Bioreductively activated reactive oxygen species (ROS) generators as MRSA inhibitors

Article abstract of DOI:10.1021/ml5001118

The number of cases of drug resistant Staphylococcus aureus infections is on the rise globally and new strategies to identify drug candidates with novel mechanisms of action are in urgent need. Here, we report the synthesis and evaluation of a series of benzo[b]phenanthridine-5,7,12(6H)-triones, which were designed based on redox-active natural products. We find that the in vitro inhibitory activity of 6-(prop-2-ynyl)benzo[b]phenanthridine-5,7,12(6H)-trione (1f) against methicillin-resistant Staphylococcus aureus (MRSA), including a panel of patient-derived strains, is comparable or better than vancomycin. We show that the lead compound generates reactive oxygen species (ROS) in the cell, contributing to its antibacterial activity.

Full text of DOI:10.1021/ml5001118

Letter
pubs.acs.org/acsmedchemlett
Bioreductively Activated Reactive Oxygen Species (ROS) Generators
as MRSA Inhibitors
Vinayak S. Khodade,† Mallojjala Sharath Chandra,† Ankita Banerjee,‡ Surobhi Lahiri,‡
Mallikarjuna Pulipeta,‡ Radha Rangarajan,‡ and Harinath Chakrapani*,†
†Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pune, 411008 Maharashtra, India
‡Vitas Pharma Research Private Limited, Technology Business Incubator, University of Hyderabad, C. R. Rao Road, Gachibowli,
Hyderabad 500046, India
S
* Supporting Information
ABSTRACT: The number of cases of drug resistant Staphylococcus
aureus infections is on the rise globally and new strategies to identify
drug candidates with novel mechanisms of action are in urgent need.
Here, we report the synthesis and evaluation of a series of
benzo[b]phenanthridine-5,7,12(6H)-triones, which were designed
based on redox-active natural products. We find that the in vitro
inhibitory activity of 6-(prop-2-ynyl)benzo[b]phenanthridine-
5,7,12(6H)-trione (1f) against methicillin-resistant Staphylococcus aureus
(MRSA), including a panel of patient-derived strains, is comparable or
better than vancomycin. We show that the lead compound generates reactive oxygen species (ROS) in the cell, contributing to its
antibacterial activity.
KEYWORDS: Drug resistance, reactive oxygen species, MRSA, superoxide radical, RecA, DNA damage
Potentiating these ROS in cancers using small molecules has
been considered as a possible drug design strategy.7 For
example, deoxynyboquinone, a natural product derivative, has
potent tumoristatic activity in animal models, and its efficacy is,
in part, dependent on generation of ROS (Chart 1).8,9 The
ecause of the high levels of morbidity and mortality, drug-
Bresistant infections have now become a major public
health problem globally.1 The pathogen Staphylococcus aureus
(S. aureus), for example, has acquired resistance to several
antibiotics including the methicillin-based drugs. Methicillin-
resistant S. aureus (MRSA) strains are fast becoming resistant to
other frontline antibiotics as well.2 The global pipeline for new
antibiotics is weak, and hence, the development of new
strategies that specifically address drug resistance is necessary.
Redox-active natural products were long considered as
innocuous byproducts of metabolism of microorganisms
without any major function.3,4 It is increasingly clear that
these secondary metabolites are produced to mediate a number
of cellular processes including gene expression, interspecies
communication, and defense.3,4 The ability of such small
molecules to keep competitors in check through the generation
of reactive oxygen species (ROS) has attracted attention for
new drug development.3,5 ROS including superoxide radical
Chart 1
potential for ROS generators to inhibit growth of certain
mycobacteria is also reported.10,11 The oxidative stress-inducing
antimalarial drug artemisinin, when conjugated with mycobac-
tin,12 and the antileprosy drug Clofazimine13 have potent
inhibitory effects on multidrug-resistant strains of Mycobacte-
rium tuberculosis. Here, we report results of design and synthesis
of natural product-inspired ROS generators and their potential
to inhibit multidrug-resistant S. aureus.
