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

Inorganica Chimica Acta 363 (2010) 2812–2818

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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

Article history: Received 30 January 2010 Received in revised form 10 March 2010 Accepted 23 March 2010 Available online 27 March 2010 Dedicated to Prof. Animesh Chakravorty on the occasion of his 75th birthday Keywords: Antithyroid drugs Protein nitration Lactoperoxidase Bovine serum albumin Cytochrome c Selenium

a b s t r a c t 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 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 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. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Nitrotyrosine, produced by the enzymatic or non-enzymatic nitration of tyrosine residues in proteins and enzymes has attracted much attention as a biomarker for oxidative and nitrative stress in various inflammatory, allergic and other diseases [1]. Recent studies suggest that the enzymatic nitration of tyrosine residues in proteins take place mainly by heme-peroxidases such as lactoperoxidase (LPO) [2], eosinophil peroxidase (EPO) [3], horseradish peroxidase (HRP) [4] and myeloperoxidase (MPO) [4] in the presence of hydrogen peroxide (H2O2) and nitrite (NO2 ). Furthermore, 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 important for the tyrosine nitration activity of peroxidases. As shown in Fig. 1, the iodination of tyrosine residues in thyroglobulin is catalyzed by the heme-enzyme thyroid peroxidase (TPO) in the presence of H2O2 and iodide [9]. Reports on the peroxidase-catalyzed

* Corresponding author. E-mail address: [email protected] (G. Mugesh). 0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2010.03.048

nitration of tyrosine residues suggest that the mechanism of nitration is similar to that of the iodination reactions as shown in Fig. 1 [2a,4,10]. Detailed mechanistic insights into the inhibition of TPO-catalyzed iodination of L-tyrosine by thiourea-based antithyroid drugs revealed that the antithyroid drugs such as MMI (1) and PTU (3) may block the thyroid hormone biosynthesis in vivo either by coordinating 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 antithyroid drugs and their analogues (1–14) exhibit significant antioxidant activity. These compounds inhibit peroxynitrite (PN)mediated nitration reactions by scavenging PN (Fig. 2) [15]. Although antithyroid drugs are known to inhibit the peroxidasecatalyzed 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

K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818

OI FeIV

Iodination of Thyroglobulin ArH

IO FeIV

H 2 O2

Fe III

ArH

LPO

+

.

NO2-/ H +

OH Fe IV

ArH

pi-cation

. NO

+

2

ArH HN

O C

HO O

O

C

C OH Fe IV

N H

N H

.

+

.

ArH O

O

O

O

C

C

N H

2

.

+

O

N H

. NO

NO 2 OH

Nitration of tyrosine residues

Fig. 1. Mechanism of peroxidase-catalyzed iodination [9] and nitration [2a,4,10] of tyrosine residues in proteins.

S

E Me

N

N

1, E = S (MMI) 2, E = Se (MSeI)

E

H

H

N

N

H

Me

O R 3, R = nPr (PTU) 4, R = Me (MTU)

E N

Me

MeO

serum albumin (BSA) [2,4,8] and cytochrome c [16]. The inhibition of tyrosine nitration was followed by SDS–PAGE and immunoblotting techniques using antibody against 3-nitro-L-tyrosine. Although cytochrome c can catalyze the self-nitration, no such nitration of cytochrome c was observed when the nitration of cytochrome 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 cytochrome 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 LPOcatalyzed nitration of the tyrosine residues in BSA. Particularly, the antithyroid drugs MMI (1), PTU (3) and MTU (4) exhibited almost a complete inhibition of BSA tyrosine nitration. However, the selenium compounds 6, 8 and 12 exhibited very weak inhibition towards the nitration of BSA (Fig. 3A). In contrast, the inhibitory activity of these compounds was completely different for the nitration of cytochrome c. For the LPO-catalyzed nitration of cytochrome 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 cytochrome c [16a]. It should be mentioned that the concentration of inhibitors required for the inhibition of tyrosine nitration in cytochrome 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 different. 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 inhibition 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

E N

7, E = S; 8, E = Se

N

N

11, E = S; 12, E = Se

N

9, E = S; 10, E = Se

E-Me Me N

N

5, E = S 6, E = Se

OMe

MeO

Me

N

2813

OMe

E Me

N

N

13, E = S; 14, E = Se

Fig. 2. Chemical structures of some antithyroid drugs and their analogues 1–14.

peroxidase-catalyzed nitration of free L-tyrosine by the antithyroid drugs and their analogues.

