Substituted Imidazole of 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5- yl)methyl]-1-piperazinyl]pyrimidine Inactivates Cytochrome P450 2D6 by Protein Adduction

detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron

Molecular Dynamics of CYP2D6 Polymorphisms in the Absence and Presence of a Mechanism-Based Inactivator Reveals Changes in Local Flexibility and Dominant Substrate Access Channels

<div><p>Cytochrome P450 enzymes (CYPs) represent an important enzyme superfamily involved in metabolism of many endogenous and exogenous small molecules. CYP2D6 is responsible for ∼15% of CYP-mediated drug metabolism and exhibits large phenotypic diversity within CYPs with over 100 different allelic variants. Many of these variants lead to functional changes in enzyme activity and substrate selectivity. Herein, a molecular dynamics comparative analysis of four different variants of CYP2D6 was performed. The comparative analysis included simulations with and without SCH 66712, a ligand that is also a mechanism-based inactivator, in order to investigate the possible structural basis of CYP2D6 inactivation. Analysis of protein stability highlighted significantly altered flexibility in both proximal and distal residues from the variant residues. In the absence of SCH 66712, *34, *17-2, and *17-3 displayed more flexibility than *1, and *53 displayed more rigidity. SCH 66712 binding reversed flexibility in *17-2 and *17-3, through *53 remained largely rigid. Throughout simulations with docked SCH 66712, ligand orientation within the heme-binding pocket was consistent with previously identified sites of metabolism and measured binding energies. Subsequent tunnel analysis of substrate access, egress, and solvent channels displayed varied bottle-neck radii. Taken together, our results indicate that SCH 66712 should inactivate these allelic variants, although varied flexibility and substrate binding-pocket accessibility may alter its interaction abilities.</p></div

Allelic Variants of CYP2D6 Analyzed in this Study^∧.

<p>amino acid alterations and functional significance as compared to allelic form CYP2D6*1.</p><p>Allelic Variants of CYP2D6 Analyzed in this Study<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108607#nt101" target="_blank">^∧</a>.</p

Total free energy of SCH 66712 binding estimated by MM-PBSA.

1<p>Corresponding errors for each value are shown in parenthesis.</p>2<p>ΔG_exp was calculated using spectral binding constants, <i>K</i>_s, and the relationship: ΔG_exp = RT ln <i>K</i>_s, as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108607#pone.0108607-Livezey1" target="_blank">[22]</a>.</p><p>Total free energy of SCH 66712 binding estimated by MM-PBSA.</p

Structural fluctuations of each CYP2D6 variant with SCH 66712 bound.

<p>Rainbow color scheme indicates degree of fluctuation with blue indicating little fluctuation to red indicating large fluctuation. The size of the backbone strand is also indicative of fluctuation with large diameter indicative of fluctuations. All structures showed a rigid core surrounding the heme with the area of the greatest flexibility in the F-G loop and helix A′ regions on the distal side. *34 was the most flexible of the variants and *53 was the most rigid.</p

Interaction of SCH 66712 with allelic variants.

<p>(A) Initial docking poses of SCH 66712 with each CYP2D6 variant. SCH 66712 was docked using AutoDock Vina as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108607#s2" target="_blank">Methods</a>. Other poses not shown include positioning of the heteroaromatic ring above the heme iron. Both positions, phenyl or heteroaromatic ring pointing toward heme, are consistent with known sites of metabolism by *1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108607#pone.0108607-Nagy1" target="_blank">[21]</a>. (B) Representative snapshots of the most prominent SCH 66712 binding mode for each variant as determined by PCA from 1000 snapshots (PCA population kernels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108607#pone.0108607.s012" target="_blank">Figure S12</a>). Additional, minor, binding modes were also identified for *1 and *17-3 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108607#pone.0108607.s013" target="_blank">Figure S13</a>. Helix I is shown in the background. For each variant, the phenyl ring of SCH 66712 points toward the heme. In *1, *17-2, and *17-3 the heteroaromatic ring of SCH 66712 is pointing to a pocket between helices I and G. In *53 and *34, SCH 66712 is oriented with the heteroaromatic ring pointing to a pocket between helices F and E.</p

Changes in root mean square fluctuations (ΔRMSF) of backbone atoms of *34 (purple), *17-2 (blue), *17-3 (green), and *53 (orange) relative to *1 (corresponds to zero line) with (A) no ligand bound and (B) with SCH 66712 bound.

<p>Positive values for ΔRMSF correlate to atoms of increased flexibility while negative values for ΔRMSF correlate with more rigid atoms compared to reference CYP2D6*1. The x-axis indicates amino acid position. Helices are indicated in blue (and labeled at the top of Panel A), β-sheets in green, and turns in pink. The F-G, C-D, and G-H loop regions show the greatest variability in flexibility between variants and *1 both with and without SCH 66712 bound. There was also a modest difference in flexibility in the meander loop region.</p