Some Anti-Inflammatory Agents Inhibit Esterase
Activities of Human Carbonic Anhydrase Isoforms
I and II: An
In Vitro Study
Zuhal Alım1, Namık Kılıncß1, Mehmet M. _Isßg€or2, B€ulent Sßeng€ul1and Sß €ukr€u Beydemir1,*
1
Department of Chemistry, Faculty of Sciences, Atatu¨rk University, 25240 Erzurum, Turkey
2
Department of Biochemistry, Faculty of Veterinary Sciences, Mustafa Kemal University, 31000 Hatay, Turkey *Corresponding author: S߀ukr€u Beydemir,
beydemir@atauni.edu.tr
Carbonic anhydrases (CAs) are known as a drug–target enzymes. The inhibitors of the enzyme are important compounds for discovering new therapeutic agents and understanding in detail protein–drug interactions at the molecular level. For this purpose, the in vitro effects of some anti-inflammatory agents such as ten-oxicam, fluorometholone acetate, and dexamethasone were investigated on esterase activity of human eryth-rocyte CA-I and CA-II in this study. hCA-I and hCA-II were purified by affinity chromatography with a yield of 47.25% and 87%, and a specific activity of 642.8 EU/ mg proteins and 5576.9 EU/mg proteins, respectively. SDS-PAGE was performed to determine the purity of the enzymes. Inhibitory effects of the drugs on hCA-I and hCA-II were determined by spectrophotometric method. IC50values for hCA-I and hCA-II were 0.198,
2.18, 11.7, 0.11, 17.5 and 14lM using tenoxicam,
fluorometholone acetate, and dexamethasone, respec-tively. For fluorometholone acetate and dexametha-sone, Ki values from Lineweaver–Burk plots were
obtained as 1.044 and 21.2lM (noncompetitive) for hCA-I and 9.98 and 8.66lM(non-competitive) for
hCA-II. In conclusion, tenoxicam, fluorometholone acetate, and dexamethasone showed potent inhibitory effects on esterase activity of hCA-I and hCA-II isozymes underin vitro conditions.
Key words: anti-inflammatory agents, carbonic anhydrase, dexamethasone, inhibition, tenoxicam, fluorometholone ace-tate
Received 6 November 2014, revised 13 January 2015 and accepted for publication 11 March 2015
Carbonic anhydrase or carbonate dehydratase (EC 4.2.1.1) is a type of zinc-containing enzyme that rapidly catalyzes the interconversion of carbon dioxide to bicarbonate. This
reaction is needful for many basic physiological and patho-physiological processes such as respiration, pH and CO2 homeostasis, electrolyte secretion in variety of tissues and organs, bone resorption, calcification, biosynthetic reac-tions (such as gluconeogenesis and lipid and urea synthe-sis), and photosynthesis (1–3). Sixteen isoenzymes have been identified in mammals until now. These isoenzymes show difference with regard to their subcellular localization, catalytic activity, and sensibility to different classes of inhibi-tors and activainhibi-tors. Thus, carbonic anhydrase isozymes perform different functions at their specific locations, and their absence or malfunction can lead to diseased states (4). CAII is the most active of these isoenzymes. Moreover, CAI and CAII are the two major CA isoenzymes present in the cytosol in erythrocytes at high concentrations (5). Inhibition and activation studies on various enzymes are important for clarification of the mechanisms of enzyme catalysis and designating new drugs (4,6,7). Inhibitors and activators of CAs were used in therapeutic applications in many diseases. For example, CAI and CAII inhibitors are used for the treatment of glaucoma and epilepsy, or as diuretic drugs (6). For instance, acetazolamide and brinzo-lamide are used for the treatment of glaucoma (8), and to-piramate is used for the treatment of epilepsy (9) and acetazolamide and methazolamide are used as diuretic drugs. Our laboratory has been obtained on the drug– enzyme interaction studies, in particular CAs (4,6,7,10–16). For example, our group synthesized novel pyrazole carb-oxamide derivatives and investigated in vitro inhibitory effects of these compounds on human CA-I and CA-II iso-zymes in the previous study (7). In another study, we examined in vitro inhibitory effects of some antibiotics (cef-triaxone sodium, imipenem, and ornidazole) on human erythrocyte CAI and CAII. These drugs exhibited inhibitory effects on the enzymes (5). Additionally, Coban et al. reported that sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I-XIV. They investigated in vi-tro and in vivo effects of sildenafil citrate on recombinant CA isozymes (17).
