
side
effects
of
the
drugs
in
clinic.
It
is
important,
however,
to
differentiate
acute
and
chronic
viral
infections
when
repurposing
BSAAs,
given
that
drug
concentra-
tions
and
duration
of
the
treatment
could
be
different,
and
therefore,
drug
safety
issues
should
be
considered.
For
phase
II,
patients
with
the
viral
disease
in
question
are
invited
to
join
the
study,
where
they
are
administered
the
BSAAs
at
the
ideal
therapeutic
doses.
Phase
III
is
the
longest
of
the
phases,
and
includes
multiple
levels
of
securities
to
the
studies,
such
as
the
use
of
placebos
and
double-blinded
studies,
to
ensure
the
data
is
as
unbiased
as
possible.
Upon
completing
phase
III,
depending
on
its
performance
and
efcacy,
BSAAs
may
end
either
being
approved
or
dropped.
The
U.S
Food
and
Drug
Administration
(FDA)
estimates
that
only
2530%
BSAA
candidates
that
enter
phase
III
are
approved
for
use
in
the
public
(U.S
Food
and
Drug
Administration,
2018).
After
approval
and
marketing
of
the
drug,
phase
IV
may
be
initiated
to
follow
up
on
the
use
of
the
drug
in
public,
to
surveil
for
rare
effects
(U.S
Food
and
Drug
Administration,
2018;
Umscheid
et
al.,
2011).
Forty-eight
safe-in-man
BSAAs
undergo
clinical
studies
as
anti-
virals.
There
are
currently
21
compounds inphase
I,
34
agents
inphase
II
and
11
compounds
in
phase
III
clinical
trials.
For
example,
nitazoxanide,
remdesivir
and
brincidofovir
are
under
clinical
inves-
tigations
against
different
viral
infections
(NCT03336619,
NCT00302640,
NCT03605862,
NCT03719586,
NCT01276756,
NCT03905655,
NCT01529073,
NCT03395405,
NCT03216967,
NCT01431326,
NCT02087306,
NCT01769170).
Twenty-one
BSAAs
were
approved
by
FDA,
EMA
or
other
agencies.
These
BSAAs
altogether
target
15
viruses.
For
example,
favipiravir,
also
known
as
T-705,
was
approved
against
FLUAV
in
Japan;
cidofovir
is
an
injectable
antiviral
medication
used
as
a
treatment
for
CMV
retinitis
in
people
with
AIDS;
ribavirin,
also
known
as
tribavirin,
is
used
for
treatment
of
RSV
and
HCV
infections;
pleconaril
is
used
against
viruses
in
the
picornavir-
idae
family,
including
enterovirus
and
rhinovirus;
and
valacy-
clovir
is
used
against
CMV,
EBV,
HBV,
HSV-1,
HSV-2
and
VZV
infections.
Twenty
BSAAs
are
undergoing
surveillance
studies
(phase
IV).
Azithromycin,
chloroquine,
cyclosporine,
ezetimibe,
mycophenolic
acid,
nitazoxanide
and
rapamycin
progressed
to
phase
IV
studies
without
approvals
from
national
or
international
authorities
(NCT01779570,
NCT02058173,
NCT02564471,
NCT00821587,
NCT03360682,
NCT02328963,
NCT02768545,
NCT01624948,
NCT01770483,
NCT02683291,
NCT01624948,
NCT01469884,
NCT03901001,
NCT01412515,
NCT02990312).
BSAA
database
We
have
developed
a
database
for
safe-in-man
BSAAs,
which
is
available
at
https://drugvirus.info/
(Figure
3).
The
drug
annota-
tions
were
obtained
from
PubChem,
DrugBank,
DrugCentral,
PubMed
and
clinicaltrials.gov
databases
(Table
S1)
(Kim
et
al.,
2019;
Ursu
et
al.,
2019;
Wishart
et
al.,
2018).
The
information
on
virus
families
was
exported
from
Virus
Pathogen
Database
and
Analysis
Resource
(Table
S2)
(Pickett
et
al.,
2012).
The
database
summarizes
activities
and
developmental
status
of
BSAAs.
We
decided
to
set
no
limits
for
EC50,
SI
and
statistical
signicance
because
the
studies
were
not
harmonized
(different
cell
lines,
assays,
end-point
measurements,
time
of
compound
addition,
etc.).
The
database
allows
interactive
exploration
of
virus-BSAA
interactions.
