A Chemical Genetic Screen for Modulators of
Asymmetrical 2,29-Dimeric Naphthoquinones
Cytotoxicity in Yeast
Ashkan Emadi1, Ashley E. Ross2, Kathleen M. Cowan3, Yolanda M. Fortenberry4, Milena Vuica-Ross3*
1Department of Internal Medicine and Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 2Department of Urology, Johns
Hopkins School of Medicine, Baltimore, Maryland, United States of America, 3Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, United
States of America, 4Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America
Background: Dimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have
shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-
neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of
binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for
enhanced or suppressed growth in the presence of binaphthoquinones.
Methodology/Principal findings: Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast
growth with IC50s in the mM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid
yeast deletion mutant library to BiQs at concentrations near their IC50. Sensitivity screens identified yeast with deletions
affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild
type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs
was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS).
Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be
further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external
NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS
production and increased sensitivity of wild type yeast to BiQ.
Conclusions/Significance: In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine
oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this
mechanism in mammalian cells.
Citation: Emadi A, Ross AE, Cowan KM, Fortenberry YM, Vuica-Ross M (2010) A Chemical Genetic Screen for Modulators of Asymmetrical 2,29-Dimeric
Naphthoquinones Cytotoxicity in Yeast. PLoS ONE 5(5): e10846. doi:10.1371/journal.pone.0010846
Editor: Xuewen Pan, Baylor College of Medicine, United States of America
Received February 5, 2010; Accepted April 26, 2010; Published May 26, 2010
Copyright: ? 2010 Emadi 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 supported by Richard Starr Ross Clinician Scientist Award (to MVR) and the JHH Department of Pathology funds (to MVR). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Ashkan Emadi owns a patent on the synthesis of the drugs studied (Inventors: Stagliano; Kenneth William, Emadi; Ashkan, Assignee:
Illinois Institute of Technology, Chicago, IL). Anti-Retroviral Multi-Quinone Compounds and Regiospecific Synthesis Thereof. United States Patent and Trademark
Office; Filed November 6, 2002; Application number: 10/288,685; U.S. Field of Search: 549/297,296,293 552/389,390,391 514/454,685; International Class: C07D
307/77; A61K 031/122; A61K 031/35; C07C 050/04. The United States Letters Patent Number 6,828,347 was issued on December 07, 2004 on behalf of Illinois
Institute of Technology. Authors declare that this patent has not generated any financial benefit. All authors are currently employed by the Johns Hopkins Medical
Institute. This patent does not affect the authors’ adherence to PLoS ONE policy on sharing data and materials. The authors agree to make freely available any
materials and information described in this publication that are reasonably requested by others for the purpose of academic, non-commercial research.
* E-mail: email@example.com
Multimeric naphthoquinones are unique molecules, which
possess a diverse array of biologic activities including antineoplas-
tic, antiprotozoal and antiviral effects . Their structures are
based on two or more naphthoquinone units linked together in
different positions. In nature, their synthesis likely involves
oxidative coupling of a common naphthol intermediate in the
process of oligomerization .
One member of this class, conocurvone, was first isolated from
the Western Australian smoke bush and has been shown to inhibit
the cytopathogenic effects of HIV-1 in human T lymphoblasts .
In an effort to synthesize anti-retrovirals, Emadi et al. previously
reported the regiocontrolled synthesis of symmetrical and
asymmetrical dimeric and trimeric naphthoquinones by using a
novel method , . Several of the dimeric naphthoquinones
(binaphthoquinones) inhibited HIV-1 integrase with ID50(con-
centration of drug required to inhibit HIV-1 mediated cytopath-
ogenicity in infected cells by 50%) ranging from 1 to 3.5
micromolar . In addition, potent activity of binapthoquinones
against non-HIV infected CEM-T4lymphoblastic leukemia cells
was observed with TD50(reduction of non-infected cell growth by
50%) ranging from 5 to 8 micromolar, suggesting the presence of
other cytotoxic mechanisms for these compounds .
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The mechanism of binaphthoquinones cytotoxicity is currently
unknown however it may be in part related to the presence of
quinone moieties. Previously, quinone cytotoxicity has been
attributed to a wide array of cellular effects including DNA and
protein modification , , , topoisomerase inhibition ,
, caspase activation , oxidative stress ,  and
endoplasmic reticulum stress . Elucidation of the cellular
components necessary to protect cells or sensitize them to
binaphthoquinones might allow for the enhanced use of these
drugs as antiretroviral, antiparasitic or antineoplastic agents. To
this end, we carried out genome wide screens for binaphthoqui-
none sensitivity and resistance in yeast. Yeast Saccharamyces cerevisiae
share conserved sequences with known and predicted human
proteins and provide a powerful model organism in which high
throughput genetic screens can be performed , , ,
. We utilized several different binaphthoquinone analogues
and developed a genetic model of their cytotoxicity by performing
a systematic high-throughput screen of a yeast isogenic deletion
mutant array for drug enhanced or suppressed growth. We then
confirmed the validity of the suggested targets genetically.
