Activation of Two Different Drugs Used in Alzheimer's Disease Treatment on Human Carbonic Anhydrase Isozymes I and II Activity: an In Vitro Study/Alzheimer Hastaliginin Tedavisinde Kullanilan Iki Farkli Ilacin Insan Karbonik Anhidraz I ve II Izoenzim Aktiviteleri Uzerindeki Aktivasyonu: In Vitro Calisma.
Carbonic anhydrases (CA) (CA, EC 126.96.36.199) are belong to family of metalloenzyme and have 16 isoforms in mammals. They catalyze from the reversible hydration of C[O.sub.2] to the bicarbonate ion and protons and are expressed as pH regulatory enzyme in most tissues especially in erythrocytes. (1-6) Many such CA isozymes which make these processes are important therapeutic targets with the potential to be inhibited/activated for the treatment of diseases such as glaucoma, edema, obesity, osteoporosis, epilepsy and cancer. (2-8) Activation of several these isoenzymes was reported to be a possible therapy for increasing of synaptic efficacy. This increase might represent the new approach for the treatment of Alzheimer's disease (AD). At the same time, it may ensure to improvement aging, spatial learning and memory therapy. (9)
AD is characterized clinically as a progressive dementia. The neurobiological mechanisms influencing the progressive impairments in memory and intellectual performance that are the hallmarks of AD are not well understood. In addition, the levels of several CA isozymes, including human carbonic anhydrase (hCAI), are diminished in patients affected by AD or in the older population. (10)
Several classes of CA activators are known. One of them is histamine. Histamine is an organic compound including nitrogen and both mediates local immune responses and acts as a neurotransmitter. It was reported to increase the activity of CA and to attend the proton shuttling process. (11) Function of CA activators is to bind at the entrance of the enzyme active site, at the same time to ease the proton transfer processes between active site and solvent system. Histidine, phenylalanine, sildenafil citrate have been shown to be potential activators of different CA isozymes. D-3,4-dihydroxyphenylalanine; dextrodopa (D-DOPA), L-Tyr, and 4-amino-L-Phe act as perfect activators for CAs like the histamine. But LHis, L-Trp, L-Adrenaline, and dopamine have been demonstrated weak activating effects for different CAs. (2,12-15)
Generally it is known that activators bind to different site from the inhibitors within the enzyme active cavity. (11,16) Also, they participate in facilitated the proton transfer processes between active site and solvent system, shuttling protons with groups which have an appropriate pKa such as the carboxylate groups. (17) Memantine is an antagonist of N-methyl-D-aspartate glutamate receptors as uncompetitively. It is proposed to treat of patients with moderate to severe AD. Additionally, benefits of memantine in AD are reported. (18,19) Memantine was chosen because of its similarity to histamine which is activator of CA isoenzymes (Figure 1). Both compounds have -NH2 group. Donepezil is a drug used in the palliative treatment of AD. It is approved for treatment in patients with mild to moderate AD. (20,21)
In light of the above information, we thought that these drugs could activate hCAI and II isoenzymes. We have purified hCAI and hCAII from human erythrocytes and analyzed the in vitro effects of these drugs memantine (1) and donepezil (2) on these isoenzymes. We used the esterase activity of hCAI and hCAII and 4-nitrophenyl acetate (NPA) as substrate. We are justified in our opinion. Because we found that memantine (1) and donepezil (2) are a potent activator of hCAI and hCAII.
RESULTS AND DISCUSSION
CA purification, assay and activation
We used a simple one step method which is the Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography for the purification of the two CA isozymes. (22,23) These isozymes have important roles in different tissues. (24-29) In many studies, they have been purified from different tissues. Theirs activity have been investigated with various chemicals, pesticides and drugs. (22,23,30-36) In this study, activities of purified hCAI and hCAII isoenzymes from human erythrocytes were determined by using the esterase activity method. And we used NPA as substrate as previous study. (36)
Activator effects of these drugs memantine (1) and donepezil (2) on enzyme activities were tested under in vitro conditions. %Activity / (drug concentration) curves was drawn (Figure 2, 3) and they was used at determination of activation constant ([K.sub.A]) values of the drugs for CAI and II isoenzymes. The [K.sub.A] values of memantine against hCAI was found to be 0.013 [micro]M which whereas that of donepezil was of 1.8 [micro]M. The [K.sub.A] values of memantine against hCAII were found to be 0.045 [micro]M whereas that of donepezil was of 3.7 [micro]M (Table 1).