It
also
includes
information
on
BSA
targets.
A
feedback
form
is
available
on
the
website.
The
database
will
be
updated
upon
request
or
as
soon
as
a
new
safe-in-man
BSAA
emerges
or
novel
activity
for
an
existing
BSAA
is
reported.
Altogether,
the
database
contains
120
approved,
investigational
and
experimental
safe-in-man
BSAAs,
which
inhibit
86
human
viruses,
belonging
to
25
viral
families.
The
BSAAs
inhibit
viral
or
host
factors
and
block
viral
replication,
reduce
the
viral
burden
to
a
level
at
which
host
immune
responses
can
deal
with
it
or
facilitate
apoptosis
of
infected
cells
(Table
S1).
Analysis
of
BSAA
targets
and
structures
(Figure
4)
revealed
that
the
most
abundant
are
nucleotide
and
nucleoside
analogues
which
inhibit
viral
RNA
and
DNA
polymerases.
Imatinib,
erlotinib,
getinib,
and
dasatinib,
which
inhibit
tyrosine
kinases,
are
the
most
abundant
host-
directed
BSAAs.
Most
of
the
host
targets
(except
Bcl-xL
protein)
are
essential
for
viral
replication
but
redundant
for
the
cell,
which
is
critical
for
reducing
putative
toxicities
associated
with
blocking
cellular
pathways.
The
limited
diversity
of
the
targets
and
scaffolds
could
slow
down
the
development
of
BSAA
concept.
P.I.
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et
al.
/
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Journal
of
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Diseases
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(2020)
268276
271
Figure
3.
Safe-in-man
broad-spectrum
antiviral
agents
(BSAAs)
and
viruses
they
inhibit.
A
snapshot
is
taken
from
https://drugvirus.info/
website.
Viruses
are
clustered
by
virus
groups.
BSAAs
range
from
the
highest
to
lowest
number
of
targeted
viruses.
Different
shadings
indicate
different
development
status
of
BSAAs.
Gray
shading
indicates
that
the
antiviral
activity
has
not
been
either
studied
or
reported.
Abbreviations:
ds,
double-stranded;
RT,
reverse
transcriptase;
ss,
single-stranded.
272
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et
al.
/
International
Journal
of
Infectious
Diseases
93
(2020)
268276
Emerging
BSAAs,
such
as
5,6-dimethoxyindan-1-one,
saliphe-
nylhalamide,
and
GS-5734
(Denisova
et
al.,
2012;
Kuivanen
et
al.,
2017;
Muller
et
al.,
2011;
Muller
et
al.,
2014;
Patil
et
al.,
2017;
Sheahan
et
al.,
2017),
whose
safety
proles
in
humans
are
not
yet
available,
are
not
included
in
the
database.
However,
they
could
serve
as
valuable
antivirals
in
the
future,
pending
the
results
of
further
pre-clinical
and
clinical
investigations.
BSAAs
as
drug
candidates
for
treatment
of
SARS-CoV-2
infections
SARS-CoV-2
is
a
novel
strain
of
coronaviruses
which
is
associated
with
a
cluster
of
cases
of
pneumonia
in
China
(Zhou
et
al.,
2020).
Coronaviruses
(CoV)
are
a
broad
family
of
virusesthat
includes
SARS-CoV,
MERS-CoV,
HCoV-229E,
HCoV-OC43,
HCoV-
NL63
and
HCoV-HKU1
strains.
HCoV-229E,
HCoV-OC43,
HCoV-
NL63
and
HCoV-HKU1
strains
are
usually
associated
with
mild,
self-limiting
upper
respiratory
tract
infections,
such
as
the
common
cold.
By
contrast,
people
infected
with
MERS-CoV,
SASRS-CoV
or
SARS-CoV-2
could
develop
severe
respiratory
illness,
and
many
of
the
infected
have
died.
No
vaccines
and
drugs
are
available
for
prevention,
prophylaxis
and
treatment
of
coronavirus
infections
in
humans
(Eurosurveil-
lance
Editorial,
2020).
However,
safe-in-man
BSAAs
could
be
effective
against
SARS-CoV-2
and
other
coronaviruses
(Figure
5).
For
example,
teicoplanin,
oritavancin,
dalbavancin,
monensin
and
emetine
could
be
repurposed
for
treatment
of
COVID-19.