Binaphthoquinones inhibit yeast growth
To establish whether yeast could be used as a model system for
binaphthoquinone cytotoxicity, we first aimed to determine the
ability of binaphthoquinones to suppress yeast growth. We selected
three different binaphthoquinones to reflect their diverse chemical
properties (Fig. 1A). Binaphthoquinone 7 (BiQ7) possesses two
chlorine (Cl) atoms on the quinone cores and a hydroxyl group at
position 5 on one of the aromatic rings . Binaphthoquinone 3
(BiQ3) is an iodo-hydroxy-binaphthoquinone with a remote
methoxyl group on the aromatic ring . Binaphthoquinone 11
(BiQ11) is a pyranylated chloro-hydroxy-binaphthoquinone .
All tested binaphthoquinones inhibited growth of wild type yeast
(strain BY4741) in liquid cultures in a concentration-dependent
manner (Fig. 1B). As the purpose of our yeast screen was to
identify drug sensitive strains, we determined the concentrations at
which approximately 50% of yeast growth is suppressed (IC50) in
the wild type strain. Our analysis showed that wild type yeast is
particularly sensitive to BiQ7 with an IC50of 0.960.2 mM while
other tested binaphthoquinones showed IC50 in the 762 mM
range (Fig. 1B).
Genome-wide growth suppression screening reveals
important roles for mitochondria and cellular respiration
in the action of binaphthoquinones
For an initial high-throughput phenotypic screen, a set of
approximately 5000 commercially available S. cerevisiae non-
essential isogenic deletion strains were arrayed onto 96 well plates
containing either DMSO (no drug control) or a sub-lethal
binaphthoquinone concentration at its IC50 . Plates were
incubated at 23uC and strain growth on DMSO- and binaphtho-
quinone-treated plates were compared over a period of 24h. The
screen was carried out in a duplicate with two different
binaphthoquinones (BiQ7 and BiQ3). As expected, wild type
yeast in these assays demonstrated a relative growth of 0.6+0.1.
Median mutant relative growth was slightly more right-shifted,
likely representing conditions in the assay to favor robust growth.
Accordingly, a cut off for decreased growth of at least 3 standard
deviations below the mean in both screens was chosen to identify
binaphthoquinone sensitive strains for further analysis (Supple-
mental Fig. S1, Supplementary Table S1). As a control we also
tested a drug previously analyzed in similar assays, 6-azauracil (6-
AU), and obtained a set of sensitive strains similar to those
previously reported (data not shown) . While the lists of
mutants hypersensitive to BiQ7 and BiQ3 contained many
overlapping genes (76 out of 128 genes) (Supplementary Table
S1), 6-AU sensitive strains had very little overlap with either
binaphthoquinone group (14 out of 128 genes) (Supplemental Fig.
S1). The most drug sensitive haploid strains to both binaphtho-
quinones are listed in Table 1.
The strains hypersensitive to binaphthoquinones in both screens
were further analyzed for distribution of all (Figures 2A, 2C and
2E) gene ontology (GO) categories among shared mutants .
To determine significantly enriched GO categories among
hypersensitive mutants, an analysis by GO::TermFinder was
carried out. This analysis strongly suggested that strains with
defects in mitochondria (p,0.0001), cellular respiration (p,0.01)
and ubiquinone biosynthetic process (p,0.05) are particularly
sensitive to binaphthoquinones. To rank these categories, we
further determined the enrichment factors (fold increase over
random enrichment) of the significant GO categories uncovered
by GO::TermFinder (Fig. 2B and 2D) . Again mitochondrial
mutants and cellular respiration mutants appear significantly
enriched (Fig. 2B and 2D). Interestingly, no functional category
Figure 1. Binaphthoquinones suppress growth of Saccharomyces cerevisiae. (A) Chemical structures of dimeric naphthoquinones. (B)
Determination of the IC50of binaphthoquinones in yeast. Log-phase cultures at an OD600of 0.1 were treated with different concentrations of
binaphthoquinones or DMSO over 24 h. Relative growth of wild-type (BY4741) strain in the presence of three of the binaphthoquinones was
measured against that of the yeast grown in the presence of DMSO. Growth curves were performed in triplicate and represent the average of three
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was significantly enriched with the closest group being oxidore-
ductases (enrichment factor of 2.34) (p=0.069).
To confirm the sensitivity of the enriched strains, we
individually measured the sensitivity of selected strains to
binaphthoquinones by spot assays and relative growth (Fig. 3).
Spot assays of tested deletion strains demonstrated increased
sensitivity of these strains to BiQs compared to wild-type yeast
(Fig. 3A). Furthermore, all tested deletion strains were more
sensitive to BiQs than the wild-type control over a range of
different concentrations, and this selectivity was lost at higher
concentrations of the drug (Fig. 3B and 3C).
