Design and synthesis of a mitochondria-targeted mimic of glutathione peroxidase, mitoebselen-2, as a radiation mitigator

Article abstract of DOI:10.1021/ml5003635

Ionizing radiation (IR) triggers mitochondrial overproduction of H₂O₂ and accumulation of lipid hydroperoxides leading to the induction of apoptotic and necroptotic cell death pathways. Given the high catalytic efficiency of the seleno-enzyme glutathione peroxidase (Gpx) toward reduction of lipid hydroperoxides and H₂O₂, we tested the potential of mitochondria-targeted derivatives of ebselen to mitigate the deleterious effects of IR. We report that 2-[[2-[4-(3-oxo-1,2-benzoselenazol-2-yl)phenyl]acetyl]amino]ethyl-triphenyl-phosphonium chloride (MitoPeroxidase 2) was effective in reducing lipid hydroperoxides, preventing apoptotic cell death, and, when administered 24 h postirradiation, increased the survival of mice exposed to whole body γ-irradiation.

Full text of DOI:10.1021/ml5003635

Letter
pubs.acs.org/acsmedchemlett
Design and Synthesis of a Mitochondria-Targeted Mimic of
Glutathione Peroxidase, MitoEbselen-2, as a Radiation Mitigator
Detcho A. Stoyanovsky,*,† Jianfei Jiang,† Michael P. Murphy,⊥ Michael Epperly,§ Xiaolan Zhang,∥
Song Li,∥ Joel Greenberger,§ Valerian Kagan,*,† and Hulya Bayır*,‡
̈
‡
§
∥
†Departments of Environmental and Occupational Health, Critical Care Medicine, Radiation Oncology, and Pharmaceutical
Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
⊥Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, U.K.
S
* Supporting Information
ABSTRACT: Ionizing radiation (IR) triggers mitochondrial overproduction of H2O2 and accumulation of lipid hydroperoxides
leading to the induction of apoptotic and necroptotic cell death pathways. Given the high catalytic efficiency of the seleno-
enzyme glutathione peroxidase (Gpx) toward reduction of lipid hydroperoxides and H2O2, we tested the potential of
mitochondria-targeted derivatives of ebselen to mitigate the deleterious effects of IR. We report that 2-[[2-[4-(3-oxo-1,2-
benzoselenazol-2-yl)phenyl]acetyl]amino]ethyl-triphenyl-phosphonium chloride (MitoPeroxidase 2) was effective in reducing
lipid hydroperoxides, preventing apoptotic cell death, and, when administered 24 h postirradiation, increased the survival of mice
exposed to whole body γ-irradiation.
KEYWORDS: Ebselen, mitochondria, H2O2, radiation, mitigators, apoptosis
xposure of eukaryotic cells to ionizing radiation (IR)
results in a burst of species with high energy (eaq−; H•,
secondary effects whereby activated inflammatory cells
massively generate reactive oxygen species (ROSs) thus
producing “friendly fire” and inducing new waves of oxidation
reactions culminating in apoptotic and nonapototic cell death.9
Among these secondary oxidants, H2O2 and lipid hydro-
peroxides (LOOH) are most prominent in setting-up the
“peroxide tone” and perpetuating enzymatic and nonenzymatic
oxidations of critical biomolecules with signaling functions. In
particular, peroxidase functions of mitochondrial intermem-
brane space hemoprotein, cytochrome c (cyt c), toward a
mitochondria-specific phospholipid, cardiolipin (CL), has been
associated with the accumulation of CL hydroperoxides
required for the execution of mitochondrial apoptosis.10 In
addition, a group of cytosolic nonheme iron proteins,
lipoxygenases (LOX), catalyze oxygenation reactions yielding
hydroperoxides of free polyunsaturated fatty acids thus
inducing necroptotic and ferroptotic cell death signals.11
E
HO•, and O2−•) and indiscriminate reactivity with biomole-
cules, including DNA, on the millisecond time scale.1 Studies
by Patt, Bacq, and others have established that aminothiols such
as cysteine and cysteamine protect mice from short-lived
radiolytic intermediates.2−4 Humans, however, do not tolerate
the doses of cysteine and cysteamine, which would be required
for analogous protection, and the early hope that these
compounds might be useful as radiation protectors (RPs) has
not been realized. These initial observations have been followed
by a research program under the auspice of the Walter Reed
Army Research Institute, which has led to the synthesis of 4400
aminothiols and their assessment as RPs.5 Structural variables
in the synthesis of RPs were the length and branching of the
carbon chain that linked NH2, SH, and OH functional groups.