•
O2−•, hydrogen peroxide H2O2, and hydroxyl radical OH are
generated as a natural consequence of respiration but can cause
cellular damage at elevated levels (Scheme 1).6
Scheme 1. Reactive Oxygen Species (ROS) and Their
Possible Cellular Effects
We proposed to synthesize and study benzo[b]-
phenanthridine-5,7,12(6H)-triones of general structure 1
Received: March 18, 2014
Accepted: May 18, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml5001118 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(Scheme 2). This scaffold contained the amide adjoining the
quinone of deoxynyboquinone and the aryl ring-like
Information) was oxidized during the reaction (Table 1). Using
this one-pot procedure, compounds 1b−1g were prepared from
2 (Table 1, entries 2−7). The use of an aromatic amine such as
aniline did not produce the desired product but instead gave an
inseparable mixture of products. The one-pot methodology
was, however, found to be compatible with benzylamines, and
1h−1n were prepared in moderate yields (Table 1, entries 8−
14).
−•
Scheme 2. Proposed Mechanism of O2 generation from 1
To test if this library of compounds had an effect on the
growth of bacterial cells, the minimum inhibitory concentration
(MIC) was determined for the series against methicillin
sensitive S. aureus (MSSA). Five of the analogues tested were
found to inhibit MSSA at 32 μg/mL or less (Table 2). The best
jadomycins,14 another class of natural products that are
known to cleave DNA in the presence of metal ions such as
Cu(II)15 presumably through the generation of hydroxyl radical
(Chart 1).16 1 is predicted to undergo bioreduction to its
Table 2. Calculated Partition Coefficients (ClogP) and
Minimum Inhibitory Concentration (MIC) against
Methicillin-Sensitive S. aureus (MSSA) of 1a−1n
a
b
entry
compd
ClogP
MIC (μg/mL)
semiquinone,17 which then is reoxidized by molecular oxygen,
−•
1
1a
1b
1c
2.63
3.16
3.69
4.66
3.40
2.73
2.82
4.40
5.28
5.11
4.32
4.14
5.28
3.70
1.0
>32
>32
>32
>32
0.5
>32
>32
>32
>32
32
forming O2
(Scheme 2). During this oxidation, 1 is
2
regenerated and becomes available for further ROS generation
via bioreduction (See Supporting Information, Scheme S3).
The synthesis of 1 can be achieved in two steps from 2-(2-
formyl-phenyl)-1,4-naphthoquinone 2 (Table 1), which in turn
3
4
1d
5
1e
6
1f
7
1g
Table 1. One-Pot Synthesis of 1a−1n by the Reaction of 2
with a Primary Amine
8
1h
9
1i
10
11
12
13
14
15
16
17
1j
1k
1l
8.0
>32
8.0
1
1m
1n
tobramycin
fosfomycin
vancomycin
8
0.5
a
b
a
Calculated using Chembiodraw Ultra 13.0. Minimum inhibitory
entry
R
compd
% yield
concentration (MIC) is defined as the lowest concentration required
to inhibit visible bacterial growth; MSSA = MSSA 29213.
1
Me
1a
1b
1c
1d
1e
1f
73
2
Et
nPr
50
3
45
inhibitor was 1f with MIC of 0.5 μg/mL against MSSA and
MRSA (Table 2, entry 6, and Table 3, entry 1). The activity was
comparable to vancomycin, the drug of choice in the clinic for
multidrug resistant MRSA infections. This compound was
identified as the lead for further studies.
4
cyclohexyl
allyl
23
5
21
b
6
propargyl
20 (37)
39
7
CH2CO2Me
benzyl
1g
1h
1i
8
39
9
2-CF3PhCH2
4-ClPhCH2
14
Table 3. MICs of 1f against Methicillin-Resistant S. aureus
(MRSA) Strains
10
11
12
13
14
1j
20
4-OMePhCH2
4-NO2PhCH2
4-CF3PhCH2
(3,4,5-triOMe)PhCH2
1k
1l
17
22
MIC
(μg/mL)
MIC
(μM)
MIC of vancomycin
1m
1n
47
entry
strain
(μg/mL)
13
a
b
1
MRSA 33591
MRSA 7419
B19506
0.5
1.6
0.5−2
1.0
Isolated yield. Addition of H2O2 (10 equiv) to the reaction mixture.