Teicoplanin,
ritavancin,
dalbavancin
and
monensin
are
approved
antibiotics,
whereas
emetine
is
an
anti-protozoal
drug.
These
drugs
have
been
shown
to
inhibit
several
corona-
as
well
as
some
other
viral
infections
.
Moreover,
chloroquine
and
remdesivir
were
shown
to
effectively
inhibit
SARS-CoV-2
infection
in
vitro.
In
addition,
clinical
investigations
into
the
effectiveness
of
lopinavir,
ritonavir,
remdesivir,
hydroxychloroquine
and
arbidol
against
COVID-19
have
started
recently
(NCT04252664;
NCT0425487;
NCT04255017;
NCT04261517,
NCT04260594).
BSAA
combinations
BSAAs
could
be
combined
with
other
antiviral
agents
to
obtain
synergistic
or
additive
effects
against
certain
viruses
(Cheng
et
al.,
2019;
Zheng
et
al.,
2018).
Several
combination
therapies
which
Figure
4.
Structure-activity
relationship
of
safe-in-man
BSAAs.
Web-application
serves
C-SPADE
was
used
to
cluster
BSAAs
based
on
their
structural
similarities
and
visualize
them
as
a
dendrogram
of
compounds
augmented
with
their
functional
annotations
(https://cspade.mm./).
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et
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Diseases
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273
include
BSAAs
(such
as
abacavir/dolutegravir/lamivudine
(Triu-
meq),
darunavir/cobicistat/emtricitabine/tenofovir
(Symtuza),
lopinavir/ritonavir
(Kaletra),
ledipasvir/sofosbuvir
and
sofosbu-
vir/velpatasvir)
became
a
standard
for
the
treatment
of
HIV
or
HCV
infections.
Several
synergistic
drug
combinations,
such
as
obato-
clax/saliphenylhalamide
and
gemcitabine/pimodivir,
could
enter
clinical
studies
and
become
effective
treatment
of
ZIKV
and
FLUAV
infections
(Fu
et
al.,
2016;
Kuivanen
et
al.,
2017).
By
contrast
to
individual
drugs,
combinations
of
23
BSAAs
could
be
used
to
target
an
even
broader
range
of
viruses
(Foucquier
and
Guedj,
2015;
Zheng
et
al.,
2018).
Such
combinations
could
serve
as
front
line
therapeutics
against
poorly
characterized
emerging
viruses
or
re-emerging
drug-resistant
viral
strains.
For
example,
a
cocktail
of
nitazoxanide,
favipiravir,
and
niclosamide
could
be
developed
for
the
treatment
of
viruses
belonging
to
11
families.
Other
activities
of
BSAAs
Fifty
BSAAs
possess
not
only
antiviral
but
also
antibacterial
activity
(Figure
4;
Table
S1)
(Schor
and
Einav,
2018).
Moreover,
13
of
the
50
agents
are
approved
as
antibiotics
(2
withdrawn).
These
agents
with
dual
activity
could
be
used
for
treatment
of
viral
and
bacterial
co-infections
or
for
the
protection
of
patients
from
the
secondary
infections.
For
example,
azithromycin
could
be
used
against
FLUAV
and
Chlamydophila
pneumoniae,
Haemophilus
inuenzae,
Mycoplasma
pneumoniae
or
Streptococcus
pneumoniae
infections
(NCT01779570)
(Mandell
et
al.,
2007).
In
addition,
BSAAs
showed
activity
against
a
wide
range
of
other
medically
important
human
pathogens,
including
fungi,
protozoa
and
parasites
(Table
S1)
(Montoya
and
Krysan,
2018),
pointing
out
that
some
pathogens
utilize
common
mechanisms
to
infect
hosts.
Moreover,
structure-activity
relationship
analysis
of
BSAAs
suggests
that
some
agents,
such
as
doxycycline,
artesunate,
omeprazole,
nitazoxanide,
suramin,
azithromycin,
minocycline
and
chloroquine,
could
have
novel
antibacterial,
antiprotozoal,
antifungal
or
anthelmintic
activities
(Figure
4).
If
conrmed,
this
could
lead
to
development
of
broad-spectrum
anti-infective
drugs.
BSAAs
could
also
serve
as
treatment
of
other
co-morbidities,
therefore,
simplifying
the
therapy
and
lowering
its
cost
(Table
S1).