Binaphthoquinones inhibit yeast growth in
nonfermentable media and depolarize the mitochondrial
Yeast are able to grow either anaerobically or aerobically by
utilizing fermentable or nonfermentable carbon sources, respec-
tively. In the presence of a fermentable carbon source, such as
glucose (dextrose), yeast preferentially adopt glycolysis to generate
energy even under aerobic conditions and can grow normally even
when mitochondrial respiration level is minimal . In order to
further determine whether binaphthoquinone toxicity occurs via
interference with mitochondrial function, we compared yeast
growth in the presence or absence of BiQ7 in a dextrose
containing media or in media where glucose was replaced with
glycerol, a nonfermentable carbon source. Yeast growth in
glycerol-containing media was severely inhibited by BiQ7
indicating that binaphthoquinones affect mitochondrial function
Enhanced sensitivity of yeast mitochondrial mutants to
binaphthoquinones as well as the observation of severe growth
inhibition in nonfermentable media in the presence of BiQ7
encouraged us to examine whether the yeast mitochondrial
membrane potential (critical to mitochondrial functions such as
oxidative phosphorylation, lipid and pyrimidine synthesis) is
binaphthoquinones led to extensive loss of membrane potential,
as evidenced by the decreased rhodamine 123 uptake into the
mitochondria (Fig. 4B). This effect was observed with yeast grown
in either glycerol or dextrose-containing media, but the degree of
membrane potential loss was greater in the glycerol-containing
media (data not shown).
bybinaphthoquinones.Treatment ofyeast with
Binaphthoquinones generate reactive oxygen species
Monoquinones are believed to generate superoxide radicals and
oxidative stress by undergoing a futile redox cycle in which
carbonyl groups are reduced by reductases to a semiquinone (Q.2)
radical and subsequently to a hydroquinone (QH2), which then
rapidly undergoes a two-step oxidation back to the parent
compound. This redox cycling and oxygen activation, in theory,
results in cytotoxic levels of ROS .
To further assess production of ROS, wild type yeast were
exposed to 0, 1, or 5 mM of binaphthoquinones followed by
incubation with dihydrorhodamine 123 (DHR 123) for 1 h.
Oxidation of DHR 123 to rhodamine 123 produces a
fluorescent signal that can be measured. Treatment of yeast
with BiQ7 or BiQ3 resulted in the oxidation of DHR 123 in a
concentration-dependent manner (Fig. 5A). We additionally
tested one of the hypersensitive strains, coq5D (a para-
benzoquinone methyltransferase, involved in ubiquinone bio-
synthesis and localized on the matrix face of the mitochondrial
inner membrane), for ROS production. ROS production was
increased in this mutant strain compared to wild type both in
the presence and absence of BiQ7 (Fig. 5B). Previously, several
groups had demonstrated that cells lacking mitochondrial DNA
(rho0) were incapable of generating reactive oxygen species via
redox mechanisms , . We hypothesized that if
BiQ toxicity was mediated through the generation of ROS,
rho0strains would demonstrate resistance. Accordingly, incu-
bation of rho0yeast with BiQ7 over a range of concentrations
Table 1. The most sensitive yeast strains to BiQ7 and BiQ3.
BiQ7* BiQ3*ORF NameBiological Process
0.002 0.002YML129C COX14 Mitochondrial membrane protein, involved in translational regulation of Cox1p and assembly of cytochrome
c oxidase (complex IV)
0.0650.038 YPL029W SUV3ATP-dependent RNA helicase, component of the mitochondrial degradosome along with the RNase Dss1p
0.0930.083 YOR285W- Protein of unknown function, localized to the mitochondrial outer membrane
0.074 0.039YOR194C TOA1TFIIA large subunit; involved in transcriptional activation, acts as antirepressor or as coactivator
0.0200.029YJL166W QCR8 Subunit 8 of ubiquinol cytochrome-c reductase complex, which is a component of the mitochondrial inner
membrane electron transport chain
0.064 0.075 YDR487CRIB3 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), required for riboflavin biosynthesis from
ribulose-5-phosphate, also has an unrelated function in mitochondrial respiration
0.0320.063YMR125WGCR3 Large subunit of the nuclear mRNA cap-binding protein complex; also involved in nuclear mRNA degradation
and telomere maintenance
0.0420.074YBR003WCOQ1 Hexaprenyl pyrophosphate synthetase, catalyzes the first step in ubiquinone (coenzyme Q) biosynthesis
0.005 0.054YML110CCOQ5 2-hexaprenyl-6-methoxy-1,4-benzoquinone methyltransferase, involved in ubiquinone (Coenzyme Q)
biosynthesis; localizes to the matrix face of the mitochondrial inner membrane in a large complex with other
ubiquinone biosynthetic enzymes
0.0360.047 YGR255CCOQ6 Putative flavin-dependent monooxygenase, involved in ubiquinone (Coenzyme Q) biosynthesis; localizes to
the matrix face of the mitochondrial inner membrane in a large complex with other ubiquinone biosynthetic
0.0550.073 YER164WCHD1Nucleosome remodeling factor that functions in regulation of transcription elongation; contains a chromo
domain, a helicase domain and a DNA-binding domain; component of both the SAGA and SLIK complexes
*Average relative growth.