2. Results and discussion 2.1. Inhibition of protein tyrosine nitration 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 effects of antithyroid drugs and their analogues on the LPO-catalyzed protein nitration reactions, we have chosen the nitration of bovine

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 + NO2 - + H2O2; (3), CO2 CO2 CO2protein + LPO + NO2 + H2O2 + MMI (1); (4), protein + LPO + NO2 + H 2O2 + 4; (5), H N H3 N+ LPO + NO2 + H2O2 + 3; (6), protein H3 N + LPO + NO2 +3 H2O2 + 5; (7), proprotein tein + LPO + NO2 + H2O2 + 6; (8) protein + LPO + NO2 + H2O2 + 8; (9) pro+ tein + LPO + NO2 + H2O2 +LPO 12. Assay condition for BSA nitration: BSA (50 lM), LPO (50 nM), NO2 (2 mM) and H-2O2 (1 mM) with or without inhibitors (12.5 lM). H 2O2 , NO2c nitration: O2 N cytochrome c (200 O2N lM), LPO NO Assay condition for cytochrome (522nM), OH NO2 (4 mM)OH and H2O2 (4 mM) with or without inhibitors (300 lM).OH Proteins were 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.

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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 cytochrome c. If the inhibition is due to the interaction of these compounds with the Fe(III)-center of LPO, the nitration of both BSA and cytochrome c should be inhibited. The absence of any noticeable 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 antithyroid drugs and related compounds. The effect of these compounds on the nitration of 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 derivative (3-nitro-L-tyrosine) was observed under initial rate conditions (Fig. S1, Supporting information), we followed the rate of formation of 3-nitro L-tyrosine in the presence and absence of different inhibitors. The IC50 values, which represent the concentrations required 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 antithyroid drugs and their selenium analogues having thione/selone moieties strongly inhibit the LPO-catalyzed nitration of L-tyrosine. Interestingly, the imidazole-based antithyroid agent MMI (1) exhibited remarkable inhibition towards the LPO-catalyzed nitration of tyrosine with an IC50 value of 0.51 ± 0.01 lM. The selenium compound MSeI (2) also showed good inhibitory activity although the IC50 value obtained for this compound (7.30 ± 0.50 lM) was almost 14 times higher than that of MMI. A similar trend in the activ-

CO2-

CO2-

H3 N

CO2H3 N

H3 N +

LPO H 2O2 , NO2

-

O2 N

O2N

OH

OH

NO2 OH

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 (lM)a

Compound

IC50 (lM)a

1, 2, 3, 4, 5 6 7

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 10 11 12 13 14

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

MMI MSeI PTU MTU

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: Ltyrosine (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.

100

% Control Activity

2814

80

60

40

(a)

(b)

(c)

(d)

20 0

5

10

15

20

25

[Inhibitor] (µM) Fig. 4. Effect of antithyroid drugs and their analogues on the inhibition of LPOcatalyzed nitration of L-tyrosine. The formation of 3-nitro-L-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).

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 derivative 4 (MTU) was found to be almost five times more potent than the n-propyl derivative 3 (PTU), which is in contrast to the inhibition of LPO-catalyzed iodination and oxidation reactions in which PTU was found to be more active than MTU [14]. 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 compound 5, in which both the nitrogen atoms are substituted, was found to be almost 45 times higher than that of MMI. Unexpectedly, 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 replacements to the corresponding selones (8 and 10) did not alter the activity significantly. The relatively higher IC50 value for compound 10 (5.5 ± 0.5 lM) as compared to compounds 6 and 8 can be ascribed 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 corresponding thione counterpart, which may overcome the steric effects. 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

Table 2 IC50 values of compounds 1, 3 and 4 for the MPO-catalyzed nitration of L-tyrosine.

R Se Me

N

Se Heat

N

35 oC-40 oC

12 , R = Me 14, R = m-OMe-Bn

Me

N

N

R

6, R = Me 8, R = m-OMe-Bn

Scheme 2. Heat-induced isomerization of compounds 12 and 14 to the corresponding selones 6 and 8 [14d,15].

