Elsevier

Biochemical Pharmacology

Volume 85, Issue 8, 15 April 2013, Pages 1182-1194
Biochemical Pharmacology

Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions

https://doi.org/10.1016/j.bcp.2013.01.024Get rights and content

Abstract

The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.

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.

References (34)

  • O. Gotoh

    Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences

    J Biol Chem

    (1992)
  • F.P. Guengerich

    Cytochrome P450 and chemical toxicology

    Chem Res Toxicol

    (2008)
  • W.E. Evans et al.

    Pharmacogenomics: tralslating functional genomics into rational therapeutics

    Science

    (1999)
  • S. Rendic

    Summary of information on human CYP enzymes: human P450 metabolism data

    Drug Metab Rev

    (2002)
  • Y. Uno et al.

    Macaque cytochrome P450: nomenclature, transcript, gene, genomic structure, and function

    Drug Metab Rev

    (2011)
  • T. Sakuma et al.

    Marmoset CYP1A2: primary structure and constitutive expression in livers

    Carcinogenesis

    (1997)
  • H. Wang et al.

    CYP2B6: new insights into a historically overlooked cytochrome P450 isozyme

    Curr Drug Metab

    (2008)
  • Cited by (0)

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