Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid drugs and their analogues

Article abstract of DOI:10.1016/j.ica.2010.03.048

In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA) and cytochrome c was studied using the antibody against 3-nitro-l-tyrosine. This study reveals that the thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds also inhibit myeloperoxidase (MPO)-catalyzed nitration of l-tyrosine. A structure-activity correlation study on the peroxidase-catalyzed nitration of l-tyrosine reveals that the presence of thione/selone moiety is important for the inhibition. Although the presence of a free N-H group adjacent to CS moiety is necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not require the presence of any free N-H group for their activity. Furthermore, experiments with different concentrations of H₂O ₂ suggest that the antithyroid drugs and related thiones inhibit the nitration reaction mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the nitration by scavenging H₂O₂ without interacting with the enzyme active site. This assumption is based on the observations that catalase effectively inhibits tyrosine nitration by scavenging H₂O₂, which is one of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration reactions, indicating the absence of superoxide radical anion (O₂^-) during the peroxidase-catalyzed nitration reactions.

Full text of DOI:10.1016/j.ica.2010.03.048

Inorganica Chimica Acta 363 (2010) 2812–2818
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid
drugs and their analogues
*
Krishna P. Bhabak, Govindasamy Mugesh
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their
analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA)
and cytochrome c was studied using the antibody against 3-nitro-L-tyrosine. This study reveals that the
thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These
compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds
Received 30 January 2010
Received in revised form 10 March 2010
Accepted 23 March 2010
Available online 27 March 2010
also inhibit myeloperoxidase (MPO)-catalyzed nitration of
L-tyrosine. A structure–activity correlation
Dedicated to Prof. Animesh Chakravorty on
the occasion of his 75th birthday
study on the peroxidase-catalyzed nitration of -tyrosine reveals that the presence of thione/selone moi-
L
ety is important for the inhibition. Although the presence of a free N–H group adjacent to C@S moiety is
necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not
require the presence of any free N–H group for their activity. Furthermore, experiments with different
concentrations of H2O2 suggest that the antithyroid drugs and related thiones inhibit the nitration reac-
tion mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the
inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the
nitration by scavenging H2O2 without interacting with the enzyme active site. This assumption is based
on the observations that catalase effectively inhibits tyrosine nitration by scavenging H2O2, which is one
of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration
reactions, indicating the absence of superoxide radical anion (O2Å-) during the peroxidase-catalyzed nitra-
tion reactions.
Keywords:
Antithyroid drugs
Protein nitration
Lactoperoxidase
Bovine serum albumin
Cytochrome c
Selenium
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
nitration of tyrosine residues suggest that the mechanism of nitra-
tion is similar to that of the iodination reactions as shown in Fig. 1
Nitrotyrosine, produced by the enzymatic or non-enzymatic
nitration of tyrosine residues in proteins and enzymes has at-
tracted much attention as a biomarker for oxidative and nitrative
stress in various inflammatory, allergic and other diseases [1]. Re-
cent studies suggest that the enzymatic nitration of tyrosine resi-
dues in proteins take place mainly by heme-peroxidases such as
lactoperoxidase (LPO) [2], eosinophil peroxidase (EPO) [3], horse-
radish peroxidase (HRP) [4] and myeloperoxidase (MPO) [4] in
the presence of hydrogen peroxide (H2O2) and nitrite (NO2À). Fur-
thermore, other heme-proteins such as hemoglobin [5], myoglobin
[6], cytochrome c [7] and even free heme/iron [8] can catalyze the
nitration of tyrosine, indicating that the presence of heme is impor-
tant for the tyrosine nitration activity of peroxidases. As shown in
Fig. 1, the iodination of tyrosine residues in thyroglobulin is cata-
lyzed by the heme-enzyme thyroid peroxidase (TPO) in the pres-
ence of H2O2 and iodide [9]. Reports on the peroxidase-catalyzed
[2a,4,10].
Detailed mechanistic insights into the inhibition of TPO-cata-
lyzed iodination of -tyrosine by thiourea-based antithyroid drugs
L
revealed that the antithyroid drugs such as MMI (1) and PTU (3)
may block the thyroid hormone biosynthesis in vivo either by coor-
dinating to TPO-Fe(III) center or by reducing the oxidized iodide
generated by TPO [11,12]. Furthermore, the inhibition studies of
TPO-catalyzed iodination reactions by MMI and PTU suggest that
MMI and PTU act as irreversible inhibitors of TPO and the related
enzyme LPO [13,14]. Very recently, we have shown that antithy-
roid drugs and their analogues (1–14) exhibit significant antioxi-
dant activity. These compounds inhibit peroxynitrite (PN)-
mediated nitration reactions by scavenging PN (Fig. 2) [15].
Although antithyroid drugs are known to inhibit the peroxidase-
catalyzed iodination and oxidation reactions, it is not known
whether these compounds can inhibit the peroxidase-catalyzed
nitration reactions. In this paper, we show for the first time that
antithyroid drugs (1, 3 and 4) and their analogues (5, 6, 8 and
12) protect against peroxidase-catalyzed nitration of proteins.