From this chemical library, only amifostine (H2O3P−S−
(CH2)2−NH−(CH2)3−NH2) has found clinical applications.6
The immediate burst of radicals upon exposure to IR is
followed by a dose-dependent and continuous mitochondrial
overproduction of reactive oxygen species.7,8 These primary
lethal effects of IR, realized predominantly in rapidly
proliferating hematopoietic cells and gut epithelial cells, initiate
Received: September 4, 2014
Accepted: November 18, 2014
© XXXX American Chemical Society
A
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Letter
a
Hence, regulation of the levels of ROSs is important for the
control of the major radiation-induced cell death pathways.12
The central regulator of LOOH is a seleno-enzyme,
glutathione peroxidase 4 (GPx4), whose deficiency leads to
cell death.12 GPx has been shown to impede mitochondrial
apoptosis via clearance of hydroperoxides of CL,13,14 while
overexpression of mitochondrial catalase has been found to
increase radioresistance in vitro and in vivo.15
Scheme 3. Synthesis of MitoPeroxidase 2
Recently, Tak and Park have reported that 2-phenyl-1,2-
benzoselenazol-3-one (ebselen; Scheme 1, 1), when adminis-
Scheme 1. Ebselen Exhibits GPx-Like Activity
a
Reagents and conditions: (a) aq. NaNO2, HCl (18%), 0 °C; (b)
Na2Se2, aq. NaOH, 40 °C (2 h), 55−63%; (c) SOCl2, DMF, reflux (1
h), >95%; (d) 2-(4-aminophenyl)acetic acid, Et3N, CH3CN, 0 °C (1
h) to 25 °C (3 h), 72−76%; (e) (Ph)3P+CH2CH2NH2, DCC, 0 °C (1
h) to 25 °C (4 h), 80−85%.
radiation mitigators (RMs). Because under conditions of
oxidative stress sufficient concentrations of antioxidants at the
sites of generation of reactive metabolites are critical to
protection from oxidative damage, we have targeted the
synthesis of ebselen derivatives that selectively compartmen-
talize to mitochondria (Scheme 2).
tered for 14 days prior to radiation, provides substantial
protection against killing and oxidative damage to mice exposed
to whole-body irradiation (WBI).16 Ebselen is a multifunctional
drug that accumulates in the endoplasmic reticulum (ER),17
reacts with cellular thiols, and mimics the activity of glutathione
peroxidase (GPx) by clearing H2O2 and LOOH (Scheme 1).18
Following recent clinical trials for the prevention and treatment
of cardiovascular diseases, arthritis, stroke, and atheroscle-
rosis,18 ebselen has been included in the National Institutes of
Health Clinical Collection,19 a chemical library of bioavailable
drugs considered clinically safe.
We have assessed the potential of the derivatives of ebselen
4-[4-(3-oxo-1,2-benzoselenazol-2-yl)phenoxy]butyl-triphenyl-
phospho-nium iodide (MitoPeroxidase 1; synthesized as
reported in ref 20; Scheme 2) and 2-[[2-[4-(3-oxo-1,2-
benzoselenazol-2-yl)phenyl]acetyl]amino]ethyl-triphenyl-phos-
phonium chloride (MitoPeroxidase 2; Scheme 3, 10) to act as
To date, several methods for delivery of drugs into
mitochondria have been developed, including their derivatiza-
tion with certain peptides or with a triphenylphosphonium
group ((Ph)3P+).21 Since mitochondria maintain a negative
potential, the positive charge of the (Ph)3P+ group drives
attached molecules inside the matrix and toward a diffusion
equilibrium, thus affording up to a thousand-fold accumulation
of the drug in mitochondria vs cytosol. The accumulation of
organic cations in mitochondria may also be mediated by
proteins such as the 2-oxoglutarate carrier.22 Previous studies
have shown that MitoPeroxidase 1 is taken up by isolated
mitochondria, catalyzes the breakdown of H2O2 in the presence
of thiols, and inhibits apoptosis induced by oxidants.20 In
agreement with the reactions presented in Scheme 1,
incubation (5 min; t = 25 °C) of MitoPeroxidase 2 (15 μM;
Figure 1A) and 6,8-bis(sulfanyl)octanoic acid (dihydrolipoic
acid; DHLA; 15 μM) in He-deaerated methanol led to the
formation of a selanyl-benzamide (Figure 1B), which readily
reduced 13S-hydroperoxy-9Z,11E-octadecadienoic acid (10
μM; ROOH; Figure 1C) to the corresponding alcohol
(ROH; tincubation = 5 min; 25 °C; Figure 1D; MS/MS spectra
of both ROOH and ROH are included in Supporting
Information). Similarly, the selanyl-benzamide reduced hydro-
peroxides of CL (Supporting Information).
Scheme 2. Design of Mitochondria-Specific Ebselen
Derivatives
Though the benzoselenazol ring is a common pharmaco-
phore for ebselen and MitoPeroxidases 1 and 2, several factors
may differentiate the pharmacological effect of these com-
pounds. In contrast to ebselen, which accumulates in the ER,
MitoPeroxidases 1 and 2 are expected to compartmentalize to
mitochondria to reach millimolar concentrations.23 In addition,
the conjugated aromatic system of MitoPeroxidase 1 is
extended to the phenolic oxygen, and thus, its Se−N bond is
more polarized. This is likely to facilitate the conversion of
B
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∼40 μM concentration, while ebselen did not exhibit any
significant toxicity in this concentration range.