For
example,
the
concomitant
actions
of
ezetimibe
and
statins
could
be
benecial
for
treatment
of
both
hypertension
and
several
viral
infections
in
patients
with
these
co-morbidities
(NCT00908011,
NCT00099684,
NCT00843661,
NCT03490097,
NCT00994773,
NCT00441493).
Conclusions
and
future
perspectives
Here
we
reviewed
a
process
of
BSAA
development
and
summarized
information
on
120
safe-in-man
agents
in
freely
available
database.
We
hope
that
further
pre-clinical
and
clinical
studies
on
BSAAs
will
be
harmonized,
and
data
collection
will
be
standardized.
Furthermore,
the
follow-up
studies
as
well
as
the
results
of
on-going,
nalized
or
terminated
clinical
trials
will
be
made
publicly
available
to
allow
prioritization
and
translation
of
emerging
and
existing
BSAAs
into
clinical
practice.
This
would
allow
BSAAs
to
play
a
pivotal
role
in
the
battle
against
emerging
and
re-emerging
viral
diseases.
Discovery
of
novel
as
well
as
repositioning
existing
safe-in-man
BSAAs
may
shorten
time
and
resources
needed
for
development
of
virus-specic
drugs
and
vaccines.
In
the
future,
BSAAs
will
have
a
global
impact
by
decreasing
morbidity
and
mortality
from
viral
and
other
diseases,
maximizing
the
number
of
healthy
life
years,
improving
the
quality
of
life
of
infected
patients
and
decreasing
the
costs
of
patient
care.
Conicts
of
interest
The
authors
declare
no
conict
of
interest.
Funding
sources
This
study
was
supported
by
the
European
Regional
Develop-
ment
Fund,
the
Mobilitas
Pluss
Project
MOBTT39
(to
D.K.).
Ethical
approval
Approval
was
not
required.
Acknowledgments
We
thank
Katarzyna
Kolasa
for
illustrations.
This
manuscript
has
been
released
as
a
pre-print
(doi:
https://10.20944/pre-
prints201910.0144.v46).
We
thank
the
European
Regional
Devel-
opment
Fund,
the
Mobilitas
Pluss
Project
MOBTT39.
Figure
5.
Safe-in-man
broad-spectrum
antiviral
agents
and
coronaviruses
they
inhibit.
A
snapshot
is
taken
from
https://drugvirus.info/
website.
Different
shadings
indicate
different
development
status
of
BSAAs.
Grey
shading
indicates
that
the
antiviral
activity
has
not
been
either
studied
or
reported.
274
P.I.
Andersen
et
al.
/
International
Journal
of
Infectious
Diseases
93
(2020)
268276
Appendix
A.
Supplementary
data
Supplementary
material
related
to
this
article
can
be
found,
in
the
online
version,
at
doi:https://doi.org/10.1016/j.ijid.2020.02.018.
References
Alabaster
V,
In
Vivo
Pharmacology
Training
Group.
The
fall
and
rise
of
in
vivo
pharmacology.
Trends
Pharmacol
Sci
2002;23:138.
Alves
MP,
VielleNJ,
Thiel
V,
Pfaender
S.
Research
models
and
tools
for
the
identication
of
antivirals
and
therapeutics
against
Zika
virus
infection.
Viruses
2018;10:.
Andersen
PI,
Krpina
K,
Ianevski
A,
Shtaida
N,
Jo
E,
Yang
J,
et
al.
Novel
antiviral
activities
of
obatoclax,
emetine,
niclosamide,
brequinar,
and
homoharringto-
nine.
Viruses
2019a;11:.
Andersen
PI,
Krpina
K,
Ianevski
A,
Shtaida
N,
Jo
E,
Yang
J,
et
al.
Novel
antiviral
activities
of
obatoclax,
emetine,
niclosamide,
brequinar,
and
homoharringto-
nine.
Viruses
2019b;11:
pii:
E964.
Barré-Sinoussi
F,
Montagutelli
X.
Animal
models
are
essential
to
biological
research:
issues
and
perspectives.
Future
Sci
OA
2015;1:
FSO63-FSO63.
Barrows
NJ,
Campos
RK,
Powell
ST,
Prasanth
KR,
Schott-Lerner
G,
Soto-Acosta
R,
et
al.
A
screen
of
FDA-approved
drugs
for
inhibitors
of
Zika
virus
infection.
Cell
Host
Microbe
2016;20:25970.
Bekerman
E,
Einav
S.
Infectious
disease.