We also provide some mechanistic details about the inhibition of
* Corresponding author.
E-mail address: mugesh@ipc.iisc.ernet.in (G. Mugesh).
0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.03.048

K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818
2813
serum albumin (BSA) [2,4,8] and cytochrome c [16]. The inhibition
of tyrosine nitration was followed by SDS–PAGE and immunoblot-
ting techniques using antibody against 3-nitro-L-tyrosine.
OI
Iodination of Thyroglobulin
IV
Fe
ArH
Although cytochrome c can catalyze the self-nitration, no such
nitration of cytochrome c was observed when the nitration of cyto-
chrome c was carried out in the absence of LPO at pH 7.5. This is in
agreement with the previous results of Kambayashi et al. that cyto-
chrome c catalyzes the self-nitration at acidic pH [16c]. Similar to
the protection of PN-mediated nitration of BSA, the antithyroid
drugs and analogues exhibited significant inhibition of the LPO-
catalyzed nitration of the tyrosine residues in BSA. Particularly,
the antithyroid drugs MMI (1), PTU (3) and MTU (4) exhibited al-
most a complete inhibition of BSA tyrosine nitration. However,
the selenium compounds 6, 8 and 12 exhibited very weak inhibi-
tion towards the nitration of BSA (Fig. 3A). In contrast, the inhibi-
tory activity of these compounds was completely different for the
nitration of cytochrome c. For the LPO-catalyzed nitration of cyto-
chrome c, although the thione-based antithyroid drugs did not
show any inhibition, the selenium analogues exhibited significant
inhibition (Fig. 3B). Unlike the nitration of BSA, dimerization of
the protein was observed during the nitration of cytochrome c. A
similar dimerization was observed previously by Radi et al. during
the PN-mediated as well as peroxidase-catalyzed nitration of cyto-
chrome c [16a]. It should be mentioned that the concentration of
inhibitors required for the inhibition of tyrosine nitration in cyto-
-
I
.
+
-
NO2/ H+
O
OH
IV
.
H2O2
ArH
+
IV
NO2
III
Fe
Fe
Fe
ArH
ArH
LPO
pi-cation
HN
O
C
HO
O
O
C
C
N
H
N
H
OH
+
IV
Fe
ArH
O
O
C
O
O
C
N
H
N
H
.
+
NO2
NO2
O
OH
Nitration of tyrosine residues
chrome c (300 lM) was much higher than that of BSA (12.5 lM)
which is in accordance with the PN-mediated nitration of these
proteins [15]. The difference in the inhibition of the LPO-catalyzed
nitration of BSA and cytochrome c by the thione-based antithyroid
drugs and their selenium analogues suggests that the mechanisms
of inhibition by these two sets of compounds are completely differ-
ent. This is in agreement with the inhibition of TPO/LPO-catalyzed
oxidation and iodination reactions by antithyroid drugs and their
selenium analogues [13,14]. However, the mechanism of the inhi-
bition of the PN-mediated nitration of protein tyrosine residues by
thiones and selones was found to be identical. In this case, both the
thiones and selones scavenge PN to produce sulfurous (H2SO3) and
selenous acid (H2SeO3) as the final products [15]. Although the
Fig. 1. Mechanism of peroxidase-catalyzed iodination [9] and nitration [2a,4,10] of
tyrosine residues in proteins.
S
E
E
H
O
3,
4,
H
H
Me
Me
Me
N
N
N
N
N
N
R
1,
2,
(PTU)
(MTU)
5,
E = S
E = S (MMI)
R = nPr
6, E = Se
E = Se (MSeI)
R = Me
MeO
OMe
MeO
E
E
Me
N
N
N
N
7, E = S; 8, E = Se
9,
10,
E = S; E = Se
OMe
E-Me
Me
E
Me
N
N
N
N
13, E = S; 14, E = Se
11, E = S; 12, E = Se
Fig. 2. Chemical structures of some antithyroid drugs and their analogues 1–14.
peroxidase-catalyzed nitration of free
drugs and their analogues.
L-tyrosine by the antithyroid
2. Results and discussion
Fig. 3. Immunoblots of the inhibition of LPO-catalyzed tyrosine nitration of BSA (A)
and cytochrome c (B). Lanes: (1), pure protein; (2), protein + LPO + NO À + H O ; (3),
-
-
-
2
2 2
CO2
CO2
CO2
protein + LPO + NO2À + H2O2 + MMI (1); (4), protein + LPOÀ+ NO2À + H2O2 + 4; (5),
2.1. Inhibition of protein tyrosine nitration
protein + LPO + NO À + H2O2 + 3; (6), protein + LPO + NO + H O2 + 5; (7), pro-
H N
H N
H N
3
3
3
2
2
2
tein + LPO + NO2À + H2O2 + 6; (8) protein + LPO + NO2À + H2O2 + 8; (9) pro-
+
tein + LPO + NO2À + H2O2 + 12. Assay condition for BSA nitration: BSA (50
l
l
M),
M).