Se R N

N R'

A: selone

Se R N

2815

N R'

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].

11–14 having N- and S/Se-substitutions. Interestingly, both the Ssubstituted 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 corresponding 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 previously that the conversion of compounds 12 and 14 to the corresponding selones 6 and 8 is due to the heat-induced isomerization in which the migration of methyl or m-methoxybenzyl 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 reaction. The effective inhibition of nitration in the presence of compounds 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 inhibition, 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 compounds 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 confirmed by a large upfield chemical shift of these compounds in the 77 Se NMR spectroscopy (Scheme 3). 2.2. Inhibition of myeloperoxidase (MPO)-catalyzed nitration of Ltyrosine Myeloperoxidase (MPO), a mammalian heme-peroxidase, is abundantly expressed in neutrophils and in certain types of macrophages. MPO is known to participate in some immune defense mechanisms through the formation of microbicidal reactive oxidants as well as diffusible radical species which play important roles in antibacterial activities [17]. In the presence of H2O2 and

Compound

IC50 (lM)a

1, MMI 3, PTU 4, MTU

8.5 ± 0.3 7.2 ± 0.1 7.4 ± 0.1

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: Ltyrosine (1.0 mM), sodium nitrite (1.0 mM), myeloperoxidase (9.3 nM) and hydrogen peroxide (0.6 mM) in phosphate buffer (100 mM), pH 7.5 at 22 °C.

chloride, MPO generates hypochlorous acid (HOCl) as a potent oxidant that exhibits antimicrobial activities. However, under pathological conditions, persistent activation of MPO may lead to adverse effects as the overproduction of HOCl may initiate oxidative damages [18]. The comparison of the crystallographic information 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 specificity of LPO and MPO. Therefore the development of therapeutically useful inhibitors of MPO has attracted considerable research interest [22]. To understand the effect of antithyroid drugs and analogues on other peroxidase-catalyzed nitration reactions apart from LPO, we have carried out myeloperoxidase (MPO)-catalyzed nitration of Ltyrosine. van der Vliet and co-workers have shown that the MPO-catalyzed nitration and oxidation of L-tyrosine was much faster as compared to the reaction catalyzed by LPO [2]. Inhibition of MPO-catalyzed nitration of L-tyrosine in the presence of thioureabased 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 antithyroid 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. 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 imidazole-based compound MMI as compared to the uracil-based compounds 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 interaction of Fe(III) with H2O2 to generate O@Fe(IV) pi-cation, the inactivation 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 successfully than PTU with H2O2, because the hydrogen-bond (hard) basicity pkHB value of MMI (2.11) is much higher than that of PTU

2816

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(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 thiones (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 coordination 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 MTU towards the MPO-catalyzed nitration of L-tyrosine is probably due to the different active site conformations in which the hydrogen 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, further experiments were carried out with different concentrations of H2O2 at a fixed concentration of L-tyrosine, sodium nitrite and 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 completely abolished in the presence of MMI and it could not be recovered 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, substitution at the free N–H group in MSeI with methyl or mmethoxybenzyl groups led to a partial recovery of the LPO activity at higher concentration of H2O2. This can be ascribed to the relatively 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-

(a)

100

105

(b)

80

90 75

60

(c) 40

(d) 20

(e) 0

% LPO Activity

% LPO Activity

tuted selones react with an excess amount of H2O2 to produce selenous 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 previously 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 L-tyrosine, the NO2 concentration was increased up to 500 lM at fixed concentrations of L-tyrosine, LPO and H2O2. Interestingly, the formation of 3-nitro-L-tyrosine was found to increase in a linear fashion over the entire range of 0–500 lM of NO2 (Fig. 6). This is due to the generation of increasing amount of nitrogen dioxide radical (NO2) at higher concentration of NO2 . The enhancement of nitration with an increasing concentration of nitrite has been observed previously for the peroxidase-catalyzed 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)+]. The produced compound I oxidizes NO2 and L-tyrosine to generate NO2 and tyrosyl radical, respectively. The coupling of NO2 with the tyrosyl radical 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 formation of any reactive oxygen species (ROS) such as superoxide anion radical (O2-) during the peroxidase-catalyzed nitration reactions. It should be noted that the formation of superoxide anion radical during the protein nitration in turn may combine with nitric oxide radical (NO) in vivo to generate another strong nitrating agent peroxynitrite (PN, ONOO ). Furthermore, the addition of superoxide dismutase may convert the produced superoxide radical anion to H2O2 and thus may enhance the rate of nitration reaction. Therefore, we have carried out the LPO-catalyzed nitration of L-tyrosine in the presence of superoxide dismutase (SOD) and catalase. Interestingly, the formation of 3-nitro-L-tyrosine was unaffected in the presence of 0.1 nM of SOD as shown in Fig. 7. In contrast, a gradual inhibition of the nitration of L-tyrosine was observed with an increasing concentration of catalase with an IC50 value of 0.03 nM. The addition of catalase reduces the H2O2 present in the assay mixture and thus prevents the formation of oxoferryl pi-cation (compound I). This is in agreement with the previous

