A new strategy based on pharmacophore-based virtual screening in adenosine deaminase inhibitors detection and in-vitro study
© Bazl et al.; licensee BioMed Central Ltd. 2012
Received: 18 September 2012
Accepted: 14 October 2012
Published: 22 October 2012
Background and the purpose of the study
Adenosine deaminase (ADA) inhibition not only may be applied for the treatment of ischemic injury, hypertension, lymphomas and leukaemia, but also they have been considered as anti- inflammatory drugs. On the other hand according to literatures, ADA inhibitors without a nucleoside framework would improve pharmacokinetics and decrease toxicity. Hence we have carried out a rational pharmacophore design for non-nucleoside inhibitors filtration.
A merged pharmacophore model based on the most potent non-nucleoside inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and natural products were generated and applied for compounds filtration. The effects of filtrated compounds based on pharmacophore and docking studies investigated on ADA by UV and Fluorescence spectroscopy techniques.
Extracted compounds were find efficiently inhibit ADA, and the most potent (2) shows an inhibition constant equal to 20 μM. Besides, Fluorescence spectroscopy studies revealed that enzyme 3D structure bear further change in lower concentrations of compound 2.
3 non-nucleoside inhibitors for ADA are presented. According to obtained results from UV and fluorescence spectroscopy, such interesting pharmacophore template with multiple approaches will help us to extract or design compound with desired properties.
KeywordsAdenosine deaminase Pharmachophore Docking Lead discovery, Inhibitor
Adenosine deaminase (ADA) is a key enzyme in the purine metabolism that hydrolyse adenosine to inosine irreversibly . This path involves in RNA, DNA, ATP synthesizes, and energy transitions reactions. This enzyme also has been found in lymphoid systems like lymph nodes, spleen and thymus . Involvement of this enzyme is clear in catabolytic paths and also its role in the protection of immune systems [3–5].
Over activity of ADA is associated with AIDS, leukemia, stresses and Parkinson [6–8]. In addition, the high value of ADA has been seen in rheumatoid arthritis . Adenosine as the substrate of ADA regulates many of physiological processes in different organisms [10, 11]. Adenosine influences deeply on hypertension, sedation and vessels dilatation . Also, it acts as nerve modulators or as neural hormones .
On the other hand, most of adenosine analogues have more importance in chemotherapy, cancer, immunology, virology and parasitology, which could be deaminated by the enzyme and deactivated through their metabolic pathway . So, inhibition of ADA can solve mentioned problems [15, 16]. Till now, suggested inhibitors have some drawbacks such as irreversibility, side effects, high inhibition constant (Ki) and toxicity on the different cells . Moreover, most of existing nucleoside inhibitors not only have difficulties in their synthesis, but also because of interfering with function of other enzymes they have been deleted from researches pathways despite of appropriate inhibition potency .
Natural sources are receiving increasing attention recent years since they were reported to have a remarkable spectrum of biological activities including antioxidant, anti-inflammatory and anti- carcinogenic activities [19–21]. On the other hand, several methods have been raised in drug discovery such as high throughput screening, docking and QSAR analysis . The designed compounds were more investigated based on sequential filters and at last selected compounds were more studied in biological tests.
Since experimental methods are time consuming, computational techniques such as docking and virtual screening (VS), help researchers to gain effective compounds in shorter time and lower costs . The aim of this project is to develop merged Pharmachophore model based on the most potent non-nucleoside inhibitor EHNA and natural products from ZINC data base compounds which have effective interaction with active site of enzyme. This model was applied for filtration of effective inhibitors for ADA from the in-house data base, and their efficiencies are determined through biological investigations.
Adenosine deaminase (from bovine spleen in 3.2 M ammonium sulfate) was purchased from Sigma (St. Louis, MO, USA). Phosphate buffer 50 mM, pH 7.5, was used as media which is comprised NaH2PO4 and Na2HPO4 and are from Merck. In addition, other material such as solvents, were purchased from this company.
