High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase

Abstract

Cytochrome P450 2C9 (CYP2C9) metabolizes many clinically important drugs including warfarin and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, found in a warfarin hypersensitive patient with Arg125Leu and Arg144Cys mutations. Here, we have investigated the molecular basis for the functional consequences of these polymorphic changes. CYP2C9.1 and CYP2C9-Arg144Cys expressed in human embryonic kidney 293 cells effectively metabolized both S-warfarin and diclofenac in NADPHdependent reactions, whereas CYP2C9-Arg125Leu or CYP2C9.35 were catalytically silent. However, when NADPH was replaced by a direct electron donor to CYPs, cumene hydroperoxide, hereby bypassing the CYP oxidoreductase (POR), all variant enzymes were active, indicating unproductive interactions between CYP2C9.35 and POR. In silico analysis revealed a decrease of the electrostatic potential of CYP2C9-Arg125Leu-POR interacting surface and the loss of stabilizing salt bridges between these proteins. In conclusion, our data strongly suggest that the Arg125Leu mutation in CYP2C9.35 prevents CYP2C9-POR interactions resulting in the absence of NADPH-dependent CYP2C9-catalyzed activity in vivo, thus influencing the warfarin sensitivity in the carriers of this allele.

In vitrofunctional analysis of 24 novel CYP2C19 variants recently found in the Chinese Han population

“…Additionally, some variants, such as R124Q and R125G, exhibited significantly decreased catalytic activities while showing a similar protein expression level to that of the wild-type protein. Previous reports have shown that substitutions of R124Q and R125H in CYP2C9 could significantly reduce the enzymatic activities of the CYP2C9 protein when expressed them in COS-7 or 293 cells (Dai et al, 2014b;Lee et al, 2014). Similarly, the enzymatic activities of CYP2C9 variants T130R (CYP2C9*26), T130M (CYP2C9*44), I327T (CYP2C9*31) and D360E (CYP2C9*5) are significantly reduced in vitro (Dai et al, 2013(Dai et al, , 2014aNiinuma et al, 2014;Wang et al, 2014).…”

Warfarin Dose Requirements in a Patient with the CYP2C9*14 Allele

“…Hence, we believe that this case adds to the accumulating data on the implications of the CYP2C9*14 allele for warfarin dosing. Genotyping was limited to the variants detected by the commercial platform used, and thus, we cannot rule out the presence of other CYP2C9 variants, such as *4 , *8 , *13 or *35 , which have been associated with reduced enzyme activity against warfarin [6, 19–20]. Additionally, no plasma or urine samples were collected from the patient to confirm that the low dose requirements were secondary to reduced warfarin clearance.…”

Whole-Exome Sequencing Reveals Defective CYP3A4 Variants Predictive of Paclitaxel Dose-Limiting Neuropathy

“…Cell lysates or microsomes, prepared as previously described (18) and containing an equal amount of total protein, were separated using 15% SDS-polyacrylamide gel (18). Membranes were probed with primary antibodies against CYP3A4 (a-hCYP3A4, 1:1,000; ref.…”