Horseradish peroxidase inhibition and antioxidant activity of ebselen and related organoselenium compounds

Article abstract of DOI:10.1016/j.bmcl.2006.07.085

Horseradish peroxidase (HRP) inhibition and glutathione peroxidase (GPx) activities of ebselen and some related derivatives are described. These studies show that ebselen and ebselen ditelluride (EbTe₂) with significant antioxidant activity, inhibit the HRP-catalyzed oxidation reactions. In addition, inhibition of lipid peroxidation and singlet oxygen quenching studies were carried out. Although the inhibition of HRP by ebselen is comparable with that of EbTe₂, the inhibitory effect on γ-radiation induced lipid peroxidation and the GPx activity of ebselen is found to be much higher than that of EbTe₂.

Full text of DOI:10.1016/j.bmcl.2006.07.085

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5334–5338
Horseradish peroxidase inhibition and antioxidant activity
of ebselen and related organoselenium compounds
Beena Mishra,a K. I. Priyadarsini,a Hari Mohana and G. Mugeshb,*
aRadiation and Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 85, India
bDepartment of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
Received 10 March 2006; revised 10 July 2006; accepted 26 July 2006
Available online 17 August 2006
Abstract—Horseradish peroxidase (HRP) inhibition and glutathione peroxidase (GPx) activities of ebselen and some related deriv-
atives are described. These studies show that ebselen and ebselen ditelluride (EbTe2) with significant antioxidant activity, inhibit the
HRP-catalyzed oxidation reactions. In addition, inhibition of lipid peroxidation and singlet oxygen quenching studies were carried
out. Although the inhibition of HRP by ebselen is comparable with that of EbTe2, the inhibitory effect on c-radiation induced lipid
peroxidation and the GPx activity of ebselen is found to be much higher than that of EbTe2.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Ebselen (2-phenyl-1,2-benzisoselazol-3(2H)-one), an
organoselenium compound, has been shown to protect
tissue against oxidative damage by reducing hydrogen
peroxide and other hydroperoxides.1 The hydrogen per-
oxide reducing ability of ebselen, therefore, mimics the
antioxidant selenoenzyme, glutathione peroxidase
(GPx).2 The catalytically active selenium in ebselen
reduces hydroperoxides at the expense of thiol. Similarly
to the GPx catalytic cycle, the selenol form of ebselen re-
acts with hydroperoxides to form a selenenic acid, which
reacts with thiol to produce the corresponding selenenyl
sulfide intermediate. This intermediate further reacts
with an additional thiol to reproduce the selenol. There-
fore, ebselen, when combined with suitable thiol com-
pounds such as glutathione (GSH), dithioerythritol, N-
acetyl cysteine, dihydrolipoate, etc.,3 can reduce hydro-
gen peroxide to water and attenuate lipid peroxidation
by reducing organic, cholestrol, cholesterol ester- and
phospholipid peroxides.4 In contrast to this reductive
detoxification of hydroperoxides, ebselen is a poor free
radical scavenger and consequently this is not regarded
as a significant component of its pharmacodynamic
activity.5
Although the biological activity of ebselen has been
extensively studied,6 the effect of ebselen and its deriva-
tives on metalloproteins have not been studied in detail.
However, a few reports have appeared in the literature,
which show that some organoselenium compounds with
reactive selenolate moiety can inhibit certain metallo-
proteins.7 Recently, the role of metal coordination in
the antioxidant activity of selenium has been reported.8
Ebselen and other related compounds have been report-
ed to be inhibitors of constitutive endothelial nitric
oxide synthase (ceNOS),9 lipoxygenases,10 and c-Jun
N-terminal kinase.11 In this paper, we report the effect
of ebselen on horseradish peroxidase (HRP) activity.
In addition, we report the GPx-like antioxidant activity
and the effect of ebselen and related derivatives on sin-
glet oxygen and lipid peroxidation (see Scheme 1).
O
O
O
Ph
Ph
N
N
N
Ph
H
H
Se
SeCH2Ph
SeCH3
EbBz
Eb
EbMe
O
O
Ph
Ph
N
N
H
H
Se
Te
)
)
Keywords: Ebselen; Antioxidant; Selenium; Horseradish peroxidase;
Lipid peroxidation.