2
0.12
0.06
0.25
0.12
1.0
0.38
0.19
0.80
0.38
3.19
1.60
1.60
1.60
1.60
3.19
1.60
3
0.5−2
1.0
4
MRSA K-1
MRSA 7425
MRSA 7386
E9902
can be synthesized from commercially available 2-bromo-1,4-
naphthoquinone (Supporting Information).14 The addition of
an amine to 2 would result in the formation of an imine, which
sets up the molecule for an intramolecular 6π-electrocyclic ring
closure to afford the alcohol 3; subsequent oxidation of 3
should give the desired lactams of structure 1 (Table 1).14
When 2 was reacted with methylamine in an open container,
we found 1a as the major product in 73% yield implying that
the intermediate alcohol (3a, R = Me, Scheme S2, Supporting
5
0.12
1.0
6
7
0.5
0.5
8
E151
0.5
0.5
9
E288
0.5
0.5
10
11
12
B21838
0.5
0.5
B853
1.0
1.0
MRSA B0085
0.5
1.0
B
dx.doi.org/10.1021/ml5001118 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
−•
First, we attempted to improve the yield of 1f. A mechanism
for the electrocyclic ring closure followed by hydration and
oxidation was proposed (Scheme S2, Supporting Information).
Aerobic oxidation presumably involved hydrogen peroxide and
when the reaction mixture was supplemented with H2O2, an
increased yield of 1f (37%, Table 1, entry 6) was observed. The
overall yield of 1f could thus be improved to 24% in two steps
from commercially available 2-bromo-1,4-naphthoquinone. In
contrast, jadomycins are synthesized in >20 steps14 while
deoxynyboquinone was prepared in seven linear steps.9
ently assess O2 production (Figure 1b).21,22 Here, the
conversion of DHE to 2-hydroxyethidium (2-OH-E+) is
indicative of O2 generation.23 During incubation of 1f in
−•
the presence of DT-D, the formation of 2-OH-E+ was observed,
−•
thus, confirming the intermediacy of O2 (Figure 1b). In
addition, H2O2, the product of 1e− transfer to O2−•, was
measured using an Amplex Red-based fluorescence assay.
Again, we found that 1f was capable of generating H2O2 in the
presence of DT-D.24
−•
Next, the possibility of 1f generating intracellular O2 in
bacteria was examined using a DHE assay.21,22 The bacterial
control showed unreacted DHE (Figure 1d), and in the
presence of 1f, the formation of 2-OH-E+ in a concentration-
Cyclic voltammetry analysis revealed that 1e− reduction
potentials (Ered) of 1f was −1.00 V (Table S1, Supporting
Information), which appears appropriate for metabolism by
bioreductive enzymes.17 This compound was found to be
unreactive with biological thiols such as glutathione (see
Supporting Information, Figure S1), possibly due to steric
hindrance created by the substituents on the quinone ring. A
luminol-based chemiluminescence assay was used to detect
O2−•, and we found generation of O2−• only in the presence of
NQO1-naphthoquinone oxidoreductase (DT-Diaphorase, DT-
D),18 a model bioreductive enzyme (Figure 1a).19,20 A HPLC-
based dihydroethidium (DHE) assay was used to independ-
−•
dependent manner was observed, suggestive of O2
production intracellularly (Figure 1d). Superoxide accumu-
lation intracellularly leads to the production of H2O2 through
dismutation.6 H2O2 diffuses out of the cell and can be measured
using Amplex Red. In this assay, extracellular H2O2 serves as a
surrogate marker for intracellular ROS levels.25 When S. aureus
cells are exposed to 1f, we find increased H2O2 confirming that
1f was capable of enhancing ROS (Figure 1e). The yield of
H2O2 produced by 1f was nearly quantitative, a testament to
efficient ROS generation by this compound (Figure 1e).
−•
A possible consequence of increased O2 and H2O2 is the
•
generation of OH. However, because of its extremely short
half-life, detection of •OH is challenging. Instead, reported
•
methods measure all elevated oxidative species including OH.
Using dichlorofluorescein-diacetate (DCFH2-DA) fluorescence
assay, the levels of oxidative species generated intracellularly in
S. aureus was estimated. The results of this assay indicate that 1f
at 0.5 μg/mL was capable of generating oxidative species
intracellularly (Figure S5, Supporting Information).
Lethality induced by elevated ROS is attributable to DNA
damage by •OH leading to single and double strand breaks.6 In
response to DNA damage, DNA rescue pathways are activated.6
Symptomatic of one such rescue pathway is the expression of
RecA, a repair protein crucial to homologous recombination.26
In the presence of 1f, we found a significant upregulation of
RecA expression suggesting that DNA damage repair response
is activated in S. aureus upon treatment with 1f (Figure 1f). We
next examined the effect of 1f on the viable colony count of
MSSA in the presence of thiourea, a •OH quencher. The
growth inhibition of 1f is reduced in the presence of thiourea,
suggesting that quenching the ROS in the cell reduces the
antibacterial effect of the compound (Figure S6, Supporting
Information). These data provide further support for a ROS
based mechanism contributing to antibacterial activity.