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showed greatly abrogated effects of the drug in this strain
We additionally tested whether the addition of the antioxidant,
N-acetyl cysteine (NAC) could neutralize the effects of BiQs. Co-
incubation of yeast with NAC resulted in decreased ROS
formation and increased relative growth of multiple BiQ sensitive
strains (Supplemental Fig. S2). While NAC may serve to diminish
binaphthoquinone cytotoxicity by neutralizing ROS through
increased glutathione production, it also could exert its actions
by interacting directly with binaphthoquinones or their derivatives
to reduce their cytotoxicity , . To test for whether direct
interaction between NAC and BiQs occurs, we performed mass
spectrometry on BiQ7 (MW 398 Da) and NAC (MW 163 Da)
both separately and after co-incubation (predicted reacted
products at 526 Da or 561 Da). A product at 526 Da was present
in the coincubated sample which could be fragmented into
components of 361 and 162 Da, suggesting that NAC can interact
directly with binaphthoquinones (data not shown).
NAD(P)H:quinone oxidoreductase type II, NDE1, plays an
important role in the action of binaphthoquinones
Our initial screens to define BiQ sensitive mutants were
designed to allow for robust yeast growth and thus be more likely
to identify drug sensitive strains. While this method was effective
for defining mutants with decreased survival, it proved limited in
the identification of BiQ resistant mutants. For example, while the
same drug sensitive strains were reproducibly found in repeat
experiments of this screen (Fig 2), strains suggested to be resistant
by this assay (3 SD above the mean) were not reproducibly
consistent. This may be due to the high ODs registered by BiQ
resistant mutants which are out of the linear range for absorbance.
Accordingly, to screen for yeast mutants resistant to the
binaphthoquinones we exposed the yeast deletion library grown
in glycerol-containing agar plates to 3 mM of BiQ7, or 7 mM of
BiQ11, 7 mM of BiQ3 or DMSO (Supplemental Table S2).
Glycerol plates were used as wild-type yeast growth on glycerol
plates is severely inhibited by exposure to BiQs thus reducing
Figure 2. Enhanced sensitivity of mitochondrial and cellular respiration mutants to binaphthoquinones. (A) Distribution of all GO
Component categories among strains hypersensitive to binaphthoquinones. (B) Enrichment factor comparison of significantly enriched cellular
component GO categories of deletion strains hypersensitive to binaphthoquinones showing a significant enrichment of mitochondrial mutants. (C)
Distribution of GO Cellular Process categories among strains hypersensitive to binaphthoquinones. (D) Enrichment factor comparison of significantly
enriched biological processes of deletion strains hypersensitive to binaphthoquinones demonstrating a significant enrichment of cellular respiration
mutants. (E) Distribution of GO Function categories among strains hypersensitive to binaphthoquinones.
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background. Only the deletion mutant nde1D, lacking a mito-
chondrial NADH dehydrogenase, showed resistance to all tested
binaphthoquinone molecules (Supplemental Table S2, Fig. 6A
and Supplemental Fig. S3). As NDE1 contains sequence
similarities to NDE2 (another mitochondrial NADH dehydroge-
nase) , , we proceeded with individual testing of mutants
lacking these genes either separately or in combination over a wide
concentration range of all three binaphthoquinones (Supplemental
Fig. S3) . The nde1D and nde1D nde2D strains showed enhanced
resistance to BiQs (Figs. 6A and Supplemental Fig. S3). In
contrast, the nde2D mutant showed no significant resistance to the
binaphthoquinones (Figs. 6A and S3). This may be explained by
the fact that at the cytosolic side of the inner mitochondrial
membrane Nde1p is the major NADH-dehydrogenase .
Interestingly, deletion of main inner membrane NADH dehydro-
genase NDI1 showed increased sensitivity to binaphthoquinones
(Supplemental Fig. S3). We therefore concluded that the presence
of some NADH dehydrogenases might enhance binaphthoqui-
none toxicity. Indeed, overexpression of NDE1 under a GPD
(glyceraldehyde 3-phosphate dehydrogenase) promoter  in the
parental BY4741 background (wild type) resulted in increased
sensitivity to binaphthoquinones (Fig. 6A, Supplemental Fig. S3D–
F). Similarly the nde1D nde2D deletion strain had diminished ROS
production as compared to wild type strain in cells treated with
5 mM of BiQ7 (Fig. 6B), while overexpression of NDE1 augmented
ROS production in the wild type strain as compared to vector
alone control when treated with 1 mM of BiQ7 (Fig. 6C).
We next tested whether the cytotoxicity of several well known
quinone-containing drugs including doxorubicin, mitomycin C
and menadione, was partially dependent on NDE1. These drugs
showed sensitivity and resistance patterns similar to that of
binaphthoquinones (Fig. 7). In contrast to binaphthoquinones,
however, the ndi1D strain was resistant to these drugs (Fig. 7A–C).
In this report, we utilized a yeast model to decipher the
cytotoxic action of binaphthoquinones. We showed that bi-
Figure 3. Validation of binaphthoquinone sensitivity of deletion strains identified in the high throughput screen. (A) Spot assays of
five binapthoquinone sensitive deletion strains. Deletion strains were spotted in five fold serial dilutions on SC-ura plates containing DMSO or BiQ7,
BiQ11 or BiQ3 at their IC50concentrations. Spot assays were performed in duplicate in two separate experiments with representative plates shown.