Histamine (3) which taken as the reference compound have the [K.sub.A] values against hCAI and hCAII of 2 [micro]M, and 125 [micro]M, respectively, being a highly potent activator against both the isoforms (Table 1). (37) The best activator of hCAI is memantine with respective [K.sub.A] of 0.013 [micro]M. Likewise, the best activator of hCAII is memantine with respective [K.sub.A] of 0.045 [micro]M. Donepezil activated hCAI almost the same rate compared with histamine. But it activated hCAII more activated than histamine (Table 1). As shown in Figure 1, memantine has bicyclic structure. Other structures are planar. Memantine has been easily interacted with the amino acids in active site of CA isoenzymes and these isoenzymes have been more active.
Memantine and donepezil acted as perfect activators for CAI and II isoenzymes like LHis, L-Adrenaline, D-DOPA, L-Tyr, and 4-amino-L-Phe. But these two isoenzymes more activated than L-Trp and dopamine have been demonstrated weak activating effects for different CAs. (2,12-15)
We reported here the first study on the activator effects of these drugs memantine (1) and donepezil (2) on the hCA esterase activity. The structures of active substances were shown in Figure 1. Consequently, memantine and donepezil are much more potent compared with histamine. These compounds may be used as leads for developing novel activators. This study will contribute to understand the relationship between CA isoenzymes and AD. Also, it will provide important information for the diagnosis of AD and its treatment.
Sepharose-4B, protein assay reagents, 4-nitrophenylacetate and chemicals for electrophoresis were purchased from Sigma-Aldrich Co. All other chemicals were analytical grade and obtained from Merck.
Purification of carbonic anhydrase
Erythrocytes suspension was obtained from the Blood Center of the Research Hospital at Erzincan University. The red cells were washed twice with 0.9% NaCl, and hemolyzed with 1.5 volumes of ice-cold water. The ghost and intact cells were removed by centrifugation at 3100 g for 30 min at 4[degrees]C. The pH of the hemolysate was adjusted to 8.7 with a solid Tris base, and applied to the prepared Sepharose 4B-L-tyrosine-sulfonamide affinity column equilibrated with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7). (22-25) The hCAI and hCAII isozymes were eluted with 1 M NaCl/25 mM [Na.sub.2]HP[O.sub.4] (pH 6.3) and 0.1 M C[H.sub.3]COONa/0.5 M Na[ClO.sub.4] (pH 5.6), respectively. The absorbance of the protein in the column effluents was determined spectrophotometrically at 280 nm. (22,23,36)
CA activation assay
CA activity was assayed by following the change in absorbance at 348 nm of NPA to 4-nitrophenylate ion over a period of 3 min at 25[degrees]C using a spectrophotometer (Shimadzu UV-VIS) according to the method described by Verpoorte et al. (38) A reference measurement was obtained by preparing the same cuvette without enzyme solution. (39) The activation effects of memantine and donepezil were examined. Different activator concentrations were used. Stock solutions of activators (10 mM) were prepared in distilled-deionized water and dilutions up to 0.1-0.9 [micro]M were done thereafter with the assay buffer. Then, %Activity / (drug concentration) curves was drawn (Figure 2, 3) and they was used at determination of [K.sub.A] values of the drugs for CA I and II isoenzymes.
The [K.sub.A] is defined similarly like the inhibition constant (K.). It is obtained with the help of the classical Michaelis-Menten equation as shown below:
[formula not reproducible]
[[A].sub.f] is the free concentration of activator and can be represented in the form of the total concentration of the enzyme ([[E].sub.t]) and activator ([[A].sub.t]). Because we work at substrate concentrations considerably lower than [K.sub.M] ([S] [much less than] [K.sub.M]), the obtained competitive steady-state equation for determining the activation constant is given by the following equation:
[formula not reproducible]
[[nu].sub.o] represents the initial velocity of the enzyme-catalyzed reaction without activator. (12,25,27)
We determined amount of protein during the purification steps according to the Bradford method. We measure it spectrophotometrically at 595 nm, using bovine serum albumin as the standard. (36,40-43) We have used ten tubes with different concentrations of albumin as shown in Figure 2. Then we mixed them with the Bradford reagent (Coomassie Brilliant Blue G-250) and measured the absorbance at 595 nm. Our unknown sample concentration was defined as [micro]g/[micro]L according to standard curve in Figure 4.
