Photoproducts of Indomethacin Exhibit
the
Decreased Hydroxyl
Radical Scavenging and Xanthine Oxidase Inhibition Activities
GI-SHIH LIEN1, CHIEN-SHU CHEN2, WEI-YU CHEN3, SHIH-HAO HUANG4, KUR-TA CHENG5, CHUN-MAO LIN5*, SU-HUI CHAO5*
1 Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei 116, Taiwan, R. O. C.
2 School of Pharmacy, China Medical University, Taichung, Taiwan, R. O. C. 3 Department of Pathology, College of Medicine, Taipei Medical University,
Taipei 11031, Taiwan, R. O. C.
4 Department of Food & Beverage Management, Taipei College of Maritime Technology, Taipei, Taiwan, R. O. C.
5 Department of Biochemistry, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan, R. O. C.
Author for correspondence Tel: 886-2-27361661 ext. 3165; Fax: 886-2-27387348;
E-mail: [email protected]
*These authors contributed equally to the work 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
ABSTRACT
Indomethacin (IN) is a widely used nonsteroidal anti-inflammatory drug (NSAID). In this study, four photoproducts of IN (IN1 through IN4) were produced and isolated from photo-irradiated IN. This study investigated the abilities of IN and its
photoproducts to scavenge hydroxyl radical and inhibit xanthine oxidase (XO). Hydroxyl radical scavenging activity was measured in vitro by electron spin
resonance (ESR) spectrometry using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. The enzyme activity was measured by continuous monitoring uric acid formation with xanthine as a substrate. The results showed that, among all the related products, IN has the strongest hydroxyl radical-scavenging effect, with IC50 = 65 µM, and XO inhibitory effect, with IC50 = 86 µM. To further understand the stereochemistry between these IN derivatives and XO, we performed computer-aided molecular modeling. IN was the most potent inhibitor, showing with the most
favorable interaction in the reactive site. The various photoproducts exhibited affinity toward XO as a result of the absence of hydrogen bonding with molybdopterin domain.
Key words: Indomethacin, Hydroxyl radical, Xanthine oxidase, Electron spin
resonance, Molecular modeling, Photoproduct 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
INTRODUCTION
Reactive oxygen species (ROS), including superoxide anion (O -2
), hydroxyl
radical (HO
) and hydrogen peroxide (H2O2), could initiate inflammation, which contributes to a number of pathological processes(1,2). Xanthine oxidase (XO) is not,only an essential enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid, is but also a critical biological source of ROS(3). An antioxidant with dual roles of ROS scavenging and XO inhibition activity could be beneficial as a protective agent in diseases associated with ROS and XO, such as gout. Indomethacin (IN) has been widely used as an nonsteroidal anti-inflammatory drug (NSAID)
because of its cyclooxygenase (COX) inhibiting inhibitory activity(4). Treatment with NSAIDs is often accompanied by adverse effects such as gastrointestinal damage and platelet dysfunction(5). Recent reports have noted the ability of NSAIDs to eliminate and inhibit free radicals(6). For example, nimesulide and its metabolite
4-hydroxynimesulie were found to protect cartilage against oxidative stress through their ROS-scavenging activity. IN has demonstrated ROS-scavenging activity(7), but has not proved effective in XO inhibition.
Photoproducts of NSAIDs might cause unexpected effects on biological systems(8,9). In our previous studies, photoproducts of IN, including the γ-lactone (IN1), decarboxylated (IN2), methyl ester (IN3), and ethyl ester (IN4) were produced by photo-irradiated synthesis(10). IN3 inhibited PGE2 and NO production, and
expressed iNOS and COX-2 in LPS-stimulated macrophage more effectively than did
IN did(11). However, light energy absorbed by IN may be converted to heat, and photolysis may be accompanied by a thermal reaction when the activation energy is reached. Amber or light-resistant containers are commonly used to prevent a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
photochemical reaction prevention. This study evaluated the HO
-scavenging activity and XO inhibition activity of IN and its photoproducts. We performed sStructure-based molecular modeling was also perform to further understand the stereochemistry between these INs derivatives and XO.MATERIALS AND METHODS
I. Chemicals and reagents
IN, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), ferrous sulfate (FeSO4•7H2O), xanthine oxidase (XO, EC 1.2.3.2.), and xanthine were obtained from Sigma
Chemical Company (St. Louis, MO, USA). Hydrogen peroxide (H2O2) was obtained
from Acros Chemical Company (Morris Plains, NJ). EDTA was obtained from Merck (Darmstadt, Germany). The mMethanol and ethanol used in the study were of HPLC grade (JT Baker, Phillipsburg, NJ).