60 45 30 15

0

80

160

240

320

400

[H2O2] (µM) Fig. 5. Plot of the %LPO activity for the nitration of L-tyrosine with an increasing concentration of H2O2. Line (a) control activity; (b) 15 lM of MseI (2); (c) 5 lM of compound 8; (d) 5 lM of compound 6; (e) 5 lM of MMI (1). The test mixture with each concentration of inhibitor was incubated for 20 min. Assay condition: Ltyrosine (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.

0 0

100

200

300

400

500

[NaNO2] (µM) Fig. 6. Plot of the %LPO activity for the nitration of L-tyrosine with an increasing concentration of sodium nitrite. The reaction mixture was incubated for 20 min. Assay condition: L-tyrosine (0.3 mM), sodium nitrite (0–500 lM), LPO (20 nM) and H2O2 (0.6 mM) in phosphate buffer (100 mM), pH 7.5 at 22 °C.

K.P. Bhabak, G. Mugesh / Inorganica Chimica Acta 363 (2010) 2812–2818

(a)

100

% LPO Activity

80

60

40

(b)

20 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 concentrations of SOD and catalase.

report on the Hemin- and MPO-catalyzed nitration of bovine serum albumin. Although SOD did not alter the nitration of BSA significantly, 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 peroxidase-catalyzed nitration reactions. 3. Conclusions 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 peroxidase-catalyzed protein nitration reactions. Inhibition of the nitration 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 thione-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 compounds exhibit the inhibition by reducing hydrogen peroxide and do not interfere with the enzyme active site. Furthermore, the inhibition of peroxidase-catalyzed nitration by commonly used antithyroid drugs and analogues may be beneficial in the treatment for hyperthyroidism as the protein tyrosine nitration may inactivate enzyme leading to various disease states. 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

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Aldrich. The sulfur analogues of antithyroid drugs (2-mercapto-1methylimidazole 1; 6-n-propyl-2-thiouracil 3; 6-methyl-2-thiouracil 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 spectrometer. 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. 4.2. LPO-catalyzed nitration assay High performance liquid chromatography (HPLC) experiments were carried out on a Waters Alliance System (Milford, MA) consisting 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 sample injection. The Alliance HPLC System was controlled with EMPOWER software (Waters Corporation, Milford, MA). The nitration assay of L-tyrosine was analyzed by reverse phase HPLC (5 lm, Grace-Vydac column, 4.6  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 nitration 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 mixture was incubated for 20 min at 22 °C before injection. The formation 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 buffer (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 absence and presence of different antithyroid drugs and analogues (12.5 lM). Upon performing the reactions, the mixture was denatured by boiling at 100 °C for 5 min in the presence of sample loading dye and subjected to polyacrylamide gel electrophoresis and immunoblotting analyses. 4.4. Nitration of cytochrome c 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 presence 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 subjected 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 separating 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 membrane was probed with rabbit polyclonal primary antibody against nitro-tyrosine (1:20,000 dilutions) in blocking solution for 2 h followed by incubation with horseradish peroxidase-conjugated donkey polyclonal anti-rabbit IgG secondary antibody (1:20,000

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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 luminol-enhanced chemiluminiscence (ECL, Amersham). 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 immunoblotting experiments. GM acknowledges DST for the award of Ramanna and Swarnajayanti fellowships and KPB thanks Indian Institute of Science for Junior Research Associate fellowship.

[10]

[11]

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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.

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