The [K.sub.A] values of memantine against hCAI was found to be 0.013 [micro]M which whereas that of donepezil was of 1.8 [micro]M. The [K.sub.A] values of memantine against hCAII were found to be 0.045 [micro]M whereas that of donepezil was of 3.7 [micro]M (Table 1).
We used the histamine as the reference compound. It has the [K.sub.A] values against hCAI and hCAII of 2 [micro]M, and 125 [micro]M, respectively. For two isoenzymes were reported that it is a highly potent activator. (37) It was reported that histamine attends the proton shuttling process and increases the activity of CA. (12)
Activation of these isoenzymes can be a potential target for drug development because of the physiological relevance of CAs. (2) CA activators may be designed as a derivative for increasing of synaptic efficacy. (37) The pharmacological effects of memantine and donepezil not yet been developed clinically for hCA I and hCA II isoenzymes. Thus, in the near future, the novel therapeutic applications will make for enzyme activators.
Conflict of Interest: No conflict of interest was declared by the author.
(1.) Imtaiyaz Hassan M, Shajee B, Waheed A, Ahmad F, Sly WS. Structure, function and applications of carbonic anhydrase isozymes. Bioorg Med Chem. 2013;21:1570-1582.
(2.) Supuran CT, Vullo D, Manole G, Casini A, Scozzafava A. Designing of novel carbonic anhydrase inhibitors and activators. Curr Med Chem Cardiovasc Hematol Agents. 2004;2:49-68.
(3.) Supuran CT, Scozzafava A. Carbonic anhydrases as targets for medicinal chemistry. Bioorg Med Chem 2007;15:4336-4350.
(4.) Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov. 2008;7:168-181.
(5.) Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors: interactions of phenols with the 12 catalytically active mammalian isoforms (CAI-XIV). Bioorg Med Chem Lett. 2008;18:1583-1587.
(6.) Ozturk Sarikaya SB, Topal F, Senturk M, Gulcin I, Supuran CT. In vitro inhibition of a-carbonic anhydrase isozymes by some phenolic compounds. Bioorg Med Chem Lett. 2011;21:4259-4262.
(7.) Nair SK, Ludwig PA, Christianson DW. Two site binding of phenol in the active site of human carbonic anhydrase II: structural implications for substrate association. J Am Chem Soc. 1994;116:3659-3660.
(8.) Casey JR. Why bicarbonate? Biochem Cell Biol. 2006;84:930-939.
(9.) Sun MK, Alkon DL. Carbonic anhydrase gating of attention: memory therapy and enhancement. Trends Pharmacol Sci. 2002;23:83-89.
(10.) Sultana R, Boyd-Kimball D, Poon HF, Cai J, Pierce WM, Klein JB, Merchant M, Markesbery WR, Butterfield DA. Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD. Neurobiology of Aging. 2006;27:1564-1576.
(11.) Briganti F, Mangani S, Orioli P, Scozzafava A, Vernaglione G, Supuran CT. Carbonic anhydrase activators: X-ray crystallographic and spectroscopic investigations for the interaction of isozymes I and II with histamine. Biochemistry. 1997;36:10384-10392.
(12.) Temperini C, Scozzafava A, Vullo D, Supuran CT. Carbonic anhydrase activators. Activation of isozymes I, II, IV, VA, VII, and XIV with l- and d-histidine and crystallographic analysis of their adducts with isoform II: engineering proton-transfer processes within the active site of an enzyme. Chemistry. 2006;12:7057-7066.
(13.) Abdulkadir Coban T, Beydemir S, Gulcin I, Ekinci D, Innocenti A, Vullo D, Supuran CT. Sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I-XIV. Bioorg Med Chem. 2009;17:5791-5795.