II. Photodegradation of indomethacin
200 mg of IN were weighed and placed in a 100 mL volumetric flask. Methanol was added to make a concentration of 5.6 mmol/LM (2.0 mg/mL). The solution was transferred to a 100-mL quartz sample vial. A Philips 400 W UV-lamp was used as a light source (HPA 400/30S UV-lamp, Philips, Belgium). Irradiation was performed done with the UV lamp mounted horizontally overhead, 30 cm from the sample for 14 days. IN1, IN2 and IN3 were isolated from the irradiated methanol sample in
methanol. IN4 was isolated from the irradiated ethanol sample in ethanol(8). The chemical structures of IN and its photoproducts are shown in Fig. 1 ( IN, 1-(4-chloro-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
benzoyl)-5-methoxy- 2-methyl-indol-3-acetic acid; IN1, 8-(4-chloro-benzoyl)-3a-hydroxy-5-methoxy-8a-methyl- 2-oxo-2,3,3a,8a-tetrahydro-1H-furo-[2,3-b]-indole; IN2, 1-(4-chloro-benzyl)-5-methoxy- 2,3-dimethyl-indol, IN3; [1-(4-chloro-benzoyl)-5-methoxy-2-indol-3-yl]-acetic acid methyl ester; and IN4, [1-(4-chloro-benzoyl)-5-methoxy-2- methyl-indol-3-yl]-acetic acid ethyl ester (11)).
III. Hydroxyl radical scavenging activity
HO
-scavenging was measured by the electron spin response (ESR) spin-trapping method. The reaction mixture contained 20 µL of 500 mM DMPO, 20 µL of 0.5 mM FeSO4, 20 µL of 1 mM EDTA, and 20 µL of the sample solution. After rapid stirring, the reaction mixture was placed into a capillary ESR tube. Recording of the ESR spectrum was started 40 seconds after the addition of 20 µL of 1 mM H2O2. Deionized water was used instead of the sample solution for the control experiments. The intensities of the DMPO-OH spin signal (secondary peak) in EPR spectrometry were used to evaluate the scavenging activity of the isolated compounds. HO
-scavenging activity was calculated according to the following formula:scavenging activity (%) = (1 – Isample / Icontrol) × 100
where Icontrol and Isample represent the intensity of the signal in the absence and presence of the sample, respectively.
IV. Electron spin resonance spectrometry
ESR detection of the spin adduct was performed detected at room temperature
using on a Bruker EMX-6/1 ESR spectrometer equipped with WIN-EPR Sim-Fonia
software (version 1.2). ESR measurement conditions were as follows: magnetic field, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
347.5±5.0 mT; microwave power, 2.0 mW; modulation frequency, 100 kHz; modulation amplitude, 5 G; and time constant, 0.6 s(12).
V. Xanthine oxidase activity assay
The enzyme activity was determined spectrophotometrically by continuously measuring uric acid formation at 295 nm with xanthine as a substrate. The XO assay consisted of a reaction mixture containing 20 mM
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 38 μM EDTA, 3 u/L XO and 50 μM xanthine. The assay was started by adding the enzyme to the reaction mixture, either with or without inhibitors. The assay mixture was incubated for 3 min at 37℃ and absorbance
readings were taken read every 5 s. The data obtained for the enzyme assays were plotted using Microsoft Office Excel 2010(13).
