Discovery of Potent Broad Spectrum Antivirals Derived
from Marine Actinobacteria
Avi Raveh1, Phillip C. Delekta2, Craig J. Dobry2, Weiping Peng2, Pamela J. Schultz1, Pennelope K.
Blakely3, Andrew W. Tai2, Teatulohi Matainaho6, David N. Irani3, David H. Sherman1,4,5, David J. Miller2,4*
1 Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Department of Internal Medicine, University of Michigan, Ann
Arbor, Michigan, United States of America, 3 Department of Neurology, University of Michigan, Ann Arbor, Michigan, United States of America, 4 Department of
Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan, United States of America, 5 Department of Chemistry and Medicinal Chemistry,
University of Michigan, Ann Arbor, Michigan, United States of America, 6 School of Medicine and Health Sciences, University of Papua New Guinea, Boroko,
Papua New Guinea
Abstract
Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although
secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial
and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this
report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to
identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from
Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New
Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against
western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a
selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral
activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function
by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis.
Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of
RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and
Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers,
improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine
encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine
microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport
is a viable target for the continued development of broadly active antiviral compounds.
Citation: Raveh A, Delekta PC, Dobry CJ, Peng W, Schultz PJ, et al. (2013) Discovery of Potent Broad Spectrum Antivirals Derived from Marine
Actinobacteria. PLoS ONE 8(12): e82318. doi:10.1371/journal.pone.0082318
Editor: Adrianna Ianora, Stazione Zoologica, Italy
Received September 12, 2013; Accepted October 29, 2013; Published December 5, 2013
Copyright: © 2013 Raveh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the National Institute of Allergy and Infectious Diseases grants U54 AI057153 (Region V “Great Lakes” Regional Center
of Excellence for Biodefense and Emerging Infectious Diseases Research Career Development and Developmental Project Awards to DJM), R21
AI076975 and AI093642 (to DJM), and the Hans W. Vahlteich Professorship (to DHS). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: milldavi@umich.edu
Introduction
system (CNS) causing encephalitis. The Alphavirus genus
within the Togaviridae family contains about 30 mosquitoborne, enveloped, positive-stranded RNA viruses, one-third of
which cause significant diseases in human and animals
worldwide [4]. The encephalitic alphaviruses, including
western, eastern, and Venezuelan equine encephalitis viruses
(WEEV, EEEV, and VEEV), directly infect neurons resulting in
CNS inflammation and neuronal destruction [5–8]. These highly
virulent pathogens can cause severe disease in humans with
fatality rates of up to 70%, as well as long-term neurological
sequelae in most survivors [9,10].
Infections caused by arthropod-borne viruses (arboviruses)
represent dramatic examples of disease reemergence [1], due
in part to significant urban growth as well as ease of worldwide
travel, thereby producing conditions that facilitate arbovirus
epidemics [2,3]. Furthermore, the threat posed by the
intentional exposure of a population center to a virulent
arbovirus has prompted the U.S. federal government to
designate numerous arboviruses as high priority biodefense
pathogens, particularly those that infect the central nervous
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Marine Microbe-Derived Antivirals
extensive library of pre-fractionated extracts derived from
marine actinomycetes [30] as starting material. This chemical
diversity enabled us to implement a drug discovery program for
antivirals effective against WEEV and related arboviruses. In
this report, we describe an efficient bioassay-guided sequential
fractionation process and purification of a natural product
molecule generated by a novel marine Streptomyces species.
We identify this compound as an antimycin A derivative,
displaying potent and broad spectrum antiviral activity. We also
provide evidence for its mechanism of action, which is
mediated in part by disruption of mitochondrial electron
transport and pyrimidine biosynthesis. Furthermore, we
demonstrate that antimycin A reduces CNS viral titers and
improves both clinical disease and survival in mice given a
lethal challenge with WEEV. These results provide clear proofof-concept that potent, broad spectrum antiviral compounds
with in vivo activity against highly pathogenic arboviruses can
be isolated and identified from natural product chemical
diversity resources.
There are currently no licensed vaccines or antiviral drugs for
alphavirus infections. Formalin-inactivated vaccines for WEEV
or EEEV and a live attenuated vaccine against VEEV (TC-83
strain) are available on an investigational drug basis, whose
use is limited primarily to laboratory personnel working with
these infectious agents. The development of alternative live
attenuated, chimeric, and DNA-based alphavirus vaccines is
being actively pursued, but the broad clinical application of
these next generation vaccines is likely years away [11].
Furthermore, the combination of active vaccination plus
antiviral therapy may be a more effective response in the
setting of an outbreak due to either natural transmission or
intentional exposure to a viral pathogen [12]. Although
numerous compounds have been reported to inhibit alphavirus
replication in cultured cells, only a select few have shown any
activity in animal models [13–16]. Thus, there is a pressing
need to identify new antiviral compounds and drug targets as
part of an effective medical countermeasures strategy to
prevent or mitigate illness, suffering, and death resulting from
infections caused by these virulent pathogens [17].
Chemical libraries containing small molecule compounds
with known structures provide a rich source of starting material
for the identification of novel antiviral agents. Indeed, our group
has used such libraries to identify a novel class of compounds
effective against neurotropic alphaviruses [13,18]. Although
these libraries can be vast in size and scope, their use is often
constrained by factors such as the cost of acquiring or
maintaining large compound collections and the limits of
synthetic and combinatorial chemistry [19]. Even the largest
small molecule libraries, often containing 106 compounds or
more, represent a vanishingly small fraction of the number of
chemically feasible drug-like molecules, which is estimated to
be on the order of 1060 to 10100 [20–22].
An alternative approach takes advantage of the complex
biosynthetic pathways of living organisms, which can produce
natural products of almost unlimited structural diversity [23].
This approach has been utilized quite effectively in the
identification and development of antimicrobial agents, as a
substantial portion of currently available drugs used clinically to
treat bacterial and fungal infections were originally derived from
microbial sources [24]. Although natural products have not
previously been used to any large extent in the development of
novel antiviral agents, there is limited precedence for this
approach [14]. Numerous secondary metabolites obtained from
microbes recovered over a range of geographical regions and
habitats have been developed as potential therapeutics, and
they are frequently the endpoint of a complex biosynthetic
system that comprises a metabolic pathway [25]. These
products have changed the face of human and veterinary
medicine over the past several decades and continue to
provide new drug leads for pharmaceutical development.
Sediment from shallow and deep water marine habitats is
proving to be a rich source of novel microorganisms,
particularly actinomycetes, whose metabolic products provide
entirely new structural diversity with broad potential clinical
applications [26–29]. For the current study, we used an
established phenotypic cell-based replicon high throughput
screen (HTS) and validation protocol [18] in conjunction with an
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Results
Primary HTS and validation of candidate extracts from
marine microbes
We previously developed, validated, and utilized a WEEV
replicon cell-based assay to complete an HTS with a defined
synthetic small molecule chemical library containing >50,000
compounds [18], leading to the development of a novel class of
inhibitors with in vivo activity against neurotropic alphaviruses
[13]. We used the same cell-based phenotypic assay to
complete an additional HTS and secondary validation with a
library of extracts derived from marine microbes isolated from
diverse geographic regions. Figure 1 illustrates our workflow for
strain purification and cultivation, extract preparation, HTS
completion with selection criteria, and secondary, tertiary, and
final validation steps. For each microbial isolate, which based
on the location and isolation procedures are primarily within the
order Actinomycetales [30], several pre-fractionated extracts
were prepared by sequential organic solvent extraction of the
resin used to adsorb the secreted metabolites.
We initially identified 37 extracts as primary hits from a
collection of 2,206 extracts, and 23 of these extracts derived
from 14 individual microbial isolates were validated in
secondary dose-response assays using the WEEV replicon
system. To further validate candidate extracts and select those
with potential broad spectrum antiviral activity, we completed
tertiary validation assays with an EEEV replicon bearing a
secreted alkaline phosphatase reporter gene. We then used a
final validation cytopathic effect (CPE) reduction assay with
several infectious viruses, including the alphaviruses WEEV
and Fort Morgan virus (FMV), the bunyavirus La Crosse virus
(LACV), and the picornavirus encephalomyocarditis virus
(EMCV). These rigorous validation steps led to the
identification of four candidate extracts derived from two
individual microbial isolates, designated LE-496-unk and
05-1015-2N. All remaining data contained in this report were
generated with isolate 05-1015-2N, which we recovered from
marine sediment collected near the southern coast of New
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Figure 1. Schematic of marine microbe-based natural product extract production, screening, and validation. Individual
steps are indicated in the left column, with explanatory comments provided on the right. The number of extracts and corresponding
number of individual strains, where appropriate, are indicated in bold type between steps.
doi: 10.1371/journal.pone.0082318.g001
Ireland province in Papua New Guinea. On the basis of 16S
rRNA sequence, phylogenetic analyses, and geographic
location where the isolate was recovered, we named this
marine microbe Streptomyces kaviengensis (Figure S1).
complex mixture of secreted bacterial secondary metabolites
containing an indeterminate number of distinct chemical
entities. To isolate and purify the active compound or
compounds responsible for the observed antiviral activity, we
developed an empiric biochemical fractionation protocol that
used the WEEV replicon assay to follow antiviral activity at
each fractionation step (Figure 2). The first step involved C18
flash chromatographic separation of the S. kaviengensisderived extract into eight fractions, which we initially used to
Isolation of a purified antiviral compound from the S.
kaviengensis-derived extract
The S. kaviengensis-derived extract we identified and
validated as possessing antiviral activity was most likely a
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confirm that inhibitory activity against WEEV replicons
represented authentic antiviral activity against infectious virus
(Figure S2). Fractions 6 and 7 contained the most potent
inhibitory activity against WEEV replicons (Figure S2A),
suppressed infectious WEEV production (Figure S2B), and
rescued WEEV-induced CPE (Figure S2C), all indicating that
the replicon assay was a valid and convenient surrogate to
follow antiviral activity during subsequent fractionation steps.
We used four sequential separation steps for the final
isolation of a purified antiviral compound from the S.
kanviengensis-derived extract: C18 flash chromatography,
LH-20 size exclusion, and two successive C18 HPLC steps
(Figure 2A). We used weight-based concentrations and
completed full dose-titration curves at each step to follow
potency, thereby demonstrating a progressive increase in
antiviral activity from initial extract to final pure active
compound (designated F7E2e) in both the replicon inhibition
assays (Figure 2B) and in the suppression of infectious WEEV
titers (Figure 2C). To quantify sequential increases in activity
and to examine potential toxicity, we calculated concentrations
that produced a 50% reduction in cell viability (CC50), a 50%
reduction in replicon activity or virus titers (IC50), or a 50%
decrease in virus-induced CPE (EC50) (Table 1). This analysis
revealed an approximate 500-fold increase in antiviral potency
from the initial extract to the final active compound, F7E2e, and
a selectivity index (CC50/IC50) for F7E2e of >550. Furthermore,
there was excellent correspondence in the IC50 and EC50
values for individual samples, with nearly identical results for
the final active compound F7E2e. Finally, HPLC and high
resolution LC-MS analyses showed that F7E2e was >98% pure
and had a molecular weight of 548.279 g/mol, which
corresponded to IC50 and EC50 values of approximately 3 nM
(Table 1).
