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.