The enzymatic nitration of protein tyrosine residues was carried
out using lactoperoxidase (LPO), which is readily available in pure
form and has been shown to promote tyrosine nitration in the
presence of H2O2 and NO2 [2]. To understand the inhibitory ef-
fects of antithyroid drugs and their analogues on the LPO-catalyzed
protein nitration reactions, we have chosen the nitration of bovine
LPO
LPO (50 nM), NO2À (2 mM) and H2O2 (1 mM) with or without inhibitors (12.5
-
H2O2, NO2
O2N
O2N
NO2
Assay condition for cytochrome c nitration: cytochrome c (200
l
M), LPO (52 nM),
OH
À
OH
OH
NO2 (4 mM) and H2O2 (4 mM) with or without inhibitors (300
l
M). Proteins were
À
incubated with nitrating mixture and inhibitors for 30 min at 22 °C and then
denatured with loading dye and subjected to gel electrophoresis followed by
immunoblotting analyses.

2814
K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818
inhibition of peroxidase-catalyzed nitration of BSA by compounds
1, 3, 4 and 5 is not surprising, it is interesting to note that these
compounds do not inhibit the LPO-catalyzed nitration of cyto-
chrome c. If the inhibition is due to the interaction of these com-
pounds with the Fe(III)-center of LPO, the nitration of both BSA
and cytochrome c should be inhibited. The absence of any notice-
able inhibition of cytochrome c nitration by compounds 1, 3, 4 and
5 is probably due to their interactions with cytochrome c, which
makes these compounds unavailable for interaction with LPO. It
should be noted that the concentration of cytochrome c (200 lM)
was much higher than that of LPO (52 nM) for the LPO-catalyzed
nitration of cytochrome c.
The difference in the inhibition of LPO-catalyzed nitration of
BSA and cytochrome c by antithyroid drugs and their analogues
prompted us to determine the relative activities of different anti-
thyroid drugs and related compounds. The effect of these com-
100
80
60
40
20
(a)
0
(c)
(b)
(d)
5
10
15
20
25
pounds on the nitration oÀf free
L-tyrosine was studied in the
presence of LPO/H2O2/NO2 system using reverse phase HPLC
method (Scheme 1). As the formation of only mono-nitro deriva-
[Inhibitor] (µM)
Fig. 4. Effect of antithyroid drugs and their analogues on the inhibition of LPO-
catalyzed nitration of -tyrosine. The formation of 3-nitro- -tyrosine was followed
by reverse phase HPLC. The amount of inhibition is expressed as a percent of the
control activity for compounds MMI (a), MTU (b), PTU (c) and MSeI (d).
tive (3-nitro-
(Fig. S1, Supporting information), we followed the rate of forma-
tion of 3-nitro -tyrosine in the presence and absence of different
L-tyrosine) was observed under initial rate conditions
L
L
L
inhibitors. The IC50 values, which represent the concentrations re-
quired to inhibit 50% of the enzyme’s activity, were determined at
various concentrations of the inhibitors. The IC50 values obtained
for different test compounds (1–14) are summarized in Table 1
and the corresponding inhibition curves obtained by plotting the
percentage control activity against the concentration of inhibitors
are given in the Supporting information.
As evident from Table 1 and Fig. 4, the thiourea-based antithy-
roid drugs and their selenium analogues having thione/selone moi-
eties strongly inhibit the LPO-catalyzed nitration of L-tyrosine.
ity was observed previously for the inhibition of PN-mediated
nitration of L-tyrosine [15]. The lesser activity of MSeI as compared
to MMI can be ascribed to the spontaneous aerial oxidation of MSeI
to the corresponding diselenide. The other two antithyroid drugs,
PTU (3) and MTU (4), were also found to be very potent inhibitors
of LPO-catalyzed nitration reactions (Table 1, Fig. 4). However,
these thiouracil-based compounds 3 and 4 were found to be
slightly less active than the imidazole-based compound 1, which
is in agreement with the previous studies on the inhibition of
LPO/TPO-catalyzed iodination reactions [13,14]. The methyl deriv-
ative 4 (MTU) was found to be almost five times more potent than
the n-propyl derivative 3 (PTU), which is in contrast to the inhibi-
tion of LPO-catalyzed iodination and oxidation reactions in which
PTU was found to be more active than MTU [14].
