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: firstname.lastname@example.org
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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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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