(14.) Nishimori I, Onishi S, Vullo D, Innocenti A, Scozzafava A, Supuran CT. Carbonic anhydrase activators: the first activation study of the human secretory isoform VI with amino acids and amines. Bioorg Med Chem. 2007;15:5351-5357.
(15.) Dessirier JM, Simons CT, Carstens MI, O'Mahony M, Carstens E. Psychophysical and neurobiological evidence that the oral sensation elicited by carbonated water is of chemogenic origin. Chem Senses. 2000;25:277-284.
(16.) Ilies MI, Banciu MD, Ilies MA, Scozzafava A, Caproiu MT, Supuran CT. Carbonic anhydrase activators: design of high affinity isozymes I, II, and IV activators, incorporating tri-/tetrasubstituted-pyridinium-azole moieties. J Med Chem. 2002;45:504-510.
(17.) Briganti F, Iaconi V, Mangani S, Orioli P, Scozzafava A, Vernaglione G, Supuran CT. A ternary complex of carbonic anhydrase: X-ray crystallographic structure of the adduct of human carbonic anhydrase II with the activator phenylalanine and the inhibitor azide. Inorg Chim Acta. 1998:295-300.
(18.) Winblad B, Poritis N. Memantine in severe dementia: results of the 9M-Best Study (Benefit and efficacy in severely demented patients during treatment with memantine). Int J Geriatr Psychiatry. 1999;14:135-146.
(19.) Wilkinson D, Andersen HF. Analysis of the effect of memantine in reducing the worsening of clinical symptoms in patients with moderate to severe Alzheimer's disease. Dement Geriatr Cogn Disord. 2007;24:138-145.
(20.) Winblad B, Black SE, Homma A, Schwam EM, Moline M, Xu Y, Perdomo CA, Swartz J, Albert K. Donepezil treatment in severe Alzheimer's disease: a pooled analysis of three clinical trials. Curr Med Res Opin. 2009;25:2577-2587.
(21.) Birks J, Harvey RJ. Donepezil for dementia due to Alzheimer's disease. Cochrane Database Syst Rev. 2006:CD001190.
(22.) Caglar S, Dilek E, Caglar B, Adiguzel E, Temel E, Buyukgungor O, Tabak A. New metal complexes with diclofenac containing 2-pyridineethanol or 2-pyridinepropanol: synthesis, structural, spectroscopic, thermalproperties, catechol oxidase and carbonic anhydrase activities. Journal of Coordination Chemistry. 2016;69:3321-3335.
(23.) Burmaoglu S, Dilek E, Yilmaz AO, Supuran CT. Synthesis of two phloroglucinol derivatives with cinnamyl moieties as inhibitors of the carbonic anhydrase isozymes I and II: an in vitro study. J Enzyme Inhib Med Chem. 2016;31(Suppl 2)208-212.
(24.) Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov. 2008;7:168-181.
(25.) Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem. 1995;64:375-401.
(26.) Ozensoy O, Arslan O, Sinan SO. A new method for purification of carbonic anhydrase isozymes by affinity chromatography. Biochemistry. 2004;69:216-219.
(27.) (a) Parkkila S, Parkkila AK, Carbonic anhydrase in the alimentary tract. Roles of the different isozymes and salivary factors in the maintenance of optimal conditions in the gastrointestinal canal. Scand J Gastroenterol. 1996;31:305-317. (b) Pastorekova S, Parkkila S, Pastorek J, Supuran CT. Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem. 2004;19:199-229.
(28.) Bulbul M, Hisar O, Beydemir S, Ciftci M, Kufrevioglu OI. The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity. J Enzyme Inhib Med Chem. 2003;18:371-375.
(29.) (a) Svastova E, Hulikova A, Rafajova M, Zat'ovicova M, Gibadulinova A, Casini A, Cecchi A, Scozzafava A, Supuran CT, Pastorek J, Pastorekova S. Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH. FEBS Lett. 2004;577:439-445. (b) Cecchi A, Hulikova A, Pastorek J, Pastorekova S, Scozzafava A, Winum JY, Montero JL, Supuran CT. Carbonic anhydrase inhibitors. Design of fluorescent sulfonamides as probes of tumor-associated carbonic anhydrase IX that inhibit isozyme IX-mediated acidification of hypoxic tumors. J Med Chem. 2005;48:4834-4841.
