P450 Oxidoreductase Variants Research Articles

Pharmacogenomics of poor drug metabolism in greyhounds: Canine P450 oxidoreductase genetic variation, breed heterogeneity, and functional characterization.

Greyhounds metabolize cytochrome P450 (CYP) 2B11 substrates more slowly than other dog breeds. However, CYP2B11 gene variants associated with decreased CYP2B11 expression do not fully explain reduced CYP2B11 activity in this breed. P450 oxidoreductase (POR) is an essential redox partner for all CYPs. POR protein variants can enhance or repress CYP enzyme function in a CYP isoform and substrate dependent manner. The study objectives were to identify POR protein variants in greyhounds and determine their effect on coexpressed CYP2B11 and CYP2D15 enzyme function. Gene sequencing identified two missense variants (Glu315Gln and Asp570Glu) forming four alleles, POR-H1 (reference), POR-H2 (570Glu), POR-H3 (315Gln, 570Glu) and POR-H4 (315Gln). Out of 68 dog breeds surveyed, POR-H2 was widely distributed across multiple breeds, while POR-H3 was largely restricted to greyhounds and Scottish deerhounds (35% allele frequencies), and POR-H4 was rare. Three-dimensional protein structure modelling indicated significant effects of Glu315Gln (but not Asp570Glu) on protein flexibility through loss of a salt bridge between Glu315 and Arg519. Recombinant POR-H1 (reference) and each POR variant (H2-H4) were expressed alone or with CYP2B11 or CYP2D15 in insect cells. No substantial effects on POR protein expression or enzyme activity (cytochrome c reduction) were observed for any POR variant (versus POR-H1) when expressed alone or with CYP2B11 or CYP2D15. Furthermore, there were no effects on CYP2B11 or CYP2D15 protein expression, or on CYP2D15 enzyme kinetics by any POR variant (versus POR-H1). However, Vmax values for 7-benzyloxyresorufin, propofol and bupropion oxidation by CYP2B11 were significantly reduced by coexpression with POR-H3 (by 34-37%) and POR-H4 (by 65-72%) compared with POR-H1. Km values were unaffected. Our results indicate that the Glu315Gln mutation (common to POR-H3 and POR-H4) reduces CYP2B11 enzyme function without affecting at least one other major canine hepatic P450 (CYP2D15). Additional in vivo studies are warranted to confirm these findings.

Exploring Novel Variants of the Cytochrome P450 Reductase Gene (POR) from the Genome Aggregation Database by Integrating Bioinformatic Tools and Functional Assays.

Cytochrome P450 oxidoreductase (POR) is an essential redox partner for steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR lead to metabolic disorders, including congenital adrenal hyperplasia, and affect the metabolism of steroids, drugs, and xenobiotics. In this study, we examined approximately 450 missense variants of the POR gene listed in the Genome Aggregation Database (gnomAD) using eleven different in silico prediction tools. We found that 64 novel variants were consistently predicted to be disease-causing by most tools. To validate our findings, we conducted a population analysis and selected two variations in POR for further investigation. The human POR wild type and the R268W and L577P variants were expressed in bacteria and subjected to enzyme kinetic assays using a model substrate. We also examined the activities of several cytochrome P450 proteins in the presence of POR (WT or variants) by combining P450 and reductase proteins in liposomes. We observed a decrease in enzymatic activities (ranging from 35% to 85%) of key drug-metabolizing enzymes, supported by POR variants R288W and L577P compared to WT-POR. These results validate our approach of curating a vast amount of data from genome projects and provide an updated and reliable reference for diagnosing POR deficiency.

