Changes in Drug Metabolizing Enzyme Cytochrome P450 CYP3A4 Activities Due to Polymorphisms in Human Cytochrome P450 Oxidoreductase

Abstract

BackgroundCytochromes P450 proteins are responsible for the metabolism of drugs and xenobiotics. All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Previously it has been shown that mutations in POR cause metabolic disorders of steroid hormone biosynthesis but less attention has been paid to drug metabolizing P450 activities. Considering the critical role of POR in activities of drug metabolizing P450 enzymes it is likely that polymorphisms and mutations in POR also affect drug metabolism. We aimed to characterize the variations identified in nonclinical individuals to access their effects on drug metabolizing cytochrome P450 CYP3A4. The variant P284T of POR (rs72557937, NM_000941.2:c.850C>A) is exclusively present in the African population, while A503V allele of POR is found in about 25% of alleles across different populations.MethodsWT and mutant POR proteins, as well as cytochrome P450s, were expressed in recombinant form in bacteria and purified. We used liposomes to embed the P450 and POR proteins and created a functional P450 metabolic system. Metabolism of Testosterone, small molecule dyes, and tracer compounds was evaluated by radioactive ligand metabolism, fluorescent substrate metabolism and colorimetric assays using chromogenic substrates. Enzyme kinetic analysis was performed using Prizm.ResultsTo study the effect of the P284T variant on POR activity, we first measured the ability of WT and P284T variant to transfer electrons from NADPH to cytochrome c, MTT or ferricyanide. Compared to WT POR, the P284T variant had ~50% activity to reduce cytochrome c and 15 % activity to reduce MTT. However, the ferricyanide reduction activity was not affected by the P284T mutation. As compared to WT POR, both FMN and FAD binding was affected due to the P284T mutation. The FMN content of POR‐P284T was 64% as compared to WT while the FAD content was reduced by 35% suggesting that these mutations do affect FMN and FAD binding to POR. The PORvariant P284T had severe loss of CYP3A4 activity with only 22.8% residual activity as compared to WT indicating a potential impact on drug metabolism. The POR common polymorphism A503V which is found in around 25% of all alleles across different genetic populations showed increased activities of drug metabolizing enzyme CYP3A4.ConclusionsThese results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to determine the genotype‐phenotype correlations of POR variants accurately. These results further affirm the critical role of POR in drug metabolism and suggest that variations in POR should also be considered during the evaluation of abnormal drug‐metabolizing cytochrome P450 activities.Support or Funding InformationThis research was supported by grants to Amit V Pandey from the Swiss National Science Foundation, Novartis Foundation for Medical Biological Research and BurgerGemeinde Bern, Switzerland.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.