Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine

Abstract

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys(132) of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

FIGURE 1.

Bioactivation of olmesartan medoxomil. A, bioactivation of olmesartan medoxomil is shown. Hydrolysis librates its active metabolite olmesartan and generates a diketone. Another possible product, RNH-8097, was not detected in in vitro reaction mixture. B, shown is the estimated mechanism of the base-catalyzed activation of olmesartan medoxomil, supporting the postulated mechanism previously published for several medoxomil ester prodrugs of carboxylic acid (39, 40).

FIGURE 2.

Enzymatic OM hydrolysis in human tissue preparations. A and B, kinetic analysis of OM hydrolysis in human liver and intestinal cytosols, respectively, is shown. Data points represent the means ± S.D. of triplicate determinations. The solid line is the best fit by nonlinear least squares regression to the Michaelis-Menten equation. The respective Eadie-Hofstee plot is presented in the inset. C, shown is the inhibitory effect of known esterase inhibitors on OM-hydrolyzing activity in human plasma, liver, and intestinal cytosols. Each inhibitor concentration was as follows: BNPP, DFP, PCMB, eserine, and PMSF, 1000 μm; EDTA, 500 μm. Data represent the means of duplicate determinations. D, shown is OM-hydrolyzing activity in human liver and intestinal preparations. Data represent the means ± S.D. of triplicate determinations.

FIGURE 3.

Identification of bands corresponding to OM-hydrolyzing activity. Protein with OM-hydrolyzing activity was purified from human liver cytosol by successive column chromatography. A, fractions of the last purification step were subjected to SDS-PAGE, and the gel was stained with a fluorescent dye (SYPRO Ruby, Bio-Rad). Arrowheads indicate 30-kDa bands correlating with OM-hydrolyzing activity. B, the OM-hydrolyzing activity in the fractions determined by HPLC and the intensities of the bands shown as arrows in A are indicated by the solid line and bars, respectively.

FIGURE 4.

Amino acid sequences of CMBL and identification of the active residue. A, the human, rat, and mouse CMBL (GenBank^TM accession number: NP_620164.1, NP_001008770.1, and NP_853619.1, respectively) are aligned. Asterisks and dots, respectively, indicate identical and homologous residues between the three sequences. Arrowheads and bold letters indicate the putative catalytic triad of human CMBL, Cys¹³², Asp¹⁷⁹, and His²¹², predicted based on the crystal structure of dienelactone hydrolase from Pseudomonas sp. B13 (34) (PDB code 1DIN, gi: 1827808) as a template with the aid of ModBase (26). Underlined letters indicate the dienlactone hydrolase family domain (pfam01738). Shadowing indicates the identified sequences by LC-MS/MS analysis of the purified protein from human liver cytosol. B and C, shown is OM-hydrolyzing activity of recombinant CMBL proteins overexpressed in E. coli system, the wild-type (WT) and two mutant constructs with each of an amino acid substitution (C132A or C132S) on the putative active site, Cys¹³². B and C show the activities at 10 μm of the substrate concentration and the kinetic analysis, respectively. Data represent the means ± S.D. of triplicate determinations. The solid line in C is the best fit by nonlinear least squares regression to the Michaelis-Menten equation. The K_m value in the wild-type construct was estimated to be 116 μm.

FIGURE 5.

Enzyme characterization of recombinant CMBL overexpressed in mammalian cell line. A, OM-hydrolyzing activity of recombinant CMBL expressed in Freestyle 293-F cells is shown. The cell lysate supernatant was used as an enzyme source for the in vitro metabolic reactions. Data represent the means ± S.D. of triplicate determinations. B, kinetic analysis of OM hydrolysis by recombinant CMBL is shown. Data points represent the means ± S.D. of triplicate determinations. The solid line is the best fit by nonlinear least squares regression to the Michaelis-Menten equation. The respective Eadie-Hofstee plot is presented in the inset. C, the inhibitory effect of known esterase inhibitors on OM-hydrolyzing activity of recombinant CMBL is shown.

FIGURE 6.

Expression of CMBL gene transcript in human tissues. The relative expression of mRNA from multiple human tissues was measured by quantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase as a reference gene and normalized to the placenta expression.

FIGURE 7.

Protein expression of native CMBL proteins in human tissues. Protein expression of native CMBL in human liver, intestine, and plasma was analyzed by Western blot. The SDS-PAGE-separated proteins were transferred electrophoretically onto a polyvinylidene difluoride membrane and stained by polyclonal anti-CMBL antibody. Open and closed arrowheads indicate the recombinant CMBL-FLAG fusion protein- and the native CMBL bands, respectively.

FIGURE 8.

Kinetic analysis of medoxomil-prodrug hydrolysis by recombinant CMBL overexpressed in mammalian cell line. Enzyme kinetics of the reactions with chemical structures of faropenem medoxomil and lenampicillin are shown in A and B, respectively. Data points represent the means ± S.D. of triplicate determinations. The solid line is the best fit by nonlinear least-squares regression to the Michaelis-Menten equation. The respective Eadie-Hofstee plot is presented in the inset. C, chemical structure of prulifloxacin.