Effects of G169R and P34S Substitutions Produced by Mutations ofCYP2D6*14on the Functional Properties of CYP2D6 Expressed in V79 Cells

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:CYP2D6 is a polymorphic enzyme that catalyzes the oxidation of various drugs. At least 40-mutant alleles of CYP2D6 have been reported. CYP2D6*14, which is one of them found in Asian populations, causes deficient activity of CYP2D6. Four amino acid substitutions, P34S, G169R, R296C, and S486T, are present in the protein encoded by CYP2D6*14 (CYP2D6 14). Among them, G169R is thought to be a definitive substitution because it is unique to CYP2D6 14. However, a previous study showed that the activity of G169R-substituted CYP2D6 was about 40% of wild-type CYP2D6, suggesting that a combination of G169R and other substitutions may be required to abolish the activity of CYP2D6. In the present study, we examined the effects of combined substitutions of G169R and P34S on the functional properties of CYP2D6 and compared them with those of a single substitution of G169R or P34S using a cDNA expression system of V79 cells. The results showed that a combined substitution of G169R and P34S reduced the activities of CYP2D6 to less than the detection limit of our analytical method for bufuralol 1-hydroxylation and dextromethorphan O-demethylation. However, these activities were not completely abolished by a single substitution of P34S or G169R. The findings suggest that simultaneous substitution of G169R and P34S is crucial for almost completely abolishing the activity of CYP2D6 at least in V79 cells, although whether the absence of metabolism is due to the absence of functional protein or catalytic incompetency remains unclear because the levels of CYP2D6 protein expressed in V79 cells were too low to be determined by difference COreduced spectra.

Comparison of Cytochrome P450 2D6 and Variants in Terms of Drug Oxidation Rates and Substrate Inhibition

“…The ratios of K i for CYP2D6.10 with respect to CYP2D6.1 for inhibitors against dextromethorphan Odemethylation were: budipine (1.3), sparteine (1.6), debrisoquine (8.1), fluoxetine (16), norfluoxetine (30), paroxetine (14), 3,4-methylenedioxymethamphetamine (MDMA) (21), and 2-methoxy-4,5-methylenedioxyamphetamine (MMDA-2) (7.1), indicating that CYP2D6.10 shows drug-specific altered susceptibility to inhibition [13]. On the other hand, Shen et al [14] reported that K i values for all inhibitors except (S)-fluoxetine and (S)-norfluoxetine toward CYP2D6.10 and CYP2D6.17 were comparable to or higher than those toward CYP2D6.1.…”

Frequency of CYP2D6*10 and *14 Alleles and their Influence on the Metabolic Activity of CYP2D6 in a Healthy Chinese Population

“…However, there are few studies on the CYP2D6*14 allele. Shiraishi et al 16 . studied the bufuralol 1′hydroxylation activity and dextromethorphan O ‐demethylation activity of recombinant CYP2D6, and found 2D6P34S was approximately one‐third of that of 2D6WT.…”

Cytochrome P450 2D6

“…In-vitro studies in both COS-1 [18] and V79 [21] cells have shown that cells transfected with CYP2D6 100C > T alone exhibit reduced function, suggesting that this mutation contributes to the reduced function of the CYP2D6*10 allele. Association studies have examined the role of this variant in contributing to generalized tonic clonic seizures seen in epilepsy (no association found) [22] and tardive dyskinesia in Chinese schizophrenic patients (weakly positive) [23].…”