Binding of a cyano- and fluoro-containing drug bicalutamide to cytochrome P450 46A1: unusual features and spectral response

Abstract

Cytochrome P450 46A1 (CYP46A1) is the cholesterol 24-hydroxylase initiating the major pathways of cholesterol removal from the brain, and bicalutamide (BIC) is a drug of choice for the treatment of progressive androgen-dependent prostate cancer. We evaluated the interactions of BIC with CYP46A1 by x-ray crystallography and by conducting solution and mutagenesis studies. Because BIC is administered to patients as a racemic mixture of the S and R isomers, we studied all three, racemic BIC as well as the S and R isomers. Co-crystallization of CYP46A1 with racemic BIC led to structure determinations at 2.1 Å resolution with the drug complexes exhibiting novel properties. Both BIC isomers bind to the CYP46A1 active site in very similar single orientation and adopt an energetically unfavorable folded conformation. This folded BIC conformation is correlated with drug-induced localized shifts of amino acid side chains in CYP46A1 and unusual interactions in the CYP46A1-BIC complex. One of these interactions involves a water molecule that is coordinated to the P450 heme iron and also hydrogen-bonded to the BIC nitrile. Due to polarization of the water in this environment, the heme elicits previously unreported types of P450 spectral responses. We also observed that access to the P450 active site was affected by differential recognition of S versus R isomers at the CYP46A1 surface arising from BIC conformational polymorphism.

Figures

FIGURE 1.

Absolute and difference (left side insets) spectra of CYP46A1 in the absence (black line) and presence (dashed colored lines) of different ligands.Right side insets show the fitting of the binding curves. Shown are P450 spectra with racBIC (orange dashed line, A), 24S-hydroxycholesterol (olive dashed line, B), imidazole (solid blue line) and racBIC (orange dashed line) (C), S-BIC (dashed purple line, D), and R-BIC (dashed green line, E). The concentration of CYP46A1 is 1 μm, and that of the ligand is 20 μm. The gray solid lines are the absorbance of BIC. Chemical structures of S-BIC (in purple) and R-BIC (in green) are also shown.

FIGURE 2.

Inhibition of CYP46A1-mediated cholesterol hydroxylation by racBIC in isolated bovine brain microsomes. Assay conditions are described under “Experimental Procedures.” The results represent the average of triplicate measurements ± S.D.

FIGURE 3.

Unbiased σA weighted |Fo| − |Fc| electron density for the R,S-BIC complex of CYP46A1 at 2.1 Å resolution contoured from 3σ to 5σ in intervals of 1σ. BIC carbon atoms are white, oxygen atoms are red, nitrogen atoms are blue, fluorine atoms are purple, and sulfur atoms are cyan; heme carbon atoms are orange; Cα atoms of the I-helix are yellow. Both BIC isomers are shown.

FIGURE 4.

Views of the CYP46A1 active site illustrating interactions with BIC isomers (A and C, S-BIC; B and D, R-BIC).A and B, interactions with the BIC chiral hydroxyl, amide nitrogen, sulfonyl oxygen, and cyano group are shown. C and D, interactions with the BIC fluorophenyl and cyanotrifluoromethylphenyl rings are shown. S- and R-BIC are shown as sticks and balls in magenta and gray, respectively, and amino acid residues in CYP46A1 are depicted as sticks in light magenta. Water molecules are shown as green spheres. Dashed black lines indicate hydrogen bonds. The heme group is in red. The nitrogen, oxygen, fluorine, sulfur, and iron atoms are in blue, red, pale cyan, yellow, and orange, respectively.

FIGURE 5.

Views of the AR binding pocket illustrating interactions with R-BIC (in dark gray). Carbon atoms of AR are shown in purple. Water molecules are shown as green spheres. Dashed black lines indicate hydrogen bonds. The nitrogen, oxygen, fluorine and sulfur atoms are in blue, red, pale cyan, and yellow, respectively.

FIGURE 6.

Conformations of BIC.A, in co-complex with CYP46A1 (both isomers are shown, S- is in magenta, and R- is in gray). B, in co-complex with the AR (37). C–F, in structures of BIC alone (–41), and G, in the computed structure (42). The nitrogen, oxygen, fluorine and sulfur atoms are in blue, red, pale cyan, and yellow, respectively.

FIGURE 7.

Superimposed views of the active sites in BIC-bound (light magenta) and ligand-free (light blue) CYP46A1 showing the amino acid residues undergoing conformational changes upon BIC binding. The putative sequence of the conformational shifts is also shown. A and B, shown are the movements induced by the initial BIC recognition on the CYP46A1 surface and subsequent BIC sliding inside the substrate access channel. C, shown are key conformational changes in the active site. D, shown are secondary adjustments in the active site. E, shown are secondary adjustments on the CYP46A1 surface. The enclosed volumes of the active site in BIC-bound and ligand-free CYP46A1 are shown as a semitransparent pink surface and blue mesh. Water molecules are shown as green spheres. Dashed black lines indicate hydrogen bonds. The heme group in BIC-bound and ligand-free CYP46A1 are in red and salmon, respectively. The nitrogen, oxygen, fluorine, sulfur, and iron atoms are in blue, red, pale cyan, yellow, and orange, respectively.

FIGURE 8.

Effect of replacements of amino acid residues outside the active site on difference spectra and apparent Kd values of the CYP46A1 mutants.Insets show the fitting of the binding curves. BIC binding properties of the CYP46A1 wild type (WT) are shown for comparison. Titrations were carried with racBIC, and S and R isomers as described under “Experimental Procedures.” The results represent the average of triplicate measurements ± S.D.

FIGURE 9.

Effect of replacements of amino acid residues inside the active site on difference spectra and apparent Kd values of the CYP46A1 mutants.Insets show the fitting of the binding curves. Titrations were carried with racBIC, S and R isomers as described under “Experimental Procedures.” The results represent the average of triplicate measurements ± S.D.