To test for synergy between 1f and other known antibiotics,
we conducted synergy time kill assays with ciprofloxacin
(Figure S7, Supporting Information) and tobramycin (Figure
S8, Supporting Information) against MRSA 33591. We found
no evidence for synergy suggesting that the mechanism of
action of 1f was distinct from these clinically-used antibiotics.
The lack of synergy shows that mechanism of action of 1f is
independent of the antibiotics tested and could represent a
novel way to inhibit bacterial growth. If the mechanism is novel,
we hypothesized that it would be able to overcome existing
resistance. Hence we tested the activity of the compound
against a panel of 11 clinical isolates of MRSA. We found the
activity to be well-conserved between the reference strain
MRSA 33591 and the clinical isolates (Table 3, entries 2−12).
Finally, we tested the growth inhibitory potential of 1f against
mammalian cells. When screened against A549 human lung
−•
Figure 1. (a) Time course of O2 generation was estimated by a
luminol-based chemiluminescence assay in the presence of DT-
diaphorase. Control (Ctrl) is 1f incubated in pH 7.4 buffer. (b)
Superoxide generated during incubation of 1f with DT-D was
estimated using a dihydroethidium (DHE) assay. Superoxide
specifically react with DHE to produce 2-hydroxyethidium (2-OH-
E+); ethidium (E+) is formed by nonspecific oxidation of DHE and is
indicative of a general increase in oxidative species. Ctrl is 1f in pH 7.4
buffer. (c) Hydrogen peroxide produced during incubation of 1f (2
μg/mL) with DT-D for 5 min was quantified by an Amplex Red-based
−•
fluorescence assay. (d) O2 generated during incubation of S. aureus
with 1f was estimated using a dihydroethidium (DHE) assay. O2
−•
specifically react with DHE to produce 2-hydroxyethidium (2-OH-E+);
ethidium (E+) is formed by nonspecific oxidation of DHE and is
indicative of a general increase in oxidative species. (e) Hydrogen
peroxide generation during incubation of S. aureus with 1f (2 μg/mL)
for 60 min as measured using an Amplex Red-based fluorescence assay.
Ctrl is untreated bacteria. (f) ROS accumulation in the cell can damage
DNA, leading to the upregulation of the DNA repair enzyme, RecA.
Quantitative real time PCR analysis of recA expression in MSSA cells
incubated with 6.7 × MIC concentrations, i.e., 3.35 μg/mL for 120
min showed a significant increase in recA levels. Ctrl is untreated
bacteria.
C
dx.doi.org/10.1021/ml5001118 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(2) Rodvold, K. A.; McConeghy, K. W. Methicillin-resistant
Staphylococcus aureus therapy: Past, present, and future. Clin. Infect.
Dis. 2014, 58, S20−S27.
(3) Dietrich, L. E. P.; Teal, T. K.; Price-Whelan, A.; Newman, D. K.
Redox-active antibiotics control gene expression and community
behavior in divergent bacteria. Science 2008, 321, 1203−1206.
(4) Price-Whelan, A.; Dietrich, L. E. P.; Newman, D. K. Rethinking
‘secondary’ metabolism: physiological roles for phenazine antibiotics.
Nat. Chem. Biol. 2006, 2, 71−78.
(5) Borrero, N. V.; Bai, F.; Perez, C.; Duong, B. Q.; Rocca, J. R.; Jin,
S.; Huigens Iii, R. W. Phenazine antibiotic inspired discovery of potent
bromophenazine antibacterial agents against Staphylococcus aureus and
Staphylococcus epidermidis. Org. Biomol. Chem. 2014, 12, 881−886.
(6) Imlay, J. A. The molecular mechanisms and physiological
consequences of oxidative stress: Lessons from a model bacterium.
Nat. Rev. Microbiol. 2013, 11, 443−454.
(7) Wondrak, G. T. Redox-directed cancer therapeutics: Molecular
mechanisms and opportunities. Antioxid. Redox Signal. 2009, 11,
3013−3069.
(8) Parkinson, E. I.; Bair, J. S.; Cismesia, M.; Hergenrother, P. J.
Efficient NQO1 substrates are potent and selective anticancer agents.
ACS Chem. Biol. 2013, 8, 2173−2183.