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Cytochrome P450 oxidoreductase variant A503V contributes to the increased CYP3A5 activity with tacrolimus in vitro

ABSTRACT Background Tacrolimus is a immunosuppressant drug in transplantation with a narrow therapeutic range and high pharmacokinetic variability. Clinical studies have revealed that POR*28 contributes enhanced tacrolimus clearance in CYP3A5 expressers. However, it remains unknown that how exactly the POR polymorphism could influence the metabolism of tacrolimus via CYP3A5 in vitro. Research design & methods A503V is an amino acid sequence variant encoded by POR*28. Wild-type (WT) and A503V POR, with WT CYP3A5 were expressed in recombinant HepG2 cells and reconstituted proteins. Michaelis constant (K m) and maximum velocity (V max) of CYP3A5 with tacrolimus as substrates were determined, and catalytic efficiency is expressed as V max/K m. Results Both WT and A503V POR down-regulated the CYP3A5 mRNA expression, and WT POR rather than A503V down-regulated the protein expression of CYP3A5 in recombinant HepG2 cells. Compared with WT POR, A503V increased metabolism of tacrolimus by CYP3A5 in both cellular and protein levels. Conclusion A503V can affect CYP3A5-catalyzed tacrolimus metabolism in vitro, which suggests that A503V has the potential to serve as a biomarker for tacrolimus treatment in transplantation recipients.

Methadone pharmacogenetics in vitro and in vivo: Metabolism by CYP2B6 polymorphic variants and genetic variability in paediatric disposition.

Methadone metabolism and clearance are determined principally by polymorphic cytochrome P4502B6 (CYP2B6). Some CYP2B6 allelic variants affect methadone metabolism in vitro and disposition in vivo. We assessed methadone metabolism by CYP2B6 minor variants in vitro. We also assessed the influence of CYP2B6 variants, and P450 oxidoreductase (POR) and CYP2C19 variants, on methadone clearance in surgical patients in vivo. CYP2B6 and P450 oxidoreductase variants were coexpressed with cytochrome b5 . The metabolism of methadone racemate and enantiomers was measured at therapeutic concentrations and intrinsic clearances were determined. Adolescents receiving methadone for surgery were genotyped for CYP2B6, CYP2C19 and POR, and methadone clearance and metabolite formation clearance were determined. In vitro, CYP2B6.4 was more active than wild-type CYP2B6.1. CYPs 2B6.5, 2B6.6, 2B6.7, 2B6.9, 2B6.17, 2B6.19 and 2B6.26 were less active. CYPs 2B6.16 and 2B6.18 were inactive. CYP2B6.1 expressed with POR variants POR.28, POR.5 and P228L had lower rates of methadone metabolism than wild-type reductase. In vivo, methadone clinical clearance decreased linearly with the number of CYP2B6 slow metabolizer alleles, but was not different in CYP2C19 slow or rapid metabolizer phenotypes, or in carriers of the POR*28 allele. Several CYP2B6 and POR variants were slow metabolizers of methadone in vitro. Polymorphisms in CYP2B6, but not CYP2C19 or P450 reductase, affected methadone clearance in vivo. CYP2B6 polymorphisms 516G>T and 983T>C code for canonical loss of function variants and should be assessed when considering genetic influences on clinical methadone disposition. These complementary translational in vitro and in vivo results inform on pharmacogenetic variability affecting methadone disposition in patients.

Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase

Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia. Genome sequencing studies have revealed the existence of a POR missense variant P228L which was linked with reduced function of some P450 enzymes. We aimed to expand the enzymatic studies of POR variant P228L for its role in human metabolism.We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed.The single point mutation P228L had reduced thermal stability indicated by a lower melting point comparing to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR comparing to WT.A single change in the amino acid sequence can affect the protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.

Subcellular Location of Tirapazamine Reduction Dramatically Affects Aerobic but Not Anoxic Cytotoxicity.

Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed ‘metabolic efficiency’) when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O2 dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.

