Browsing by Subject "Aryl Hydrocarbon Hydroxylases"

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    Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution
    (Public Library of Science, 2015) Schmidt, Eva M.; Wiek, Constanze; Parkinson, Oliver T.; Roellecke, Katharina; Freund, Marcel; Gombert, Michael; Lottmann, Nadine; Steward, Charles A.; Kramm, Christof M.; Yarov-Yarovoy, Vladimir; Rettie, Allan E.; Hanenberg, Helmut; Department of Pediatrics, IU School of Medicine
    CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5-exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.
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    CYP2B6 pharmacogenetics-based in vitro-in vivo extrapolation of efavirenz clearance by physiologically based pharmacokinetic modeling
    (ASPET, 2013-12) Xu, Cong; Quinney, Sara K.; Guo, Yingyying; Hall, Stephen D.; Li, Lang; Desta, Zeruesenay; Medicine, School of Medicine
    Efavirenz is mainly cleared by CYP2B6. The CYP2B6*6 allele is associated with lower efavirenz clearance. Efavirenz clearance was predictable using in vitro data for carriers of the CYP2B6*1/*1 genotype, but the prediction in carriers of the CYP2B6*6 allele was poor. To test the hypothesis that incorporation of mechanism of reduced efavirenz metabolism by the CYP2B6*6 allele can predict the genetic effect on efavirenz pharmacokinetics, in vitro-in vivo extrapolation of efavirenz clearance was performed by physiologically based pharmacokinetic modeling (Simcyp Simulator; Simcyp Ltd., Sheffield, UK) using data obtained from expressed CYP2B6.1 and CYP2B6.6 as well as human liver microsomes (HLMs) with CYP2B6*1/*1, *1/*6, and *6/*6 genotypes. Simulated pharmacokinetics of a single 600-mg oral dose of efavirenz for individuals with each genotype was compared with data observed in healthy subjects genotyped for the CYP2B6*6 allele (n = 20). Efavirenz clearance for carriers of the CYP2B6*1/*1 genotype was predicted reasonably well using HLM data, but the clearance in carriers of the CYP2B6*6 allele was underpredicted using both expressed and HLM systems. Improved prediction of efavirenz clearance was obtained from expressed CYP2B6 after recalculating intersystem extrapolation factors for CYP2B6.1 and CYP2B6.6 based on in vitro intrinsic clearance of bupropion 4-hydroxylation. These findings suggest that genetic effect on both CYP2B6 protein expression and catalytic efficiency needs to be taken into account for the prediction of pharmacokinetics in individuals carrying the CYP2B6*6/*6 genotype. Expressed CYP2B6 proteins may be a reliable in vitro system to predict effect of the CYP2B6*6 allele on the metabolism of CYP2B6 substrates.