P31 Allelic Variants of CYP2D6 have Altered Drug Metabolite Profile, Kinetics, and Susceptibility to Mechanism-based Inactivation

Laura Lowe Furge , Kalamazoo College, Kalamazoo, MI
Sarah M. Glass , Kalamazoo College, Kalamazoo, MI
Christi Cho , Kalamazoo College, Kalamazoo, MI
Cydney M. Martell , Kalamazoo College, Kalamazoo, MI
Jacqueline M. Mills , Kalamazoo College, Kalamazoo, MI
Alexandria K. Oswalt , Kalamazoo College, Kalamazoo, MI
Sabrina Leddy , Kalamazoo College, Kalamazoo, MI
The metabolite profiles, kinetics and susceptibility to inactivation of four allelic variants of CYP2D6 were analyzed in the current study. The variants included three variants of CYP2D6 with a series of distal mutations (*34, *17-2, *17-3) and one ultra-metabolizer (*53), and one active-site mutant (CYP2D6-T309AT). The prototypical substrates bufuralol and dextromethorphan were used in analyses as well as the known mechanism-based inactivator SCH66712. Decreased enzyme efficiencies for metabolism of dextromethorphan and bufuralol were observed for *34, *17-2, and *17-3 while *53 showed increased activity consistent with its designation as an ultra-metabolizer. The active site mutant CYP2D6-T309A displayed similar kinetic values as reference CYP2D6*1. Both *53 and CYP2D6-T309A showed altered metabolite profiles as compared to *1, particularly with dextromethorphan as substrate. These findings support the roles of Phe120 (changed to Ile in *53) and Thr309 in determining product regio-selectivity. Likewise, though *1 was inactivated by SCH66712, *53 and T309A were less susceptible to inactivation. These findings support the designation of T309A as the nucleophilic target for inactivation of CYP2D6 by SCH66712 and suggest Phe120 may also play a role in inactivation. The inactivation of *1 and *53 by SCH66712 was further investigated through determination of the partition ratio. Comparison of the partition ratio values showed *53 with partition ratio of ~25 turns over more product before being inactivated than *1 with partition ratio of ~3. The decreased rate of inactivation of *53 suggests that inactivation may be dependent on time within and positioning of inactivator in the active site. Furthermore, the reduction in inactivation of *53 and CYP2D6-T309A may be due to altered product regio-selectivity. Cumene hydroperoxide was used as an oxygen surrogate to determine the role of oxygen activation in altered product formation and inactivation with *53 and CYP2D6-T309A. (Support: NIH [LLF] 1R15-GM086767-02,-03; by the Cook, Heyl, Hutchcroft, and Varney funds of Kalamazoo College, and by a grant to Kalamazoo College from the HHMI [52006304]).