Abstract
Purified L-asparaginase in combination with other anticancer drugs like pyrimidine derivatives is administered usually in the body to treat ALL. In the present study, L-asparaginase was purified from Erwinia carotovora up to 247.6 fold and its catalytic properties were studied in the presence of eight different dihydropyrimidine (DHP) derivatives, out of eight derivatives only two viz 4(2'-hydroxy phenyl) -6methyl -2thioxo)-1 -N benzilydene 1, 4 dihydropyrimidine 5 carboxylic acid ethyl ester (P1) and 4 -(2'-hydroxy-5'-chlorophenyl)-5-acetyl-6-methyl-2 pyrimidinone (P2) were found to be activators of L-asparaginase. Their catalytic effect was assayed at optimum pH 8.6 and at temperature 35°C in the absence and presence of derivatives P1 and P2 (20-40 μM) at 0.020.1 mM concentration of asparagine. It was found that derivatives below the concentration 5 μg/ml have no effect on the activity. Derivative P1 is found to be a strong activator of the asparaginase activity that was reflected by an increase in the Vmax (1.75 fold by P1 and 2.80 fold by P2 respectively) and decrease in the Km (0.91 fold by P1 and 0.81 fold by P2 respectively). The activation of asparaginase is explained by suppressing the cooperativity for the substrate, producing hyperbolic kinetics with Km of 0.080 mM and by 3 fold increase in the Vmax of the enzyme. The activation by derivative P1 and P2 were additive, at optimal or suboptimal concentrations of both activators (up to 30 μg/ ml). The DHP derivatives were further analyzed for quantitative SAR study (QSAR) by using PASS, online software to determine their Pa value. Toxicity and drug relevant properties were analyzed by PALLAS software in terms of their molecular weight and log p values. The results showed both the derivatives P1 and P2 are positive modulators of asparaginase activity and may support the development of novel combination therapy for the treatment of Leukemia and solid blood tumors.
Highlights
L-asparaginase (L-asparagine amino hydrolase, E.C.3.5.1.1) catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia [1,2,3,4,5]
The synthesized DHP derivatives were characterized by IR and NMR spectral analysis. 4-(2′-Hydroxy phenyl) - 6 -methyl - 2- thioxo-1N - benzilydene - 1, 4 -dihydropyrimidine - 5 carboxylic acid ethyl ester (P1) M .P. 217oC, yield 85 %. (Scheme 1) IR (KBr, Vmax, cm-1) : 3340, 3210, 1730,1595,1480,1115,1182,1080. 1H NMR (DMSO-d6): 9.8 (s,1H, NH, D2O exchangeable ) 7.45 - 7.90 ( m, 9H, Ar - H), 9.30 (s,1H, N= C-H), 2.15 (s, 3H, CH3), 4.l0 (q, 2H, -COOCH2CH3), 3.95 (t, 3H, -COOCH2 CH3), 5.15 (s, 1H,4-C-H), 10.50 (s, 1H, OH, D2O exchangeable)
The present investigation on the catalytic activity of L-asparaginase was prompted by our interest in the reactivity and hypersensitivity of L-asparaginase during therapeutic use
Summary
L-asparaginase (L-asparagine amino hydrolase, E.C.3.5.1.1) catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia [1,2,3,4,5]. Lasparaginase has been a clinically acceptable anti tumor agent for the effective treatment of acute lymphoblastic leukemia (ALL) and lymph sarcoma [10, 11]. The treatment protocols of ALL and lymph sarcoma do not employ only L-asparaginase as a single agent; instead, it is always a part of multiple agent regimens and combined with drugs having definitive immunosuppressive effects [20].An immunosuppressive agent prevents the immune system from reaction with appearance of foreign protein (asparaginase) in patientsblood circulation [21]. The diversified activities of DHP derivatives specially antitumor and antileukemic properties of asparaginase prompted us to synthesize different pyrimidine derivatives, to extract and purify E. carotovora Lasparaginase and to study its catalytic activity in absence and presence of 4- (2'-hydroxy phenyl)-6-methyl-2- thioxo)-1 -N- benzilydene 1, 4 - dihydropyrimidine - 5 -carboxylic acid ethyl ester (P1) and 4-(2'-hydroxy-5'chlorophenyl)-5-acetyl-6-methyl-2pyrimidinone (P2), the dihydropyrimidine derivatives
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