Combating
emerging
viral
threats.
Science
2015;348:2823.
Boonham
N,
Kreuze
J,
Winter
S,
van
der
Vlugt
R,
Bergervoet
J,
Tomlinson
J,
et
al.
Methods
in
virus
diagnostics:
from
ELISA
to
next
generation
sequencing.
Virus
Res
2014;186:2031.
Boonyasuppayakorn
S,
Reichert
ED,
Manzano
M,
Nagarajan
K,
Padmanabhan
R.
Amodiaquine,
an
antimalarial
drug,
inhibits
dengue
virus
type
2
replication
and
infectivity.
Antiviral
Res
2014;106:12534.
Bosl
K,
Ianevski
A,
Than
TT,
Andersen
PI,
Kuivanen
S,
Teppor
M,
et
al.
Common
nodes
of
virushost
interaction
revealed
through
an
integrated
network
analysis.
Front.
Immunol
2019;4:2186.
Bulanova
D,
Ianevski
A,
Bugai
A,
Akimov
Y,
Kuivanen
S,
Paavilainen
H,
et
al.
Antiviral
properties
of
chemical
inhibitors
of
cellular
anti-apoptotic
Bcl-2
proteins.
Viruses
2017;9:.
Cairns
DM,
Boorgu
D,
Levin
M,
Kaplan
DL.
Niclosamide
rescues
microcephaly
in
a
humanized
in
vivo
model
of
Zika
infection
using
human
induced
neural
stem
cells.
Biol
Open
2018;7:.
Carter
M,
Shieh
J.
Chapter
14
cell
culture
techniques.
In:
Carter
M,
Shieh
J,
editors.
Guide
to
research
techniques
in
neuroscience.
Second
edition
San
Diego:
Academic
Press;
2015.
p.
295310.
Chaves
Valadao
AL,
Abreu
CM,
Dias
JZ,
Arantes
P,
Verli
H,
Tanuri
A,
et
al.
Natural
plant
alkaloid
(emetine)
inhibits
HIV-1
replication
by
interfering
with
reverse
transcriptase
activity.
Molecules
2015;20:1147489.
Cheng
YS,
Williamson
PR,
Zheng
W.
Improving
therapy
of
severe
infections
through
drug
repurposing
of
synergistic
combinations.
Curr
Opin
Pharmacol
2019;48:928.
DALYs,
G.B.D.,
Collaborators,
H.
Global,
regional,
and
national
disability-adjusted
life-years
(DALYs)
for
359
diseases
and
injuries
and
healthy
life
expectancy
(HALE)
for
195
countries
and
territories,
1990-2017:
a
systematic
analysis
for
the
Global
Burden
of
Disease
Study
20 17.
Lancet
2018;392:1859922.
De
Clercq
E,
Li
G.
Approved
antiviral
drugs
over
the
past
50
years.
Clin
Microbiol
Rev
2016;29:695747.
de
Clercq
E,
Montgomery
JA.
Broad-spectrum
antiviral
activity
of
the
carbocyclic
analog
of
3-deazaadenosine.
Antiviral
Res
1983;3:1724.
de
Graaf
M,
Herfst
S,
Schrauwen
EJ,
van
den
Hoogen
BG,
Osterhaus
AD,
Fouchier
RA.
An
improved
plaque
reduction
virus
neutralization
assay
for
human
meta-
pneumovirus.
J
Virol
Methods
2007;143:16974.
Deans
RM,
Morgens
DW,
Okesli
A,
Pillay
S,
Horlbeck
MA,
Kampmann
M,
et
al.
Parallel
shRNA
and
CRISPR-Cas9
screens
enable
antiviral
drug
target
identication.
Nat
Chem
Biol
2016;12:3616.
Debing
Y,
Neyts
J,
Delang
L.
The
future
of
antivirals:
broad-spectrum
inhibitors.
Curr
Opin
Infect
Dis
2015;28:596602.
Denisova
OV,
Kakkola
L,
Feng
L,
Stenman
J,
Nagaraj
A,
Lampe
J,
et
al.
Obatoclax,
saliphenylhalamide,
and
gemcitabine
inhibit
inuenza
a
virus
infection.
J
Biol
Chem
2012;287:3532432.
Disease
GBD,
Injury
I,
Prevalence
C.