Tenoxicam, fluorometholone acetate, and dexamethasone were selected to observe the in vitro inhibitory effects on human erythrocyte carbonic anhydrase I and II isozymes in this study.
Tenoxicam is a non-steroidal anti-inflammatory drug in the oxicam group (18). Its effects are generally believed to be mediated by the inhibition of cyclooxygenase and subsequent prostaglandin formation. Tenoxicam is used for the treatment of chronic rheumatic disorders such as rheumatoid arthritis, osteoarthritis, and ankylosing spon-dylitis (19). Fluorometholone acetate and dexamethasone are corticosteroid drugs. They prevent the release of substances in body that cause inflammation (20). In our study, we found that the activities of human erythro-cyte carbonic anhydrase I and II isozymes were reduced by very low concentrations of these drugs.
Methods and Materials
ChemistryCNBr-activated Sepharose 4B, protein assay reagents, and chemicals for electrophoresis were obtained from Sigma-Aldrich Co. (Sigma-Sigma-Aldrich Chemie GmbH, Sweden). Para-aminobenzene sulfonamide and L-tyrosine were from E. Merck (Merck KGaA, Darmstadt, Germany). All other chemi-cals used were of analytical grade and were obtained from either Sigma-Aldrich or Merck. Tenoxicam, fluorometholone acetate, and dexamethasone were obtained from local phar-maceutical manufacturing companies.
Purification of carbonic anhydrase isozymes from human erythrocytes by affinity chromatography
Fresh human blood samples were obtained from the blood centre of the research hospital at Ataturk University, and hemolysate of erythrocytes was obtained from these samples according to the previous study (5). The hemoly-sate was centrifuged at 11 5009 g for 30 min at 4 °C. The pH of the hemolysate was adjusted to 8.7 with solid Tris. Sepharose-4B L-tyrosine affinity chromatography col-umn was prepared according to our previous studies (21). The pH-adjusted human erythrocyte hemolysate was applied to the Sepharose 4B-L-tyrosine-sulfanilamide affin-ity column preequilibrated with 25 mM Tris–HCl/0.1 M Na2SO4 (pH 8.7). All procedures were performed at 4°C (5).
During purification by affinity chromatography was mea-sured the absorbance at 280 nm for each elution. Thus, the presence of protein was determined in the elution
tubes. Subsequently, CO2 hydratase activities were assayed for each protein-containing tube (22,23).
CA enzyme activity assay
The activity of CA isozymes can be assayed by two differ-ent methods: the first is CO2 hydratase activity, which is the physiological activity of the CA, and the second one is the esterase activity that can be performed in vitro and fol-lowed spectrophotometrically at 348 nm. The hydratase activity was carried out according to the method described by Wilbur and Anderson (23). This activity was calculated in terms of enzyme unit (EU) using the equation: (t0 tc)/ tc, where t0and tcare the times change in pH of the non-enzymatic and the non-enzymatic reactions, respectively. On the other hand, CA enzyme uses p-nitrophenyl acetate as a substrate esterase activity in in vitro cases. Esterase activity of the enzyme was assayed as increase in absor-bance at 348 nm (24).
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
After the purification of CA isozymes from human erythro-cytes, purity of the enzyme was verified by 3–8% discon-tinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis in our previous studies (5,16,25).
Protein determination
Protein quantity was determined at 595 nm spectrophoto-metrically according to the Bradford method during the purification steps using bovine serum albumin as the stan-dard (26).
