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Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity.

ABSTRACT: 1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.

SUBMITTER: Fernley HN

PROVIDER: S-EPMC1271245 | biostudies-other | 1967 Sep

REPOSITORIES: biostudies-other

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Project description:The presence of alkaline phosphatases has been observed in several species and has been known to play a crucial role in various biological functions. Higher expressions of alkaline phosphatase have been found in several multifactorial disorders and cancer patients, which has led it to be an interesting target for drug discovery. A strong structural similarity exists between intestinal alkaline phosphatases (IAPs) and tissue-nonspecific alkaline phosphatases (TNAPs), which has led to the discovery of only a few selective inhibitors. Therefore, a series of 22 derivatives of 6-(chloromethyl)-4-(4-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1) and ethyl 6-(chloromethyl)-4-(2-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2) were synthesized to evaluate the anticancer potential of these compounds against breast cancer. The compounds were characterized through spectral and elemental analyses. The inhibitory effect of dihydropyrimidinone derivatives on alkaline phosphatases was evaluated using the calf alkaline phosphatase assay. The antioxidant activity of these compounds was performed to study the radical scavenging effect. In silico molecular docking and molecular dynamic simulations were performed to elucidate the binding mode of active compounds. Moreover, the two-dimensional qualitative-structure-activity relationship (2D-QSAR) was performed to study the structural requirements for enzyme inhibition. The calf alkaline phosphatase inhibitory assay revealed significant inhibition of the enzyme by compound 4d with IC50 1.27 μM at 0.1 mM concentration as compared to standard KH2PO4 having IC50 2.80 μM. The compounds 4f, 4e, and 4i also showed very good inhibition with IC50 values of 2.502, 2.943, and 2.132 μM, respectively, at the same concentration. The antioxidant assay revealed efficient radical scavenging activity of compounds 4f, 4e, and 4g at 100 μg/mL with IC50 values of 0.48, 0.61, and 0.75 μg/mL, respectively. The molecular docking and simulation studies revealed efficient binding of active compounds in the active binding site of the target enzyme. The final QSAR equation revealed good predictivity and statistical validation having R 2 = 0.958 and Q 2 = 0.903, respectively, for the generated model. The compound 4d showed the highest inhibitory activity with stable binding modes acting as a future lead for identifying alkaline phosphatase inhibitors. The molecular simulations suggested the stable binding of this compound, and the QSAR studies revealed the importance of autocorrelated descriptors in the inhibition of alkaline phosphatase. The investigated compounds may serve as potential pharmacophores for potent and selective alkaline phosphatase inhibitors. We intend to further investigate the biological activities of these compounds as alkaline phosphatase inhibitors.

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Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity.

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