(B,C) BiQ7 (B) or BiQ3 (C) sensitivity of several deletion strains identified in the high-throughput screen. Log-phase cultures at an OD600of 0.1 were
treated with different concentrations of binaphthoquinones or DMSO over 24h. Relative growth of wild-type and deletion strains in the presence of
BiQ7 (B) or BiQ3 (C) was measured against that of the yeast grown in the presence of DMSO. Growth curves were performed in triplicate and
represent the average of three experiments. *=p,0.05 for a difference in the mean compared to wild type (growth compared to wild type was
significantly impaired in all deletion mutants tested at 0.3, 0.6 and 1.2 mM of BiQ7 and 1.25, 2.5 and 5 mM of BiQ3).
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naphthoquinones are capable of depolarizing the mitochondrial
membrane and that interference with cellular respiration increases
the sensitivity of the cells to binaphthoquinones. Furthermore, we
demonstrated that binaphthoquinones generate ROS and that the
toxic effects of many binaphthoquinones is dependent on
NAD(P)H:quinone oxidoreductase type II, NDE1.
Figure 5. Binaphthoquinones generate reactive oxygen species in yeast. (A) Binaphthoquinones generate ROS in a concentration
dependent manner. Yeast wild type strain was treated with DMSO, BiQ7 or BiQ3 at increasing concentrations for 2h and incubated with DHR 123.
Experiments were performed three times with similar results. (B). Enhanced generation of ROS in the BiQ hypersensitive mutant coq5D in the
presence and absence of binaphthoquinone. Yeast wild type and coq5D cells were treated with 1 mM BiQ7 or DMSO for 2h and incubated with DHR
123. The experiment was performed three times with similar results. (C) Mitochondrial deficient yeast, rho0, are resistant to binapthoquinones. Log-
phase cultures of wild-type or rho0yeast at an OD600of 0.1 were treated with DMSO or different concentrations of BiQ7 for 24 (wild type) and 48
(rho0) h. Growth curves were performed in triplicate and represent the average of three experiments. P,0.05 for differences between wild-type and
rho0yeast at all concentrations of drug.
Figure 4. Binaphthoquinones inhibit yeast respiratory growth by depolarizing the mitochondrial membrane. (A) BiQ7 inhibits yeast
growth in nonfermentable media. Yeast growth is severely repressed in glycerol-containing media in the presence of 1 mM BiQ7 (p,0.001 for
differences between all experimental categories except dextrose with drug and glycerol alone). (B) Binaphthoquinones depolarize mitochondrial
membrane. The wild type yeast was treated at IC50concentrations of binaphthoquinones (1, 5 and 5mM of BiQ7, BiQ11 and BiQ3 respectively) for 2h
and then incubated with rhodamine 123. The peak shift toward the left represents a decrease of fluorescence signal of rhodamine 123 in the yeast,
indicating the loss of mitochondrial membrane potential in the presence of BiQs as compared to no drug (DMSO) control.
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Our screen results underscore the importance of intact cellular
respiration in diminishing the negative effects of binaphthoqui-
nones on cellular growth. Among the most sensitive strains, several
are involved in ubiquinone synthesis. Ubiquinone is a mono-
benzoquinone present in the mitochondria of most eukaryotic cells
as a part of the electron transport chain which participates in
aerobic cellular respiration . Ubiquinone possesses antioxidant
properties and disruption of ubiquinone synthesis leads to
increased ROS production as seen in our binaphthoquinones
sensitive mutants . Disruption of electron flow, such as would
occur in ubiquinone deficient cells, would be expected to result in
a backlog of electrons along the NADH utilizing electron transport
pathway and this likely synergizes with increased ROS production
by biquinones to cause cytotoxicity. Accordingly we observed
substantial increases in ROS production compared to wild type
when the ubiquinone synthesis mutant coq5D was treated with
To determine whether binaphthoquinones affect the mitochon-
drial membrane potential, we studied the ability of BiQs to
depolarize mitochondrial membrane potential in both dextrose
and glycerol-containing media. All tested BiQs were capable of
depolarizing mitochondrial membrane in both media, though the
effect was stronger in nonfermentable media. Attenuation of the
effect of BiQs on depolarizing mitochondrial membrane potential
by dextrose is possibly due to the partial repression of the activity
of respiration enzymes, including NDE1, by glucose .
Interestingly, at IC50 concentrations of binaphthoquinones the
inhibition of respiratory chain is most noticeable in BiQ11. This
might be due to the presence of the pyran ring in this molecule
which increases its lipophilicity.
Figure 6. NDE1 plays a critical role in the action of binaphthoquinones. (A) Dependence of yeast sensitivity to BiQs on NDE1 in glucose and
glycerol containing media. Spot assays of wild-type, nde1nde2D, nde1D, nde2D and wild type yeast overexpressing NDE1 driven by a GPD promoter
(PGPD-NDE1) were spotted in five fold serial dilutions on plates containing glycerol or glucose and either DMSO or BiQ7, BiQ11 or BiQ3 at their IC50
concentrations. Spot assays were performed in duplicate in two separate experiments with representative plates shown. (B) Decreased generation of
ROS in the nde1Dnde2D mutant following exposure to 5 mM BiQ7. (C) Increased generation of ROS in a yeast overexpressing NDE1 (PGPD-NDE1)
following exposure to 1 mM BiQ7. Yeast strains were treated with binaphthoquinones or DMSO for 2h and incubated with DHR 123. The experiment
was performed three times with similar results.