2
2
EbSe2
EbTe2
*
Corresponding author. Tel.: +91 80 2293 2825; fax: +91 80 2360
0683; e-mail: mugesh@ipc.iisc.ernet.in
Scheme 1. Ebselen and related derivatives.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.085

B. Mishra et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5334–5338
5335
The inhibition of horseradish peroxidase (HRP) was
0.24
0.18
studied by using spectrophotometric method.12 The
IC50 values for the inhibition of HRP by ebselen and
other related compounds are summarized in Table 1.
Interestingly, ebselen inhibited the HRP activity with
an IC50 value of 16.9 1.4 lM, indicating that ebselen
may strongly inhibit peroxidase-catalyzed oxidation
reactions. The Se-methylated and Se-benzylated deriva-
tives of ebselen (EbMe and EbBz, respectively), on the
other hand, do not show any significant inhibition.
The ditelluride (EbTe2) was found to be almost as po-
tent as ebselen in the inhibition. These observations re-
veal that the oxidation of selenium or tellurium centres
in ebselen and ditelluride (EbTe2) may be responsible
for the inhibition. At a concentration of 100 lM, the
methyl and the benzyl derivatives inhibited only 25.5%
and 33.1%, of the HRP activity, respectively. On the
other hand, the diselenide (EbSe2) exhibited a significant
inhibition, although the inhibitory activity of this com-
pound was found to be much lower than that of ebselen
and the ditelluride (see Fig. 1).
b
c
0.12
0.06
0.00
d
a
0.00
0.02
0.04
0.06
0.08
0.10
Ebselen derivative, mM
Figure 1. Absorbance at 645 nm (ABTSÅꢀ) in presence of different
concentration of ebselen and its derivatives (a, Eb; b, EbMe; c, EbBz;
d, EbTe2).
20
16
12
8
b
To understand the effect of H2O2 and ABTS2ꢀ on the
HRP inhibition, we carried out some experiments in
which the concentration of one of the substrates (perox-
ide or ABTS2ꢀ) was varied while keeping other one con-
stant (Figs. S1 and S2, supporting information). The
Lineweaver–Burk plots obtained by increasing the
H2O2 concentration reveal that the inhibition of HRP
by ebselen is different from that of EbSe2 and EbTe2.
While the H2O2 concentration does not have much effect
on the inhibition of HRP by ebselen, the inhibition by
EbSe2 and EbTe2 appears to be reversed by increasing
the peroxide concentration.
a
c
4
0
0
500
1000
1500
2000
1/[GSH], M-1
Figure 2. Lineweaver–Burk plots for the reduction of H2O2 by ebselen
and its derivatives (a, Eb; b, EbMe; c, EbBz).
Recently, it has been shown that Fe-containing peroxi-
dases such as lactoperoxidase can be inhibited by orga-
noselenium compounds that exhibit significant
glutathione peroxidase-like antioxidant activities.7
Therefore, ebselen may inhibit HRP by reducing hydro-
gen peroxide or by reacting with the oxidized enzyme,
because ebselen is a well-known GPx mimic that reduces
hydrogen peroxide and organic peroxides by using thiol
co-substrates.2,3 To this end, we have investigated the
GPx activity13 of ebselen and related derivatives
(Fig. 2) and correlated the GPx activity of these com-
pounds with their HRP inhibitory activities. While the
HRP inhibition activities of ebselen and EbTe2 correlate
well with their GPx activities, the methyl (EbMe) and
benzylic (EbBz) compounds, which show significant
GPx activity, do not show any appreciable HRP inhibi-
tion. This indicates that the HRP inhibition by selenium
or tellurium compounds cannot be directly correlated
with their GPx-like behaviour, since the GPx activity
of selenium/tellurium compounds also depend on the
reactivity of these compounds with thiols in addition
to their reactions with peroxide. It has been shown that
the GPx activity of selenium compounds not only de-
pends on the reactivity of the selenol intermediates to-
wards hydrogen peroxide, but also depends on the
reactivity of the selenenyl sulfide intermediates towards
thiols.3a As there is no thiol present in the HRP inhibi-
tion experiments, only the reactivity of the selenium/tel-
lurium compounds towards hydrogen peroxide may
determine the HRP inhibition potency of the test
compounds.