In vitro inhibition studies
Carbonic anhydrase activities were assayed by following the changes in absorbance at 348 nm spectrophotometri-cally. The esterase activity method was used for inhibition studies. 4-nitrophenyl acetate (NPA) is the substrate in this method. We screened the conversion from 4-nitrophenyl acetate (NPA) to 4-nitrophenylate over a period of 3 min at 25°C in the spectrophotometer. The enzymatic reaction contained 50 mM Tris–SO4 buffer (pH= 7. 4), 3 mM 4-nitrophenyl acetate, and enzyme solution in a total volume of 1 mL. A reference measurement was taken by the same cuvette without enzyme solution. The inhibitory
Table 1: Results of purification of hCA-I and hCA-II from human erythrocytes Purification steps Activity (EU/mL) Total volume (mL) Protein (mg/mL) Total protein (mg) Total activity (EU) Specific activity (EU/mg) Yield (%) Purification factor Hemolysate 200 50 19.1 950.5 10 000 10.52 100 1 hCA-I 450 10.5 0.70 7.35 4725 642.8 47.25 61.1 hCA-II 1450 6 0.26 1.56 8700 5576.9 87 530
effects of tenoxicam, fluorometholone acetate, and dexa-methasone on the activity of purified carbonic anhydrase enzyme from human erythrocyte were tested. These experiments were performed three times for five different drug concentrations. Control activity in the absence of drug was ascertained to be 100%. A percent activity
ver-sus drug concentration graph was plotted and IC50values were calculated from these curves. Kivalues of the drugs were calculated by measuring enzyme activity at three dif-ferent drug concentrations with five difdif-ferent substrate concentrations. Ki constant and inhibition type Linewe-aver–Burk curves were used.
Results and Discussion
Carbonic anhydrases (CAs), a family of zinc metalloen-zyme, were present in almost all tissues. The enzyme converts carbon dioxide to bicarbonate in a reversible reaction, primarily in red blood cells and other animal tissues. Due to the reaction, CA has important role in respiration and transport of CO2/bicarbonate between lungs and other tissues. Additionally, CA acts for pH regulation, CO2 homeostasis, and electrolyte secretion in a variety of tissues and organs. Also biosynthetic reac-tions such as gluconeogenesis and lipid and urea syn-thesis, bone resorption, calcification, tumorigenicity and many other physiological or pathological processes are carried out by CA isozymes (1–3). Carbonic anhydrase isozymes, particularly CAI and CAII, have a critical role in red blood cells (RBCs). The two major CA isozymes (CA-I and CA-II) are present at high concentrations in the cytosol of erythrocytes, and even CA-II has the Figure 1: Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis analysis of purified hCA-I and hCA-II. (Lane 1 is standard proteins (kD); 1, Escherichia coli b-galactosidase (116), rabbit phosphorylase B (97), bovine serum alb€umin (66), chicken ovalbumin (45), and bovine carbonic anhydrase (29) were used as standards. Lane 2, human CA-I and Lane 3, human CA-II).
A
B D
C E
Figure 2: (A–C) Activity% versus drug concentration regression analysis graphs for hCA-I in the presence of different drug concentrations. (D) (for hCA-I): Lineweaver–Burk graph of fluorometholone acetate using three different drug concentrations for the determination of Kiand inhibition type. (E) (for hCA-I): Lineweaver–Burk graph of dexamethasone using three different drug concentrations for the determination of Kiand inhibition type.
highest turnover rate of all CAs (5). RBC is the oxygen transporter for almost all tissues and also transports the CO2to lung. The important reaction is catalyzed by CAs as described previously.