Changes in Steroids, Xenobiotics, and Drug‐Metabolism in Humans by Genetic Variations in NADPH Cytochrome P450 Oxidoreductase

A broad spectrum of human diseases, including disordered steroid metabolism, are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR) [1, 2]. Human POR is a diflavin reductase that transfers electrons from NADPH to small molecules, non‐P450 redox partners, and cytochrome P450 proteins in the endoplasmic reticulum [2]. Cytochrome P450 proteins perform an extensive range of reactions, including the metabolism of steroids, drugs, and other xenobiotics. Therefore, genetic variations in POR change several metabolic pathways by changing the activities cytochromes P450 [2]. Due to this unique role of POR in metabolism, it was believed for a long time that genetic defects in POR are unlikely, and indeed a POR knockout mouse is embryonically lethal. However, in 2004, the first human patients with defects in POR were reported, and since then, over 200 different variations in POR have been found [3, 4].Recently, we have characterized POR variations from non‐clinical samples [5]. By analyzing the POR sequences from the 1000 genome and other sequencing projects, we identified potentially disease‐causing variations and characterized these by functional studies using recombinant proteins produced in bacteria, yeast, and mammalian cells [6, 7].Identification of severe effects of POR mutations on both the drug and the steroid metabolizing cytochromes P450 indicates that likely pathogenic mutations may be found in non‐clinical population. Their combination as compound heterozygotes or homozygous may severely impact both steroid and drug metabolism by modifications of cytochrome P450 activities. Variations in POR need to be evaluated individually. Changes in drug and steroid metabolism due to genetic variations can be addressed using personalized metabolic profiling and supplementation using modified dosages of drugs and steroids.Support or Funding InformationSupported by grants from the Swiss National Science Foundation (31003A‐134926) & Novartis Foundation for Medical‐Biological Research (18A053) to AVP.

Impact of Common Polymorphic Variant A503V in POR on Drug Metabolism: Implications for POR Deficiency

A broad spectrum of human diseases, including abnormalities in steroidogenesis, is caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR) [1, 2]. Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. Therefore, genetic variations in POR can impact many different metabolic pathways by changing the activities of cytochromes P450. In 2004 the first human patients with defects in POR were reported with symptoms of ambiguous genitalia and bone malformations, and over 200 variations in POR are known.By analyzing the POR sequences from genomics databases, we focused on the common variant in POR A503V, which is often present in heterozygous form in many PORD patients. We expressed the A503V variant of POR in bacteria and characterized it by functional studies using purified recombinant proteins. The POR, as well as P450 Proteins, were expressed in E. coli BL21(DE3) and purified by ion‐exchange, affinity, and metal chelate chromatography for activity assays as described in our earlier publications [3, 4]. Activities of cytochrome P450 enzymes were tested with lipids into which P450 and P450 reductase proteins were embedded and assayed using fluorogenic substrates on a microplate spectrofluorometer.We found a positive effect on many drug‐metabolizing enzyme activities due to the A503V variant in POR. The activity of CYP2C9 was increased to 180% of the WT POR, and CYP2C19 showed 243% of WT activity with the A503V variant of POR. Most remarkable was the impact of the A503V variant on CYP3A5 activity, which was increased by 421% compared to WT POR.Effect on drug metabolism in patients with PORD requires careful considerations of cytochrome P450 mediated activities. While many PORD patients have low or reduced drug metabolism due to defects in POR, the presence of the A503V allele can alter the overall effect on drug metabolism. Significantly, tacrolimus, a drug given during organ transplant, can be metabolized several folds faster due to the presence of the A503V variant of POR. The presence of the A503V allele is likely beneficial in patients with POR deficiency.Support or Funding InformationThis work was supported by the Swiss National Science Foundation (31003A‐134926), the Novartis Foundation for Medical‐Biological Research (18A053), and a grant from Burgergemeiende Bern to AP.

Stereoselective Bupropion Hydroxylation by Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase Genetic Variants.