Table 1. Antioxidant profile of ebselen and its related derivatives
a
1O2+substrate k (·106 Mꢀ1 sꢀ1
)
Lipid peroxidation
IC50, 280 Gy
GPx activity
Km (·10ꢀ3
GPx activity
Vmax (lM minꢀ1
)
HRP inhibition (IC50
)
(lM)
)
Eb
4.16 0.12
14.7 3.3
25 lM
100 lM
75 lM
19% at 200 lM
175 lM
13.15
5.25
2.2
182.9
37.5
39.5
—
16.9 1.4
>100
>100
MeEb
BzEb
EbSe2
EbTe2
6.65 0.25
Does not scavenge at 1 mM
9.9 0.41
—
50.0 1.0
17.3 1.7
2.3
31.2
a The IC50 value is the concentration at which lipid peroxidation is inhibited by 50%.

5336
B. Mishra et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5334–5338
In order to find out the effect of ebselen and related
derivatives on lipid peroxidation and singlet oxygen,
we have carried out singlet oxygen quenching and lipid
peroxidation experiments.14,15 The extent of c-radia-
tion-induced lipid peroxidation in liposomes was moni-
tored as thiobarbituric acid reactive substances
(TBARS) in the absence and the presence of different
concentration of ebselen and its derivatives. The perox-
idation was found to be inhibited in the presence of
ebselen and related compounds. At a constant c-radia-
tion dose of 280 Gy, inhibition of peroxidation was fol-
lowed at different concentration (0.025–0.150 mM) of
ebselen and its derivatives. The TBARS formation was
found to decrease with increasing concentration of ebse-
len and its derivatives. As shown in Figure 3, ebselen is
found to be the most potent antioxidant having IC50 val-
ue of 25 lM. In the series, ebselen diselenide (EbSe2)
was found to be the least effective. It showed only 19%
protection to the liposomes at 200 lM. The IC50 values
for EbMe, EbBz and EbTe2 were found to be 100, 75,
and 175 lM, respectively. When aqueous liposomal
solutions were exposed to c-radiation, the hydroxyl rad-
icals produced during water radiolysis, induced lipid
peroxidation in the liposomes.16 Inhibition by ebselen
derivatives may be due to the reaction with either
hydroxyl radicals or peroxyl radicals produced during
lipid peroxidation. Figure 3 shows the inhibition of
TBARS formation by ebselen and its derivatives at a
given concentration (50 lM).
bly due to their great sensitivity to light and air. Howev-
er, in recent years, the biological importance of
tellurium has attracted considerable attention, as evi-
denced by several model studies on the antioxidant
and photochemotherapeutic properties of synthetic
organotellurium compounds.17
The higher efficiency of ebselen as compared to that of
its sulfur analogue in the prevention of lipid peroxida-
tion initiated via radicals, on the thiol reduction of ferric
cytochrome c and reduction of peroxynitrite are signifi-
cant examples where, the redox properties of selenium
plays an important role.6a However, basic structural
modification in organoselenium compound can also al-
ter their redox activity. Therefore, we carried out singlet
oxygen quenching studies in the presence of different
ebselen derivatives. The energy of the singlet oxygen is
22.5 kcal/mol above the ground state of the triplet oxy-
gen.18 Depending upon the substrates, singlet oxygen re-
acts either by electron transfer or by addition across the
double bond to form endoperoxides.18 The latter reac-
tion is found to be crucial step in lipid peroxidation. Sin-
glet oxygen in absence of any quencher has a lifetime of
66.0 1.0 ls in dicholoroethane. However, in the pres-
ence of different concentration of ebselen and its deriva-
tives, the lifetime of the singlet oxygen was found to
decrease. The bimolecular rate constant for the reaction
between singlet oxygen and ebselen derivatives (Eq. 2)
was calculated from the Stern–Volmer plots (Fig. 4).