Interestingly, the imidazole-based antithyroid agent MMI (1)
exhibited remarkable inhibition towards the LPO-catalyzed nitra-
tion of tyrosine with an IC50 value of 0.51 0.01
compound MSeI (2) also showed good inhibitory activity although
the IC50 value obtained for this compound (7.30 0.50 M) was al-
most 14 times higher than that of MMI. A similar trend in the activ-
lM. The selenium
l
The replacement of N–H group in MMI by an N–Me substituent
led to a decrease in the inhibitory activity as the IC50 value for com-
pound 5, in which both the nitrogen atoms are substituted, was
found to be almost 45 times higher than that of MMI. Unexpect-
edly, the replacement of sulfur in 5 by selenium (compound 6)
led to a dramatic increase in the inhibitory activity. It should be
noted that the replacement of N–H group in MMI by an N–Me
group reduces the activity of parent compound, whereas such
replacement in the selenium analogue enhances the potency of
parent molecule. This is in agreement with our previous report
on the PN-mediated nitration reactions [15]. The replacement of
methyl group in compound 5 by m-methoxybenzyl group further
reduced the activity as the IC50 values for compounds 7 and 9 were
much higher than that of compound 5. However, similar replace-
ments to the corresponding selones (8 and 10) did not alter the
activity significantly. The relatively higher IC50 value for compound
-
-
-
CO2
CO2
CO2
H3N
O2N
H3N
H3N
O2N
+
LPO
-
H2O2, NO2
NO2
OH
OH
OH
À
Scheme 1. LPO-catalyzed nitration of L-tyrosine in the presence of LPO/H2O2/NO2
system.
Table 1
IC50 values of compounds 1–14 for the LPO-catalyzed nitration of L-tyrosine in the
presence of H2O2 and sodium nitrite.
Compound
IC50
(
l
M)a
Compound
IC50
(l
M)a
10 (5.5 0.5 lM) as compared to compounds 6 and 8 can be as-
cribed to the incorporation of two sterically hindered non-polar
m-methoxybenzyl groups in the imidazole ring that reduces the
solubility of the compound in assay buffer. However, the effect of
sterically bulky m-methoxybenzyl substituent was found to be
more pronounced in the corresponding sulfur analogue 9. This is
probably due to the relatively larger size of selenium atom and
more polarizability of selone moiety as compared to the corre-
sponding thione counterpart, which may overcome the steric
effects.
1, MMI
2, MSeI
3, PTU
4, MTU
5
0.51 0.01
7.30 0.50
7.52 0.01
1.49 0.20
23.10 1.50
0.59 0.03
38.70 0.20
8
9
2.20 0.10
137.00 9.90
5.50 0.50
138.60 4.60
56.60 1.20
119.90 4.80
20.90 0.80
10
11
12
13
14
6
7
a
The IC50 values were determined from inhibition plots obtained by plotting the
percentage control activity against the concentration of inhibitors. The test mixture
with each concentration of inhibitor was incubated for 20 min. Assay condition:
L-
tyrosine (0.3 mM), sodium nitrite (0.3 mM), LPO (20 nM) and H2O2 (0.6 mM) in
phosphate buffer (100 mM), pH 7.5 at 22 °C.
To understand the effect of thione/selone moieties present in
the antithyroid drugs and analogues, we have used compounds

K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818
2815
Table 2
R
IC50 values of compounds 1, 3 and 4 for the MPO-catalyzed nitration of L-tyrosine.
Se
Se
Compound
IC50 (l
M)a
Heat
35 oC-40 oC
Me
R
Me
N
N
N
N
1, MMI
3, PTU
4, MTU
8.5 0.3
7.2 0.1
7.4 0.1
6
12
, R = Me
, R = Me
8, R = m-OMe-Bn
14, R = m-OMe-Bn
a
The IC50 values were determined from inhibition plots obtained by plotting the
percentage control activity against the concentration of inhibitors. The test mixture
with each concentration of inhibitor was incubated for 20 min. Assay condition:
Scheme 2. Heat-induced isomerization of compounds 12 and 14 to the corre-
sponding selones 6 and 8 [14d,15].
L-
tyrosine (1.0 mM), sodium nitrite (1.0 mM), myeloperoxidase (9.3 nM) and hydro-
gen peroxide (0.6 mM) in phosphate buffer (100 mM), pH 7.5 at 22 °C.
Se
Se
chloride, MPO generates hypochlorous acid (HOCl) as a potent oxi-
dant that exhibits antimicrobial activities. However, under patho-
logical conditions, persistent activation of MPO may lead to
adverse effects as the overproduction of HOCl may initiate oxida-
tive damages [18]. The comparison of the crystallographic informa-
tion between the structures of LPO and MPO suggest a considerable
amount of difference in the overall shape of the channels around
the heme group [21]. This may alter the substrate binding specific-
ity of LPO and MPO. Therefore the development of therapeutically
useful inhibitors of MPO has attracted considerable research inter-
est [22].
R
N
N
R'
R
N
N
R'
A: selone
B: zwitterion
2, R = Me; R' = H; 6, R, R' = Me
8, R = Me; R' = m-OMe-Bn
10, R, R' = m-OMe-Bn
Scheme 3. The possible tautomeric structures of compounds 2, 6, 8 and 10. These
compounds exist predominantly in their zwitterionic forms B, which may only have
a partial C–Se double bond character [14d,15].
