Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1

Abstract

Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by docking at-RA in the active site and comparing the results to known metabolic profiles of at-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å 3 and 977 Å 3 , respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.

Comparison of the ligand binding site of CYP2C8 with CYP26A1 and CYP26B1: a structural basis for the identification of new inhibitors of the retinoic acid hydroxylases

“…Portions of the protein structure are not displayed for added clarity. ) based on homology modeling are somewhat smaller than the volume of the active site measured from the crystal structure of CYP2C8 (1438 Å 3 ), though it would appear they are large enough to accommodate larger xenobiotic compounds, similar to other CYP isoforms 56,60 . In order to test the hypothesis of whether CYP26A1 and CYP26B1 were capable of binding xenobiotics with a similar pharmacophore profile as CYP2C8, a set of known CYP2C8 inhibitors was screened for inhibition activity against CYP26A1 and CYP26B1.…”

“…CYP26A1 and CYP26B1 homology models based on the crystal structure of CYP120 (pdb 2VE3) were designed using Prime modeling software (Schrodinger LLC, New York) as previously described 60 . In brief, CYP120 was chosen as a template based on its 33% sequence identity and 53% positive sequence coverage with CYP26A1 and 34% sequence identity and 54% positive sequence coverage with CYP26B1.…”

“…Inhibitors of CYP26 activity were included in the screening set as positive controls. In vitro screening conditions consisted of 10 mM inhibitor, 5 nM CYP26A1 or CYP26B1, 25 nM purified human cytochrome P450 reductase, and 200 nM tazarotenic acid, a compound which has recently been shown to be a substrate of CYP26 60 . The final volume of the incubation was 50 mL.…”

“…Tazarotenic acid (Figure 1) has recently been identified as a xenobiotic substrate of CYP26A1 and CYP26B1 60 . Prior to utilizing the tazarotenic acid assay to screen new compounds for inhibition of CYP26A1 and CYP26B1, IC 50 values were generated for a test set of known CYP26 inhibitors (Figure 2) using tazarotenic acid as the probe substrate and compared to previously published results obtained when 9-cis-retinoic acid Figure 2.…”

“…The RMSD value for the CYP26B1 protein structure and CYP2C8 was 4.624. Visual examination of the active sites of the three cytochrome P450 isozymes revealed carboxylic acid binding residues located in comparable regions of the active site of CYP2C8 (Gly98, Asn99, Ser100), CYP26A1 (Arg 86, Arg90) and CYP26B1 (Tyr372, Arg373) that have been suggested to interact with the carboxylic acid moiety of 9-cis-retinoic acid, at-RA, or tazarotenic acid 57,60,76,77 . In order to rationalize the ligand binding of the known CYP2C8 inhibitors in the active sites of either CYP26A1 or CYP26B1, a number of computational docking experiments were performed.…”