Molecular cloning, expression, and characterization of CYP2D17 from cynomolgus monkey liver.
The cynomolgus monkey is a species used in drug-safety evaluation and biotransformation studies by the pharmaceutical industry. Relatively little is known, however, about the catalytic activities and specificities of cytochromes P450 (CYP) in this species. As a first step in characterizing monkey CYPs, a cDNA was cloned by reverse-transcriptase PCR from cynomolgus monkey liver mRNA using oligonucleotide primers based on the human CYP2D6 sequence. The full-length cDNA (called CYP2D17) encoded a 497-amino-acid protein that is 93% identical to human CYP2D6 and 90% identical to marmoset CYP2D19. The CYP2D17 cDNA was cloned into a baculovirus expression vector, and microsomes prepared from CYP2D17-infected insect cells were used to determine the catalytic properties of the recombinant enzyme. The recombinant CYP2D17 results were compared to data generated with monkey liver microsomes, human liver microsomes, and recombinant CYP2D6 and demonstrated catalytic similarity using probe substrates and inhibitors. Recombinant CYP2D17 catalyzed the oxidation of bufuralol to 1'-hydroxybufuralol and dextromethorphan to dextrorphan, reactions shown to be mediated by CYP2D6 in humans; the apparent K(m) values for bufuralol and dextromethorphan were 1 and 0.8 microM, respectively. Moreover, both of these reactions were more strongly inhibited by quinidine than by quinine. A more complete understanding of the substrate specificities and activities of monkey CYPs will be advantageous in delineating species differences in metabolite profiles and metabolic activation of new chemical entities in the pharmaceutical industry.
Mankowski
D C
DC
Pfizer Central Research, Eastern Point Road, Groton, Connecticut, 06340, USA.
Laddison
K J
KJ
Christopherson
P A
PA
Ekins
S
S
Tweedie
D J
DJ
Lawton
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002144:Callithrix; D003001:Cloning, Molecular; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008252:Macaca fascicularis / Q000235:genetics* / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D019389:Cytochrome P-450 CYP2D6; C105144:cytochrome P-450 CYP2D19 (marmoset)