CAs have been purified from many sources till now. This enzyme was first identified in red blood cells of cows. After that it has been isolated from many organisms such as humans, various animals, bacteria, and plant tissues (2,27,28). Many scientists are still studying on activities of carbonic anhydrases and their purification from various sources. Moreover, they perform considerable experiments about three-dimensional structure of the CA and targeting studies on this enzyme (2,17,29,30). Many CA isozymes are well known as therapeutic targets with the potential inhibition or activation effects for the treatment of disorders such as edema, glaucoma, obesity, cancer, epilepsy, and osteoporosis (31). Also, it has been investigated the effects of various chemicals, pesticides, and other toxic molecules on CA activity (5). As an example, it is reported that defi-ciency of carbonic anhydrase II is the leading defect in the syndrome of osteoporosis, cerebral calcification, and renal tubular acidosis (32). Therefore, the effects of inhibition and activation of the drugs on CA activity are vital for patients with CA deficiency. As can be seen here, the inhibitors and activators of CA isozymes have been utilized in the treatment of many diseases particularly in humans and CA is a target enzyme of all xenobiotics including drugs. However, many enzymes may be drug– target such as glucose 6-phosphate dehydrogenase, paraoxonase,
and sorbitol dehydrogenase (10–12,16,33). Drugs and other chemicals can have adverse or good effects on human enzymes as mentioned previously.
Thus, drug–enzyme interaction studies have become impor-tant for scientists all over the world. Particularly, our labora-tory has been specialized on drug–enzyme interaction studies. Several important drug–target enzymes have been purified from tissues of various sources and we investigated the in vitro and in vivo effects of the drugs and other chemi-cals on these enzyme activities in our laboratory. For instance, interaction between carbonic anhydrase and silde-nafil citrate was investigated in our previous study. Sildesilde-nafil citrate is a phosphodiesterase-5 (PDE5) inhibitor widely used for the treatment of erectile dysfunction. Authors found that sildenafil citrate activated the mammalian carbonic an-hydrase isoforms I–XIV. They found that sildenafil citrate was a potent activator for several CA isozymes such as CA-I, CA-VA, and CA-VI (KAs in the range of 1.08–6.54 lM), the activation of CA-I, CA-VA, and CA-VI was slightly less (KAs of 13.4–16.8 lM), the activation of CA-II, CA-IX, CA-XIII, and XIV were 27.5–34.0 lM and also the least activatable CA isozymes were VII and XII (KAs of 72.9–73.0 lM). After that authors gave the sildenafil citrate to Sprague Dawley rats (1 mg/kg body weight) orally. They observed that the drug caused an inhibitory effect in animals in the range of 60– 85% treated at 3–5 h after application; subsequently, it showed activation effect (17). In another study, paraoxonase 1 enzyme, which is closely associated with atherosclerosis, was purified from human serum and examined the in vitro
A
B D
C E
Figure 3: (A–C) Activity% versus drug concentration regression analysis graphs for hCA-II in the presence of different drug concentrations. (D) (for hCA-II): Lineweaver– Burk graph of fluorometholone acetate using three different drug concentrations for the determination of Kiand inhibition type. (E) (for hCA-II): Lineweaver–Burk graph of dexamethasone using three different drug concentrations for the determination of Ki and inhibition type.
effects of some cardiovascular therapeutics such as digoxin, metoprolol tartrate, verapamil, diltiazem, amioda-rone, dobutamine, and methylprednisolone in our labora-tory. Authors found that all drugs used in the in vitro experiments inhibited the enzyme activity at low concentra-tion. Particularly, digoxin had the maximum inhibition rate (14). Additionally, the effects of some intravenous anesthet-ics, analgesanesthet-ics, antibiotanesthet-ics, and calcium channel blockers on human serum PON-1 activity were investigated by our group (10,11,13,15). Possible effects of commonly used some antineoplastic drugs on sorbitol dehydrogenase (SDH), as another drug–target enzyme, were investigated. We observed that the antineoplastic drugs, dacarbazine, methotrexate, epirubicin hydrochloride, calcium folinate, gemcitabine hydrochloride, and oxaliplatin showed inhibitory effects in in vitro, but dacarbazine was the strongest
inhibi-tor for liver SDH enzyme activity compared to the other drugs (16).