3
1O2 ! O2 þ hm ð1270 nmÞ
ð1Þ
The higher activity of the organotellurium compound
(EbTe2) as compared to the ebselen diselenide derivative
(EbSe2) indicates that peroxide-decomposing capacity as
well as chain-breaking ability play role to inhibit c-radi-
ation induced lipid peroxidation in liposomes under the
conditions used. These observations are further support-
ed by the singlet oxygen quenching studies. Although
replacement of sulfur by selenium is known to modify
the biological activities considerably, a similar role of
tellurium in biosystems has not been discovered proba-
kq
1O2 þ Ebselen-derivative ! Product
ð2Þ
In these studies, methyl ebselen (EbMe) was found to be
the most active compound with a bimolecular rate con-
stant of 1.47 0.33 · 107 Mꢀ1 sꢀ1 and ebselen diselenide
was found to be the least active, which did not show any
noticeable activity even at 1 mM concentration. Thus,
all the derivatives except ebselen diselenide (EbSe2)
showed singlet oxygen quenching ability and the
quenching rate constant was in the order of
3.5
90
1.0
3.0
2.5
2.0
1.5
1.0
60
30
0
2.0
1.5
d
d
0.8
1.0
blank a
b
c
d
0.5
b
a
0.6
0.4
0.2
0.0
0.00 0.03 0.06 0.09
c
Substrate, mM
c
b
a
0.00
0.05
0.10
0.15
0.20
Ebselen derivatives, mM
0.000
0.001
0.002
0.003
0.004
Figure 3. Effect of different concentration of ebselen and its derivative
(0.025–0.15 mM) on the amount of TBARS formed on c-irradiation-
induced lipid peroxidation of liposomes at 280 Gy. Inset shows the
relative protection of 50 lM of ebselen and its derivative on the c-
radiation-induced lipid peroxidation (a, Eb; b, EbMe; c, EbBz; d,
EbTe2).
Substrate, mM
Figure 4. Stern–Volmer plot for quenching of singlet oxygen by
ebselen and its derivatives (condition: Solvent: dichloroethane Sensi-
tizer: Hypocrellin-A excitation wavelength: 532 nm). a, Eb; b, EbMe;
c, EbBz; d, EbTe2.

B. Mishra et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5334–5338
5337
ꢁ107–106 Mꢀ1 sꢀ1. It has been reported that singlet oxy-
gen reacts with selenium compounds to form the corre-
sponding selenoxide.17 This observation is consistent
with the involvement of a charge transfer complex in
the deactivation of singlet oxygen.19 The singlet oxygen
reacts mainly with the selenium atom and the reactivity
of ebselen and its derivative with singlet oxygen mainly
depends upon the electron density on the chalcogen
atom. In the case of methyl derivative, due to the elec-
tron-donating effect of methyl group, the density on
the selenium atom increases, while in case of the benzyl
derivative the electron-withdrawing effect on the benzyl
group decreases the electron density on the selenium
atom. The rate constant also depends upon the one elec-
tron reduction potential of the radical. In the series,
methyl ebselen has the lowest reduction potential
(E = 1.43 V vs NHE)20 and has high reactivity with the
singlet oxygen, whereas ebselen diselenide has high
reduction potential and has least reactivity. Although
ebselen and its derivatives seem to react with free radi-
cals or singlet oxygen at different rates, their contribu-
tion to the total antioxidant capacity should be
proportional to their respective physiological concentra-
tions. Thus from the rate constant it can be inferred that
very high concentration (ꢁ10ꢀ3 M) of the compounds
are required to scavenge the singlet oxygen efficiently
inside the cells.
Pharmacol. 1985, 34, 1185; (f) Haenen, R. M. M.; De
Rooij, B. M.; Vermeulen, N. P. E.; Bast, A. Mol.
Pharmacol. 1990, 37, 412.
4. (a) Coffey, M. D.; Cole, R. A.; Colles, S. M.; Chisolm, G.
M. J. Clin. Invest. 1995, 96, 1866; (b) Christison, J.; Sies,
H.; Stocker, R. Biochem. J. 1994, 304, 341; (c) Maiorino,
M.; Roveri, A.; Ursini, F. Arch. Biochem. Biophys. 1992,
295, 404.