To understand the effect of antithyroid drugs and analogues on
other peroxidase-catalyzed nitration reactions apart from LPO, we
11–14 having N- and S/Se-substitutions. Interestingly, both the S-
substituted compounds (11 and 13) were found to be relatively
less active as inhibitors of LPO-catalyzed nitration reactions. The
Se-substituted compounds (12 and 14) exhibited some inhibition
although the activities were much lower than that of the corre-
sponding selones 6 and 8, respectively. This is in agreement with
our previous observations on the LPO-catalyzed iodinations and
PN-mediated nitration reactions [14d,15]. It has been shown previ-
ously that the conversion of compounds 12 and 14 to the corre-
have carried out myeloperoxidase (MPO)-catalyzed nitration of
tyrosine. van der Vliet and co-workers have shown that the
MPO-catalyzed nitration and oxidation of -tyrosine was much fas-
ter as compared to the reaction catalyzed by LPO [2]. Inhibition of
MPO-catalyzed nitration of -tyrosine in the presence of thiourea-
L-
L
L
based antithyroid drugs suggest that antithyroid drugs MMI, PTU
and the corresponding methyl analogue MTU exhibit excellent
inhibition potencies (Table 2). However, the IC50 values of MMI,
is almost identical to that of PTU and MTU. This is in contrast to
the LPO-catalyzed nitration reactions (Table 1) in which MMI
exhibited much better activity than PTU and MTU. It should be
noted that the activity of MMI was found to be higher than that
of PTU and MTU for the TPO/LPO-catalyzed iodination as well as
oxidation reactions as reported earlier [13,14]. This study suggests
that the inhibition of peroxidase-catalyzed reactions by the anti-
thyroid drugs depends on the nature of peroxidase used for the
reaction and possibly the strength of binding of drugs with the
Fe(III)-center of the enzyme.
sponding selones
6
and
8
is due to the heat-induced
isomerization in which the migration of methyl or m-methoxyben-
zyl group takes place from selenium center to nitrogen at 35–40 °C
as shown in Scheme 2 [14d,15]. However, the migration was slow
at temperatures below 35 °C and only small amounts of the N,N’-
disubstituted compounds (6 and 8) were generated under the
experimental conditions.
This study suggests that the presence of thione/selone moiety is
important for the inhibition towards LPO-catalyzed nitration reac-
tion. The effective inhibition of nitration in the presence of com-
pounds 6 and 8 and the very weak inhibition in the presence of
compounds 5 and 7 suggest that the presence of a free N–H group
is required for the sulfur compounds to exhibit an efficient inhibi-
tion, whereas the selenium analogues do not strictly require a free
N–H moiety for their activity. The higher activities of selenium
compounds as compared to their sulfur analogues may arise from
their zwitterionic nature. It has been shown previously that com-
pounds 2, 6, 8 and 10 exist predominantly in their zwitterionic
forms in which the selenium atom carries a large negative charge
[14d,15]. The zwitterionic nature of these selones was further con-
firmed by a large upfield chemical shift of these compounds in the
77Se NMR spectroscopy (Scheme 3).
2.3. Mechanistic insights
The mechanism by which antithyroid drugs and their analogues
inhibit the peroxidase-catalyzed nitration is important to explain
the different inhibition properties of these compounds towards
the nitration of L-tyrosine. The inhibition of TPO and LPO has been
extensively studied by different research groups and these results
suggest that MMI and PTU act as irreversible inhibitors of TPO and
LPO as they block the TPO/LPO-Fe(III)-center at the enzyme active
site via thione coordination [11–14]. The higher activity of imidaz-
ole-based compound MMI as compared to the uracil-based com-
pounds PTU and MTU can be explained on the basis of drug
coordination with the Fe(III)-center at the active site of TPO. As
the activation of iron center in TPO proceeds through an interac-
tion of Fe(III) with H2O2 to generate O@Fe(IV) pi-cation, the inacti-
vation of TPO may occur via a competitive coordination of the drug
to Fe(III)-center, which is further assisted by the hydrogen bonding
interactions with a proximal histidine residue at the active site
[14]. Under these conditions, MMI might compete more success-
fully than PTU with H2O2, because the hydrogen-bond (hard) basi-
city pkHB value of MMI (2.11) is much higher than that of PTU
2.2. Inhibition of myeloperoxidase (MPO)-catalyzed nitration of
tyrosine
L-
Myeloperoxidase (MPO), a mammalian heme-peroxidase, is
abundantly expressed in neutrophils and in certain types of macro-
phages. MPO is known to participate in some immune defense
mechanisms through the formation of microbicidal reactive oxi-
dants as well as diffusible radical species which play important
roles in antibacterial activities [17]. In the presence of H2O2 and

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K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818
(ꢀ1.32) [12,19]. Furthermore, the importance of free N–H moiety
in thiourea-based compounds is reflected in their IC50 values for
the inhibition of nitration reactions as the N,N’-disubstituted thi-
ones (5, 7 and 9) were found to be almost inactive as compared
to the parent compound MMI (1). The presence of free N–H group
in MMI, PTU and MTU would assist the stronger binding of the drug
with proximal histidine residue leading to an effective drug coordi-
nation with the Fe(III)-center. As such a hydrogen bonding is not
possible for compounds 5, 7 and 9, these compounds may weakly
bind to the iron center. The comparable activities of MMI, PTU and
tuted selones react with an excess amount of H2O2 to produce sel-
enous acid (H2SeO3) with the elimination of imidazole derivative
as confirmed by NMR and ESI-mass spectral analyses (data not
shown). The formation of selenous acid was also observed previ-
ously in the reaction of MSeI and compounds 6, 8 and 10 with an
excess amount of peroxynitrite [15].