We confirmed the antiviral activity of F7E2e against WEEV in
single-step growth assays using BE(2)-C human neuronal cells
infected with virus at a multiplicity of infection (MOI) = 10
(Figure 3). For these experiments we used F7E2e at 100 ng/ml
(~200 nM) and included mycophenolic acid as a positive
control,
as
this
cellular
inosine
5’-monophosphate
dehydrogenase inhibitor effectively suppresses alphavirus
replication [31]. Both mycophenolic acid and F7E2e reduced
infectious WEEV production by 12 h post-infection (hpi) and
resulted in a stable 10-fold reduction in virus titers at 24-48 hpi
(Figure 3A). We also measured viral RNA accumulation by
qRT-PCR (Figure 3B) and northern blotting (Figure 3C), and
found that F7E2e suppressed the accumulation of both viral
genomic and subgenomic RNA in infected cells.
To further characterize the antiviral activity of F7E2e, we
examined its activity against WEEV in several cell lines derived
from various mammalian species and tissues (Figure 4 and
Table 2). We infected cells at a low inoculum (MOI = 0.1) and
determined the optimal time point to harvest supernatants for
WEEV titer analysis. The temporal pattern of WEEV production
varied between cell lines, with maximal production of infectious
virions at 24 hpi for BE(2)-C and Vero cells, 24 to 48 hpi for
BHK-21 and HEK293 cells, and 48 to 72 hpi for CHO, Huh-7,
SH-SY5Y, and U87 cells (Figure 4A). We subsequently
infected cells with WEEV, simultaneously treated with either
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Figure 2. Sequential fractionation and purification of an
active antiviral compound in the extract derived from S.
kaviengensis.
(A) Schematic of the four sequential
fractionation steps used to obtain a purified antiviral compound
from S. kaviengensis. Chromatographic method and total
fractions from each separation step are shown in italics type.
The individual fraction at each step that had the highest
antiviral activity in the replicon assay was used for subsequent
fractionation steps. The final purified compound is referred to
as F7E2e.
(B) Antiviral activity of the initial S. kaviengensis extract and
sequential fractions analyzed with WEEV replicons. Results are
presented as the percent untreated control transfected cells
and represent the mean ± SEM from at least three analyses of
fractions from one purification experiment. Similar results were
obtained from a second independent purification experiment
using the steps outlined in Figure 2A.
(C) Antiviral activity of the initial S. kaviengensis extract and
select sequential fractions analyzed with infectious WEEV in
BE(2)-C neuronal cells. Cells were infected with WEEV at an
MOI = 0.1, simultaneously treated with the indicated weightbased concentration of the indicated material (whole extract or
specific fraction), and virus production was measured by
plaque assay at 24 hpi. Results are presented as infectious
virion concentration in tissue culture supernatants and
represent the mean ± SEM from at least three analyses of
fractions from one purification experiment. Similar results were
obtained from a second independent purification experiment
using the steps outlined in Figure 2A.
doi: 10.1371/journal.pone.0082318.g002
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Marine Microbe-Derived Antivirals
Table 1. Quantitative antiviral potency values for S.
kaviengensis extract and select fractions.
Toxicity
Replicon LUC
WEEV titers
WEEV CPE
Sample1
(CC50)
assay (IC50)
(IC50)
reduction (EC50)
Extract
>10,0002
900 ± 52
968 ± 11
1551 ± 95
Step 1 – F7
>10,000
19.7 ± 2.2
31.2 ± 7.5
9.5 ± 0.5
>1,000
1.8 ± 0.3
1.8 ± 0.2
1.7 ± 0.1
Step 4 –
F7E2e
(3.3 ± 0.5 nM)
(3.3 ± 0.3
nM)
(3.2 ± 0.3 nM)
1Correspond to samples in Figure 2A purification schematic.
2 Values in ng/ml represent the sample concentration that produced either a 50%
reduction in replicon LUC activity or WEEV titers or a 50% increase in virusinfected cell viability at 24 hpi compared to untreated controls. For toxicity values,
the highest concentration used in the titration assays is shown. Results are
presented as the mean ± SEM from at least three independent experiments. Molar
IC50 and EC50 values for F7E2e are given in parentheses and are based on a MW
of 548.279 g/mol determined by LC-MS.
doi: 10.1371/journal.pone.0082318.t001
mycophenolic acid or F7E2e, and harvested supernatants at
the appropriate times post-infection for individual cell lines to
analyze virus titers (Figure 4B). While both compounds
suppressed infectious WEEV production in all cell lines tested,
the magnitude of suppression varied between cell lines from
approximately 200- to 9,000-fold for mycophenolic acid and 15to 5,000-fold for F7E2e (Table 2). This variance was not due to
differential compound toxicity (Figure S3) or intrinsic
characteristics of the cell lines, such as the expression of drug
efflux pumps, as the pattern of suppression varied between
compounds. For example, although both compounds potently
suppressed WEEV production in HEK293 cells, their activity in
CHO, Huh-7, and SH-SY5Y cells was quite divergent (Table 2).
Taken together, these results indicated that we had
successfully isolated and purified a compound produced by S.
kaviengensis with potent antiviral activity against WEEV.
Figure 3.
Purified antiviral compound from S.
kaviengensis suppresses WEEV RNA replication and virus
production in single-step growth assays. (A) Infectious
virion production. BE(2)-C cells were infected with WEEV at an
MOI = 10, treated with DMSO, 25 μM mycophenolic acid
(MPA), or 100 ng/ml (~200 nM) purified compound F7E2e, and
virus titers is tissue culture supernatants were determined by
plaque assay at 6, 12, 24, and 48 hpi. Plaque assay sensitivity
was 102 pfu/ml. Results represent the mean ± SEM from three
independent experiments. *p-value < 0.05 compared to DMSOtreated controls for both MPA- and F7E2e-treated samples.
(B) Quantitative RT-PCR analysis of WEEV RNA accumulation.
Cells were infected and treated as above in (A), total RNA was
harvested at the indicated time points, and primers
corresponding to either the nsP1 or E1 WEEV genome were
used to amplify and quantify either genomic (nsP1) or genomic
plus subgenomic (E1) RNA accumulation. Results are
presented as WEEV RNA levels relative to infected DMSOtreated control cells, and represent the mean ± SEM from six
independent experiments. p-value < 0.001* or 0.0001**
compared to DMSO-treated controls.
(C) Northern blot analysis of WEEV RNA accumulation. Mockinfected cells (lane 1) or cells infected and treated as above in
(A) with DMSO (lane 2), MPA (lane 3) or F7E2e (lane 4) were
harvested at 12 hpi, and total RNA was analyzed by Northern
blotting with a strand-specific 32P-labelled riboprobe that
detected both positive-sense genomic and subgenomic viral
RNA (vRNA). The location and relative size of genomic and
subgenomic vRNA are shown on the right, and the ethidium
bromide-stained 28S rRNA band is shown as a loading control.
Representative results from one of three independent
experiments are shown.
Antimycin A derivatives produced by Streptomyces
species are potent antivirals against WEEV serogroup
alphaviruses
We confirmed the molecular structure of F7E2e by an
extensive array of 1D and 2D NMR techniques and high
resolution MS analysis, ultimately determining that the antiviral
compound was antimycin A1a (Figure 5A and Table S1). We
determined the structure of a second purified compound from
S. kaviengensis (F7E2f) as antimycin A10a, which also
displayed potent antiviral activity against WEEV replicons with
an IC50 of approximately 3 nM (data not shown). Antimycins are
a family of secondary metabolites produced by Streptomyces
species, consisting of a 3-formylaminosalicylic acid linked via
an amide bond to a nine-membered cyclic dilactone moiety,
plus two alkyl side chains that vary in length and composition
(Figure 5A). These natural products were first isolated in 1949
[32], and have been shown to possess antifungal [33–35] and
some antiviral [36–38] activity. The antimycins block the
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doi: 10.1371/journal.pone.0082318.g003
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Figure 4. Purified antiviral compound from S. kaviengensis has potent but variable antiviral activity against WEEV in a
wide range of cell lines. (A) Time course of WEEV production in various mammalian cell lines. Individual cell lines were infected
with WEEV at an MOI = 0.1 and infectious virus titers in tissue culture supernatants were determined at 24, 48, and 72 hpi by
plaque assay. WEEV-infected MDBK, HeLa, and A549 cells were also examined but showed no significant virion production by 72
hpi (data not shown). Results represent the mean ± SEM from three independent experiments.
(B) Antimycin A activity against WEEV in various mammalian cell lines. The indicated cell lines were infected as described above,
simultaneously treated with DMSO control, 25 μM mycophenolic acid (MPA), or 100 ng/ml (~200 nM) F7E2e, and infectious virus
titers in tissue culture supernatants were determined at the indicated time post-infection. The drug concentrations used for treatment
were 50- to 100-fold the IC50 values for each compound in the WEEV replicon assay (see Table 1 and Figure 5). Results represent
the mean ± SEM from three independent experiments. p-value < 0.05* or 0.005** compared to DMSO-treated controls.
doi: 10.1371/journal.pone.0082318.g004
mitochondrial electron transport chain (mETC) and hence
cellular respiration by binding the Qi site of cytochrome C
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reductase in complex III [39]. Thus, antimycins can have
profound effects on cellular physiology, including significant
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Marine Microbe-Derived Antivirals
methoxyantimycin A3, which has been developed as a
potential anticancer agent due to potent inhibition of the antiapoptotic protein, Bcl-xL [42,43]. Modification of the 3formylaminosalicyclic acid moiety disrupts antimycin A binding
to cytochrome C reductase in complex III and abrogates
inhibition of the mETC and cellular respiration, thereby
reducing its toxicity [44,45]. However, 2-methoxyantimycin A3
was completely ineffective in suppressing WEEV replicon
activity (Figure 5F), suggesting that mETC inhibition played a
central role in the antiviral activity of antimycin A.
Table 2. WEEV titer reduction mediated by S.
kaviengensis-derived compound F7E2e is cell linedependent.