Bioactivation of the antidepressant and smoking cessation drug bupropion is catalyzed predominantly by CYP2B6. The metabolite hydroxybupropion derived from t-butylhydroxylation is considered to contribute to the antidepressant and smoking-cessation effects of the parent drug. Bupropion hydroxylation is the canonical in vitro and in vivo probe for CYP2B6 activity. P450 also requires obligate partnership with P450 oxidoreductase (POR). Human CYP2B6 and POR genes are highly polymorphic. Some CYP2B6 variants affect bupropion disposition. This investigation evaluated the influence of several human CYP2B6 and POR genetic variants on stereoselective bupropion metabolism, using an insect cell coexpression system containing CYP2B6, POR, and cytochrome b 5 Based on intrinsic clearances (Clints), relative activities for S,S-hydroxybupropion formation were in the order CYP2B6.4 > CYP2B6.1 > CYP2B6.17 > CYP2B6.5 > CYP2B6.6 ≈ CYP2B6.26 ≈ CYP2B6.19 > CYP2B6.7 > CYP2B6.9 > > CYP2B6.16 and CYP2B6.18; relative activities for R,R-hydroxybupropion formation were in the order CYP2B6.17 > CYP2B6.4 > CYP2B6.1 > CYP2B6.5 ≈ CYP2B6.19 ≈ CYP2B6.26 > CYP2B6.6 > CYP2B6.7 ≈ CYP2B6.9 > > CYP2B6.16 and CYP2B6.18. Bupropion hydroxylation was not influenced by POR variants. CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Though Vmax and Km varied widely among CYP2B6 variants, stereoselectivity was preserved, reflected by similar Clint(S,S-hydroxybupropion)/Clint(R,R-hydroxybupropion) ratios (1.8-2.9), except CYP2B6.17, which was less enantioselective. Established concordance between human bupropion hydroxylation in vitro and in vivo, together with these new results, suggests additional CYP2B6 variants may influence human bupropion disposition. SIGNIFICANCE STATEMENT: Bupropion pharmacokinetics, metabolism, and clinical effects are affected by the CYP2B6*6 polymorphism. Other expressed CYP2B6 polymorphisms had diminished (*5, *6, *7, *9, *19, *26) or defective (*16, *18) in vitro bupropion hydroxylation. P450 oxidoreductase genetic variants had no effect on metabolism, suggesting no clinical consequence of this polymorphism. These CYP2B6 polymorphisms may portend diminished in vivo bupropion hydroxylation and predict additional clinically important variant alleles.

Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20–50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

Isoform‐Dependent Effects of Cytochrome P450 Oxidoreductase Polymorphisms on Drug Metabolism by Cytochrome P450 Enzymes in Dogs

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 (CYP) enzymes. We have recently discovered three polymorphisms in the dog POR coding region resulting in one (POR‐H2 and POR‐H4) or two (POR‐H3) amino acid substitutions. In humans, POR mutations can have CYP isoform‐dependent effects on CYP enzyme activity depending on the POR amino acid residue that is substituted.1 Genotyping of 2,040 DNA samples collected from 65 different dog breeds revealed a heterogeneous distribution of POR alleles across breeds. The POR‐H2 allele was found in 19 of 65 breeds and was most common in Rottweilers and Doberman Pinschers (51% and 19% allele frequencies, respectively). The POR‐H3 allele was found in 8 of 65 breeds and was most common in Scottish Deerhounds and Greyhounds (36% and 35% allele frequencies, respectively). The POR‐H4 allele was rare, only being found in 5 of 2,040 dogs. Greyhounds have been shown to metabolize substrates of CYP2B11 (but not other CYPs including CYP2D15) much more slowly compared with mixed‐breed dogs.2 Preliminary studies identified polymorphisms in the CYP2B11 gene that only partially explained reduced CYP2B11 metabolism in Greyhounds. Consequently, we hypothesized that the newly discovered canine POR variants found in Greyhounds would reduce CYP metabolism in a CYP isoform‐dependent manner. The aim of this study was to determine the functional impact of the canine POR variants on drug metabolism by CYP2B11 and CYP2D15 in vitro. In silico molecular structure modelling predicted that POR‐H3 and POR‐H4 but not POR‐H2 would have altered function. The three POR variants and wild‐type were expressed alone or co‐expressed with CYP2B11 or CYP2D15 using an insect cell system. Enzyme kinetic experiments were conducted to determine the impact of the mutations on POR function and CYP‐catalyzed reactions. The POR variants were not significantly different from wild‐type POR in protein expression or function (cytochrome c reduction) when expressed alone. POR variants also did not significantly alter CYP2D15 protein level or catalytic activities or CYP2B11 protein level when co‐expressed. However, POR‐H3 and POR‐H4 (but not POR‐H2) significantly reduced CYP2B11 catalytic efficiency (CLint) by 37% and 69%, respectively, compared to wild‐type POR. This was due to reduced maximal velocities (Vmax) but not substrate affinities (Km). POR‐H3 and POR‐H4 appear to reduce CYP activities in a CYP isoform‐dependent manner. Further investigations are needed to determine POR variant effects on other CYP isoforms and redox partners. The POR‐H3 and POR‐H4 variants are likely to contribute to slower CYP2B11 drug metabolism in Greyhounds and possibly related sighthound breeds (such as Scottish Deerhounds), but results must be confirmed in vivo.Support or Funding InformationAmerican Kennel Club Canine Health Foundation (Grants 2242 and 2529) and the William R. Jones Endowment at Washington State University College of Veterinary Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