Crystal structure 1KRM from bovine for ADA was extracted as raw enzyme structure with 80% identity to applied enzyme in biological tests. Autogride4.2 and Autodock4.2 were used for calculation of grid maps and docking, respectively. AutoDockTools 1.5.4 was used for preparing input files. In this study the compounds were docked on ADA with the grid-box of 126 Å (x, y and z) with the spacing of 0.375 Å. Docking calculation parameters were set to these values: number of Lamarckian job =100; initial population =100; maximum number of energy evaluations =25×105; maximum generations =27000; mutation rate of 0.02; a crossover rate of 0.80. For better understanding of interactions and access to respective pharmacophore, LIGSCOT3.0 program was used.
Biological tests have been done on synthesized compounds from Tehran University of Medical Sciences in-house library. All of biological samples were prepared with double distilled water. The adenosine stock solution prepared and was used in concentration range of 0.01-0.4 mM. The concentration of enzyme in the assay mixture 50 mM sodium phosphate buffer, was 0.9 nM with a final volume of 1ml. Enzymatic activities were assayed by UV–vis spectrophotometry (Shimadzu-3100, Japan) based on Kaplan Method to follow the decrease in absorbance at 265 nm resulting from the conversion of adenosine to inosine . Determination of enzyme activity in the presence of different concentration of substrate was showed that the increasing concentration of substrate from 10 to 124 μM enhanced the enzyme activity. But this activity reached to zero in the higher concentration of substrate (more than 400 μM). This effect may be due to suicide-like mechanism in higher concentration of adenosine.
Fluorescence study has been done with fluorescent spectroscopy (Hitachi MPF-4, Japan), which its cell length is 1cm. In this method, the exciting wavelength was adjusted in 290 nm and the emission was studied in range of 300–400 nm for possible enzyme 3-D structure changes. In this experiment, different concentrations of inhibitors (5–100 μM) were investigated in the presence of ADA (0.01unit) by final volume of 400 μL with phosphate buffer.
To validate the method in prediction of binding energy and possible interaction between ligands and receptor, at first existing ligand in crystal structure was re-docked in 1KRM. After superimposition RMSD <0.1 was gained and showed that this technique and applied parameters would be applicable for next predictions.
Extraction of compounds from Tehran University of Medical Sciences database
Summary of binding energies, theoretical K i , IC 50 and inhibition constants of new inhibitors for calf intestinal adenosine deaminase
According to importance of enzyme inhibition in treatment of some diseases , in this research we use a new approach in better understanding and filtering of specific ADA inhibitors. After pharmacophore generation, docking was performed in order to rank the in-house compounds on the basis of their ability to form favorable interactions within the active site of ADA. This approach not only helps us to filter compounds according to their similarity to most potent non-nucleoside existing inhibitor, but also helps us to extract compounds with natural template. As it shown in Figure 2, imidazole derivative compounds properly stabilize in the binding pocket of ADA with different kinds of interaction such as, hydrophobic, electrostatic, H-bond and π-π stacking. Previously potent inhibitors with imidazole ring have been reported for ADA inhibition . From Table 1, the calculated ΔG° energies are so low which shows effectiveness of compounds in inhibition. Indeed, resulted Ki from biological tests improves this subject. Among, compound 2 shows better interaction with lower binding energy with enzyme −9.59 kcal mol-1.
ADA is inhibited by three filtrated compounds through competitive mechanism, which is in agreement with docking studies. This mechanism can attribute to size of molecule which able compounds penetrate in enzyme active site and make appropriate interactions. Changing in temperature can affect on the reaction rate and enzyme activity. As can be seen in Table 1, resulted Ki from inhibition investigation was higher than expected from docking studies according to their binding energies. This is probably due to difference in temperature was used in biological test, 37°C which enzyme has maximum activity in, and docking approach which subjected to 25°C according to Gibbs free energy equation. However, calculated binding energies and experimental Ki values are following logical trend (r2=0.99). According to fluorescence intensities for three obtained inhibitors, ADA shows sever structural changes in lower concentration of 2 (10 μM). Florescence, kinetic study and appropriate interaction of compound 2 in enzyme active site have confirmed its potency as competitive ADA inhibitor.