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Furthermore, binaphthoquinones were found to be capable of
inducing oxidative stress in yeast cells by increasing ROS
production. Additionally, binaphthoquinone cytotoxicity was
abolished in mitochondrial DNA deficient yeast strains which
lack critical respiratory chain catalytic subunits and have
diminished ROS production .
Figure 7. The effects of several quinone based drugs on the relative survival of NDE1 mutants. (A,D) doxorubicin, (B,E) mitomycin C, (C,F)
menadione. Log-phase cultures at an OD600of 0.1 were treated with different concentrations of BiQ7 or DMSO over 17 h. Relative growth of wild-
type and mutant strains in the presence of binaphthoquinones was measured against that of the yeast grown in the presence of DMSO. Growth
curves were performed in triplicate and represent the average of three experiments. *P,0.05 for differences between wild-type and NDE1 over-
expressing or mutant yeast.
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A screen for deletion mutants resistant to binaphthoquinones
identified NDE1, a mitochondrial external NADH dehydrogenase
(a type II NAD(P)H:quinone oxidoreductase) that catalyzes the
oxidation of cytosolic NADH. Nde1p and Nde2p function
primarily to provide cytosolic NADH to the mitochondrial
respiratory chain , . Our findings suggest NDE1 is
involved in bioactivation of BiQs. Deletion of NDE1 increased
resistance of yeast to binaphthoquinone action, while overexpres-
sion of NDE1 has an opposite effect. Interestingly, and in contrast
to nde1D, the ndi1D strain demonstrated sensitivity to BiQs and this
was not reproduced in relationship to other quinone containing
drugs (doxorubicin, mitomycin C and menadione). It is possible
that binaphthoquinones, due to their larger size and varied
hydrophilicity are less accessible to the internal side of the
mitochondrion. In addition, NDE1 may play a unique role in the
bioactivation of BiQs. Indeed, another oxidoreductase, LOT6, has
been previously described as having the ability to detoxify
quinones in yeast but in our studies, growth of the lot6D mutant
did not differ significantly from the wild type control when
exposed to a range of binaphthoquinone concentrations (data not
In contrast to the mitochondria of fungi and plants, mammalian
mitochondria do not harbor external NADH dehydrogenases and
instead depend on redox shuttle mechanisms to couple the
oxidation of cytosolic NADH to internal NADH dehydrogenases
. Despite this, the main enzymes involved in quinone
metabolism in human cells belong to the NAD(P)H:quinone
acceptor oxidoreductase (NQO) gene family and, with respect to
binaphthoquinone metabolism, may function similarly to NDE1.
For example, in last two decades, attention has been given to beta-
lapachone, an ortho-naphtoquinone antineoplastic drug, and
other quinone based drugs which take advantage of NQO1
overexpression in human cancers and selectively target them ,
, , . NQO1 reduces beta-lapachone to an unstable
hydroquinone that rapidly undergoes a two-step oxidation back to
fully oxidized parent molecule, perpetuating a futile redox cycle.
Deficiency or inhibition of NQO1 renders cells resistant to beta-
lapachone . Additionally, our preliminary data in human cells
overexpressing NQO1 show increased sensitivity of these cells to
both beta-lapachone and binaphthoquinones (unpublished results).
Based on these findings, we propose that fully oxidized
binaphthoquinones undergo enzymatic reduction by NAD(P)H:-
quinone oxidoreductases to fully reduced form of bi-hydro-
naphthoquinones. Subsequently, through a series of oxidation
steps, hydronaphthoquinones convert to semiquinones and
ultimately back to oxidized binaphthoquinones. These stepwise
oxidations culminates in ROS generation (Fig. 8).
Compared to other binaphthoquinones and quinones tested in
this study, BiQ7 shows the most potent cytotoxic activity. Several
observations might explain this phenomenon. Because BiQ7
possesses a hydroxyl group at the 5-position, it is able to form
an intramolecular hydrogen bond with the carbonyl group’s
oxygen. Interestingly, previous studies of monomeric naphthoqui-
nones with a hydroxyl group at position 5 on the aromatic ring
have also shown an increase in toxicity toward rat hepatocytes
compared to other naphthoquinones . There are several other
potential factors that might have contributed to the higher potency
of BiQ7 and quinone drugs with hydroxyl group at 5-position: 1)
increased efficiency of redox cycling in BiQ7, 2) increased stability
of the semiquinone derived from 5-hydroxy-1,4-naphthoquinone
as compared to 1,4-naphthoquinone, which may lead to a higher
semiquinone concentration and thereby a higher rate of
autoxidation, 3) stabilization of other BiQs after deprotonation
of their core hydroxyl groups, which BiQ7 lacks, and donation of
the electrons from the deprotonized oxygen to the quinone ring to
form tautomers, and 4) better utilization of oxidoreductases ,
In conclusion, here we use high throughput screens in yeast to
elucidate the basic cellular mechanisms mediating binaphthoqui-
none cytotoxicity. We find that treatment with binaphthoquinones
depolarizes mitochondrial membranes and results in the genera-
tion of ROS. Accordingly, binaphthoquinone cytotoxicity can be
abrogated in yeast mitochondrial DNA deficient mutants.