(9) Bair, J. S.; Palchaudhuri, R.; Hergenrother, P. J. Chemistry and
biology of deoxynyboquinone, a potent inducer of cancer cell death. J.
Am. Chem. Soc. 2010, 132, 5469−5478.
carcinoma cells, the compound showed a 50% growth
inhibitory concentration (GI50) of 8.6 μM (Supporting
Information). The selectivity index (SI) (GI50/MIC) for
MRSA 33951 is >5, which is favorable for further development.
Thus, taken together, our investigations reveal 1f as a potent
MRSA inhibitor with a unique mechanism of action that
involves enhancement of ROS levels in cells. Several redox-
active analogues of 1f (Table S1, Supporting Information) were
capable of undergoing bioreduction to generate superoxide in
buffer (Figure S3, Supporting Information) and increase
hydrogen peroxide levels in MRSA (Figure S4, Supporting
Information) but were poor S. aureus inhibitors (Table
2).3,4,25,27−29 Hence, generation of ROS by these redox-active
small molecules appears necessary but not sufficient for
inhibiting MRSA growth (Figure S4, Supporting Informa-
tion).3,4 It is noteworthy that even small structural
modifications to Jadomycins resulted in significant differences
in DNA damaging capability,30 and SCH538415 (Chart 1), the
structural analogue with an additional N-methyl group had a
10-fold lower potency in comparison with deoxynyboquinone.9
In summary, we report a natural product-inspired redox-
active small molecule that is able to overcome drug resistance in
MRSA. The in vitro potency of this compound is comparable
or better than that of vancomycin, the drug of last resort for
such infections.
(10) Dharmaraja, A. T.; Alvala, M.; Sriram, D.; Yogeeswari, P.;
Chakrapani, H. Design, synthesis and evaluation of small molecule
reactive oxygen species generators as selective Mycobacterium tuber-
culosis inhibitors. Chem. Commun. 2012, 48, 10325−10327.
ASSOCIATED CONTENT
* Supporting Information
■
̀
(11) Vilcheze, C.; Hartman, T.; Weinrick, B.; Jacobs, W. R.
S
Mycobacterium tuberculosis is extraordinarily sensitive to killing by
a vitamin C-induced Fenton reaction. Nat. Commun. 2013, 4, 1881.
(12) Miller, M. J.; Walz, A. J.; Zhu, H.; Wu, C.; Moraski, G.;
Preparative procedures, assay protocols, NMR spectra, and
other experimental data. This material is available free of charge
via the Internet at http://pubs.acs.org.
Mollmann, U.; Tristani, E. M.; Crumbliss, A. L.; Ferdig, M. T.;
̈
Checkley, L.; Edwards, R. L.; Boshoff, H. I. Design, synthesis, and
study of a mycobactin−artemisinin conjugate that has selective and
potent activity against Tuberculosis and Malaria. J. Am. Chem. Soc.
2011, 133, 2076−2079.
(13) Yano, T.; Kassovska-Bratinova, S.; Teh, J. S.; Winkler, J.;
Sullivan, K.; Isaacs, A.; Schechter, N. M.; Rubin, H. Reduction of
clofazimine by mycobacterial Type 2 NADH: Quinone oxidoreductase
a pathway for the generation of bactericidal levels of reactive oxygen
species. J. Biol. Chem. 2011, 286, 10276−10287.
(14) Shan, M.; Sharif, E. U.; O’Doherty, G. A. Total synthesis of
jadomycin A and a carbasugar analogue of jadomycin B. Angew. Chem.,
Int. Ed. 2010, 49, 9492−9495.
(15) Khodade, V. S.; Dharmaraja, A. T.; Chakrapani, H. Synthesis,
reactive oxygen species generation and copper-mediated nuclease
activity profiles of 2-aryl-3-amino-1,4-naphthoquinones. Bioorg. Med.
Chem. Lett. 2012, 22, 3766−3769.
AUTHOR INFORMATION
Corresponding Author
*(H.C.) E-mail: harinath@iiserpune.ac.in.
Funding
The authors thank IISER Pune and the Department of
Biotechnology, India (BT/PR6798/MED/29/636/2012) for
financial support. V.S.K. and M.S.C. acknowledge fellowships
from Council for Scientific and Industrial Research (CSIR) and
Department of Science and Technology (DST), respectively.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(16) Jakeman, D. L.; Bandi, S.; Graham, C. L.; Reid, T. R.; Wentzell,
J. R.; Douglas, S. E. Antimicrobial activities of jadomycin B and
structurally related analogues. Antimicrob. Agents Chemother. 2009, 53,
1245−1247.
