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

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In Vitro Enzyme Kinetics.

The enzyme kinetic parameters describing the formation of tazarotenic acid sulfoxide and hydroxytazarotenic acid were determined for CYP26A1 and CYP26B1. Enzyme kinetic parameters were determined using Michaelis-Menten kinetics and are reported in Table 9-3 and shown in Figure 9-12. In general, incubations with CYP26B1 resulted in slightly higher Km and kcat values as compared to incubations conducted with CYP26A1. Intrinsic clearance values (calculated as Vmax / Km) were slightly higher for CYP26A1 as compared to CYP26B1 owing primarily to the difference in Km values between the two enzymes. Both enzymes appeared to favor formation of the hydroxylated metabolite of tazarotenic acid. Formation of tazarotenic acid sulfone from tazarotenic acid sulfoxide was linear through a substrate concentration of 50 μM and as such no kinetic parameters were determined for this metabolic pathway.

Tazarotenic Acid Phenotyping.

Previous reports evaluating the enzymes responsible for tazarotenic acid metabolism in vitro have implicated CYP2C8, FMO1 and FMO3 in the formation of tazarotenic acid sulfoxide (Attar et al., 2003). Using an expanded drug metabolizing enzyme panel and clinically relevant concentrations of tazarotenic acid, additional enzymes were identified that may contribute to the metabolism of tazarotenic acid. The highest rates of tazarotenic acid sulfoxide formation were observed for CYP26A1 and CYP26B1, followed by CYP2C8 and CYP3A7 (Figure 9-13A). Formation of the sulfoxide metabolite was also observed in incubations with CYP2C9, CYP2J2, CYP3A4, CYP3A5 and aldehyde oxidase. Minor contributions were noted for CYP1A2 and CYP2B6. No metabolite formation was observed in incubations with FMO1, FMO3 or FMO5. The hydroxylated metabolite of tazarotenic acid was formed primarily by CYP26A1 and CYP26B1, with additional contributions from CYP2C8, CYP3A5 and CYP3A7 (Figure 9-13B). Similar to the formation of tazarotenic acid sulfoxide, trace amounts of the hydroxylated metabolite were also observed in incubations with the majority of the enzymes evaluated in the panel.

Homology Modeling and Computational Docking Simulations.

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 (Foti et al., 2016). The crystal structure of CYP2C8 was obtained from the RCSB Protein Data Bank (pdb 1PQ2). The CYP26A1 or CYP26B1 homology models were superimposed on the CYP2C8 crystal structure using the Super script within Pymol (Schrodinger LLC, New York; http://www.pymolwiki.org/index.php/Super). Structural similarity was determined by calculating the root mean square deviation (RMSD) between the protein structures. Amino acid residues 494 – 512 from the CYP26B1 homology model were not included in the RMSD calculation. Computational docking of clotrimazole (CYP26A1 and CYP26B1), zafirlukast (CYP26A1) or candesartan cilexetil (CYP26B1) was accomplished using an induced fit docking algorithm which incorporated decreased van der Waals radii, spatial repositioning of non-rigid protein side chains and additional energy minimization functions post-ligand docking (Sherman et al., 2006a; Sherman et al., 2006b). Compounds for docking simulations were chosen based on inhibition potency as well as their potential (or lack thereof) for type II azole-heme interactions. Docking parameters required the center of mass of the inhibitors to be positioned within a 1728 Å3 grid which was designed to be approximately 3 Å above the protoporphyrin ring system using Glide (Schrodinger LLC, New York). The OPLS_2005 force field constraints used by LigPrep (Schrodinger LLC, New York) were used to prepare the energy minimized structures of clotrimazole (hypothesized to bind through type II ligand interactions) and candesartan cilexetil and zafirlukast (two compounds not hypothesized to interaction through type II binding) prior to docking. Binding orientations obtained from the computational docking experiments were evaluated and scored using GlideScore and eModel, which incorporates aspects of the GlideScore, ligand score and grid score into the final assessment of the plausibility of the docking results.

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In Vitro Inhibition Assays.