Furthermore, we demonstrate the dependency of binaphthoqui-
none cytotoxicity on NDE1 and the ability to sensitize yeast to
binaphthoquinones by overexpression of this enzyme. These
mechanisms are likely paralleled in mammalian cells and
manipulation of these pathways may allow for enhanced use of
these drugs as therapeutic agents.
Materials and Methods
naphthalenyl-1,4,19,49-tetraone (binaphthoquinone #7), 3-Hy-
(binaphthoquinone #3) and 8-(3-Chloro-1,4-dioxo-1,4-dihydro-
7,10-dione (binaphthoquinone #11)] were synthesized and
characterized by us as previously described and dissolved in
DMSO. N-acetylcysteine (NAC), doxorubicin, menadione, mito-
mycin C, Rhodamine 123 and 123-dihydrorhodamine (123 DHR)
were purchased from Sigma–Aldrich (St. Louis, MO).
Yeast Strains, Media, and Genetic Manipulations
Haploid deletion mutants were generated by the International
Deletion Consortium and were obtained from American Type
leu2D0 met15D0 ura3D0) was used as wild type strain. Yeast were
grown on synthetic complete (SC) media supplemented with amino
acids and lacking uracil (2% glycerol was substituted for glucose as a
carbon source as indicated). An isogenic rho0mutant was generated
by ethidium bromide treatment as described previously .
Drug Sensitivity Screen and Individual Growth Rate
The deletion mutant arrays (DMA) containing a yeast artificial
chromosome carrying the URA3 marker were propagated on SC
medium lacking uracil. The DMA were transferred by hand with a
model MC96 96-pin replicator (Dan-Kar Corp, Woburn, MA)
into 96-well plates containing 100 microliters of liquid media and
were grown overnight at 23uC to saturation. Saturated cultures
compensated for differences in growth rate between strains, to
ensure that roughly equal amounts of cells were deposited on the
agar plates. After 24h, the DMA were pinned onto 96-well plates
and were allowed to grow until the OD600of 0.01 was reached at
which point media with either DMSO or BiQs in a final
concentration of of IC50were added. Growth rates were measured
by determination of OD600 as a function of time by using a
VersaMax plate reader (Molecular Device, Sunnyvale, CA).
Growth curves were carried out in triplicate and curves shown
are averages of three experiments with error bars representing one
STD. Individual growth rate was carried out in a similar manner
with a wider spectrum of drug concentrations. DMSO was used as
a no drug control. Where indicated, 100 mg/mL NAC was added.
PLoS ONE | www.plosone.org10 May 2010 | Volume 5 | Issue 5 | e10846
Spot assays were performed by growth of yeast strains to an OD600
of 1 and then serially spotting them at 5 fold dilutions onto glucose
or glycerol containing plates at indicated concentrations of BiQs.
For further information, refer to the Saccharomyces Genome
Database (SGD), which can be accessed at www.yeastgenome.
com. For gene ontology analyses, we used the GO annotation for
yeast gene products curated by SGD. Specifically, we determined
significantly enriched GO annotations among the genes within
each subcluster using GO::TermFinder (http://www.yeastgen-
ome.org/cgi-bin/GO/goTermFinder.pl) and GO::SlimMapper
. Enrichment factor was calculated by using the formula (a/b)/
(c/d), where a is the number of mutants in a particular GO category
with decreased growth, b is the total number of genes with
decreased growth, c is the total number of genes in a particular GO
category and d is the total number of genes across all GO
categories. We defined significant enrichment as at least 3 fold
above random enrichment (enrichment factor of 1) .
Drug Resistance Screen
DMA were arrayed onto glycerol containing plates with DMSO
or BiQ at the indicated concentration at high density by using a
96-pin tool that spots 20 nanoliter or 250 cells. Strains were pinned
in duplicate with a density of approximately 864 per plate. Pins
were flame sterilized. Plates were grown at 23uC and strains were
scored for growth, slow growth, or no growth, compared with no
drug over a period of 4 days.
Gene deletion and ectopic expression
The double mutant nde1D nde2D was generated by deleting the
open reading frame of NDE2 from the nde1D strain (BY4741) by
using homologous recombination . Gene deletion was
confirmed by PCR. The amplified open reading frame of NDE1
gene was cloned into a GPD-promoter driven expression vector,
p415GPD. Yeast transformation was done by the lithium acetate
method. Transformed cells were selected on SC-leucin plates.
Sequences for primers used for generation of knock-out strain and
expression vector are available upon request. The identity of all
strains used for individual experiments were confirmed by uptag
and downtag amplification and sequencing as described previously
Assay of the electrochemical potential
After treatment in 20 mM HEPES buffer (pH 7.4) containing
50 mM glucose, 1 ml of the yeast suspension was incubated with
2 mM Rh123 (rhodamine 123) for 30 minutes at at 23u Celsius,
washed, and then resuspended in 100 microliter PBS. Propidium
iodide (PI) was then added 10 minutes prior to analysis by flow
cytometry. Mitochondrial electrochemical potential is correlated
to the fluorescence intensity of Rho123 (with decreased fluores-
cence signifying loss of the mitochondrial electrochemical
potential). Flow cytometry was performed using a FACS Calibur
(Becton Dickinson, San Jose, California, United States) with
excitation at 488nm and emission read using a 525–550nm filter
(FL1). PI stained, dead cells were excluded from analysis.
