Propranolol hydroxylation and N-desisopropylation by cytochrome P4502D6: studies using the yeast-expressed enzyme and NADPH/O2 and cumene hydroperoxide-supported reactions.
We have studied the enantioselectivity and regioselectivity of ring-hydroxylation and N-desisopropylation of R(+)- and S(-)-propranolol in microsomes from yeast expressing cytochrome P4502D6 (CYP2D6), using both NADPH and molecular oxygen (NADPH/O2) and cumene hydroperoxide-supported reactions. With NADPH/O2-supported reactions, CYP2D6 catalyzed 4- and 5-ring-hydroxylation, as well as N-desisopropylation of propranolol, although Vmax was considerably greater for ring-hydroxylation, compared with N-desisopropylation. The R/S ratios for KM and Vmax were less than unity for all three pathways. In contrast, using cumene hydroperoxide-supported reactions, CYP2D6 catalyzed 4- and 5-ring-hydroxylation, and there was negligible N-desisopropylation of propranolol. The R/S ratio for KM was less than unity, but the R/S ratio for Vmax was close to unity. The cumyl group of cumene hydroperoxide did not seem to be a selective inhibitor of N-desisopropylation, because i) cumyl alcohol (a nonalkylhydroperoxide analog of cumene hydroperoxide) did not inhibit N-desisopropylation in NADPH/O2-supported reactions, and ii) the use of t-butyl hydroperoxide (a noncumyl alkylhydroperoxide) to support CYP2D6 catalysis resulted in ring-hydroxylation, but not N-desisopropylation. At a propranolol concentration near KM, quinidine inhibited both ring-hydroxylation and N-desisopropylation in an equipotent manner in NADPH/O2-supported reactions. However, in cumene hydroperoxide-supported reactions, the IC50 of inhibition of ring-hydroxylation by quinidine was an order of magnitude less potent than in NADPH/O2-supported reactions. Our study shows that recombinant CYP2D6 cannot only catalyze 4- and 5-ring-hydroxylation of propranolol, but also N-desisopropylation. The lack of propranolol N-desisopropylation observed in cumene hydroperoxide-supported reactions highlights the need for caution when using alkyhydroperoxides to study CYP2D6 catalysis.
Bichara
N
N
Department of Medicine, University of Melbourne, West Heidelberg, Victoria, Australia.
Ching
M S
MS
Blake
C L
CL
Ghabrial
H
H
Smallwood
R A
RA
D001555:Benzene Derivatives / Q000378:metabolism*; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D009249:NADP / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011433:Propranolol / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D001555:Benzene Derivatives; D009249:NADP; D011433:Propranolol; D019389:Cytochrome P-450 CYP2D6; C007164:cumene hydroperoxide; D010100:Oxygen