WEEV titer fold-reduction1
Cell line
Species
Tissue/cell type
Mycophenolic acid
BE(2)-C
Human
Neuron
179
F7E2e
14
BHK-21
Hamster
Kidney fibroblast
920
1,391
HEK293
Human
Kidney epithelium
5,107
4,968
Vero
Primate
Kidney epithelium
1,419
166
CHO
Hamster
Ovarian epithelium
5,665
42
Huh-7
Human
Hepatocyte
2,030
57
SH-SY5Y
Human
Neuron
8,568
59
U87
Human
Astrocyte
8,156
463
Antimycin A1a purified from S. kaviengensis and
commercial antimycin A standard induce similar
transcriptional responses indicative of mitochondrial
dysfunction
The previous identification of the mETC complex III as the
target for antimycin A [39], and the WEEV replicon results with
the 2-methoxyantimycin A3 derivative (Figure 5F), provided
strong evidence for its potential mechanism of action. However,
mETC inhibition likely has pleotropic effects within cells, and
the nanomolar IC50 values we identified for antimycin A (Figure
5 and Table 1) were substantially lower than the concentrations
often used for cell-based assays [36,38,42,46]. Thus, to assess
the cellular response to antimycin A treatment we conducted
genome-wide transcriptional microarray analyses. This
approach provided further evidence supporting the
identification of F7E2e derived from S. kaviengensis by
comparative transcriptional response analyses with commercial
antimycin A. We identified 1,120 up-regulated and 35 downregulated genes in BE(2)-C cell treated with F7E2e (Table S2),
and 976 up-regulated and 45 down-regulated genes in the
same cells treated under identical conditions with commercial
antimycin A (Table S3). There was a 61% concordance in the
gene sets between the two treatment groups when all genes
regulated ≥2-fold were compared, which increased to 73%
concordance when only genes regulated ≥3-fold were analyzed
(Table S4). There were 757 genes co-regulated in BE(2)-C
cells treated with F7E2e or commercial antimycin A, and there
was a significant correlation (R = 0.96) in the magnitude of
transcriptional changes of individual genes in cells treated with
either of the two compounds (Table S4 and Figure S4).
To further analyze the cellular changes induced in BE(2)-C
cells by F7E2e or commercial antimycin A, we conducted in
silico analyses with differentially regulated genes that were
assigned to known cellular pathways using Ingenuity Pathway
Analysis software. We identified 46 canonical pathways
preferentially modulated after treatment with F7E2e and 45
preferentially modulated after treatment with commercial
antimycin A, with 32 pathways overlapping between the two
treatments (Table S5). Table 3 lists the ten canonical pathways
co-modulated by both F7E2e and commercial antimycin A that
showed the highest significance. The most significantly
associated pathway, by an overwhelming margin, was the
mitochondrial dysfunction pathway, which included numerous
mETC components co-regulated after treatment with F7E2e or
commercial antimycin A (Table S6). The strong correlation in
cellular responses between treatments supported the
molecular identification of F7E2e isolated from S. kaviengensis
1 Values represent the average decrease in infectious WEEV production in cells
treated with 25 μM mycophenolic acid or 100 ng/ml (~200 nM) S. kaviengensisderived compound F7E2e. Tissue culture supernatants were harvested at 24 hpi
for BE(2)-C, BHK-21, HEK293, and Vero cells, and 48 hpi for CHO, Huh-7, SHSY5Y, and U87 cells (see Figure 4).
doi: 10.1371/journal.pone.0082318.t002
cytotoxicity at moderate to high doses. Indeed, antimycin A is
the active ingredient of Fintrol, a piscicide commonly used in
fishery management [40].
To verify that antimycin A functions as an antiviral against
WEEV, we examined the activity of an authentic standard
(Sigma-Aldrich A8674) in the replicon assay (Figure 5B).
Commercial antimycin A showed potent inhibitory activity
against WEEV replicons, with an IC50 of approximately 3 nM,
which was almost 100-fold more potent than mycophenolic acid
(Figure 5B, closed symbols). Although antimycin A showed
cellular toxicity similar to mycophenolic acid, cell viability
measured by 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) signal was never less than 50% of control even
at the highest concentrations tested (Figure 5B, open symbols).
We obtained similar results using trypan blue exclusion and cell
counting to measure cytotoxicity (data not shown). We further
investigated the antiviral activity of antimycin A in BE(2)-C cells
infected with WEEV (Figure 5C) or FMV (Figure 5D), a lower
virulence WEEV serogroup alphavirus that can be safely
handled under BSL2 conditions [41]. Antimycin A reduced
WEEV and FMV titers by 10- to 100-fold, with IC50 values
similar to those obtained in the replicon assay (Figure 5B) and
with compound F7E2e (Table 1). Antimycin A was also
approximately 100-fold more potent than mycophenolic acid in
suppressing WEEV and FMV titers, similar to the replicon
assay results, although the maximal level of suppression for
WEEV titers was greater with mycophenolic acid (Figure 5C).
The commercial antimycin A standard is a mixture of
congeners isolate from Streptomyces species with four major
components (manufacturer’s product literature). We verified
this by HPLC (Figure 5E), purified the four major peaks, and
confirmed their structures by NMR (Table S1). We identified
the four major components as antimycins A1a, A2a, A3a, and
A4a (Figures 5A and 5E), all of which showed potent individual
activity against WEEV replicons (Figure 5F). We also examined
the antiviral activity of a commercial analogue, 2-
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Figure 5. Antimycin A derivatives produced by Streptomyces have potent antiviral activity against WEEV serogroup
alphaviruses. (A) Molecular structure of antimycin A. Core structure is shown at the top, and the individual R1 and R2 constituents
of derivatives A1a, A2a, A3a, A4a, and A10a are shown below the core structure. Specific atom designations correspond to the
NMR results in Table S1.
(B) Antiviral activity of commercial antimycin A (AA) and mycophenolic acid (MPA) analyzed with WEEV replicons. Dose titration
results for both replicon activity (closed symbols) and viability (open symbols) are presented as the percent untreated control cells
and represent the mean ± SEM from at least five independent experiments. Calculated IC50 values for anti-replicon activity are
shown on the graph for both compounds, and an average MW of 550 g/mol was used to estimate molar concentrations for
commercial antimycin A.
(C and D) Antiviral activity of commercial AA and MPA analyzed with infectious WEEV (C) or FMV (D) in BE(2)-C neuronal cells.
Cells were infected with WEEV (MOI = 0.1) or FMV (MOI = 1), treated simultaneously with compounds at the indicated
concentrations, and virus production was measured by plaque assay at 24 hpi. Results are presented as infectious virion
concentration in tissue culture supernatants and represent the mean ± SEM from at least three independent experiments.
Calculated IC 50 values are shown on the graph for both compounds, and for commercial antimycin A these values were determined
as described above in (B). The dashed reference lines represent results from infected cells treated with DMSO control.
(E) HPLC separation of individual antimycin A derivatives from commercial stock compound. Only the select portion of an HPLC
tracing that contained the four most prominent peaks is shown, and the various grey scale tracings represent different absorbance
wavelengths. The identification of individual antimycin A derivatives represented by the four most prominent peaks is shown, where
structures were determined by NMR analysis of purified fractions (see Table S1).
(F) Antiviral activity of individual antimycin A derivatives analyzed with WEEV replicons. Dose titration results are presented as the
percent untreated control cells and represent the mean ± SEM from at least four independent experiments. Calculated IC50 values
for individual derivatives are shown on the graph, and were calculated using MWs of 548.63, 534.61, 520.58, and 506.55 g/mol for
antimycins A1a, A2a, A3a, and A4a, respectively. The methoxy group in 2-methoxyantimycin A3 (MeO-AA3) is located at the 6’
position in the core antimycin structure shown in Figure 5A. ND, not determined.
doi: 10.1371/journal.pone.0082318.g005
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Table 3. Cellular pathways modulated in BE(2)-C cells treated with S. kaviengensis-derived compound F7E2e or commercial
antimycin A.
p-value from IPA analysis
Ingenuity Canonical Pathway1
Molecular/Cellular Functions
S. kaviengensis F7E2e Antimycin A
Mitochondrial dysfunction
Free radical scavenging; Cellular function and maintenance
2.0 x 10-20
1.3 x 10-13
Colanic acid building block synthesis
Energy production
0.00056
0.0033
2-ketoglutarate dehydrogenase complex
Energy production; Lipid metabolism
0.00058
0.00040
TCA cycle II (eukaryote)
Free radical scavenging; Small molecule biochemistry; Lipid metabolism
0.0010
0.000006
2-oxobutanoate degradation I
Small molecule biochemistry; Lipid metabolism; Vitamin/mineral metabolism
0.0014
0.00095
Arginine biosynthesis IV
Cellular growth and proliferation; Amino acid metabolism
0.0026
0.0018
Superpathway of cholesterol biosynthesis
Small molecular biochemistry; Lipid metabolism
0.0037
0.0020
Role of BRCA1 in DNA damage response
DNA replication, recombination, and repair; Cell death and survival
0.0042
0.0066
Ascorbate recycling (cytosolic)
Energy production; Lipid metabolism
0.0081
0.0063
Mismatch repair in eukaryotes
DNA replication, recombination, and repair
0.0087
0.0055
1 The listed pathways were identified as significantly associated with transcriptional profile changes induced in BE(2)-C cells treated for 24 h with 100 ng/ml (~200 nM)
F7E2e or commercial antimycin A purchased from Sigma-Aldrich, using Ingenuity Pathway Analysis (IPA) software. Only those pathways identified with p-values < 0.01 for
both treatments are shown. A complete list of all IPA pathways associated with treatment using either compound at a threshold p-value of < 0.05 is provided in Table S5.
doi: 10.1371/journal.pone.0082318.t003
low nanomolar range (Figure 6B, open bars). The proton
ionophore, CCCP, was less active and inhibited replicon
activity with an IC50 of approximately 3 μM, whereas replicon
inhibition IC50 values for the complex II inhibitor,
thenoyltrifluoroacetone, and the complex IV inhibitor, cyanide,
were in the low millimolar range. Eukaryotic mETC activity is
responsible for ATP production under aerobic conditions, and
therefore the antiviral activity of inhibitors may result from a
decrease in energy production and non-selective viral
suppression. To examine this potential confounding effect of
mETC inhibition, we determined IC50 values for global ATP
suppression and CC50 values for toxicity (Figure 6B, crosshatched bars), and calculated ATP IC50/replicon IC50 ratios for
each inhibitor. For thenoyltrifluoroacetone and cyanide, there
were no significant differences in IC50 or CC50 values for
replicon inhibition, ATP production suppression, or cytotoxicity.
In contrast, CCCP had a ratio of 56, rotenone and oligomycin
had ratios of 1,638 and 2,281, respectively, and myxothiazole
and antimycin A had the largest ratios of 16,717 and 47,980,
respectively. These results indicated that some, but not all,
mETC inhibitors disrupted WEEV replicon activity in the
absence of global ATP suppression or overt cellular toxicity,
and that complex III inhibitors had the most potent and
selective activities.
We further examined the effect of mETC suppression of
WEEV replicon activity using combination treatments to
examine possible synergy or antagonism between inhibitors.
We used WEEV replicons and pairwise combination treatments
with antimycin A and rotenone, myxothiazole, oligomycin, or
CCCP to calculate Chou-Talaley parameters and combination
index values [47]. We found no synergy with any combination
treatment, near additive effects with antimycin A and rotenone,
myxothiazole, or oligomycin, but strong antagonism between
antimycin A and CCCP (Table S7). Complex I and III in the
mETC are both significant sources of reactive-oxygen species
(ROS) [48,49], whereas mitochondrial uncoupling by proton
gradient disruption can either prevent or enhance mitochondrial
as antimycin A1a. Furthermore, although we cannot exclude
the modulation of non-mitochondrial pathways as potential
mechanisms, these results suggested that mETC disruption
was a primary mechanism behind the antiviral activity of
antimycin A.