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.

Genetic variants and Expression of Cytochrome p450 Oxidoreductase Predict Postoperative Survival in Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma.

Our current study investigates the prognostic values of genetic variants and mRNA expression of cytochrome p450 oxidoreductase (POR) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). A total of 19 candidate single nucleotide polymorphisms (SNPs) located in the exons of POR were genotyped using Sanger sequencing from 476 HBV-related HCC patients who underwent hepatectomy between 2003 and 2013. The mRNA expression of POR in 212 patients with HBV-related HCC was obtained from GSE14520 dataset. Survival analysis was performed to investigate the association of POR variants and mRNA expression with overall survival (OS) and recurrence-free survival (RFS). Nomograms were used to predict the prognosis of HBV-related HCC patients. Gene set enrichment analysis (GSEA) was used to investigate the mechanism of POR in HBV-related HCC prognosis. The polymorphism POR-rs1057868 was significantly associated with HBV-related HCC OS (CT/TT vs. CC, hazard ratio [HR] = 0.69, 95% confidence interval [CI] = [0.54, 0.88], P = 0.003), but not significantly associated with RFS (CT/TT vs. CC, P = 0.378). POR mRNA expression was also significantly associated with HBV-related HCC OS (high vs. low, HR = 0.61, 95% CI = [0.38, 0.97], P = 0.036), but not significantly associated with the RFS (high vs. low, P = 0.201). Two nomograms were developed to predict the HBV-related HCC OS. Furthermore, GSEA suggests that multiple gene sets were significantly enriched in liver cancer survival and recurrence, as well as POR-related target therapy in the liver. In conclusion, our study suggests that POR-rs1057868 and mRNA expression may serve as a potential postoperative prognosis biomarker in HBV-related HCC.

Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography-mass spectrometry. Michaelis-Menten kinetic parameters were determined. For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

Common Polymorphisms of CYP2B6 Influence Stereoselective Bupropion Disposition

Bupropion hydroxylation is a bioactivation and metabolic pathway, and the standard clinical CYP2B6 probe. This investigation determined the influence of CYP2B6 allelic variants on clinical concentrations and metabolism of bupropion enantiomers. Secondary objectives evaluated the influence of CYP2C19 and P450 oxidoreductase variants. Healthy volunteers in specific cohorts (CYP2B6*1/*1, CYP2B6*1/*6, CYP2B6*6/*6, and also CYP2B6*4 carriers) received single-dose oral bupropion. Plasma and urine bupropion and hydroxybupropion was quantified. Subjects were also genotyped for CYP2C19 and P450 oxidoreductase variants. Hydroxylation of both bupropion enantiomers, assessed by plasma hydroxybupropion/bupropion AUC ratios and urine hydroxybupropion formation clearances, was lower in CYP2B6*6/*6 but not CYP2B6*1/*6 compared with CYP2B6*1/*1 genotypes, and numerically greater in CYP2B6*4 carriers. CYP2C19 and P450 oxidoreductase variants did not influence bupropion enantiomers hydroxylation or plasma concentrations. The results show that clinical hydroxylation of both bupropion enantiomers was equivalently influenced by CYP2B6 allelic variation. CYP2B6 polymorphisms affect S-bupropion bioactivation, which may affect therapeutic outcomes.

