Effect of dihydropyrimidine derivatives on kinetic parameters of E.carotovora L-asparaginase

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)-6-methyl-2-thioxo)-1-N-benzilydene-1, 4-dihydropyrimidine-5-carboxylic acid ethyl ester (P 1) and 4-(2'-hydroxy-5'-chlorophenyl)-5-acetyl-6-methyl-2 pyrimidinone (P 2) 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 P 1 and P 2 (20-40 µM) at 0.02-0.1 mM concentration of asparagine. It was found that derivatives below the concentration 5 µg/ml have no effect on the activity. Derivative P 1 is found to be a strong activator of the asparaginase activity that was reflected by an increase in the V max (1.75 fold by P 1 and 2.80 fold by P 2 respectively) and decrease in the K m (0.91 fold by P 1 and 0.81 fold by P 2 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 V max of the enzyme. The activation by derivative P 1 and P 2 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 P 1 and P 2 are positive modulators of asparaginase activity and may support the development of novel combination therapy for the treatment of Leukemia and solid blood tumors.