Sublethal and selective disruption of mETC function inhibits
virus replication in part due to suppression of de novo
pyrimidine synthesis.
We focused subsequent mechanism of action studies
designed to understand the antiviral activity of antimycin A on
the mETC (Figure 6). Figure 6A shows a schematic of the
mETC, which resides in the mitochondrial inner membrane and
is responsible for oxidative phosphorylation, respiration, and
aerobic ATP production in eukaryotes. There are three enzyme
complexes
responsible
for
electron
transport
and
electrochemical proton gradient maintenance: NADHcoenzyme Q (CoQ) reductase (complex I), CoQ-cytochrome C
reductase (complex III), and cytochrome C oxidase (complex
IV). Two additional enzyme complexes also participate in
oxidative phosphorylation: succinate-CoQ reductase (complex
II), which links the mETC to the citric acid cycle via reduction of
CoQ, and ATP synthase, also referred to as complex V, which
is ultimately responsible for protein gradient-driven ATP
production. There are known inhibitors for all five enzyme
complexes, which are shown at their site of action in Figure 6A.
Furthermore, there are two well-described complex III
inhibitors: antimycin A and myxothiazole, which block the Qi
and Qo sites of cytochrome C reductase, respectively [39]. The
proton ionophore carbonyl cyanide 3-chlorophenylhydrazone
(CCCP) also disrupts mETC activity via bypassing proton efflux
through the ATP synthase complex.
We used the seven inhibitors shown in Figure 6A to examine
the impact of suppression at different sites within the mETC on
WEEV replicon activity (Figure 6B). The complex I inhibitor,
rotenone, the complex III inhibitors, antimycin A and
myxothiazole, and the ATP synthase inhibitor, oligomycin, all
potently inhibited WEEV replicon activity with IC50 values in the
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Figure 6. Disruption of mitochondrial electron transport suppresses WEEV replication. (A) Schematic of mETC enzyme
complexes. The known targets for the inhibitors shown in italics are indicated by the cross bars. Cyt c, cytochrome C; CoQ,
coenzyme Q.
(B) Antiviral activity and toxicity of mETC inhibitors. Cells were treated with increasing concentrations of the indicated inhibitors, and
replicon inhibition, total cellular ATP production, and cytotoxicity were measured in separate assays. Results are presented as IC50
or CC50 values for the indicated parameter, and represent the mean ± SEM from at least three independent experiments. The
numerical values on the graph indicate fold-differences in IC50 values between replicon inhibition and ATP production suppression
for the indicated select compounds. For rotenone, the comparison was made with CC50 values, since we were unable to calculate
reliable IC50 values for ATP production suppression.
(C) Complementation assays with select mETC inhibitors and WEEV replicons. Cells were treated with 100 μM of the indicated
supplement or antioxidant and antimycin A (AA), CCCP, or mycophenolic acid (MPA) at 2X or 5X replicon IC50 concentrations, and
replicon activity was measured 16-20 h later. Results represent the mean ± SEM from four independent experiments. p-value <
0.05* or 0.005** compared to supplement- or antioxidant-only treated controls. 2-MPG, N-(2-mercaptopropionyl)glycine.
(D) Complementation assay with antimycin A and infectious virus. BE(2)-C cells were infected with FMV at an MOI = 1, treated
simultaneously with 100 μM of the indicated supplement or antioxidant and control DMSO or antimycin A at 5X replicon IC50
concentration, and viral titers in tissue culture supernatants were measured at 24 hpi. Results represent the mean ± SEM from four
independent experiments. **p-value < 0.005 compared to inhibitor-treated controls without supplementation (open bars).
doi: 10.1371/journal.pone.0082318.g006
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ROS production in a dose-dependent and tissue- or cell typespecific manner [49–51]. These observations suggested that
antimycin A-CCCP antagonism was due to differential effects
on ROS generation. We were unable to detect significant ROS
generation using either compound at concentrations up to 5X
the IC50 for replicon inhibition, whereas 1,000-fold higher
antimycin A concentrations readily induced detectable ROS
(data not shown), as previously reported [46].
We subsequently used complementation assays and the
glutathione
analogue
antioxidant
N-(2mercaptopropionyl)glycine (2-MPG) to examine the possible
functional significance of low level ROS generation on
antimycin A- or CCCP-mediated inhibition of WEEV replicon
activity (Figure 6C). We also used uridine and guanosine for
complementation experiments, as cellular pyrimidine and
purine biosynthesis pathways are potential antiviral drug
targets [52–54], and de novo pyrimidine biosynthesis is tightly
linked to the mETC via dihydroorotate dehydrogenase
(DHODH) [55]. Uridine, guanosine, and 2-MPG had no impact
on cell viability or replicon activity in the absence of mETC
inhibitors (data not shown). As expected, guanosine rescued
mycophenolic acid-induced WEEV replicon suppression
[31,54], whereas neither uridine nor 2-MPG produced
significant complementation. For antimycin A-treated cells, only
uridine rescued WEEV replicon activity, whereas only 2-MPG
rescued activity in CCCP-treated cells. We obtained similar
results using another antioxidant, N-acetyl cysteine (data not
shown). Finally, we confirmed the replicon complementation
results using infectious virus, where we found that uridine
supplementation rescued antimycin A-mediated suppression of
FMV production in BE(2)-C cells (Figure 6D). These results
suggested that antimycin A and CCCP inhibited WEEV replicon
activity via distinct mechanisms, and that suppression of de
novo pyrimidine synthesis was partially responsible for the
antiviral activity of antimycin A.
although the titer magnitude varied by almost 10,000-fold from
106 pfu/ml for EMCV to 1010 pfu/ml for VEEV (Figure 7A).
Antimycin A suppressed the production of infectious VEEV,
LACV, and EMCV in BE(2)-C cells (Figure 7B, left graph), and
both VEEV and LACV in Vero cells (Figure 7B, right graph).
The lack of antimycin A activity against VSV in either cell type
or EMCV in Vero cells was not due to intrinsic cellular or viral
resistance, as mycophenolic acid suppressed virus production
for all four viruses in both cell lines. Antimycin A also
suppressed recombinant GFP-SeV replication at multiple
inocula in both Vero (Figure 7C) and BE(2)-C cells (data not
shown), and potently inhibited HCV replicon activity in Huh-7
cells (Figure 7D). Taken together, these results indicated that
antimycin A had broad spectrum antiviral activity against a wide
range of RNA viruses.
Antimycin A improves clinical disease severity,
reduces mortality, and decreases CNS viral titers in
mice infected with WEEV
Despite the anticipated in vivo toxicity of mETC inhibitors, the
high selectivity index of antimycin A in vitro (~48,000, Figure
6B) raised the possibility that we might be able to discern a
therapeutic window in vivo. Thus, we examined the antiviral
activity of antimycin A in mice infected with WEEV (Figure 8).
The recombinant Cba87 strain of WEEV is highly pathogenic in
mice [56], and initial dose-titration experiments indicated that a
subcutaneous inoculum of 103 pfu routinely produced high
mortality in C57BL/6 mice by 14 days after infection with a
mean time to death (MTD) of approximately 11 days (data not
shown). Antimycin A toxicity is species-dependent and varies
widely, from an oral LD50 of less than 0.2 mg/kg for fish to
greater than 50 mg/kg for mice [57]. The intraperitoneal LD50
for mice is 1-2 mg/kg [57,58], and therefore we tested three
doses: 1 mg/kg, 0.2 mg/kg, and 0.02 mg/kg delivered via
intraperitoneal injection twice daily for 7 days starting on the
day of infection. Initial experiments showed that 1 mg/kg
antimycin A accelerated WEEV-induced disease with an MTD
of approximately 7 days (data not shown). However, when
antimycin A doses were reduced by 5- and 50-fold, we were
able to discern a modest therapeutic effect. Vehicle-treated
control mice showed 100% mortality by 12 days after infection,
whereas both clinical disease severity (Figure 8A) and overall
survival (Figure 8B) were improved in mice treated with the 0.2
mg/kg antimycin A dose. The lower dose had less clinical
effect. We also examined the impact of antimycin A on CNS
virus titers in infected mice. Initial experiments showed that
CNS titers peaked 5-7 days after infection with WEEV in
untreated mice (data not shown). Mice treated with 0.2 mg/kg
antimycin A showed a >10-fold reduction in WEEV CNS titers
(Figure 6C), while the lower dose resulted in a smaller titer
reduction that approached statistical significance (p-value =
0.065). These results indicated that antimycin A was effective
in improving clinical disease, prolonging survival, and reducing
CNS virus titers in mice with WEEV encephalitis.
Antimycin A has broad spectrum antiviral activity
against a range of RNA viruses
The initial HTS and validation protocol involved a step that
tested extracts in CPE-reduction assays against several
viruses, including WEEV, FMV, LACV, and EMCV (Figure 1),
which increased the probability of selecting compounds with
broad spectrum antiviral activity. The identification of
antimycins A1a and A10a as active antiviral components in the
S. kaviengensis extract confirmed this approach, as dengue
virus [37], influenza virus [38], and porcine reproductive and
respiratory syndrome virus [36] are all susceptible to antimycin
A inhibition. However, these published studies are limited in
scope, and therefore we analyzed the breadth of antimycin A
antiviral activity against a panel of RNA viruses from the
Togaviridae (VEEV), Bunyaviridae (LACV), Picornaviridae
(EMCV), Rhabdoviridae (vesicular stomatitis virus, VSV),
Paramyxoviridae (Sendai virus, SeV), and Flaviviridae
(hepatitis C virus, HCV) families (Figure 7). For VEEV, LACV,
EMCV, and VSV we used infectious non-recombinant virus and
measured the effect of antimycin A on virus production in
BE(2)-C and Vero cells. In initial time course experiments, all
four viruses showed peak production by 24 hpi at an MOI = 0.1,
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Figure 7. Antimycin A has broad spectrum antiviral activity against RNA viruses. (A) Time course of virion production. BE(2)C (left graph) or Vero (right graph) cells were infected with the indicated viruses at an MOI = 0.1 and virus titers in tissue culture
supernatants were determined by plaque assay at 24, 48, and 72 hpi. The input virus concentration was 104 pfu/ml.
(B) Antimycin A activity against VEEV, LACV, EMCV, and VSV. BE(2)-C (left graph) or Vero (right graph) cells were infected with
the indicated viruses at an MOI = 0.1, treated simultaneously with DMSO, 25 μM mycophenolic acid (MPA), or 200 nM antimycin
(AA), and virus titers in tissue culture supernatants were determined by plaque assay at 24 hpi. Results represent the mean ± SEM
from three independent experiments. p-value < 0.05* or 0.005** compared to DMSO-treated controls.
