Abstract
In the present study, we investigated the esterase-like activity of human serum albumin (HSA) and the mechanism by which it hydrolyzes, and thereby activates, olmesartan medoxomil (CS-866), a novel angiotensin II receptor antagonist. CS-866 has previously been shown to be rapidly hydrolyzed in serum in which HSA appeared to play the most important role in catalyzing the hydrolysis. We found that the hydrolysis of CS-866 by HSA followed Michaelis-Menten kinetics. Compared with the release of p-nitrophenol from p-nitrophenyl acetate (PNPA), CS-866 showed lower affinity to HSA and a lower catalytic rate of hydrolysis. Thermodynamic data indicated that PNPA has a smaller value of activation entropy (ΔS) than CS-866; consequently, PNPA is more reactive than CS-866. Ibuprofen and warfarin acted as competitive inhibitors of hydrolysis of CS-866, whereas dansyl-l-asparagine, n-butyl p-aminobenzoate, and diazepam did not. These findings suggest that the hydrolytic activity is associated to parts of site I and site II for ligand binding. All chemically modified HSA derivatives (Tyr-, Lys-, His-, and Trp-modifications) had significantly lower reactivity than native HSA; Lys-HSA and Trp-HSA had especially low reactivity. All the mutant HSAs tested (K199A, W214A, and Y411A) exhibited a significant decrease in reactivity, suggesting that Lys-199, Trp-214, and Tyr-411 play important roles in the hydrolysis. Results obtained using a computer docking model are in agreement with the experimental results, and strongly support the hypotheses that we derived from the experiments.
Footnotes
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (11694298 for M.O.) and was also supported in part by Grant-in-Aid for Scientific Research, Encouragement of Young Scientists (B) (13771414 for N.Y.) from Japan Society for the Promotion of Science.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.006163.
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ABBREVIATIONS: CS-866, olmesartan medoxomil [(5-methyl-2-oxo-1,3-dioxolen-4-yl) methoxy-4-(1-hydroxyl-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methylimi-dazol-5-carboxylate]; RNH-6270, olmesartan; PNPA, p-nitrophenyl acetate; HSA, human serum albumin; rHSA, recombinant HSA; n-butyl p-AB, n-butyl-p-aminobenzoate; DNSA, dansyl-l-asparagine; ΔGT, free energy differences; ΔGS, free energy change for the initial reaction of albumin and substrate; ΔG, activation free energy; ΔH, activation enthalpy change; ΔS, activation entropy change.
- Received June 21, 2005.
- Accepted August 26, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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