5. Schewe, T. Gen. Pharmacol. 1995, 26, 1153.
6. (a) Mugesh, G.; du Mont, W. W.; Sies, H. Chem. Rev.
2001, 101, 2125; (b) Nogueira, C. W.; Zeni, G.; Rocha, J.
B. T. Chem. Rev. 2004, 104, 6255.
7. (a) Roy, G.; Nethaji, M.; Mugesh, G. J. Am. Chem. Soc.
2004, 126, 2712; (b) Roy, G.; Mugesh, G. J. Am. Chem.
Soc. 2005, 127, 15207.
8. Battin, E. E.; Perron, N. R.; Brumaghim, J. L. Inorg.
Chem. 2006, 45, 499.
9. (a) Wang, J.-F.; Komarov, P.; Sies, H.; de Groot, H.
Biochem. J. 1991, 279, 311; (b) Wang, J.-F.; Komarov, P.;
Sies, H.; de Groot, H. Hepatology 1992, 15, 1112.
10. Tabuchi, Y.; Sugiyama, N.; Horiuchi, T.; Furusawa, M.;
Furuhama, K. Eur. J. Pharmacol. 1995, 272, 195.
11. Yoshizumi, M.; Kogame, T.; Suzaki, Y.; Fujita, Y.; Kyaw,
M.; Kirima, K.; Ishizawa, K.; Tsuchiya, K.; Kagami, S.;
Tamaki, T. Br. J. Pharmacol. 2002, 136, 1023.
12. HRP inhibition was measured by using ABTS2ꢀ as
substrate. The initial rates were determined by following
the decrease in absorbance at 645 nm (due to ABTSÅꢀ) in
the presence and absence of different concentration of
ebselen and its derivatives. Briefly, the solution contained
HRP enzyme (70 nM) mixed with 25 lM ABTS2ꢀ in the
absence and presence of different concentration of seleni-
um compounds (0.06–0.1 mM). The reaction was initiated
by addition of hydrogen peroxide (10 lM). The activity
without test compound was set to 100%.
n summary, we have shown that ebselen and ebselen
ditelluride (EbTe2) inhibit the HRP activity and their
inhibitory potency was found to be much higher than
that of the corresponding methyl and benzyl derivatives.
The oxidation of selenium or tellurium in these com-
pounds may account for their efficient inhibition
properties.
13. Glutathione peroxidase activity was determined by GSH–
GSSG coupled assay by using glutathione reductase
(0.3 U/ml) as ancillary enzyme in the presence of NADPH
(0.25 mM). Different concentrations of glutathione (0.5–
6 mM) in the presence of catalytic amount of ebselen
derivative (0.025 mM) and hydrogen peroxide (1 mM) as
substrate were used to measure the catalytic activity. In
the assay, GSH is oxidized to GSSG, which is reduced
back to GSH by glutathione reductase and NADPH. The
rate of reaction is monitored by following decrease in the
absorbance at 340 nm due to the decrease in the concen-
tration of NADPH in presence of different concentration
of thiols. The absorbance at 340 nm as a function of time
is fitted to an exponential function, which gives the
observed first-order rate constant. From this rate constant
the initial rate (v) was calculated by using 6220 Mꢀ1 cmꢀ1
as the extinction coefficient for NADPH. The kinetic
parameters such as Km and Vmax were calculated from the
double reciprocal plots (Lineweaver–Burk plots) of initial
rate as a function of substrate concentration. For the GPx
activity, the rates were corrected for the background
reaction between H2O2 and GSH. Due to poor solubility
of the organoselenium compounds in water, the com-
pounds were dissolved in methanol. In most of the
experiments, 2% of methanol was added to the buffer
solution to obtain a homogeneous system.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.07.085.
References and notes
1. (a) Yang, C.-F.; Shen, H.-M.; Ong, C.-N. Biochem.
Pharmacol. 1999, 57, 273; (b) Ramakrishnan, N.; Kali-
nich, J. F.; McClain, D. E. Biochem. Pharmacol. 1996, 51,
1443; (c) Parnham, M. J.; Leyck, S.; Kuhl, P.; Schalkwijk,
J.; Van den Berg, W. B. Int. J. Tissue React. 1987, 9, 45.