(C) Antimycin A activity against SeV. Vero cells were infected with GFP-SeV at the indicated MOI, treated simultaneously with
DMSO or 200 nM antimycin A, and GFP fluorescence was measured at 48 hpi (left graph) and 72 hpi (right graph). There was
minimal detectable fluorescence above baseline at 24 hpi (data not shown). Results represent the mean ± SEM from three
independent experiments. **p-value < 0.005 compared to DMSO-treated controls. Similar results were obtained with BE(2)-C cells
(data not shown).
(D) Antimycin A activity against HCV. Huh-7 cells expressing a stable Renilla LUC-containing HCV replicon were incubated with
decreasing concentrations of antimycin A in the absence of selection, and LUC activity was measured 24 h later. The calculated IC 50
value for antimycin A in the HCV replicon system is shown on the graph. Results represent the mean ± SEM from three independent
experiments.
doi: 10.1371/journal.pone.0082318.g007
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Figure 8. Antimycin A improves clinical disease and survival and reduces CNS titers in mice infected with WEEV. (A and
B) Clinical disease severity and survival in WEEV-infected mice. C57BL/6 mice were infected with 103 pfu WEEV, treated twice daily
with DMSO or the indicated dose of antimycin A via intraperitoneal injection, and both clinical disease (A) and mortality (B) were
monitored for 14 days post-infection. Representative results from one of two independent experiments are shown (N = 7-8 mice per
group). *p-value < 0.05 compared to DMSO-treated mice.
(C) Virus titers in the CNS of WEEV-infected mice. Mice were infected and treated as described above, and virus titers in brain were
determined at 6 days post-infection. N = 4 mice per group. **p-value < 0.01 compared to DMSO control.
doi: 10.1371/journal.pone.0082318.g008
Discussion
Natural products have been a cornerstone of drug discovery
throughout the history of medicine, and have been a
particularly rich source of novel therapeutics to treat infectious
diseases and cancer [24]. Although natural products have not
been used extensively in the search for new antiviral agents,
several compounds derived from marine organisms over the
past decade have been shown to contain some degree of
antiviral activity [59–61]. Medicinal plant extracts have also
been used to identify potential novel antiviral compounds,
including some targeted against WEEV [62]. Of particular
relevance to the results in this report, several seco-pregnane
steroids produced by the Chinese herbs Strobilanthes cusia
and Cynanchum paniculatum have in vitro and in vivo activity
against several alphaviruses [14]. However, in contrast to our
results with S. kaviengensis-derived antimycin A demonstrating
broad spectrum activity, these plant-derived steroids, whose
precise target and molecular mechanism of action remain
unknown, selectively suppress subgenomic RNA synthesis and
show restricted antiviral activity limited to alphavirus-like RNA
viruses [14].
We chose an unbiased phenotypic cell-based assay rather
than a targeted assay against a specific viral or cellular protein
to select and validate extracts and guide fractionation in an
effort to broaden the potential targets and mechanisms of
action for candidate antivirals. The use of a phenotypic cellbased antiviral assay also increases the probability of selecting
compounds having a cellular rather than a viral target, thereby
potentially broadening the antiviral spectrum and presenting a
higher barrier against the development of viral resistance [63].
Although the phenotypic approach has been successful in the
discovery and development of pharmaceuticals [64], there are
The immense biosynthetic capability of microorganisms
presents an unrivaled opportunity to explore the boundaries of
chemical space in the discovery of novel therapeutic agents. In
this report, we took advantage of this capability and used
marine microbe-derived extracts combined with a phenotypic
cell-based bioassay-guided fractionation process to identify
potential novel antiviral compounds. We drew six main
conclusions from our studies: (i) the marine actinomycetes S.
kaviengensis produced a readily isolated antiviral compound,
antimycin A1a, which displayed potent activity against WEEV
RNA replication and virion production; (ii) antiviral activity
extended to at least five closely related antimycin A analogues,
although a 2-methoxy derivative with no mETC inhibitory
activity was completely inactive as an antiviral compound; (iii)
disruption of host mETC activity, either with antimycin A or
several unrelated mETC complex inhibitors, suppressed WEEV
replication; (iv) the molecular mechanism whereby antimycin A
disrupted WEEV replication through mETC inhibition involved,
in part, suppression of de novo pyrimidine synthesis; (v)
antimycin A showed broad in vitro antiviral activity against
alphaviruses, bunyaviruses, picornaviruses, paramyxoviruses,
and flaviviruses; and (vi) antimycin A was active in vivo in
WEEV-infected mice, albeit with a narrow therapeutic window.
These results demonstrate that marine Streptomyces produce
metabolites with potent antiviral activity, and that sequential
fractionation of complex microbe-derived mixtures guided by a
phenotypic cell-based assay is a viable approach to broad
spectrum antiviral drug discovery.
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therapies directed against neurotropic pathogens. Detailed
structure-activity analyses focused on the antiviral activity of
antimycin A have not been conducted, and therefore it would
be premature to exclude further development or repurposing of
this compound or its derivatives for therapeutic use as broad
spectrum antiviral agents. Studies are currently in progress to
define the structural requirements for antimycin A activity
against WEEV in vitro and in vivo, and to examine potential
synergistic activity with established neuroprotective agents or
other unrelated compounds active against WEEV [13].
Furthermore, the observation that antimycin A, myxothiazole,
rotenone, CCCP, and oligomycin all disrupted WEEV
replication suggests that the cellular mETC is a candidate drug
target for the development of broadly active antiviral
compounds. There is ample evidence supporting mitochondrial
targeting in drug discovery and development, and the mETC
has been shown to be an eminently druggable target.
Atovaquone, an antimalarial drug in clinical use, specifically
inhibits the parasite mETC, which along with pyrimidine
biosynthesis is the target of several additional antimalarial
compounds currently under development [68]. Furthermore,
human mETC-targeted drugs represent an important and
growing component of potential novel anticancer therapies, as
malignant cells are often more sensitive to mitochondrial
destabilization [69]. Although the goals of therapy for cancer
and infectious diseases can be quite divergent, there is also
striking precedence for their inadvertent convergence with
respect to drug discovery [70,71].
In summary, we describe a comprehensive and successful
antiviral drug discovery program from the initial isolation of a
novel marine Streptomyces species to the bioassay-directed
purification, identification, in vitro characterization, and in vivo
validation of a secondary metabolite with potent and broad
spectrum antiviral activity. Our studies highlight the tremendous
potential of harnessing the chemical diversity inherent in
natural products derived from marine microbes as source
material for antiviral drug discovery.
several potential disadvantages of these assays, including an
often lengthy and complex series of studies to determine
molecular mechanism of action once a functional chemical
entity has been purified and validated. We had the advantage
of isolating an antiviral compound from S. kaviengensis whose
molecular activity and cellular target have already been
described. Although the general molecular mechanism for
antimycin A activity has been known for decades, the
downstream consequences of mETC disruption are numerous
and include changes in cellular processes that could have
unpredictable effects on virus replication. For example, mETC
inhibition with nanomolar concentrations of antimycin A or
myxothiazole suppresses cellular autophagy [65], a cellular
recycling process associated with the replication of several
viruses [66]. Furthermore, mETC inhibition can generate
mitochondrial ROS or suppress ATP production, both of which
could directly or indirectly inhibit virus replication. We found no
evidence for global ROS generation in cells treated with the
same concentrations of antimycin A that actively suppressed
virus replication, and antioxidants did not reverse antimycin Amediated antiviral activity. We also found no correlation
between global ATP production and the antiviral activity of
antimycin A, in contrast to its proposed mechanism for
suppression of influenza virus replication [38]. However, we
cannot exclude the impact of antimycin A on localized ATP
levels at subcellular sites of viral RNA synthesis, which may be
a more important determinant of antiviral activity [67].
We were able to rescue antimycin A-mediated antiviral
activity with uridine supplementation, suggesting that de novo
pyrimidine synthesis, a process tightly linked to mETC activity
via DHODH [55], was partially responsible for the antiviral
activity of antimycin A against WEEV. This observation is
consistent with the identification of specific small molecule
DHODH inhibitors as a potent broad spectrum antiviral
compounds [52], including activity against WEEV [53].
However, we did not observe antimycin A activity against VSV,
even though other groups have shown potent activity of
specific DHODH inhibitors against this rhabdovirus in cultured
cells [52,53]. Since we did not complete full titration analyses
with VSV and antimycin A, we cannot exclude lower potency
activity that may have been missed by our experimental
approach. In addition, we demonstrate in vivo activity of
antimycin A in mice infected with WEEV, whereas DHODH
inhibitors are not active in mice [53]. These discrepancies may
be due to virus-specific effects or differences in
pharmacokinetic parameters between small molecule DHODH
inhibitors and antimycin A. Alternatively, antimycin A-mediated
suppression of de novo pyrimidine biosynthesis may only
partially account for its potent antiviral activity, and modulation
of additional cellular pathways downstream of mETC inhibition
may also be involved.
The direct clinical utility of unmodified antimycin A as an
antiviral agent will likely be limited by toxicity. However, some
antimycin A derivatives have favorable pharmacokinetic
parameters, including a prolonged terminal half-life in serum
[43]. In addition, both our in vivo results in WEEV-infected mice
and early toxicology studies [58] suggest some level of
antimycin A penetration into the CNS, a crucial requirement for
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Materials and Methods
Ethics statement
All animals were housed and used on-site under specific
pathogen-free conditions in an approved animal BSL3 facility in
strict accordance with guidelines set by the National Institutes
of Health and protocols approved by the University of Michigan
Committee on the Use and Care of Animal and the Institutional
Biosafety Committee (Protocol number 10055-2).
Marine microbe culture, fermentation, and extract
preparation
Marine microbes were collected, isolated, and extracts for
HTS were prepared as previously described [30]. We initially
completed optimization experiments to maximize antiviral
compound production using small scale cultures with six
different media (ISP2, X1, A3M, A3M spiked with Rhodococcus
erythropolis [72], ISP2 without NaCl, and ISP2 agar). ISP2
media resulted in the best production of biologically active
extracts with antiviral activity (data not shown), and therefore
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Marine Microbe-Derived Antivirals
above to produce approximately 12 mg of the same molecule
that was 0.0085% of the initial dried crude extract. This pure
compound was identified by NMR as antimycin A1a. In
addition, from the second extract another pure compound was
isolated, fraction F7E2f (tR = 98.3 min, 8.3 mg, 0.0059% of the
initial dried crude extract), which was identified by NMR as
antimycin A10a (see Figure 5A) and also showed potent
antiviral activity with a WEEV replicon assay IC50 of
approximately 3 nM. (data not shown). Low resolution LC-MS
analyses of HPLC fractions were completed using a Shimadzu
2010 EV APCI spectrometer, and high-resolution APCI-MS
spectra were measured using an Agilent Q-TOF HPLC-APCIMS. NMR spectra were acquired in a Varian INOVA 600 MHz
NMR Facility.
was chosen for large scale production. An oatmeal plate (6%
oat meal, 1.25% agar, NaCl) was streaked from a glycerol
stock and incubated 5 days. Seed cultures of 3 ml ISP2 media
(1% malt extract, 0.4% yeast extract, 0.4% dextrose, 3% NaCl)
were inoculated with a loop full of vegetative cells from an
oatmeal plate culture of S. kaviengensis and incubated with
shaking (200 rpm) at 28°C for 5 days, and subsequently
transferred to 100 ml cultures for the same incubation
conditions.