To understand the effect of sodium nitrite concentration on the
nitration of
500 M at fixed concentrations of
estingly, the formation of 3-nitro-
in a linear fashion over the entire range of 0–500 l
L
-tyrosine, the NO2À concentration was increased up to
l
L
-tyrosine, LPO and H2O2. Inter-
-tyrosine was found to increasÀe
M of NO2
L
MTU towards the MPO-catalyzed nitration of
L-tyrosine is probably
due to the different active site conformations in which the hydro-
gen bonding interactions may not play important roles in the
binding of drug with Fe(III)-center. In contrast to the inhibition
of LPO-catalyzed nitration of BSA, the thiones (MMI, PTU and
MTU) do not appear to have any effect on the nitration of
cytochrome c (Fig. 3B) as most of the inhibitors are bound to or
altered/inactivated by cytochrome c.
To understand the difference in the activities of thiones and the
corresponding selones on LPO-catalyzed nitration reactions, fur-
ther experiments were carried out with different concentrations
(Fig. 6). This is due to the generation of increasing amount of nitro-
gen dioxide radical (ÅNO2) at higher concentration of NO2À. The
enhancement of nitration with an increasing concentration of ni-
trite has been observed previously for the peroxidase-catalyzed
À
Å
nitration of tyrosine residues [2,8]. The oxidation of NO2 to NO2
is catalyzed by the intermediates generated in the reaction of
LPO with H2O2. The Fe(III)-center of the enzyme first reacts with
H2O2 in a two electron process to generate the oxoferryl pi-cation
radical known as compound I [O@Fe(IV)+Å]. The produced com-
À
pound I oxidizes NO2 and
L
-tyrosine to generate ÅNO2 and tyrosyl
Å
of H2O2 at a fixed concentration of
L
-tyrosine, sodium nitrite and
radical, respectively. The coupling of NO2 with the tyrosyl radical
inhibitors. It has been reported earlier that the selenium analogue
of antithyroid drug MMI (2) inhibits the LPO-catalyzed iodination
and oxidation reactions by reacting with H2O2, which is entirely
different from that of MMI and other thiourea-based antithyroid
drugs such as PTU and MTU [14]. The LPO-catalyzed nitration with
an increasing concentration of H2O2 reveals that the LPO activity
can be recovered almost completely in the presence of MSeI at
higher concentration of H2O2. In contrast, the activity was com-
pletely abolished in the presence of MMI and it could not be recov-
ered even at very high concentration of H2O2 (Fig. 5). These
observations suggest that, unlike MMI, the selenium analogue
MSeI (2) inhibits the LPO activity mainly by reducing H2O2 and
does not interfere with the enzyme active site. Furthermore, sub-
stitution at the free N–H group in MSeI with methyl or m-
methoxybenzyl groups led to a partial recovery of the LPO activity
at higher concentration of H2O2. This can be ascribed to the rela-
tively slower reactivity of N,N’-disubstituted selones towards
H2O2 as compared to the parent compound MSeI containing a free
N–H group. It should be noted that MSeI and other N,N’-disubsti-
leads to the formation of 3-nitro-L-tyrosine [2a,4,8,10,20].
As most of the heme-containing enzymes are involved in the
activation or the reduction of oxygen, it is important to see the for-
mation of any reactive oxygen species (ROS) such as superoxide
anion radical (O2Å-) during the peroxidase-catalyzed nitration reac-
tions. It should be noted that the formation of superoxide anion
radical during the protein nitration in turn may combine with ni-
tric oxide radical (NOÅ) in vivo to generate another strong nitrating
agent peroxynitrite (PN, ONOOÀ). Furthermore, the addition of
superoxide dismutase may convert the produced superoxide radi-
cal anion to H2O2 and thus may enhance the rate of nitration reac-
tion. Therefore, we have carried out the LPO-catalyzed nitration of
L
-tyrosine in the presence of superoxide dismutase (SOD) and cat-
alase. Interestingly, the formation of 3-nitro- -tyrosine was unaf-
fected in the presence of 0.1 nM of SOD as shown in Fig. 7. In
contrast, a gradual inhibition of the nitration of -tyrosine was ob-
L
L
served with an increasing concentration of catalase with an IC50
value of ꢀ0.03 nM. The addition of catalase reduces the H2O2 pres-
ent in the assay mixture and thus prevents the formation of oxofer-
ryl pi-cation (compound I). This is in agreement with the previous
(a)
100
105
90
75
60
45
30
15
0
(b)
80
60
(c)
40
(d)
20
(e)
0
0
80
160
240
320
400
[H2O2] (µM)
0
100
200
300
400
500
[NaNO2] (µM)
Fig. 5. Plot of the %LPO activity for the nitration of
concentration of H2O2. Line (a) control activity; (b) 15
compound 8; (d) 5 M of compound 6; (e) 5 M of MMI (1). The test mixture with
each concentration of inhibitor was incubated for 20 min. Assay condition:
tyrosine (0.3 mM), sodium nitrite (0.3 mM), LPO (20 nM) and H2O2 (0.6 mM) in
phosphate buffer (100 mM), pH 7.5 at 22 °C.