We next assessed the radio-mitigative properties of the three
benzoselenazols. Exposure of MEC to γ-irradiation (10 Gy)
resulted in ∼28% cell death (Figure 2B), whereas treatment
with MitoPeroxidases 1 and 2 (but not ebselen), prior to or 30
min postirradiation, afforded radioprotection/mitigation. Mito-
Peroxidase 1 exerted ∼25% radioprotection/mitigation, while at
concentrations higher than 10 μM its toxicity prevailed the
radioprotective effect. In contrast, 20 μM MitoPeroxidase 2
afforded ∼50% radioprotection/mitigation. In this model
system, MitoPeroxidase 2 acted as a potent inhibitor of
radiation-induced activation of caspase 3 (Figure 3), an
Figure 3. MitoPeroxidase 2 impedes the activity of caspase 3 in MEC.
Cells were exposed to IR (10 Gy) and then treated with 20 μM
MitoPeroxidase 2 as indicated in Figure 2. Caspase 3 activity was
determined by EnzChek Caspase 3 Assay Kit (Z-DEVD-AMC
substrate; Life Technologies, Grand Island, NY). The results represent
the mean + SD (n = 3).
Figure 1. Mass-spectral analysis of the thiol-dependent reduction of
ROOH by MitoPeroxidase 2. DHLA reduced MitoPeroxidase 2 (A) to
selanyl-benzamide (B), which reacted with ROOH (C) to afford ROH
(D). The mass spectra in A and B exhibit isotopic distribution
characteristics for the Se isotopes.20
executioner caspase in apoptosis. Treatment of irradiated
MEC with 3-hydroxypropyl(triphenyl)phosphonium chloride,
which structurally mimics the triphenylphosphonium moieties
of MitoPeroxidases 1 and 2, did not afford any radiomitigation
(data not shown).
We further assessed whether the in vitro radiomitigative
effect of MitoPeroxidase 2 was translated into an in vivo effect.
Groups of 15 mice were treated with MitoPeroxidase 2 i.v. at 24
h after WBI. As shown in Figure 4, at two different radiation
doses, administration of MitoPeroxidase 2 afforded an increase
in survival.
MitoPeroxidase 1 to a selenenyl sulfide (reaction 1 → 2), which
may result in an increased toxicity due to enhanced oxidation of
cellular thiols.24 However, weakening of the Se···OC<
interaction in 3 by the electron-withdrawing effect of the
phenolic oxygen may increase the GPx activity of the parent
benzoselenazol.25,26
Figure 2A depicts the toxicity of ebselen and MitoPerox-
idases 1 and 2 in mouse embryonic cells (MEC). In contrast to
ebselen and MitoPeroxidase 2, the toxicity of MitoPeroxidase 1
sharply increased in the concentration range of 10 to 20 μM.
Comparable toxicity with MitoPeroxidase 2 was observed at
In conclusion, the data presented herein indicate that
MitoPeroxidase 2 acts as a potent radiation mitigator. Our
data complement previous studies on the medicinal chemistry
of ebselen within the context of radiomitigation upon late, 24 h
Figure 2. Toxicity (A) and radiomitigative properties (B) of ebselen
and MitoPeroxidases 1 and 2. (A,B) Cell death was assessed flow-
cytometrically by analysis of the externalization of phosphatidylserine.
(B) Cells were exposed to IR and then treated with selenazols. The
results represent the mean SD (n = 3; *p < 0.05).
Figure 4. Radiomitigative properties of MitoPeroxidase 2 administered
24 h after exposure to γ-rays in mice (n = 15 mice per group).
C
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ASSOCIATED CONTENT
■
S
* Supporting Information
Details of biological assays, chemical synthesis, and structural
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: 412-383-6910. E-mail: dstoyanovsky@gmail.com.
*Tel: 412-6925164. E-mail: hub22@pitt.edu.
*Tel: 412-624-9479. E-mail: kagan@pitt.edu.
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cultured cells and mice. Free Radical Biol. Med. 2009, 46, 2009.
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Harris, H. H.; Lai, B.; Vogt, S.; Giles, G. I. Synchrotron radiation
induced X-ray emission studies of the antioxidant mechanism of the
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Funding
This work was supported by NIH Grant U19AI068021,
HL114453 and NS061817, and Human Frontier Science
Program RGP0013.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
GPx, glutathione peroxidase; ER, endoplasmic reticulum; IR,
ionizing radiation; RMs, radiation mitigators; RPs, radiation
protectors; WBI, whole-body irradiation; MEC, mouse
embryonic cells
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