For large scale production, 25 ml of the seed cultures were
transferred to a 2.8 L Fernbach flask containing 1.5 L of ISP2
and incubated on a rotary shaker (200 rpm) at 28°C for 4 days.
This process was performed twice with 16 Fernbach flasks
(total culture 24 L each time). After 4 days the cultures were
harvested by centrifugation and the resulting cell-free
supernatant was subjected to solid phase extraction using 20 g
Amberlite XAD-16N resin per L of culture. The resin was
separated by filtration and subjected to organic extraction using
different organic solvents. The first large scale culture was
extracted three times: twice with methanol and once with 1:1
methanol:ethyl acetate mixture to provide a final yield of 8.2 g
dried crude extract. The second large scale culture was
sequentially extracted with methanol, ethyl acetate, acetone,
and hexanes to provide a final yield of 14.0 g dried crude
extract.
Inhibitors and Antioxidants
Ribavirin, mycophenolic acid, antimycin A, myxothiazole,
rotenone, thenoyltrifluoroacetone, oligomycin, CCCP, 2-MPG,
and N-acetyl cysteine were purchased from Sigma-Aldrich (St.
Louis, MO), and 2-methoxyantimycin A3 was purchased from
Enzo Life Sciences (Farmingdale, NY).
Viruses
The Cba-87 strain of WEEV was generated from the cDNA
clone pWE2000 as previously described [73]. EMCV and the
CM4-146 strain of FMV were purchased from the American
Type Culture Collection (Manassas, VA). The human/1960
strain of LACV and the TC-83 vaccine strain of VEEV were
obtained from Robert Tesh (University of Texas Medical
Branch, Galveston, TX), and the Indiana-1 strain of VSV was
obtained from Katherine Spindler (University of Michigan, Ann
Arbor, MI). GFP-tagged SeV was obtained from Valery
Grdzelishvili (University of North Caroline Charlotte, Charlotte,
NC) and has been previously described [74]. All experiments
with infectious WEEV were performed under BSL3 conditions
in accordance with University of Michigan Institutional Biosafety
Committee, Centers for Disease Control, and National
Institutes of Health guidelines.
Streptomyces-derived antiviral compound purification
and structure determination
Organic extracts from S. kaviengensis were evaporated to
dryness, dissolved in 100 ml H2O, and loaded onto a C18 silica
gel column (30 x 2.6 cm, YMC Gel ODS-A, 12 nm, S-150 µm).
The C18 column was eluted with a stepwise gradient of H2O/
acetonitrile (100:0 → 0:100) to give eight fractions, designated
F1 through F8, and column wash with ethyl acetate. All
fractions were concentrated under vacuum and assayed for
antiviral activity using the WEEV replicon assay. Fraction F7
(15:85 H2O/acetonitrile) showed the highest potency, whereas
fractions F5, F6, and F8 had lesser potency. Fraction F7 (143
mg) was subjected to a Sephadex LH-20 column in 1:1
chloroform/methanol to obtain eight fractions, designated F7A
through F7H, and a column wash, designated F7I. Two
fractions, F7D and F7E, showed the most activity. Fraction F7E
(12.4 mg) was separated on a reversed phase HPLC column
(Waters XBridge, 250 x 20 mm, 5 mm, 17:83 H20/acetonitrile,
DAD at 210, 238 and 254 nm, flow rate 5 ml/min) eluted with
1:9 H20/acetonitrile to give eight fractions, designated F7E1
through F7E8. Fraction F7E2 showed the most potent antiviral
activity, and was further separated using a different reversed
phase HPLC column (Alltech Econosil C18, 5 mm x 250 mm,
22.5 mm, 17:83 H20/acetonitrile, DAD at 210, 238 and 254 nm,
flow rate 5 ml/min), to yield six fractions, designated F7E2a
through F7E2f. HPLC separations were performed with an
Agilent 1100 HPLC system.
Fraction F7E2e (tR = 47.3 min, 2.5 mg, 0.003% of the initial
dried crude extract) was a pure compound, determined by LCMS analysis, and was highly active in the WEEV replicon assay
with an IC50 of approximately 3 nM (see Table 1). The second
extract was fractionated using the same procedure described
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Cell culture
BE(2)-C, BHK-21, Vero, CHO, SH-SY5Y, and U87 cells were
purchased from the American Type Culture Collection. HEK293
cells were obtained from David Markovitz (University of
Michigan, Ann Arbor, MI), Huh-7 cells were obtained from
Raymond Chung (Massachusetts General Hospital, Boston,
MA), BHK-21 cells stably expressing bacteriophage T7 RNA
polymerase (BSR-T7 cells) were obtained from Klaus
Conzelman (Max von Pettenkofer-Institut, Munich, Germany)
and Sonja Gerrard (University of Michigan, Ann Arbor, MI),
BHK-21 cells stably expressing an EEEV replicon were
obtained from Ilya Frolov (University of Alabama - Birmingham,
Birmingham, AL), and Huh-7 cells stably expressing an HCV
replicon were obtained from Nobuyuki Kato (Okayama
University, Okoyama, Japan). BSR-T7 cells were cultured as
previously described [13], and all other cells were cultured in
Dulbecco’s Modified Eagle Medium containing 5% bovine
growth serum, 1% sodium pyruvate, 0.1 mM non-essential
amino acids, 10 U/ml penicillin, and 10 μg/ml streptomycin.
15
December 2013 | Volume 8 | Issue 12 | e82318
Marine Microbe-Derived Antivirals
Virus replication assays
Fischer’s exact test. The association with a particular canonical
pathway was considered significant if the p-value was < 0.05.
WEEV replicon assays used the plasmid pWR-LUC and
BSR-T7 cells and measured firefly LUC accumulation as a
surrogate marker for viral RNA replication as previously
described [13,18]. The HTS with the microbe-derived extract
library was completed in a 384-well plate format as previously
described [18]. Infectious titers for all viruses were determined
by plaque assay on Vero cell monolayers as previously
described [73] with the following modifications. Cells were
overlaid with a 1.2% (wt/vol) colloid suspension of AviCell
R-581 (FMC Biopolymer, Philadelphia, PA) in complete media
rather than agarose, and cells were harvested at 36-40 hpi for
WEEV, VEEV, VSV and 48-52 hpi for EMCV and LACV. GFPSeV replication was determined by GFP accumulation as
previously described [74]. Quantitative RT-PCR was done as
previously described [18,74,75], and the sequences for the 18S
rRNA and WEEV envelope glycoprotein 1 primers have been
previously published [75]. The sequences for the forward and
reverse WEEV nsP1 qRT-PCR primers were 5’GCAGTCCATGCACCGACA-3’
and
5’GGCTGGTACATACGTACA-3’, respectively. Northern blotting
with 32P-labeled riboprobes was done as previously described
[73]. HCV replicon assays used Renilla LUC accumulation and
were done in stable expressing Huh-7 cells as previously
described [76].
Animal infection and treatment experiments
Female C57BL/6 mice (5-6 weeks of age) were purchased
from the Jackson Laboratory (Bar Harbor, ME). Mice were
housed on a 10/14 h light/dark cycle in ventilated cages
containing no more than five animals per cage, and food and
water were available ad libitum. For WEEV infection, mice were
inoculated subcutaneously with 103 pfu WEEV suspended in
100 μl PBS. Antimycin A was solubilized in DMSO as a stock
solution at 100 mM and diluted in PBS to generate working
solutions for intraperitoneal injections into infected mice on a
twice-daily dosing schedule for 7 days following virus
inoculation. Mice were followed for an additional 7 days after
treatment was completed, and all remaining animals were
euthanized at 14 days after infection. Daily weights were
obtained on all mice, and clinical scoring was done using a 1 to
5 scale as previously described [13]. Virus titers in brain tissue
were determined by plaque assay as described above.
Statistical analysis
We used a two-tailed Student’s t-test assuming equal
variances for routine comparative analyses, and we performed
statistical analyses on log10-transformed virus titer data.
Microarray and pathway statistical analyses are described
above. Differences in survival among cohorts of WEEVinfected mice were measured using a log-rank (Mantel-Cox)
test. In all cases, differences at a p-value < 0.05 were
considered significant.
Cell viability, ATP, and ROS assays
Cell viability after viral infection or drug treatment was
determined by MTT assay as previously described [73]. Total
cellular ATP levels were determined using the ATPlite assay
(PerkinElmer, Waltham, MA) according to the manufacturer’s
instructions. Cellular ROS levels were determined with the
general oxidative stress indicator 5(6)-chloromethyl-2’,7’dichlorodihydrofluoroscein diacetate (CM-H2DCFDA) according
to the manufacturer’s instructions (Life Technologies, Grand
Island, NY).
Supporting Information
Figure S1.
05-1015-2N
phylogenetic analysis.
(PDF)
Microarray and pathway analysis
identification
and
Figure S2.
Validation of WEEV replicon-guided
fractionation as a surrogate for antiviral activity.
(PDF)
Transcriptome analyses were done on three independent
sets of cultures for each comparison using Affymetrix Human
U133 Plus 2.0 microarray chips as previously described [74].
Complete original data files have been deposited in the Gene
Expression Omnibus database (www.ncbi.nlm.nih.gov/geo)
under accession number GSE44541. The Genomatix
ChipInspector software package (Genomatix Software Inc.,
Ann Arbor, MI) was used for primary microarray data analysis.
The following parameters were chosen to identify sets of
differentially regulated transcripts: (i) false-discovery rate of
1%; (ii) three probe minimum coverage; and (iii) expression
level log2 change ≥ 1 (2-fold) compared to control. The list of
genes preferentially up- or down-regulated in BE(2)-C cells
treated with F7E2e or commercial antimycin A were analyzed
using Ingenuity Pathway Analysis software (Ingenuity Systems,
Redwood, CA). Significance was measured by determining the
ratio of the number of genes from the data set that map to a
particular canonical pathway to the total number of genes for
that pathway, and calculating a subsequent p-value using a
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strain
Figure S3. Effect of mycophenolic acid and purified
antiviral compound F7E2e from S. kaviengensis on
viability and proliferation of various cultured cell lines.
(PDF)
Figure S4. Correlation between transcriptional responses
in BE(2)-C cells treated with S. kaviengensis–derived
F7E2e or commercial antimycin A.
(PDF)
Table S1. NMR data for structure elucidation of antimycin
A derivatives dissolved in CDCl3.
(DOCX)
Table S2.
BE(2)-C transcriptional response
kaviengensis-derived compound F7E2e.
16
to
S.
December 2013 | Volume 8 | Issue 12 | e82318
Marine Microbe-Derived Antivirals
(XLSX)
(DOCX)
Table S3. BE(2)-C transcriptional response to commercial
antimycin A.