Altered Steroid and Drug Metabolism by a Cytochrome P450 Oxidoreductase Variant Found in Apparently Normal Population

Background and AimsA broad spectrum of human diseases, including abnormalities in steroidogenesis, are caused by mutations in the cytochrome P450 oxidoreductase (POR) (1–3). POR transfers electrons from NADPH to small molecules, non‐P450 redox partners and microsomal cytochrome P450 proteins. Our aim was to check if POR variations from non‐clinical samples (1000 genomes database), can be disruptive. The Y607C variant of POR (rs72557954, NM_000941.2:c. 1820A>G) has been reported in population studies and is prevalent in South Asians (4), but was predicted to be likely pathogenic. We performed detailed enzymatic and biochemical characterizations of Y607C variant of POR to study its effect on different substrate and redox partners.MethodsWe analysed the ability of POR wild type (WT) and Y607C variant to reduce ferricyanide, MTT, cytochrome c and drug, and steroid metabolizing cytochrome 450 enzymes. The POR WT and Y607C were expressed and produced as recombinant proteins while CYP19A1 and CYP3A4 were produced as His‐tagged recombinant proteins and purified by affinity chromatography. The effect of mutation on cofactor (FAD/FMN) binding and activity under varying substrate and cofactor conditions was performed.ResultsWe found varied effects of Y607C mutation on activities with different partners. As compared to WT, Y607C variant showed 66% cytochrome c and 91 % ferricyanide reduction activity but had only 13 % MTT reduction activity. The POR Y607C did not affect flavin content but NADPH binding was severely affected. With varying NADPH concentration, Y607C showed ~95% decrease in supporting CYP19A1 and CYP3A4 activity. This mutation was later identified in patients with POR deficiency.ConclusionsIdentification of severe effects of this mutation on both drug and steroid metabolizing cytochrome P450s indicates that likely pathogenic mutations may be found in apparently normal (non‐clinical) population. Their combination as compound heterozygotes or homozygous may lead to severe impact on both steroid and drug metabolism by modification of POR redox partner activities. Variations in POR need to be evaluated individually. Most importantly, advanced identification of disease causing variants in POR will help in understanding the POR deficiency in patients if the same mutations are later identified.Support or Funding InformationThis research was funded by grants to Amit V Pandey from the Swiss National Science Foundation (31003A_134926).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Altered metabolism by a P450 oxidoreductase variant found in apparently normal population

Altered metabolism by a P450 oxidoreductase variant found in apparently normal population

Polymorphisms in cytochrome P450 oxidoreductase and its effect on drug metabolism and efficacy.

Cytochrome P450 oxidoreductase (POR) has played a potential role in the metabolism of drugs and steroids by supplying electrons to microsomal cytochrome P450 (CYP) enzymes. More than 200 different POR mutations and polymorphisms causing more than 130 amino acid changes in the POR protein have been reported since 2004. A503V is a common amino acid sequence variant encoded by POR*28, whereas A287P and R457H are the most common disease-causing mutations in Europeans and Asians, respectively. Polymorphisms in the POR gene can affect POR activity, CYP-mediated drug metabolism activities, and the efficacy of several clinically used drugs. The effects of POR variants on CYP activities are substrate dependent. In this review, recent research on the effects of POR genetic polymorphisms on drug metabolism and therapy has been summarized and discussed, which can contribute to the rational use of drugs in clinic and the development of personalized medicine.

Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase.

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These 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 accurately determine the genotype-phenotype correlations of POR variants.