Human CYP2C Subfamily Research Articles

Insights into the Structural Determinants of Substrate Binding to Human Cytochromes P450 2C9 and 2C18

Xenobiotic metabolizing cytochrome P450 (CYP) enzymes constitute the major enzyme family in drug metabolism with increasing importance in pharmacogenetics, and more recently to pharmacogenomics. The highly polymorphic CYP2C9 metabolizes over 15% of clinical drugs that include ibuprofen, diclofenac, warfarin, losartan, tolbutamide, and glimepiride. Whereas CYP2C18 represents the least characterized enzyme with little information regarding substrates, the active site and the overall structure, despite the high sequence identity (more than 80%) with other human CYP2C subfamily of enzymes. Sequence alignment of CYP2C9 and 2C18 revealed interesting differences in amino acid substitutions, which allowed analysis of the active site of two enzymes of the same subfamily with distinct information regarding substrate specificity. A homology model of CYP2C18 created using the most recent crystal structure of CYP2C9‐losartan complex revealed key residue substitutions that significantly reduced the volume of the active site. In particular, the larger side chain substitutions L113, F201, F237 and N365 in CYP2C18 protrude the active site and play an important role in ligand interaction. Molecular docking studies using a diverse range of CYP2C9 substrates demonstrated remarkably altered orientation in the CYP2C18 active site indicative of differences in substrate preference compared to CYP2C9. The results suggest the active site architecture of CYP2C18 may not allow binding of larger or bulkier substrates as seen with other human CYP2C enzymes. Overall, the analysis of amino acid differences in the active site, the access channel and the recently identified potential substrate recognition site at the periphery provide important insights into the role of distinct side chain substitutions among related CYP enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Med25 is required for estrogen receptor alpha (ERα)-mediated regulation of human CYP2C9 expression

The CYP2C subfamily of cytochrome P450 enzymes is an important class of drug metabolizing enzymes in human liver. CYP2C9 is the most abundant member of the human CYP2C subfamily in liver and metabolizes ∼15% of the therapeutic drugs as well as other xenobiotics and endogenous compounds. A number of nuclear receptors including xenobiotic-sensing receptors such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glucocorticoid receptor (GR) as well as liver enriched receptors hepatic nuclear factor 4α (HNF4α) and the estrogen receptor α (ERα) regulate CYP2C9 expression. Here, we show that Med25, a variable component of Mediator complex, enhanced ligand dependent ERα-mediated transcriptional activation of CYP2C9 promoter and interacts with activated ERα by 17β-estradiol through its C-terminal LXXLL motif. In conclusion, Med25 is identified as a new coactivator of ERα that is required for ERα-mediated regulation of CYP2C9 expression.

Investigation of enzyme selectivity in the human CYP2C subfamily: homology modelling of CYP2C8, CYP2C9 and CYP2C19 from the CYP2C5 crystallographic template.

Homology modelling of human CYP2C subfamily enzymes, CYP2C8, CYP2C9 and CYP2C19, based on the rabbit CYP2C5 crystal structure template is reported. The relatively high sequence homologies (75-80%) between the rabbit CYP2C5 and human CYP2C subfamily enzymes tend to indicate that the resulting structures should prove adequate models of these major catalysts of human drug metabolism. Selective substrates of all three human CYP2C enzymes are found to fit closely within the putative active sites in a manner which is consistent with site-directed mutagenesis experiments and known positions of substrate metabolism. The specific interactions between substrates and enzymes can be used to rationalize the variation in substrate binding affinity and generate QSAR models for both inhibition and metabolism via CYP2C family enzymes, yielding a generally good agreement with experimental binding data obtained from Km values, with correlation coefficients (R values) of between 0.97 and 0.99 depending on the QSAR equation produced.

Clinical relevance of genetic polymorphisms in the human CYP2C subfamily.

The human CYP2Cs are an important subfamily of P450 enzymes that metabolize approximately 20% of clinically used drugs. There are four members of the subfamily, CYP2C8, CYP2C9, CYP2C19, and CYP2C18. Of these CYP2C8, CYP2C9, and CYP2C19 are of clinical importance. The CYP2Cs also metabolize some endogenous compounds such as arachidonic acid. Each member of this subfamily has been found to be genetically polymorphic. The most well-known of these polymorphisms is in CYP2C19. Poor metabolizers (PMs) of CYP2C19 represent approximately 3-5% of Caucasians, a similar percentage of African-Americans and 12-100% of Asian groups. The polymorphism affects metabolism of the anticonvulsant agent mephenytoin, proton pump inhibitors such as omeprazole, the anxiolytic agent diazepam, certain antidepressants, and the antimalarial drug proguanil. Toxic effects can occur in PMs exposed to diazepam, and the efficacy of some proton pump inhibitors may be greater in PMs than EMs at low doses of these drugs. A number of mutant alleles exist that can be detected by genetic testing. CYP2C9 metabolizes a wide variety of drugs including the anticoagulant warfarin, antidiabetic agents such as tolbutamide, anticonvulsants such as phenytoin, and nonsteroidal anti-inflammatory drugs. The incidence of functional polymorphisms is much lower, estimated to be 1/250 in Caucasians and lower in Asians. However, the clinical consequences of these rarer polymorphisms can be severe. Severe and life-threatening bleeding episodes have been reported in CYP2C9 PMs exposed to warfarin. Phenytoin has been reported to cause severe toxicity in PMs. New polymorphisms have been discovered in CYP2C8, which metabolizes taxol (paclitaxel). Genetic testing is available for all of the known CYP2C variant alleles.