VI. 3D computational docking modeling
The X-ray crystal structure of bovine xanthine oxidase (XO) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb, PDB code 3NVY), and the C-chain of this protein was used for docking studies of IN and its photoproducts. Small molecules were removed from the C-chain, hydrogen atoms were added, and the resultant protein structure was used in the docking simulation. The 3D structures of compounds were built and optimized by energy minimization using the MM2 force field and a minimum RMS gradient of 0.05 in the software Chem3D 6.0
(CambridgeSoft Corp. Cambridge, MA). Docking simulation was performed using the GOLD 5.0 programon an HP xw6600 workstation with Intel Xeon E5450/3.0 GHz Quadcores as the processors. The GOLD program utilizes a genetic algorithm (GA) to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
perform flexible ligand docking simulations. In the present study, for each of the 30 independent GA runs, a maximum number of 100,000 GA operations were performed on a single population of 100 individuals. Operator weights for crossover, mutation, and migration were set to be 95, 95, and 10, respectively. The GoldScore fitness function was applied for scoring the docking poses of compounds. The docking region was defined to encompass the active site of XO. The best docking solution (with the highest GOLD fitness score) for a compound was chosen to represent the predicted binding mode to the active site of XO(14).
1 2 3 4 5 6 7 8 9 10
RESULTS
Among the oxygen radicals, HO
is the most reactive and severely damages adjacent biomolecules. We investigated the HO
-scavenging abilities of the four photoproducts (IN1 through IN4)) by using ESR spectrometry. HO
was generated by the Fenton reaction and resulted in a four-line ESR spectrum (DMPO-OHadducts). Figure Fig. 2A shows the HO
suppression activity of the isolated photoproducts IN1through IN4 with a concentration of 100 µM. IN1 showed comparable activity to IN, while IN2, IN3, and IN4 did not exhibit substantial suppressive activity in this assay. The formation of the DMPO-OH spin adduct was significantly inhibited by 150 µM IN and was completely inhibited by 250 µM IN. IN and its photoproducts demonstrated HO
-scavenging activity that increased in a dose-dependent manner (Figure Fig. 2B). The results are shown as the inhibition scavenging activity (%). The IC50 was determined from the regression line obtained by the least-squares method, as shown in Table 1. IN had a stronger hydroxyl radical-scavenging effect with IC50 = 65 µM than the related photoproducts. The IC50 of IN1, IN3, and IN4 were 84, 110, and 120 µM, respectively. IN2 did not display significant inhibitory activity against HO
with IC50 > 500 M (Figure. 2C).Table 1 shows the IC50 values for XO inhibition by IN and its photoproducts. IN had the most potent inhibitory effect on XO, with IC50 = 86 µM. The IC50 values of IN2 and IN3 were 93.3 and 88.8 µM, respectively, while IN1 and IN4 did not exhibit a significant inhibitory effect, with IC50 > 100 µM. All of the photoproducts exhibited a weaker inhibitory effect than IN did.
To provide a further insight into the inhibitory effects of IN on XO, and to explain the structure-activity relationships, a molecular model was created for IN docking on XO (Figure Fig. 3). We focused on IN dockings in the molybdopterin domain of XO. In co-crystal structure of salicylate-XO(15) , both carboxylated atoms of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
salicylate were close to the guanidinium group of Arg 880, to the hydroxyl side chain of Thr 1010, and to Glu 1261 via water. The aromatic inhibitor was aligned parallel to the ring of Phe 914. The phenyl of Phe 1009 was aligned perpendicular to the ring of the inhibitor or substrate. Molecular docking studies of IN and its photoproducts into the active site of XO were performed using the docking program GOLD 5.0 and the crystal structure of bovine XO (PDB code 3NVY). IN docking in the molybdopterin domain of XO displayed a similar position and interaction to salicylate binding. The p-chlorobenzoyl of IN had a stacking force with Phe 914, and an indole ring stretched to the space surrounded by several residues including Phe1009, Val1011, Leu648, Leu873, and Leu1014. The carboxylate group of IN can interact with Arg880 by forming a salt bridge that is a combination of electrostatic interaction and hydrogen-bonding (Figure Fig. 3, left panel). The binding energy of IN also proved more potent in XO docking than the other photoproducts (right panel), which is consistent with the results of enzyme activity. The photoproducts might have shown less affinity for XO than IN because of the absence of salt bridge between IN and the molybdopterin domain.