L
-tyrosine with an increasing
l
M of MseI (2); (c) 5 M of
l
l
l
Fig. 6. Plot of the %LPO activity for the nitration of
concentration of sodium nitrite. The reaction mixture was incubated for 20 min.
Assay condition: -tyrosine (0.3 mM), sodium nitrite (0–500 M), LPO (20 nM) and
H2O2 (0.6 mM) in phosphate buffer (100 mM), pH 7.5 at 22 °C.
L-tyrosine with an increasing
L-
L
l

K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818
2817
Aldrich. The sulfur analogues of antithyroid drugs (2-mercapto-1-
methylimidazole 1; 6-n-propyl-2-thiouracil 3; 6-methyl-2-thiou-
racil 4) were obtained from TCI (Tokyo Kasei, Japan) company.
Methanol was obtained from Merck and dried before use following
standard procedure. All other chemicals were of the highest purity
available. 1H (400 MHz), 13C (100.56 MHz), and 77Se (76.29 MHz)
NMR spectra were obtained on a Bruker 400 MHz NMR spectrom-
eter. Chemical shifts are cited with respect to SiMe4 as internal (1H
and 13C), and Me2Se as external (77Se) standards. Compounds 2
[14a] and 5–14 [14b,15] were synthesized following the literature
procedures.
(a)
100
80
60
40
20
4.2. LPO-catalyzed nitration assay
(b)
High performance liquid chromatography (HPLC) experiments
were carried out on a Waters Alliance System (Milford, MA) con-
sisting of a 2695 separation module, a 2996 photodiode-array
detector and a fraction collector. The assays were performed in
1.8 mL sample vials and a built-in autosampler was used for sam-
ple injection. The Alliance HPLC System was controlled with EM-
POWER software (Waters Corporation, Milford, MA). The nitration
0.00
0.02
0.04
0.06
0.08
0.10
[Inhibitor] (nM)
Fig. 7. Plot of the %LPO activity for the nitration of L-tyrosine with an increasing
concentration of catalase and superoxide dismutase. Line (a) superoxide dismutase;
(b) catalase, IC50 = 0.03 nM. The reaction mixture was incubated for 20 min. Assay
condition: L-tyrosine (0.3 mM), sodium nitrite (0.3 mM), LPO (20 nM) and H2O2
(0.6 mM) in phosphate buffer (100 mM), pH 7.5 at 22 °C with variable concentra-
assay of L-tyrosine was analyzed by reverse phase HPLC (5 lm,
Grace-Vydac column, 4.6 Â 250 mm) using gradient elution
(100% water with 0.1% TFA to 60% water with 0.1% TFA and 40%
acetonitrile with 0.1% TFA over 12 min). In the LPO-catalyzed nitra-
tions of SOD and catalase.
report on the Hemin- and MPO-catalyzed nitration of bovine ser-
um albumin. Although SOD did not alter the nitration of BSA signif-
icantly, the treatment of catalase completely abolished the
nitration of BSA as confirmed by chemiluminescence detection
[8]. These experiments suggest the absence of superoxide anion
radical during the LPO-catalyzed nitration reaction and thus it
may preclude the in vivo generation of peroxynitrite (PN) during
the peroxidase-catalyzed nitration of protein tyrosine residues.
This study further confirms that H2O2 is essential for the peroxi-
dase-catalyzed nitration reactions.
tion assay the test mixture contained L-tyrosine (0.3 mM), sodium
nitrite (0.3 mM), lactoperoxidase (20 nM) and hydrogen peroxide
(0.6 mM) in phosphate buffer (100 mM) of pH 7.5 and the test mix-
ture was incubated for 20 min at 22 °C before injection. The forma-
tion of 3-nitro-L-tyrosine was monitored at 275 nm.
4.3. Nitration of BSA
Bovine serum albumin (BSA) (50 lM) in 50 mM phosphate buf-
fer (pH 7.5) was incubated for 30 min at 22 °C with LPO (50 nM),
sodium nitrite (2 mM) and hydrogen peroxide (1 mM) in the ab-
sence and presence of different antithyroid drugs and analogues
3. Conclusions
(12.5 lM). Upon performing the reactions, the mixture was dena-
This study suggests that the thiourea-based antithyroid drugs
such as MMI, PTU and MTU exhibit effective inhibition towards
LPO- and MPO-catalyzed nitration reactions. These compounds
and some of their sulfur and selenium analogues inhibit the perox-
idase-catalyzed protein nitration reactions. Inhibition of the nitra-
tured by boiling at 100 °C for 5 min in the presence of sample load-
ing dye and subjected to polyacrylamide gel electrophoresis and
immunoblotting analyses.