(XLSX)
Acknowledgements
We thank the University of Papua New Guinea (PNG), PNG
BioNet, and the PNG Department of the Environment and
Conservation for permission to collect research samples. We
thank Daniel Peltier, Kathryn Castorena, Martha Larsen,
Patricia Lopez, Douglas Burr, George Chipala, Eli Benchell
Eisman, Sung Ryeol Park, and the University of Michigan
Center for Chemical Genomics for technical assistance, and
Robert Tesh, Katherine Spindler, Valery Grdzelishvili,
Raymond Chung, David Markovitz, Sonja Gerrard, Klaus
Conzelman, Ilya Frolov, and Nobuyuki Kato for providing
reagents.
Table S4.
Comparison of BE(2)-C transcriptional
responses to S. kaviengensis-derived compound F7E2e or
commercial antimycin A.
(XLSX)
Table S5. Ingenuity Pathway Analysis (IPA) of BE(2)-C
transcriptional responses to S. kaviengensis-derived
compound F7E2e or commercial antimycin A.
(XLSX)
Author Contributions
Table S6. Ingenuity Pathway Analysis (IPA) mitochondrial
dysfunction pathway genes upregulated in BE(2)-C cells
stimulated with S. kaviengensis-derived compound F7E2e
or commercial antimycin A.
(XLSX)
Conceived and designed the experiments: AR PCD WP AWT
DNI DHS DJM. Performed the experiments: AR PCD CJD WP
PJS PKB AWT DNI DJM. Analyzed the data: AR PCD WP
AWT DNI DHS DJM. Contributed reagents/materials/analysis
tools: TM. Wrote the manuscript: AR PCD DNI DHS DJM.
Table S7. Synergy-antagonism assay results of antimycin
A and other mitochondrial electron transport chain
inhibitors.
References
12. Bronze MS, Greenfield RA (2003) Therapeutic options for diseases due
to potential viral agents of bioterrorism. Curr Opin Investig Drugs 4:
172-178. PubMed: 12669378.
13. Sindac JA, Yestrepsky BD, Barraza SJ, Bolduc KL, Blakely PK et al.
(2012) Novel inhibitors of neurotropic alphavirus replication that
improve host survival in a mouse model of acute viral encephalitis. J
Med Chem 55: 3535-3545. doi:10.1021/jm300214e. PubMed:
22428985.
14. Li Y, Wang L, Li S, Chen X, Shen Y et al. (2007) Seco-pregnane
steroids target the subgenomic RNA of alphavirus-like RNA viruses.
Proc Natl Acad Sci U S A 104: 8083-8088. doi:10.1073/pnas.
0702398104. PubMed: 17470783.
15. Julander JG, Smee DF, Morrey JD, Furuta Y (2009) Effect of T-705
treatment on western equine encephalitis in a mouse model. Antiviral
Res 82: 169-171. doi:10.1016/j.antiviral.2009.02.201. PubMed:
19428608.
16. Julander JG, Bowen RA, Rao JR, Day C, Shafer K et al. (2008)
Treatment of Venezuelan equine encephalitis virus infection with (-)carbodine. Antiviral Res 80: 309-315. doi:10.1016/j.antiviral.
2008.07.002. PubMed: 18675850.
17. Reichert E, Clase A, Bacetty A, Larsen J (2009) Alphavirus antiviral
drug development: scientific gap analysis and prospective research
areas. Biosecur Bioterror 7: 413-427. doi:10.1089/bsp.2009.0032.
PubMed: 20028250.
18. Peng W, Peltier DC, Larsen MJ, Kirchhoff PD, Larsen SD et al. (2009)
Identification of thieno[3,2-b]pyrrole derivatives as novel small molecule
inhibitors of neurotropic alphaviruses. J Infect Dis 199: 950-957. doi:
10.1086/597275. PubMed: 19239364.
19. Thompson LA, Ellman JA (1996) Synthesis and applications of small
molecule libraries. Chem Rev 96: 555-600. doi:10.1021/cr9402081.
PubMed: 11848765.
20. Dobson CM (2004) Chemical space and biology. Nature 432: 824-828.
doi:10.1038/nature03192. PubMed: 15602547.
21. Schneider G, Fechner U (2005) Computer-based de novo design of
drug-like molecules. Nat Rev Drug Discov 4: 649-663. doi:10.1038/
nrd1799. PubMed: 16056391.
22. Lipinski C, Hopkins A (2004) Navigating chemical space for biology and
medicine. Nature 432: 855-861. doi:10.1038/nature03193. PubMed:
15602551.
23. Verdine GL (1996) The combinatorial chemistry of nature. Nature 384:
11-13. doi:10.1038/384011a0. PubMed: 8895593.
1. Weaver SC, Reisen WK (2010) Present and future arboviral threats.
Antiviral Res 85: 328-345. doi:10.1016/j.antiviral.2009.10.008. PubMed:
19857523.
2. Enserink M (2007) Infectious diseases. Chikungunya: no longer a third
world disease. Science 318: 1860-1861. doi:10.1126/science.
318.5858.1860. PubMed: 18096785.
3. Nash D, Mostashari F, Fine A, Miller J, O'Leary D et al. (2001) The
outbreak of West Nile virus infection in the New York City area in 1999.
N Engl J Med 344: 1807-1814. doi:10.1056/NEJM200106143442401.
PubMed: 11407341.
4. Griffin DE (2001) Alphaviruses. In: DM KnipePM HowleyDE GriffinRA
LambMA Martin. Fields Virology. fourth ed. Philadelphia: Lippincott
Williams & Wilkins. pp. 917-962.
5. Aguilar MJ (1970) Pathological changes in brain and other target
organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus. Infect Immun 2:
533-542. PubMed: 16557874.
6. Zlotnik I, Peacock S, Grant DP, Batter-Hatton D (1972) The
pathogenesis of western equine encephalitis virus (WEE) in adult
hamsters with special reference to the long and short term effects on
the CNS of the attenuated clone 15 variant. Br J Exp Pathol 53: 59-77.
PubMed: 5014245.
7. Liu C, Voth DW, Rodina P, Shauf LR, Gonzalez G (1970) A
comparative study of the pathogenesis of western equine and eastern
equine encephalomyelitis viral infections in mice by intracerebral and
subcutaneous inoculations. J Infect Dis 122: 53-63. doi:10.1093/infdis/
122.1-2.53. PubMed: 4914943.
8. Steele KE, Twenhafel NA (2010) REVIEW PAPER: pathology of animal
models of alphavirus encephalitis. Vet Pathol 47: 790-805. doi:
10.1177/0300985810372508. PubMed: 20551475.
9. Deresiewicz RL, Thaler SJ, Hsu L, Zamani AA (1997) Clinical and
neuroradiographic manifestations of eastern equine encephalitis. N
Engl J Med 336: 1867-1874. doi:10.1056/NEJM199706263362604.
PubMed: 9197215.
10. Earnest MP, Goolishian HA, Calverley JR, Hayes RO, Hill HR (1971)
Neurologic, intellectual, and psychologic sequelae following western
encephalitis. A follow-up study of 35 cases. Neurology 21: 969-974.
doi:10.1212/WNL.21.9.969. PubMed: 5106260.
11. Wolfe DN, Florence W, Bryant P (2013) Current biodefense vaccine
programs and challenges. Hum Vaccin Immunother 9: 1-7. doi:
10.4161/hv.23606. PubMed: 23442580.
PLOS ONE | www.plosone.org
17
December 2013 | Volume 8 | Issue 12 | e82318
Marine Microbe-Derived Antivirals
24. Newman DJ, Cragg GM (2012) Natural products as sources of new
drugs over the 30 years from 1981 to 2010. J Nat Prod 75: 311-335.
doi:10.1021/np200906s. PubMed: 22316239.
25. Walsh CT (2004) Polyketide and nonribosomal peptide antibiotics:
modularity and versatility. Science 303: 1805-1810. doi:10.1126/
science.1094318. PubMed: 15031493.
26. Bhatnagar I, Kim SK (2012) Pharmacologically prospective antibiotic
agents and their sources: a marine microbial perspective. Environ
Toxicol Pharmacol 34: 631-643. doi:10.1016/j.etap.2012.08.016.
PubMed: 23121870.
27. Gerwick WH, Fenner AM (2013) Drug discovery from marine microbes.
Microb Ecol 65: 800-806. doi:10.1007/s00248-012-0169-9. PubMed:
23274881.
28. Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W et al. (2010) The
odyssey of marine pharmaceuticals: a current pipeline perspective.
Trends Pharmacol Sci 31: 255-265. doi:10.1016/j.tips.2010.02.005.
PubMed: 20363514.
29. Manivasagan P, Venkatesan J, Sivakumar K, Kim SK (2013) Marine
actinobacterial metabolites: Current status and future perspectives.
Microbiol Res 168: 311-332. doi:10.1016/j.micres.2013.02.002.
PubMed: 23480961.
30. Magarvey NA, Keller JM, Bernan V, Dworkin M, Sherman DH (2004)
Isolation and characterization of novel marine-derived actinomycete
taxa rich in bioactive metabolites. Appl Environ Microbiol 70:
7520-7529.
doi:10.1128/AEM.70.12.7520-7529.2004.
PubMed:
15574955.
31. Malinoski F, Stollar V (1981) Inhibitors of IMP dehydrogenase prevent
Sindbis virus replication and reduce GTP levels in Aedes albopictus
cells. Virology 110: 281-289. doi:10.1016/0042-6822(81)90060-X.
PubMed: 6111860.
32. Dushee BR, Leben C, Keitt CW, Strong FM (1949) The isolation and
properties of antimycin A. J Am Chem Soc 71: 2436-2437. doi:10.1021/
ja01175a057.
33. Hosotani N, Kumagai K, Nakagawa H, Shimatani T, Saji I (2005)
Antimycins A10~A16, seven new antimycin antibiotics produced by
Streptomyces spp. SPA-10191 and SPA-8893. J Antibiot (Tokyo) 58:
460-467.
34. Xu LY, Quan XS, Wang C, Sheng HF, Zhou GX et al. (2011)
Antimycins A(19) and A(20), two new antimycins produced by marine
actinomycete Streptomyces antibioticus H74-18. J Antibiot (Tokyo) 64:
661-665. doi:10.1038/ja.2011.65. PubMed: 21847131.
35. Seipke RF, Barke J, Brearley C, Hill L, Yu DW et al. (2011) A single
Streptomyces symbiont makes multiple antifungals to support the
fungus farming ant Acromyrmex octospinosus. PLOS ONE 6: e22028.
doi:10.1371/journal.pone.0022028. PubMed: 21857911.
36. Karuppannan AK, Wu KX, Qiang J, Chu JJ, Kwang J (2012) Natural
compounds inhibiting the replication of porcine reproductive and
respiratory syndrome virus. Antiviral Res 94: 188-194. doi:10.1016/
j.antiviral.2012.03.008. PubMed: 22487208.
37. Shum D, Smith JL, Hirsch AJ, Bhinder B, Radu C et al. (2010) Highcontent assay to identify inhibitors of dengue virus infection. Assay
Drug Dev Technol 8: 553-570. doi:10.1089/adt.2010.0321. PubMed:
20973722.