Torsemide metabolism by CYP2C9 variants and other human CYP2C subfamily enzymes.

Torsemide metabolism by CYP2C9 variants and other human CYP2C subfamily enzymes.

Genetic polymorphism of the CYP2C subfamily

Cytochromes P450 (CYP) are a superfamily of hemoproteins that metabolize various foreign compounds. The human CYP2C subfamily is one of the subfamilies of the CYP2 family and it consists of four members of CYP isoforms, CYP2C8, CYP2C9, CYP2C18 and CYP2C19. A well-characterized genetic polymorphism occurs in CYP2C19, which is associated with the 4'-hydroxylation of S-mephenytoin. There are two phenotypes, extensive metabolizers and poor metabolizers (PM) of mephenytoin. The frequency of PM in the Japanese population is 20%, while only 3% of Caucasians are PM of mephenytoin. Two defective alleles, designated as CYP2C19*2 and CYP2C19*3, have been described, and the latter mutation has been detected only in Oriental populations. Recently, an allelic variant of CYP2C9 that causes substitution of Leu359 for Ile359 has been shown to be associated with the decreased metabolic clearance of various therapeutic agents including warfarin, tolbutamide and phenytoin. The frequency of this variant allele in the Japanese population is 2%, while those of the Caucasians are 6-9%. Although the role of CYP2C18 in the drug metabolism remains obscure, we have recently found that defective alleles of CYP2C19*3 and CYP2C18m1 are completely linked, suggesting that PM of CYP2C19 with CYP2C19*3 alleles is a PM of CYP2C18 and vice versa.

S16-3 - Genetic polymorphism of human CYP2C subfamily

S16-3 - Genetic polymorphism of human CYP2C subfamily

Molecular modelling of human CYP2C subfamily enzymes CYP2C9 and CYP2C19: rationalization of substrate specificity and site-directed mutagenesis experiments in the CYP2C subfamily

1. The results of molecular modelling of human CYP2C isozymes, CYP2C9 and CYP2C19, are reported based on an alignment with a bacterial form of the enzyme, CYP102. 2. The three-dimensionalstructures of the CYP2C enzymes are consistent with known experimental evidence from site-directed mutagenesis, antibody recognition and regiospecificity of substrate metabolism. 3. The variations in substrate specificity between CYP2C9 and CYP2C19 can be rationalized in terms of single amino acid residue changes within the putative active site region, of which I99H appears to be the most significant.

N-oxidation of irsogladine by the CYP2C subfamily in the rat, dog, monkey and man

1. The metabolism of irsogladine (ISG) was studied in hepatic microsomes from the rat, dog, monkey and man, and marked species differences were observed in N-oxidation of ISG. The rank order of the activity of the N-oxidation was shown to be man < monkey < dog < rat. 2. Anti-NADPH-P450 reductase antibody inhibited the formation of the N-oxidized metabolite of ISG (ISG-N-oxide) in hepatic microsomes from rats by 74%. AntiCYP2C11 antibody also inhibited the formation of ISG-N-oxide in hepatic microsomes from rat by 73%, whereas anti-CYP2E1, 3A2 and 4A1 antibody did not inhibit Noxidation. Thus, CYP2C11 in the rat is at least partially responsible for the N-oxidation of ISG in the rat. 3. Anti-CYP2C11 antibody also inhibited the formation of ISG-N-oxide in hepatic microsomes from the dog and monkey by 61 and 46% respectively. Therefore, a isoform(s) similar to CYP2C11 partially contributed to the N-oxidation of ISG in the dog and monkey. In contrast, human CYP2C9, a member of the human CYP2C subfamily, did not catalyse the N-oxidation of ISG. 4. These findings show that the marked species difference in the N-oxidation of ISG is caused by the difference in the catalytic properties of CYP2C among the species examined.

Biochemistry and molecular biology of the human CYP2C subfamily.

The cytochromes P450 (CYP) are a superfamily of hemoproteins which metabolize foreign chemicals as well as a number of endogenous compounds such as steroids. The human CYP2C subfamily appears to principally metabolize a number of clinically used drugs. Four members of this subfamily have been identified in humans: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. CYP2C9 is important in the metabolism certain of therapeutically used drugs including the anticoagulant drug warfarin and a number of nonsteroidal antiinflammatory drugs. A number of allelic variants of CYP2C9 exist in humans, but the effects of these allelic variants on metabolism in vivo remain to be determined. A well-characterized genetic polymorphism occurs in the 2C subfamily which is associated with the metabolism of the anticonvulsant drug mephenytoin. In population studies, individuals can be segregated into extensive and poor metabolizers of mephenytoin. Poor metabolizers are unable to 4'-hydroxylate the S-enantiomer of mephenytoin. There are marked interracial variations in the frequency of the poor metabolizer phenotype which represents 3-5% of Caucasians, but 18-23% of Oriental populations. The mechanism of this polymorphism has been recently elucidated. The enzyme responsible for S-mephenytoin metabolism has been shown to be CYP2C19, and two defects in the CYP2C19 gene have been described in poor metabolizers. The first defect, CYP2C19m1, consists of the creation of an aberrant splice site in exon 5. This defect accounts for approximately 75-85% of Caucasian and Japanese poor metabolizers. A second defect, CYP2C19m2, has been found only in Oriental populations and accounts for the remaining 25% of poor metabolizers in Japanese populations. The availability of genotyping tests for this polymorphism will enhance the assessment of the role of this pathway in clinical studies.