This study has showed the effective application of docking and pharmacophore studies in introducing new inhibitors for target receptor. Designed merged pharmacophore results in extraction of effective compounds from in-house 540 compounds, and subjected them to experimental phase for inhibition evaluations. The resulted three compounds from VS studies showed inhibitory effects through competitive mechanism on ADA. Compound 2 can be further optimized and used as a lead compound in new inhibitor design with more potency.
This work was supported by grant from the Research Council of Tehran University of Medical Sciences.
- Lupidi G, Marmocchi F, Venardi G, Cristalli G: Functional residues at the active site of bovine brain adenosine deaminase. Biochem Mol Biol Int. 1997, 43: 1339-1352.PubMed
- Brady TG, O'Connell WA: Purification of adenosine deaminase from the superficial mucosa of calf intestine. Biochim Biophys Acta. 1962, 62: 216-229. 10.1016/0006-3002(62)90035-5.View ArticlePubMed
- Ford H, Dai F, Mu L, Siddiqui MA, Nicklaus MC, Anderson L, Marquez VE, Barchi JJ: Adenosine deaminase prefer s a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies. Biochemistry. 2000, 39: 2581-2592. 10.1021/bi992112c.View ArticlePubMed
- Wang Z, Quiocho FA: Complexes of adenosine deaminase with two potent inhibitors. Biochemistry. 1998, 37: 8314-8324. 10.1021/bi980324o.View ArticlePubMed
- Fox IH, Kelley WN: The role of adenosine and 2' -deoxyadenosine in mammalian cells. Annu Rev Biochem. 1978, 47: 655-686. 10.1146/annurev.bi.47.070178.003255.View ArticlePubMed
- Cristalli G, Costanzi S, Lambertucci C, Lupidi G, Vittori S, Volpini R, Camaioni E: Adenosine deaminase: functional implications and different classes of inhibitors. Med Res Rev. 2001, 21: 105-128. 10.1002/1098-1128(200103)21:2<105::AID-MED1002>3.0.CO;2-U.View ArticlePubMed
- Murray JL, Loftin KC, Munn CG, Reuben JM, Mansell PWA, Hersh EM: Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocytes from patients with acquired immune deficiency syndrome. Blood. 1985, 65: 1318-1324.PubMed
- Miyahara S, Komori T, Shizuya K, Nomura J: Changes of serum adenosine deaminase activity induced by stress in rats. Human Psychopharm. 1998, 13: 325-327. 10.1002/(SICI)1099-1077(199807)13:5<325::AID-HUP3>3.0.CO;2-7.View Article
- Sparks HV, Bardenheuer H: Regulation of adenosine formation by the heart. Circ Res. 1986, 58: 193-201. 10.1161/01.RES.58.2.193.View ArticlePubMed
- Kuno M, Seki N, Tsujimoto S, Nakanishi I, Kinoshita T, Nakamura K, Terasaka T, Nishio N, Sato A, Fujii T: Anti-inflammatory activity of non-nucleoside adenosine deaminase inhibitor FR234938. Eur J Pharmacol. 2006, 534: 241-249. 10.1016/j.ejphar.2006.01.042.View ArticlePubMed
- Klohs WD, Kraker AJ: Pentostatin: Future directions. Pharmacol Rev. 1992, 44: 459-477.PubMed
- Edmundo C, Antonio Luiz P, Carlos Faria Santos A: Adenosine for vasoreactivity testing in pulmonary hypertension: A head-to-head comparison with inhaled nitric oxide. Resp Med. 2010, 104 (Edmundo C, Antonio Luiz P, Carlos Faria Santos A): 606-611.