38. Hui EK, Nayak DP (2001) Role of ATP in influenza virus budding.
Virology 290: 329-341. doi:10.1006/viro.2001.1181. PubMed:
11883197.
39. Trumpower BL (1990) The protonmotive Q cycle. Energy transduction
by coupling of proton translocation to electron transfer by the
cytochrome bc1 complex. J Biol Chem 265: 11409-11412. PubMed:
2164001.
40. Lennon RE (1973) Antimycin A, a piscicidal antibiotic. Adv Appl
Microbiol 16: 55-96. doi:10.1016/S0065-2164(08)70023-6. PubMed:
4584680.
41. Bianchi TI, Avilés G, Sabattini MS (1997) Biological characteristics of
an enzootic subtype of western equine encephalomyelitis virus from
Argentina. Acta Virol 41: 13-20. PubMed: 9199709.
42. Tzung SP, Kim KM, Basañez G, Giedt CD, Simon J et al. (2001)
Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3.
Nat Cell Biol 3: 183-191. doi:10.1038/35055095. PubMed: 11175751.
43. Wang H, Li M, Rhie JK, Hockenbery DM, Covey JM et al. (2005)
Preclinical pharmacology of 2-methoxyantimycin A compounds as
novel antitumor agents. Cancer Chemother Pharmacol 56: 291-298.
doi:10.1007/s00280-004-0978-8. PubMed: 15883820.
44. Tokutake N, Miyoshi H, Satoh T, Hatano T, Iwamura H (1994)
Structural factors of antimycin A molecule required for inhibitory action.
Biochim
Biophys
Acta
1185:
271-278.
doi:
10.1016/0005-2728(94)90241-0. PubMed: 8180232.
45. Miyoshi H, Tokutake N, Imaeda Y, Akagi T, Iwamura H (1995) A model
of antimycin A binding based on structure-activity studies of synthetic
PLOS ONE | www.plosone.org
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
18
antimycin A analogues. Biochim Biophys Acta 1229: 149-154. doi:
10.1016/0005-2728(94)00185-8. PubMed: 7727495.
Park WH, Han YW, Kim SH, Kim SZ (2007) An ROS generator,
antimycin A, inhibits the growth of HeLa cells via apoptosis. J Cell
Biochem 102: 98-109. doi:10.1002/jcb.21280. PubMed: 17372917.
Chou TC (2006) Theoretical basis, experimental design, and
computerized simulation of synergism and antagonism in drug
combination studies. Pharmacol Rev 58: 621-681. doi:10.1124/pr.
58.3.10. PubMed: 16968952.
Murphy MP (2009) How mitochondria produce reactive oxygen species.
Biochem J 417: 1-13. doi:10.1042/BJ20082215. PubMed: 19061483.
Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE (2009)
Mitochondria and reactive oxygen species. Free Radic Biol Med 47:
333-343.
doi:10.1016/j.freeradbiomed.2009.05.004.
PubMed:
19427899.
Brennan JP, Southworth R, Medina RA, Davidson SM, Duchen MR et
al. (2006) Mitochondrial uncoupling, with low concentration FCCP,
induces ROS-dependent cardioprotection independent of KATP
channel activation. Cardiovasc Res 72: 313-321. doi:10.1016/
j.cardiores.2006.07.019. PubMed: 16950237.
Tahara EB, Navarete FD, Kowaltowski AJ (2009) Tissue-, substrate-,
and site-specific characteristics of mitochondrial reactive oxygen
species generation. Free Radic Biol Med 46: 1283-1297. doi:10.1016/
j.freeradbiomed.2009.02.008. PubMed: 19245829.
Hoffmann HH, Kunz A, Simon VA, Palese P, Shaw ML (2011) Broadspectrum antiviral that interferes with de novo pyrimidine biosynthesis.
Proc Natl Acad Sci U S A 108: 5777-5782. doi:10.1073/pnas.
1101143108. PubMed: 21436031.
Wang QY, Bushell S, Qing M, Xu HY, Bonavia A et al. (2011) Inhibition
of dengue virus through suppression of host pyrimidine biosynthesis. J
Virol 85: 6548-6556. doi:10.1128/JVI.02510-10. PubMed: 21507975.
Leyssen P, Balzarini J, De Clercq E, Neyts J (2005) The predominant
mechanism by which ribavirin exerts its antiviral activity in vitro against
flaviviruses and paramyxoviruses is mediated by inhibition of IMP
dehydrogenase.
J
Virol
79:
1943-1947.
doi:10.1128/JVI.
79.3.1943-1947.2005. PubMed: 15650220.
Löffler M, Jöckel J, Schuster G, Becker C (1997) Dihydroorotateubiquinone oxidoreductase links mitochondria in the biosynthesis of
pyrimidine nucleotides. Mol Cell Biochem 174: 125-129. doi:10.1023/A:
1006859115450. PubMed: 9309676.
Schoepp RJ, Smith JF, Parker MD (2002) Recombinant chimeric
western and eastern equine encephalitis viruses as potential vaccine
candidates. Virology 302: 299-309. doi:10.1006/viro.2002.1677.
PubMed: 12441074.
Herr F, Greselin E, Chappel C (1967) Toxicology studies of antimycin,
a fish eradicant. Trans Am Fish Soc 96: 320-326. Available online at:
doi:10.1577/1548-8659(1967)96[320:TSOAAF]2.0.CO;2
Hamilton PB, Carroll FI, Rutledge JH, Mason JE, Harris BS et al.
(1969) Simple isolation of antimycin A1 and some of its toxicological
properties. Appl Microbiol 17: 102-105. PubMed: 5774751.
Mayer AM, Rodríguez AD, Berlinck RG, Hamann MT (2007) Marine
pharmacology in 2003-4: marine compounds with anthelmintic
antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial,
antiplatelet, antiprotozoal, antituberculosis, and antiviral activities;
affecting the cardiovascular, immune and nervous systems, and other
miscellaneous mechanisms of action. Comp Biochem Physiol C Toxicol
Pharmacol 145: 553-581. doi:10.1016/j.cbpc.2007.01.015. PubMed:
17392033.
Mayer AM, Rodríguez AD, Berlinck RG, Hamann MT (2009) Marine
pharmacology in 2005-6: Marine compounds with anthelmintic,
antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial,
antiprotozoal, antituberculosis, and antiviral activities; affecting the
cardiovascular, immune and nervous systems, and other miscellaneous
mechanisms of action. Biochim Biophys Acta 1790: 283-308. doi:
10.1016/j.bbagen.2009.03.011. PubMed: 19303911.
Mayer AM, Rodríguez AD, Berlinck RG, Fusetani N (2011) Marine
pharmacology in 2007-8: Marine compounds with antibacterial,
anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal,
antituberculosis, and antiviral activities; affecting the immune and
nervous system, and other miscellaneous mechanisms of action. Comp
Biochem Physiol C Toxicol Pharmacol 153: 191-222. doi:10.1016/
j.cbpc.2010.08.008. PubMed: 20826228.
Sabini MC, Escobar FM, Tonn CE, Zanon SM, Contigiani MS et al.
(2012) Evaluation of antiviral activity of aqueous extracts from
Achyrocline satureioides against western equine encephalitis virus. Nat
Prod Res 26: 405-415. doi:10.1080/14786419.2010.490216. PubMed:
20623427.
December 2013 | Volume 8 | Issue 12 | e82318
Marine Microbe-Derived Antivirals
63. Lin K, Gallay P (2013) Curing a viral infection by targeting the host: The
example of cyclophilin inhibitors. Antiviral Res 99: 68-77. doi:10.1016/
j.antiviral.2013.03.020. PubMed: 23578729.
64. Swinney DC, Anthony J (2011) How were new medicines discovered?
Nat Rev Drug Discov 10: 507-519. doi:10.1038/nrd3480. PubMed:
21701501.
65. Ma X, Jin M, Cai Y, Xia H, Long K et al. (2011) Mitochondrial electron
transport chain complex III is required for antimycin A to inhibit
autophagy. Chem. Biol 18: 1474-1481.
66. Dreux M, Chisari FV (2010) Viruses and the autophagy machinery. Cell
Cycle 9: 1295-1307. PubMed: 20305376.
67. Ando T, Imamura H, Suzuki R, Aizaki H, Watanabe T et al. (2012)
Visualization and measurement of ATP levels in living cells replicating
hepatitis C virus genome. RNA - PLOS Pathog 8: e1002561.
68. Rodrigues T, Lopes F, Moreira R (2010) Inhibitors of the mitochondrial
electron transport chain and de novo pyrimidine biosynthesis as
antimalarials: The present status. Curr Med Chem 17: 929-956. doi:
10.2174/092986710790820660. PubMed: 20156168.
69. Rohlena J, Dong LF, Ralph SJ, Neuzil J (2011) Anticancer drugs
targeting the mitochondrial electron transport chain. Antioxid Redox
Signal 15: 2951-2974. doi:10.1089/ars.2011.3990. PubMed: 21777145.
70. Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN et al.
(1985) 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that
inhibits the infectivity and cytopathic effect of human T-lymphotropic
virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad
Sci U S A 82: 7096-7100. doi:10.1073/pnas.82.20.7096. PubMed:
2413459.
PLOS ONE | www.plosone.org
71. Horwitz JP, Chua J, Noel M (1964) Nucleosides. V. The
monomesylates of 1-(2'-deoxy-β-D-lyxofuranosyl) thymine. J Org Chem
29: 2076-2078. doi:10.1021/jo01030a546.
72. Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS et al. (2013)
Ribosomally synthesized and post-translationally modified peptide
natural products: overview and recommendations for a universal
nomenclature. Nat Prod Rep 30: 108-160. doi:10.1039/c2np20085f.
PubMed: 23165928.
73. Castorena KM, Peltier DC, Peng W, Miller DJ (2008) Maturationdependent responses of human neuronal cells to western equine
encephalitis virus infection and type I interferons. Virology 372:
208-220. doi:10.1016/j.virol.2007.10.025. PubMed: 18022665.
74. Peltier DC, Simms A, Farmer JR, Miller DJ (2010) Human neuronal
cells possess functional cytoplasmic and TLR-mediated innate immune
pathways influenced by phosphatidylinositol-3 kinase signaling. J
Immunol 184: 7010-7021. doi:10.4049/jimmunol.0904133. PubMed:
20483728.
75. Peltier DC, Lazear HM, Farmer JR, Diamond MS, Miller DJ (2013)
Neurotropic arboviruses induce interferon regulatory factor 3-mediated
neuronal responses that are cytoprotective, interferon independent, and
inhibited by western equine encephalitis virus capsid. J Virol 87:
1821-1833. doi:10.1128/JVI.02858-12. PubMed: 23192868.
76. Ikeda M, Abe K, Dansako H, Nakamura T, Naka K et al. (2005) Efficient
replication of a full-length hepatitis C virus genome, strain O, in cell
culture, and development of a luciferase reporter system. Biochem
Biophys Res Commun 329: 1350-1359. doi:10.1016/j.bbrc.
2005.02.138. PubMed: 15766575.
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