CYP2C8 in vitro IC50 values were obtained from previously reported literature sources and are noted in Table 10-1 (Walsky et al., 2005; Nath et al., 2010; VandenBrink et al., 2011). An initial single point inhibition screen (n = 3) was then used to estimate the inhibition potency of the set of known CYP2C8 inhibitors against CYP26A1 or CYP26B1. Talarazole and AM80 were included in the screening set as positive controls for CYP26 inhibition. In vitro screening conditions consisted of 10 μM 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 (Foti et al., 2016). The final volume of the incubation was 50 μL. Screening incubations were performed in triplicate and were pre-warmed at 37ºC for 3 minutes prior to addition of 1 mM NADPH (final concentration). Incubations were terminated after 10 minutes with three volumes (v/v) of 100 nM tolbutamide in acetonitrile and centrifuged for 20 minutes at 1240 x g. A portion of the resulting supernatant was transferred for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Table of contents :

1. Table of Contents
1. Table of Contents
2. Acknowledgements
3. Funding Sources
4. List of Tables
5. List of Figures
6. List of Abbreviations
7. Chapter I: Introduction to Retinoic Acid Signaling and Cytochrome P450 
7.1. Introduction
7.2. Retinoic Acid Signaling.
7.3. Cytochrome P450.
7.4. Role of CYP26.
7.5. CYP26 Pharmacology.
7.6. CYP26 Homology Modeling.
8. Aims and Scope.
9. Chapter II: Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1
9.1. Introduction
9.2. Materials and Methods.
9.2.1. Materials.
9.2.2. Sequence Verification and Expression of CYP26B1.
9.2.3. IC50 Determination for Retinoic Acid Receptor Agonists.
9.2.4. Homology Modeling.
9.2.5. Metabolic Profiling.
9.2.6. Enzyme Kinetics.
9.2.7. Tazarotenic Acid Phenotyping.
9.2.8. LC-MS/MS Analysis.
9.2.9. Data Analysis.
9.3. Results.
9.3.1. Homology Modeling.
9.3.2. Metabolic Profile.
9.3.3. In Vitro Enzyme Kinetics.
9.3.4. Tazarotenic Acid Phenotyping.
9.4. Discussion
10. Chapter III: 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
10.1. Introduction
10.2. Materials and Methods.
10.2.1. Materials.
10.2.2. Homology Modeling and Computational Docking Simulations.
10.2.3. In Vitro Inhibition Assays.
10.2.4. Spectral Binding Determination.
10.2.5. Assessment of In Vitro Free Fraction.
10.2.6. In Vitro Stability of Candesartan Cilexetil.
10.2.7. Calculation of Cmax,u / IC50.
10.2.8. Liquid Chromatography – Mass Spectrometry Analysis.
10.3. Results
10.3.1. Evaluation of tazarotenic acid sulfoxide formation as a probe substrate of CYP26.
10.3.2. In Vitro Inhibition Screening and IC50 Determination.
10.3.3. Computational Docking Simulations
10.3.4. Spectral Binding Studies.
10.3.5. Calculation of Cmax,u / IC50.
10.4. Discussion.
11. Chapter IV: Contribution of CYP26 to the Metabolism and Clearance of Retinoic Acid Receptor Agonists and Antagonists
11.1. Introduction.
11.2. Materials and Methods.
11.2.1. Materials.
11.2.2. In Vitro Clearance of Retinoic Acid Receptor Agonists and Antagonists by Recombinant CYP26s, CYP2C8 and CYP3A4.
11.2.3. Adapalene Phenotyping.
11.2.4. Metabolite Identification of Adapalene and Des-Adamantyl Adapalene in Recombinant CYP26s.
11.2.5. Computational Docking of Adapalene and Des-Adamantyl Adapalene in CYP26A1, CYP26B1 and CYP26C1 homology models.
11.2.6. LC-MS/MS Analysis.
11.2.7. Data Analysis.
11.3. Results
11.3.1. In Vitro Clearance of Retinoic Acid Receptor Agonists and Antagonists by Recombinant CYP26s, CYP2C8 and CYP3A4.
11.3.2. Metabolite Identification of Adapalene and Des-Adamantyl Adapalene in Recombinant CYP26s.
11.3.3. Computational Docking of Adapalene and Des-Adamantyl Adapalene in CYP26A1, CYP26B1 and CYP26C1 homology models.
11.3.4. Adapalene Phenotyping.
11.4. Discussion
12. Chapter V: General Conclusions
13. References
14. Vita

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