2. (a) Griffiths, H. R.; Dowling, E. J.; Sahinoglu, T.; Blake,
D. R.; Parnham, M.; Lunec, J. Agents Actions 1992, 36,
107; (b) Hayashi, M.; Slater, T. F. Free Radic. Res.
Commun. 1986, 2, 179; (c) Filipovska, A.; Kelso, G. F.;
Brown, S. E.; Beer, S. M.; Smith, R. A. J.; Murphy, M. P.
J. Biol. Chem. 2005, 280, 24113; (d) Mareque, A. M.; Faez,
J. M.; Chistiaens, L.; Kohnen, S.; Deby, C.; Hoebeke, M.;
Lamy, M.; Deby-Dupont, G. Redox Rep. 2004, 9, 81.
3. (a) Sarma, B. K.; Mugesh, G. J. Am. Chem. Soc 2005, 127,
11477; (b) Rodell, T. C. Crit. Rev. Oxid. Stress Aging
2003, 2, 1344; (c) Arteel, G. E.; Sies, H. Free Radic. Biol.
Med. 1993, 14, 313; (d) Sies, H. Methods Enzymol. 1995,
14. Kinetics of singlet oxygen reactions with ebselen and its
derivatives was studied using transient luminescence
spectrometer (TL900, Edinburgh Instruments, UK). Sin-
glet oxygen was generated by photoexcitation of hypoc-
rellin-A (absorbance at 532 nm ꢁ0.5) in dichloroethane by
using second harmonic (532 nm) of Nd-YAG laser.
Hypocrellin-A is a well-known dye, which generates
singlet oxygen in high yield in dichloroethane. After laser
252, 341; (e) Muller, A.; Gabriel, H.; Sies, H. Biochem.
¨

5338
B. Mishra et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5334–5338
excitation, the decay profile of singlet oxygen emission was
measured by standard Fricke dosimetry. At a total
absorbed dose of 280 Gy in N2O/O2-purged liposomal
solution in absence and presence of the complex at pH
7.4 (phosphate buffer). The extent of lipid peroxidation
was estimated in terms of thiobarbituric acid reactive
substances (TBARS) using 15% w/v trichloroacetic acid,
0.375% w/v Thiobarbituric acid (TBA), 0.25 N hydro-
chloric acid, 0.05% w/v butylated hydroxyl toluene
(BHT) as TBA reagent measuring the absorbance at
532 nm (e532 = 1.56 · 105 Mꢀ1 cmꢀ1).
monitored at 1270 nm with the help of liquid nitrogen
cooled germanium detector. Change in the lifetime of
singlet oxygen was monitored in the presence of different
concentration of (0.1–4 mM) test compound to determine
the singlet oxygen-quenching rate constant as per the
Stern–Volmer equation
sꢀ1 ¼ sꢀ0 1 þ kq½test compoundꢂ;
where s and s0 is the lifetime of the singlet oxygen in pres-
ence and absence of quencher. kq is the bimolecular rate
constant for the quenching of singlet oxygen and the ebse-
len derivative. In this study, it is important to maintain the
concentration of hypocrellin-A constant.
16. von Sonntag, C. The Chemical Basis of Radiation Biology;
Taylor and Francis: London, 1987.
17. Nogueira, C. W.; Zeni, G.; Rocha, J. B. T. Chem. Rev.
2004, 104, 6255, and references therein.
18. Schaap, A. P. In Singlet Molecular Oxygen; Dowden,
Hutchinson and Ross: Strondsburg, PA, 1976.
19. Scurlock, R.; Rougee, M.; Bensasson, R. V.; Evers, M.;
Dereu, N. Photochem. Photobiol. 1991, 54, 733.
20. Schoneich, C.; Narayanaswami, V.; Asmus, K. D.; Sies,
H. Arch. Biochem. Biophys. 1990, 282, 18.
15. Lipid peroxidation studies were carried out in phospha-
tidyl choline liposomes. Lamellar phosphatidyl choline
liposomes of 150 nm size, determined by light scattering
studies, were suspended in pH 7.4 phosphate buffer and
subjected to c-radiation. c-radiolysis was carried out
using 60Co c-source with a dose rate of 48 Gy/min as