- Mewshaw JP, Myrick FT, Wakefield DA, Hooper BJ, Harris JL, McCreedy B, Borroto-Esoda K: Dioxolane guanosine, the active for m of the prodrug diaminopurine dioxolane, is a potent inhibitor of drug-resistant HIV-1 isolates from patients for whom standard nucleoside therapy fails. J Acquir Immune Defic Syndr. 2002, 29: 11-20.View ArticlePubMed
- Glaze RI: Adenosine deaminase inhibitors: Their role in chemotherapy and immunosuppression. Cancer Chemo Pharmacol. 1980, 4: 227-235.
- Ghosh KS, Maiti TK, Debnath J, Dasgupta S: Inhibition of ribonuclease A by polyphenols present in green tea. Proteins. 2007, 69: 566-580. 10.1002/prot.21484.View ArticlePubMed
- Balentine D: Tea and health. Crit Rev Food Sci Nutr. 1997, 37: 691-692. 10.1080/10408399709527796.View Article
- Mitchell BS, Koller CA, Heyn R: Inhibition of adenosine deaminase activity results in cytotoxicity to T lymphoblasts in vivo. Blood. 1980, 56: 556-559.PubMed
- Dreosti IE, Wargovich MJ, Yang CS: Inhibition of carcinogenesis by tea: The evidence from experimental studies. Crit Rev Food Sci Nutr. 1997, 37: 761-70. 10.1080/10408399709527801.View ArticlePubMed
- Lee SK, Mbwambo ZH, Chung H, Luyengi L, Gamez EJ, Mehta RG, Kinghorn AD, Pezzuto JM: Evaluation of the antioxidant potential of natural products. Comb Chem High Throughput Screen. 1998, 1: 35-46.PubMed
- Gautam R, Jachak SM: Recent developments in anti-inflammatory natural products. Med Res Rev. 2009, 29: 767-820. 10.1002/med.20156.View ArticlePubMed
- Elson CE, Maltzman TH, Boston JL, Tanner MA, Gould MN: Anti-carcinogenic activity of d-limonene during the initiation and promotion/progression stages of DMBA-induced rat mammary carcinogenesis. Carcinogenesis. 1988, 9: 331-332. 10.1093/carcin/9.2.331.View ArticlePubMed
- Tang YT, Marshall GR: Virtual screening for lead discovery. Methods Mol Biol. 2011, 716: 1-22. 10.1007/978-1-61779-012-6_1.View ArticlePubMed
- Azizian H, Nabati F, Sharifi A, Siavoshi F, Mahdavi M, Amanlou M: Large-scale virtual screening for the identification of new Helicobacter pylori urease inhibitor scaffolds. J Mol Model. 2011, 18: 2917-2927.View ArticlePubMed
- Ataie G, Safarian S, Divsalar A, Saboury AA, Moosavi-Movahedi AA, Ranjbar B, Cristalli G, Mardanian S: Kinetic and structural analysis of the inhibition of adenosine deaminase by acetaminophen. J Enz Inhib Med Chem. 2004, 19: 71-78. 10.1080/14756360310001632741.View Article
- Porter DJ, Abushanab E: Kinetics of inhibition of calf intestinal adenosine deaminase by (+)- and (−)-erythro-9-(2-hydr oxy-3-nonyl)adenine. Biochemistry. 1992, 35: 8216-8220.View Article
- Alijanianzadeh M, Saboury AA, Ganjali MR, Hadi-Alijanvand H, Moosavi-Movahedi AA: The inhibitory effect of ethylenediamine on mushroom tyrosinase. Int J Biol Macromol. 2012, 50: 573-577. 10.1016/j.ijbiomac.2012.01.030.View ArticlePubMed
- Terasaka T, Tsuji K, Kato T, Nakanishi I, Kinoshita T, Kato Y, Kuno M, Inoue T, Tanaka K, Nakamura K: Rational design of non-nucleoside, potent, and orally bioavailable adenosine deaminase inhibitors: Predicting enzyme conformational change and metabolism. J Med Chem. 2005, 48: 4750-4753. 10.1021/jm050413g.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.