(30.) Celik I, Camas H, Arslan O, Kufrevioglu OI. The effect of some pesticides on human and bovine erythrocyte carbonic anhydrase enzyme activities in vitro. J Environ Sci Health. 1996;31:2651-2657.
(31.) Vitale AM, Monserrat JM, Castilho P, Rodriguez EM. Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae), Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1999;122:121-129.
(32.) Gervais MR, Tufts BL. Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish. Comp Biochem Physiol A. 1999;23:343-350.
(33.) Hochster RM, Kates M, Quastel JH. Metabolic Inhibitors (ed). Academic Press; New York; 1973:66-82.
(34.) Ozdemir H, Uguz MT. In vitro effects of some anaesthetic drugs on lactoperoxidase enzyme activity. J Enzyme Inhib Med Chem. 2005;20:491-495.
(35.) Christensen GM, Olson D, Riedel B. Chemical effects on the activity of eight enzymes: a review and a discussion relevant to environmental monitoring. Environ Res. 1982;29:247-255.
(36.) Bayram E, Senturk M, Kufrevioglu OI, Supuran CT. In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase isozymes I and II. Bioorg Med Chem. 2008;16:9101-9105.
(37.) Bertucci A, Zoccola D, Tambutte S, Vullo D, Supuran CT. Carbonic anhydrase activators. The first activation study of a coral secretory isoform with amino acids and amines. Bioorg Med Chem. 2010;18:2300-2303.
(38.) Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human carbonic anhydrases B and C. J Biol Chem. 1967;242:4221-4229.
(39.) Innocenti A, Scozzafava A, Parkkila S, Pucceti L, De Simone G, Supuran CT. Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates. Bioorg Med Chem Lett. 2008;18:2267-2271.
(40.) Dilek EB, Kufrevioglu OI, Beydemir S. Impacts of some antibiotics on human serum paraoxonase 1 activity. J Enzyme Inhib Med Chem. 2013;28:758-764.
(41.) Dilek E, Caglar S. Effects of mono and dinuclear copper (II) complexes derived from non-steroidal anti-inflammatory drug naproxen on human serum paraoxanase1 (PON1) activity. Int J Pharm Chem. 2015;5:189-195.
(42.) Dilek E, Polat MF. In Vitro Inhibition Of Three Different Drugs Used In Rheumatoid Arthritis Treatment On Human Serum Paraoxanase 1 Enzyme Activity. Protein Pept Lett. 2016;23:3-8.
(43.) Caglar S, Dilek E, Hamamci Alisir S, Caglar B. New copper (II) complexes including pyridine-2,5-dicarboxylic acid: synthesis, spectroscopic, thermal properties, crystal structure and how these complexes interact with purified PON 1 enzyme, Journal of Coordination Chemistry. 2016;69:321-325.
Erzincan University, Faculty of Pharmacy, Department of Biochemistry, Erzincan, Turkey
(*) Correspondence: E-mail: firstname.lastname@example.org, Phone: +90 530 696 84 60
ORCID ID: orcid.org/0000-0002-3629-5168
Received: 05.09.2016, Accepted: 02.11.2016
Table 1. Activation constants of hCA isozymes I, and II with memantine, donepezil and histamine Compound [K.sub.A] C[micro]M) hCAI hCAII Memantine 0.013 0.045 Donepezil 1.8 3.7 Histamine 2 125 [K.sub.A]: Activation constant, hCA: Human carbonic anhydrase
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||ORIGINAL ARTICLE|
|Publication:||Turkish Journal of Pharmaceutical Sciences|
|Date:||Aug 1, 2017|
|Previous Article:||Antibacterial, Antitubercular and Antiviral Activity Evaluations of Some Arylidenehydrazide Derivatives Bearing Imidazo[2,1-b]thiazole...|
|Next Article:||In Vitro Evaluation of the Toxicity of Cobalt Ferrite Nanoparticles in Kidney Cell/Kobalt Ferrit Nanopartikullerinin Bobrek Hucresi Uzerine...|