Global,
regional,
and
national
incidence,
prevalence,
and
years
lived
with
disability
for
354
diseases
and
injuries
for
195
countries
and
territories,1990-2017:
a
systematic
analysis
for
the
Global
Burden
of
Disease
Study
2017.
Lancet
2018;392:1789858.
Eurosurveillance
Editorial
T.
Note
from
the
editors:
World
Health
Organization
declares
novel
coronavirus
(2019-nCoV)
sixth
public
health
emergency
of
international
concern.
Euro
Surveill
2020;.
Fang
J,
Sun
L,
Peng
G,
Xu
J,
Zhou
R,
Cao
S,
et
al.
Identication
of
three
antiviral
inhibitors
against
Japanese
encephalitis
virus
from
library
of
pharmacologically
active
compounds
1280.
PLoS
One
2013;8:e78425.
Ferreira
AC,
Reis
PA,
de
Freitas
CS,
Sacramento
CQ,
Villas
Boas
Hoelz
L,
Bastos
MM,
et
al.
Beyond
members
of
the
aviviridae
family,
sofosbuvir
also
inhibits
chikungunya
virus
replication.
Antimicrob
Agents
Chemother
2019;63:.
Fink
SL,
Vojtech
L,
Wagoner
J,
Slivinski
NSJ,
Jackson
KJ,
Wang
R,
et
al.
The
antiviral
drug
arbidol
inhibits
Zika
virus.
Sci
Rep
2018;8:8989.
Fischer
C,
Torres
MC,
Patel
P,
Moreira-Soto
A,
Gould
EA,
Charrel
RN,
et
al.
Lineage-
specic
real-time
RT-PCR
for
yellow
fever
virus
outbreak
surveillance,
Brazil.
Emerg
Infect
Dis
2017;23:186771.
Foucquier
J,
Guedj
M.
Analysis
of
drug
combinations:
current
methodological
landscape.
Pharmacol
Res
Perspect
2015;3:e00149.
Fu
Y,
Gaelings
L,
Soderholm
S,
Belanov
S,
Nandania
J,
Nyman
TA,
et
al.
JNJ872
inhibits
inuenza
A
virus
replication
without
altering
cellular
antiviral
responses.
Antiviral
Res
2016;133:2331.
Gopinath
S,
Kim
MV,
Rakib
T,
Wong
PW,
van
Zandt
M,
Barry
NA,
et
al.
Topical
application
of
aminoglycoside
antibiotics
enhances
host
resistance
to
viral
infections
in
a
microbiota-independent
manner.
Nat
Microbiol
2018;3:61121.
Habjan
M,
Penski
N,
Spiegel
M,
Weber
F.
T7
RNA
polymerase-dependent
and
-independent
systems
for
cDNA-based
rescue
of
Rift
Valley
fever
virus.
J
Gen
Virol
2008;89:215766.
Haese
NN,
Broeckel
RM,
Hawman
DW,
Heise
MT,
Morrison
TE,
Streblow
DN.
Animal
models
of
Chikungunya
virus
infection
and
disease.
J
Infect
Dis
2016;214:S4827.
Howard
CR,
Fletcher
NF.
Emerging
virus
diseases:
can
we
ever
expect
the
unexpected?.
Emerg
Microbes
Infect
2012;1:e46.
HuangL, Yang
M,Yuan Y,
LiX,Kuang E.Niclosamide
inhibitslytic
replicationof Epstein-
Barr
virus
by
disrupting
mTOR
activation.
Antiviral
Res
2017;138:6878.
Hulseberg
CE,
Feneant
L,
Szymanska-de
Wijs
KM,
Kessler
NP,
Nelson
EA,
Shoemaker
CJ,
et
al.
Arbidol
and
other
low-molecular-weight
drugs
that
inhibit
lassa
and
Ebola
viruses.
J
Virol
2019;93:.
Ianevski
A,
Andersen
PI,
Merits
A,
Bjoras
M,
Kainov
D.
Expanding
the
activity
spectrum
of
antiviral
agents.
Drug
Discov
Today
2019;24:12248.
Ianevski
A,
Zusinaite
E,
Kuivanen
S,
Strand
M,
Lysvand
H,
Teppor
M,
et
al.
Novel
activities
of
safe-in-human
broad-spectrum
antiviral
agents.
Antiviral
Res
2018;154:17482.
Iwasawa
C,
Tamura
R,
Sugiura
Y,
Suzuki
S,
Kuzumaki
N,
Narita
M,
et
al.
Increased