In this study, we aimed to determine in vitro inhibitory effects of some anti-inflammatory drugs on human erythro-cyte carbonic anhydrase I and II isozymes. For this, CA isozymes (CA-I and CA-II) were purified from human ery-throcytes by Sepharose-4B L-tyrosine affinity chromatog-raphy. The purification results for CA-I were 47.25% yield, and 642.8 EU/mg specific activity. CA-II was obtained at 87% yield, and 5576.9 EU/mg specific activity. The purifi-cation results are shown in Table 1. This indicates that CA-II has a higher specific activity compared with CA-I. Additionally, purity of enzymes was controlled using SDS-PAGE. Purified CA-I and CA-II isozymes had single bands at 29 kDa (Figure 1). After these purification steps, the
Table 2: IC50and Kivalues for tenoxicam, fluorometholone acetate, and dexamethasone
Anti-inflammatory agents hCA-I IC50 hCA-I Ki Inhibition type hCA-II IC50 hCA-II Ki Inhibition type Tenoxicam 0.198lM – – 0.11lM – –
Fluorometholone acetate 2.18lM 1.044lM Non-competitive 17.5lM 9.98lM Non-competitive Dexamethasone 11.7lM 21.2lM Non-competitive 14lM 8.66lM Non-competitive
A B
D
C
Figure 4: (A–C) Molecular structure of tenoxicam, fluorometholone acetate, and dexamethasone, (D) possible schematic representation of the interaction of tenoxicam with the carbonic anhydrase active site.
in vitro effects of tenoxicam, fluorometholone acetate, and dexamethasone as anti-inflammatory drugs on the ester-ase activity of purified isozymes were investigated. Ester-ase activity is one of the non-physiological reactions under in vitro conditions catalyzed by carbonic anhydrase. This method is based on the principle of hydrolysis of p-nitro-phenyl acetate by CA. Formed p-nitrophenol is determined at 340 nm, spectrophotometrically (24). Thus, tenoxicam, fluorometholone acetate, and dexamethasone showed inhibitory effects on esterase activities of hCA-I and hCA-II. It is well known that IC50and Kivalues are used to deter-mine inhibition levels of the enzymes. In the present study, IC50 values of tenoxicam, fluorometholone acetate, and dexamethasone were found at micromolar level for hCA-I and hCA-II isoenzymes (Figures 2 and 3, Table 2). IC50 values for fluorometholone acetate and dexamethasone are also supported by the Kivalues. The inhibition types of the isozymes were determined as non-competitive from Lineweaver–Burk graphs (Figures 2 and 3, Table 2). As can be seen here, each of the three drugs is the potent inhibitor of the human CA isozymes. However, tenoxicam was more effective inhibitor for each CA isozymes com-pared with fluorometholone acetate and dexamethasone. The inhibition type of the tenoxicam could not be deter-mined experimentally. We considered that tenoxicam had very high inhibitory effect, and thus, it may be linked to the enzyme’s active site. Schematic representation of the pos-sible interaction of tenoxicam with the hCA-II active site is shown in Figure 4. Fluorometholone acetate and dexa-methasone inhibited enzyme activity in a non-competitive manner. A non-competitive inhibitor shows its inhibitory effect by decreasing turnover rate or catalytic activity of the enzyme. These obtained results may help to better understand the details of carbonic anhydrase–drug inter-actions at the molecular level.
Conclusions
In conclusion, human erythrocyte CA-I and CA-II iso-zymes were purified in one simple step with high specific activity. Tenoxicam, fluorometholone acetate, and dexa-methasone showed in vitro inhibitory effects on hCA-I and hCA-II activity at low concentrations. Tenoxicam had the strongest inhibitory effects on isozymes compared with the other drugs. The inhibition of the various enzymes in tissues may cause several disorders, but may be also the opposite. Sometimes, enzyme inhibition may need for the treatment of some diseases. In this regard, enzyme inhibition studies are very critical in the new drug development. It is known that structures of the chemical compounds and drug molecules may be chan-ged by the cytochrome P450 system in the liver and metabolites, which may affect the target enzyme differ-ently. Hence, in vivo studies are necessary in the future to understand the effect of mechanism of the enzyme– drug interaction.
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