Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions
Graphical abstract
Introduction
In the drug development process, it is important to understand the toxicities of a candidate as well as its pharmacological effects in the early stages. Though the availability of various kinds of recombinant human drug-metabolizing enzymes makes it possible to predict a fairly accurate metabolic profile for a drug candidate, a total safety evaluation of the candidate should be performed in vivo using experimental animals. The animal data obtained can be extrapolated into humans. Therefore, the choice of experimental animals is a key point to obtaining a reliable estimation of the possible toxicity of drug candidates. From this view point, monkeys are thought to be appropriate animal models of humans.
The common marmoset is a promising experimental animal species for safety evaluation and metabolism of drug candidates because of its small body size, and easy of handling and breeding, clear advantages over cynomolgus monkeys and rhesus monkeys which are too big to handle and have poor fertility. However, cumulative data on drug-metabolizing enzymes are rather scarce for common marmosets.
Cytochrome P450 (CYP) is a key enzyme in the oxidation of a number of exogenous and endogenous compounds including drugs [1], [2]. Major human drug-metabolizing type CYP enzymes are CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, accounting for more than 90% of oxidative drug metabolism [3], [4]. In cynomolgus monkeys, many kinds of drug-metabolizing type CYP enzymes are expressed in various organs or tissues, and a total of 23 CYP cDNAs in the CYP1 to 4 families have been registered in GenBank [5]. In marmosets, cDNA nucleotide sequences and deduced amino acid sequences for eight drug-metabolizing type CYP enzymes [CYP1A2 (accession number D86475), CYP2C8 (AB242600), CYP2D19 (D29822), CYP2D30 (AY082602), CYP2E1 (D86477), CYP3A4 (D31921), CYP3A5 (EF589801) and CYP3A90 (EF589800)] have been registered in GenBank to date. Previous studies have characterized the enzymatic functions of marmoset CYP1A2 [6], [7], CYP2C8 [8], CYP2D19 [9], [10], [11] and CYP2D30 [10] expressed in various heterologus expression systems, but no reliable experimental data have been published on the enzymatic functions of recombinant marmoset CYP2E1 or CYP3A enzymes so far.
In the present study, we focused on CYP2B enzymes in marmoset livers, because only very little information has been reported on the marmoset CYP2B family. Among the nine major hepatic human drug-metabolizing type CYP enzymes, CYP2B6 accounts for 2–6% of total hepatic CYP content and 8–10% of drug oxidation catalyzed by CYP enzymes [12]. Human CYP2B6 (humCYP2B6) catalyzes the oxidation of clinically prescribed drugs such as bupuropion (BUP) [13], an antidepressant, efavirenz (EFV) [14], an anti-HIV drug, and cyclophosphamide [15], an anticancer drug. Furthermore, insecticides such as chlorpyrifos [16] and endosulfan-α [17] are also oxidized by CYP2B6. Therefore, humCYP2B6 is a unique enzyme that contributes to the oxidation not only of clinically important drugs but also of agricultural chemicals [12].
In contrast, the enzymatic properties of cynologus monkey CYP2B6 (cynCYP2B6) have not been fully elucidated, though its nucleotide and deduced amino aid sequences have been revealed (GenBank accession No. DQ074793). Moreover, there is little information about a marmoset ortholog of humCYP2B6 except for the existence of proteins in marmoset liver microsomal fractions revealed by immunoblot analyses [9], [18]. The characterization of marmoset CYP enzymes as orthologs of human enzymes is a great help to further understanding the drug-metabolizing capacity of marmosets, which promotes their usefulness as an experimental animal in the research field of drug metabolism and toxicity especially in drug development. If the enzymatic functions of a marmoset enzyme are different from those of the human ortholog, the search for the molecular mechanism(s) causing the difference may bring about the elucidation of enzyme reaction mechanism(s). We thus conducted the present study to clone a cDNA encoding a novel marmoset ortholog of humCYP2B6, express its protein in insect cells and characterize its enzymatic properties, which were compared with those of humCYP2B6 and cynCYP2B6.
Section snippets
Materials
7-Ethoxy-4-(trifluoromethyl)coumarin (7-ETC) was obtained from Anaspec Inc. (Fremont, CA); 7-hydroxy-4-(trifluoromethyl)coumarin was from Sigma–Aldrich (St. Louis, MO); BUP hydrochloride and hydroxybupuropion were from Toronto Research Chemicals (North York, ON, Canada); and EFV and 8-hydroxyefavirenz were from Santa Cruz Biotechnology (Santa Cruz, CA). Pooled liver microsomal fractions from humans, cynomolgus monkeys and common marmosets were purchased from BD Biosciences (San Jose, CA). The
cDNA cloning
cDNA encoding humCYP2B6 contained in the pENTR/d-TOPO plasmid was subcloned into the pGEM-T Easy vector, while cDNA encoding cynCYP2B6 was cloned into the pGEM-T Easy vector from total RNA of the liver of a male adult cynomolgus monkey. The nucleotide sequences were confirmed via sequencing to be the same as those of humCYP2B6 (GenBank accession number AC023172.1) and cynCYP2B6 (DQ074793). Because there was no information on a marmoset ortholog of human CYP2B6, we employed 3′- and 5′-RACE
Discussion
The present study focused on the properties of marmoset and cynomolgus monkey CYP2B6s. As a first step we conducted to clone a cDNA encoding a marmoset ortholog of human CYP2B6 employing 3′- and 5′-RACE methods, succeeding in the cDNA cloning and the protein expression in the insect cells. When we consulted with Dr. Nelson of the Cytochrome P450 Nomenclature Committee on the naming of a novel marmoset CYP2B enzyme, he recommended us to use the name of “CYP2B6”. In the home page of Dr. Nelson (//drnelson.uthsc.edu/biblioA.html%232B
Conflict of interest
The authors declare that there are no conflicts of interest.
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