br Introduction Adenosine deaminase ADA also

Introduction Adenosine deaminase (ADA), also known as adenosine aminohydrolase, is a key enzyme involved in the purine metabolism that converts adenosine to inosine irreversibly (Lupidi et al., 1997). It is a zinc containing metalloenzyme present in both prokaryotes and eukaryotes. In humans ADA is normally present in all tissues and its activity is highest in the lymphoid system (Brady and O’Connell, 1962; Cortés et al., 2015). ADA plays an important role in adenosine homeostasis and nucleic FPH1 metabolism. ADA can also interact with CD26 and adenosine receptors which triggers immune responses and various cellular activities (Dong et al., 1996; Gracia et al., 2008; Saura et al., 1996). ADA deficiency is also associated with severe combined immune deficiency (SCID) (Giblett et al., 1972). Over-activity of ADA is associated with many diseases such as inflammation and some type of cancers (Flinn et al., 2000; Law et al., 2003). Due to its role in cellular proliferation and differentiation, ADA inhibitors are being considered as highly promising candidates for the development of anti-inflammatory, anti-proliferative and immunosuppressive drugs (Robert, 1980). Several ADA inhibitors were proposed from natural (Lee et al., 2009) and synthetic sources (La Motta et al., 2009; Terasaka et al., 2004). Most of the proposed inhibitors have some disadvantages such as side effects, irreversibility and cell toxicity (Mitchell et al., 1980). Currently, pentostatin is the only ADA inhibitor being used for the treatment of leukemia (Flinn et al., 2000). Novel and effective ADA inhibitors with low toxicity and high therapeutic value for clinical applications are the need of the time. Flavonoids are secondary metabolites from plants known to possess various pharmacological activities. Flavonoids such as Quercetin, Myricetin and Kaempferol inhibit the activity of ADA (Melzig, 1996). Hibifolin is a flavonoid isolated from Helicteres isora (Ramesh and Yuvarajan, 1995), It has a chemical structure similar to that of the flavanoids mentioned above and hence, the compound has been selected for the present study. The activity of hibifolin on ADA was studied by the methods of enzyme inhibition assays and isothermal titration calorimetry (ITC). The mode of binding of the compound was analyzed using molecular modeling and docking.
Materials and methods ADA was purified from bovine spleen using the method reported in the literature (Pfrogker, 1967). The specific activity of the purified enzyme was around 13.5 units/mg of protein. Amino acid sequences of human and bovine ADA are 91% identical and active site residues are the same (Kinoshita et al., 1999; Ugochukwu et al., 2009). Hibifolin, adenosine and other chemicals were purchased from Sigma-Aldrich. Solutions and reagents were prepared using double distilled water.
Results and discussion
Conclusion In this study, the inhibitory activity of a plant derived flavonoid, hibifolin on ADA has been investigated. ADA is considered as a potential target for anti-proliferative drugs because of its role in purine metabolism. It is a promising target for anti-inflammatory drugs because of the key role in maintaining adenosine concentration in the body. Most of the proposed inhibitors are nucleoside analogues. The main drawbacks of the nucleoside inhibitors are the difficulty in their chemical synthesis and also their interaction with other enzymes in the body (Dreosti et al., 1997). Therefor it has great importance to discover novel non nucleotide inhibitors from easily available sources. The present study reveals that hibifolin can significantly inhibit the catalytic activity of ADA and hence, it could be used as a promising lead compound for the development of anti-inflammatory and anti-proliferative drugs.
Acknowledgements
Introduction Adenosine deaminase (adenosine aminohydrolase, EC3.5.4.4, ADA) plays a relevant role in intracellular and extracellular nucleotide metabolism and specifically participates in the irreversible hydrolytic deamination of adenosine (AD) and deoxyadenosine to produce inosine and 2′-deoxyinosine, respectively, and ammonia (Fig. 1) [1,2]. Accumulating evidence indicates that dysfunctional ADA in human body is closely related to many important diseases, such as tuberculosis [3], diabetes [4,5], liver disorders [6,7], and cancer [[8], [9], [10], [11]]. Given its significance in pathology, ADA is an important target for drug development and disease detection. Elevated ADA levels in the sera or tissue of cancer patients have important implications. For instance, cancer cells require high ADA activity to metabolize AD for maintaining their division, whereas high AD concentrations can inhibit cell division [12]. Therefore, an effective treatment strategy is to restrain the deamination function and to elevate the AD level by inhibiting ADA. 2′-deoxycoformycin (dCF), a potent inhibitor of ADA, has been officially approved as a drug by the FDA for the treatment of acute lymphoblastic leukemia [13]. However, the proposed inhibitors exhibit some disadvantages, such as irreversibility, side effects, high inhibition constant (Ki), and toxicity on different cells [14]. In recent years, natural sources have received growing attention, and most ADA inhibitors have been isolated from natural plants or structurally modified from natural compounds. Therefore, identification of ADA inhibitors as potential drug candidates has been a major topic in the field of drug discovery.