4.4. Nitration of cytochrome c
tion of free L-tyrosine reveals that the presence of thione/selone
moiety is important for the inhibition. Although the presence of
a free N–H group adjacent to C@S moiety is necessary for most
of the thione compounds to inhibit the LPO-catalyzed nitration,
the corresponding selenium analogues do not require the presence
of free N–H group for their activity. Detailed experiments with the
variation of hydrogen peroxide concentration suggest that the thi-
one-based antithyroid drugs and analogues inhibit the nitration
reaction mainly by the coordination of thione moiety with
Fe(III)-center of enzyme active site. However, the selenium com-
pounds exhibit the inhibition by reducing hydrogen peroxide and
do not interfere with the enzyme active site. Furthermore, the inhi-
bition of peroxidase-catalyzed nitration by commonly used anti-
thyroid drugs and analogues may be beneficial in the treatment
for hyperthyroidism as the protein tyrosine nitration may inacti-
vate enzyme leading to various disease states.
Cytochrome c (0.2 mM) in 50 mM phosphate buffer (pH 7.5)
was incubated for 30 min at 22 °C with LPO (52 nM), sodium nitrite
(4 mM) and hydrogen peroxide (4 mM) in the absence and pres-
ence of different antithyroid drugs and analogues (0.3 mM). Upon
performing the reactions, the mixture was denatured by boiling
at 100 °C for 5 min in the presence of sample loading dye and sub-
jected to polyacrylamide gel electrophoresis and immunoblotting
analyses.
4.5. Electrophoretic analysis
Gel was prepared with 10% and 15% polyacrylamide with 6%
stacking gel for BSA and cytochrome c, respectively. The gel was
run in the running buffer of pH 8.3 with glycine and SDS. After sep-
arating the proteins, the gel was analyzed by immunoblotting
experiments. The proteins were transferred to a PVDF membrane
and the non-specific binding sites were blocked by 5% non-fat
skimmed milk in PBST (blocking solution) for 2 h. Then the mem-
brane was probed with rabbit polyclonal primary antibody against
nitro-tyrosine (1:20,000 dilutions) in blocking solution for 2 h fol-
lowed by incubation with horseradish peroxidase-conjugated don-
key polyclonal anti-rabbit IgG secondary antibody (1:20,000
4. Experimental
4.1. Materials and methods
Lactoperoxidase from bovine milk was purchased from Fluka
Chemical Co. L-tyrosine, 3-nitro-L-tyrosine were obtained from

2818
K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818
Werner’s the Thyroid, 1991, pp. 51–97.;
(c) A. Taurog, M.L. Dorris, D.R. Doerge, Arch. Biochem. Biophys. 315 (1994)
82;
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(e) D.R. Doerge, Xenobiotica 25 (1995) 761;
dilutions) for another 2 h. The probed membrane was then washed
three times with blocking solution with 0.1% Tween 20 after each
step and finally the immunoreactive protein was detected by lumi-
nol-enhanced chemiluminiscence (ECL, Amersham).
(f) A. Taurog, M.L. Dorris, D.R. Doerge, Arch. Biochem. Biophys. 330 (1996) 24;
(g) J. Ruf, P. Carayon, Arch. Biochem. Biophys. 445 (2006) 269.
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Acknowledgement
This study was supported by the Department of Science and
Technology (DST), Government of India, New Delhi, India. The
authors thank Mr. Debasish Bhowmick for his help in immunoblot-
ting experiments. GM acknowledges DST for the award of Ramanna
and Swarnajayanti fellowships and KPB thanks Indian Institute of
Science for Junior Research Associate fellowship.
(b) C. Raby, J.F. Lagorce, A.C. Jambut-Absil, J. Buxeraud, G. Catanzano,
Endocrinology 126 (1990) 1683.
[12] Although there is no definite evidence for the coordination of thione moiety of
the antithyroid drugs to the iron center, this model is given based on the fact
that iron–sulfur coordination is very common in metalloenzymes and
hydrogen bonding of heme ligands with a distal histidine residues appears
to be common in peroxidases. For coordination of antithyroid drugs to iron
center, see: R. Bassosi, N. Niccolai, C. Rossi, Biophys. Chem. 8 (1978) 61.
[13] (a) A. Taurog, Endocrinology 98 (1976) 1031;
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ica.2010.03.048.
(b) H. Englar, A. Taurog, C. Luthy, M.L. Dorris, Endocrinology 112 (1983) 86;
(c) A. Taurog, M.L. Dorris, F.S. Guziec Jr., Endocrinology 124 (1989) 30 (and
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