DISCUSSION
NSAIDs are the principal therapeutic agents to treat inflammatory disorders, and inhibit COX, which catalyzes the formation of prostaglandin precursors from
arachidonic acid(16). ROS has been demonstrated to be a mediator of inflammation(17). Thus, drugs with inflammatory activity commonly display ROS-eradicating activity, typically by down-regulating the production of TNFα and iNOS or directly
scavenging ROS. Ikeda et al. demonstrated the superoxide-scavenging activity of IN(6), and Costa et al. have reported the scavenging activity of H2O2(7). The results of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
our study revealed that IN demonstrates HO
-scavenging activity (Figure Fig. 2, Table 1). Other reactive species, including peroxyl radical (ROO
), HO
, O-2 ,
H2O2, and HOCl, as well as the reactive nitrogen species (RNS), nitric oxide (NO), and peroxynitrite anion (ONOO-), also play vital roles in the inflammatory process and pathophysiologies (18). IN counteracts oxidative stress, which accounts for its enhanced efficacy in anti-inflammation.
XO produces uric acid and ROS during the catabolism of purines(3). An excess of the uric acid can lead to gout, which is responsible for oxidative damage to living tissues. NSAIDs are currently the preferred treatment for acute gout attacks, given their effects on the suppression of inflammation coupled with their analgesic properties. Among these drugs, IN is considered the most potent(19-21). The main objective of acute gout therapy is the rapid, safe resolution of pain and the restoration of functional ability. In treatment of chronic gout, XO inhibitors, such as allopurinol, are the most effective in reducing serum urate in patients who are overproducers or underexcretors of uric acid(22-24). The experimental data indicated that IN exhibited an inhibitory effect on XO (Table 1). Furthermore, computer-aided molecular modeling provided an insight into this observation. This finding implies that NSAIDs may contribute polyfunctional curative effects in gout treatment, including those of anti-inflammation, antioxidant and XO inhibition. The photoproducts exhibited less potent activity than the parental IN. This suggests that IN should not be exposed to light during storage because since the photoproducts would exhibit reduced efficacy.
In conclusion, this study was performed to clarify the beneficial effects of IN and its photoproducts in inflammatory diseases where ROS and XO are pathogenically involved. Our results indicate that the anti-inflammatory and analgesic agent IN is an HO
scavenger. IN also binds to the active site of XO to block catalytic activity, and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25results in reduced ROS production. Therefore, IN partially accounts for the reduced anti-inflammatory effects of various photoproducts.
ACKNOWLEDGMENTS
This study was supported by grants from the National Science Council (NSC 101-2320-B-038-023) and Taipei Medical University-Wan
Fang Hospital (99TMU-WFH-02-3). 1 2 3 4 5 6 7 8
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Figure Legends
Figure 1. The structures of IN and its photoproducts (IN1through IN4).
Figure 2. The scavenging activity of IN and its photoproducts against HO
. Equal amounts of IN and the photoproducts IN1 through IN4 (100 µM) were contained in the Fenton reaction for ESR spectrometry (A). ESR spectra of DMPO-OH adduct obtained from the Fenton reaction with IN and IN1 at various concentrations (0-200 M) (B). Inhibitory activities of IN and its photoproducts against the Fenton reaction generated hydroxyl radical (C). Figure 3. The molecular model of indomethacin binding to the active site of xanthineoxidase. The protein structure is represented as a ribbon. IN (yellow) and some interacting amino acid residues (cyan) are shown as sticks. Hydrogen atoms are omitted for clarity. The red dashed lines indicate salt bridge interactions (left panel). GOLD fitness scores of indomethacin ant its photoproducts were obtained using the GOLD 5.0 program (right panel).
Table 1. The hydroxyl radical scavenging and XO inhibition activities of IN and its photoproducts
Compounds Scavenging activity (IC50, µM) XO inhibition activity (IC50, µM) IN 65 86 IN1 84 > 100 IN2 500 93.3 IN3 110 88.8 IN4 120 > 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Fig. 1. N H3CO C O Cl 2 3 3a 7a 4 5 6 7 9 1' 2' 3' 4' 5' 6' O O HO CH3 1 8a N H3CO R C O Cl CH3 2 3 3a 7a 4 9 5 8 6 7 1' 2' 3' 4' 5' 6'
INR= CH2COOHIN2R=CH3IN3R=
CH2COOCH3IN4R= CH2COOC2H5 IN1
1 2 3 4 5 6 7
Fig. 2A. Control IN IN1 IN2 IN3 IN4 15 G 1 2 3 4 5 6 7
Fig. 2B. Control 100 µM 150 µM 200 µM IN IN1 1 2 3 4 5 6 7 8
Fig. 2C. 1 2 3 4 5 6 7 8 9
Fig. 3. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