The authors are grateful to Prof. Lakshmi Gorthi, Department
of Microbiology, Nizam’s Institute of Medical Sciences,
Hyderabad, India for providing us with MRSA strains.
(17) Hillard, E. A.; de Abreu, F. C.; Ferreira, D. C. M.; Jaouen, G.;
Goulart, M. O. F.; Amatore, C. Electrochemical parameters and
techniques in drug development, with an emphasis on quinones and
related compounds. Chem. Commun. 2008, 2612−2628.
(18) Sharma, K.; Iyer, A.; Sengupta, K.; Chakrapani, H. INDQ/NO, a
bioreductively activated nitric oxide prodrug. Org. Lett. 2013, 15,
2636−2639.
ABBREVIATIONS USED
■
MIC, minimum inhibitory concentration; GI50, 50% growth
inhibitory concentration; ROS, reactive oxygen species; SI,
selectivity index; DHE, dihydroethidium; DT-D, DT-diaphor-
ase
(19) Trung Pham, H.; Marquetty, C.; Pasquier, C.; Hakim, J.
Luminol assay for microdetermination of superoxide dismutase
activity: Its application to human fetal blood. Anal. Biochem. 1984,
142, 467−472.
(20) Maruyama, A.; Kumagai, Y.; Morikawa, K.; Taguchi, K.;
Hayashi, H.; Ohta, T. Oxidative-stress-inducible qorA encodes an
NADPH-dependent quinone oxidoreductase catalysing a one-electron
reduction in Staphylococcus aureus. Microbiology 2003, 149, 389−398.
REFERENCES
■
(1) Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman,
K.; Ray, S.; Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.;
Craig, A. S.; Zell, E. R.; Fosheim, G. E.; McDougal, L. K.; Carey, R. B.;
Fridkin, S. K. Invasive methicillin-resistant Staphylococcus aureus
infections in the united states. J. Am. Med. Assoc. 2007, 298, 1763−
1771.
D
dx.doi.org/10.1021/ml5001118 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(21) Georgiou, C. D.; Papapostolou, I.; Grintzalis, K. Superoxide
radical detection in cells, tissues, organisms (animals, plants, insects,
microorganisms) and soils. Nat. Protoc. 2008, 3, 1679−1692.
(22) Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. Detection of 2-
hydroxyethidium in cellular systems: A unique marker product of
superoxide and hydroethidine. Nat. Protoc. 2008, 3, 8−21.
(23) Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.;
Vasquez-Vivar, J.; Kalyanaraman, B. Detection and characterization of
the product of hydroethidine and intracellular superoxide by HPLC
and limitations of fluorescence. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
5727−5732.
(24) Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. A
stable nonfluorescent derivative of resorufin for the fluorometric
determination of trace hydrogen peroxide: Applications in detecting
the activity of phagocyte NADPH oxidase and other oxidases. Anal.
Biochem. 1997, 253, 162−168.
(25) Dharmaraja, A. T.; Chakrapani, H. A Small molecule for
controlled generation of reactive oxygen species (ROS). Org. Lett.
2014, 16, 398−401.
(26) Chen, Z.; Yang, H.; Pavletich, N. P. Mechanism of homologous
recombination from the RecA-ssDNA/dsDNA structures. Nature
2008, 453, 489−494.
(27) Price-Whelan, A.; Dietrich, L. E. P.; Newman, D. K. Pyocyanin
alters redox homeostasis and carbon flux through central metabolic
pathways in Pseudomonas aeruginosa PA14. J. Bacteriol. 2007, 189,
6372−6381.
(28) Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.;
Collins, J. J. A common mechanism of cellular death induced by
bactericidal antibiotics. Cell 2007, 130, 797−810.
(29) Liu, Y.; Imlay, J. A. Cell death from antibiotics without the
involvement of reactive oxygen species. Science 2013, 339, 1210−1213.
(30) Cottreau, K. M.; Spencer, C.; Wentzell, J. R.; Graham, C. L.;
Borissow, C. N.; Jakeman, D. L.; McFarland, S. A. Diverse DNA-
cleaving capacities of the jadomycins through precursor-directed
biosynthesis. Org. Lett. 2010, 12, 1172−1175.
E
dx.doi.org/10.1021/ml5001118 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX