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 Cys132 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.
Highlights
In any biological organisms, the hydrolysis of endogenous and exogenous compounds is catalyzed by an extremely large variety of enzymes collectively known as hydrolases that catalyze numerous hydrolytic reactions of various bonds and have an essential role in biological activity in multiple sites [1]
The activities in both liver and intestinal cytosolic fractions were partially inhibited by 1000 M BNPP, a carboxylesterase inhibitor, and were strongly inhibited by 1000 M PCMB, a free thiol modifier, whereas they were not inhibited by the addition of the following esterase inhibitors: 1000 M diisopropyl fluorophosphate (DFP) and PMSF, irreversible serine
In this work we have identified human CMBL as a bioactivating hydrolase of the prodrug-type angiotensin receptor blocker (ARB), Olmesartan medoxomil (OM)
Summary
H3C O purchased from Toronto Research Chemicals Inc. (Ontario, Canada) and Hangzhou-Hetd Industry Co., Ltd. (Hangzhou, China), respec-. H3C O purchased from Toronto Research Chemicals Inc. (Ontario, Canada) and Hangzhou-Hetd Industry Co., Ltd. Bis-p-nitrophenylphosphate (BNPP) and pchloromercuribenzoate (PCMB) were purchased from Tokyo Chemical Industry Co., Ltd. Ride (PMSF), diisopropyl fluorophosphate (DFP) and EDTA were purchased from Wako Pure Chemical. Ride (PMSF), diisopropyl fluorophosphate (DFP) and EDTA were purchased from Wako Pure Chemical. (Osaka, Japan)
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