Flow cytometric analysis of ROS production
To assess production of radical oxygen species, cells were
exposed to BiQs for 2h at 23u Celsius, washed and then incubated
with dihydrorhodamine 123 (DHR 123) for 1 h at 23u Celsius and
then propidium iodide (PI) for 10 minutes. Cells were analyzed by
a FACS Calibur at excitation and emission settings of 488 and
525–550 nm (filter FL1), respectively. PI stained, dead cells, were
excluded from analysis.
Figure 8. Proposed mechanism of binaphthoquinones redox cycling. Our data suggest that binaphthoquinones undergo oxidoreductase-
dependent redox cycling which results in ROS generation and cytotoxic action of these molecules. Binaphthoquinones are reduced to the
hydroquinone forms, which are unstable and through four one-electron oxidation steps converts back to the parent molecule. Molecular oxygens act
as electron acceptors and convert to superoxide radicals.
PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10846
Masses of BiQ7 and NAC either invidually or after coincuba-
tion at room temperature for 2 hours was determined by MALDI-
TOF and QSTAR mass spectrometry performed by the Johns
Hopkins Proteomics Core Facility .
following exposure to drugs. (A) BiQ3, (B) BiQ7 and (C) 6-AU.
Yeast deletion mutant array log-phase cultures at an OD600of 0.1
were treated with DMSO or BiQ3, BiQ7, or 6-azauracil (6-AU) at
5mM, 1mM or 2 mM respectively over 24 h. Relative growth of
wild-type (BY4741) strain in the presence of drugs were measured
against that of the yeast grown in the presence of DMSO. Growth
curves were performed in duplicate. The number of mutants at
indicated relative growth values was plotted. Broken lines indicate
values 3 SD below the mean. (D) Venn’s diagram of hypersensitive
mutants shared between BiQ3, BiQ7 and 6-AU.
Found at: doi:10.1371/journal.pone.0010846.s001 (0.28 MB
Relative growth distribution of yeast mutants
cytotoxicity by treatment with N-acetylcysteine (NAC). (A)
Abrogation of BiQ dependent ROS generation by coincubation
with NAC. Yeast wild type strain was treated with BiQ7 at
increasing concentrations in the presence or absence of NAC for
2h and then incubated with DHR 123 for 1h. Experiments were
performed three times with similar results. (B) Suppression of yeast
growth by binaphthoquinone is neutralized by addition of NAC.
Decreased growth of wild type and sensitive yeast mutants was
rescued by addition of 100 mg/mL of NAC. Log-phase cultures at
an OD600of 0.1 were treated with different concentrations of
BiQ7 or DMSO over 24h. Relative growth of wild-type and
mutant strains in the presence of BiQ7 was measured against that
of the yeast grown in the presence of DMSO. Growth curves were
performed in triplicate and represent the average of three
experiments. *p,0.05 for differences between wild-type and
NDE1 over-expressing or mutant yeast.
Found at: doi:10.1371/journal.pone.0010846.s002 (0.58 MB
Neutralization of BiQ free radical generation and
while overexpression of NDE1 enhances sensitivity to BiQs. (A,D)
BiQ7 (B,E) BiQ3, (C,F) BiQ11. Log-phase cultures at an OD600
of 0.1 were treated with different concentrations of corresponding
binaphthoquinones or DMSO over 24h. Relative growth of wild-
type and mutant strains in the presence of binaphthoquinones was
measured against that of the yeast grown in the presence of
DMSO. Growth curves were performed in triplicate and represent
the average of three experiments. *p,0.05 for differences between
wild-type and mutant yeast.
Found at: doi:10.1371/journal.pone.0010846.s003 (0.39 MB
Deletion of nde1 nde2 enhances resistance to BiQs
concentrations of BiQ7 and BiQ3. BiQ7 and BiQ3 hypersensi-
tivity in the deletion strains was measured by assessing growth
defect as compared to DMSO by measuring OD600 (complete
description in Results section of the text). References that describe
haploid deletion strains that are sensitive to BiQ7 and BiQ3 are
described in the text (see Results section for detailed description).
Gene functions and cellular component of corresponding yeast
proteins were obtained from SGD.
Found at: doi:10.1371/journal.pone.0010846.s004 (0.05 MB
Yeast deletion mutants hypersensitive to the IC50
nones. Yeast deletion strains resistant to BiQ7, BiQ11 or BiQ3 at
3, 7 and 7 mM concentrations respectively are listed.
Found at: doi:10.1371/journal.pone.0010846.s005 (0.02 MB
Yeast deletion mutants resistant to binaphthoqui-
We thank the Johns Hopkins Proteomics Core Facility for performing mass
spectrometry and for their help with data interpretation.
Conceived and designed the experiments: MVR. Performed the experi-
ments: AE MVR. Analyzed the data: AE AER KMC MVR. Contributed
reagents/materials/analysis tools: AE YMF MVR. Wrote the paper: AER
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