In silico and intuitive predictions of CYP46A1 inhibition by marketed drugs with subsequent enzyme crystallization in complex with fluvoxamine

Abstract

Cytochrome P450 46A1 (cholesterol 24-hydroxylase) is an important brain enzyme that may be inhibited by structurally distinct pharmaceutical agents both in vitro and in vivo. To identify additional inhibitors of CYP46A1 among U.S. Food and Drug Administration-approved therapeutic agents, we used in silico and intuitive predictions and evaluated some of the predicted binders in the enzyme and spectral binding assays. We tested a total of 298 marketed drugs for the inhibition of CYP46A1-mediated cholesterol hydroxylation in vitro and found that 13 of them reduce CYP46A1 activity by >50%. Of these 13 inhibitors, 7 elicited a spectral response in CYP46A1 with apparent spectral K(d) values in a low micromolar range. One of the identified tight binders, the widely used antidepressant fluvoxamine, was cocrystallized with CYP46A1. The structure of this complex was determined at a 2.5 Å resolution and revealed the details of drug binding to the CYP46A1 active site. The NH(2)-containing arm of the Y-shaped fluvoxamine coordinates the CYP46A1 heme iron, whereas the methoxy-containing arm points away from the heme group and has multiple hydrophobic interactions with aliphatic amino acid residues. The CF(3)-phenyl ring faces the entrance to the substrate access channel and has contacts with the aromatic side chains. The crystal structure suggests that only certain drug conformers can enter the P450 substrate access channel and reach the active site. Once inside the active site, the conformer probably further adjusts its configuration and elicits the movement of the protein side chains.

Figures

Fig. 1.

A summary of the first round of in silico predictions. The docking scores of the 784 computed binders are shown as gray vertical lines. The effects of drugs on CYP46A1 activity are also shown: black lines correspond to the drugs that altered CYP46A1 activity by ≤20%, and cyan and magenta lines are the pharmaceutical agents that inhibited CYP46A1 by >20% (actives) and 50% (hits), respectively. The magenta dashed line indicates the inhibition cutoff limit for the hits. Drugs tested in our previous studies and the present work are shown in the regular and stroked fonts, respectively. Data on all tested drugs are given in Supplemental Table 2.

Fig. 2.

A summary of the second round of in silico predictions and intuitive predictions. The docking scores are shown as gray vertical lines and effects on enzyme activity as lines in black (no CYP46A1 inhibition), cyan (>20% inhibition), and magenta (>50% of inhibition). The magenta dashed line indicates the inhibition cutoff limit for the hits. Drugs tested in our previous studies and during the first round are shown in the regular font and drugs tested in this round are in the stroked font. Data on all tested drugs are given in Supplemental Table 2.

Fig. 3.

FLV inhibition of CYP46A1-mediated cholesterol hydroxylation in isolated bovine brain microsomes. Assay conditions are described under Materials and Methods. The results represent the average of triplicate measurements ± S.D. Some of the error bars are not seen because they are smaller than the symbol size.

Fig. 4.

View of the CYP46A1 active site illustrating interactions with FLV. FLV is in dark gray, and amino acid residues in contact with FLV are in light gray. Dashed black lines indicate hydrogen bonds. The heme group is in red. The nitrogen, oxygen, fluorine, and iron atoms are in blue, red, pale cyan, and orange, respectively. The enclosed volume of the active site is shown as a semitransparent surface.

Fig. 5.

Superimposed views of the active sites in FLV-bound (light gray) and ligand-free (light green) CYP46A1 showing the amino acid residues undergoing conformational changes upon FLV binding. The active site volumes (semitransparent surface in FLV-bound and mesh in ligand-free CYP46A1) are also shown. FLV is in dark gray. The heme group in FLV and ligand-free CYP46A1 are in red and raspberry, respectively. Coloring of atoms is the same as in Fig. 4.

Fig. 6.

Superimposed views of FLV-bound (light gray) and TCP-bound (pink) CYP46A1 showing that the same amino acid residues undergo conformational changes upon drug binding. The active site volumes (semitransparent surface in FLV-bound and mesh in TCP-bound CYP46A1) are also shown. Two active site water molecules, 613 and 614, in TCP-bound CYP46A1 are shown as green dotted spheres. The dashed black line indicates an additional hydrogen bond between FLV-NH2 and CYP46A1. FLV is in dark gray, and TCP is in magenta. The heme groups in FLV- and TCP-bound CYP46A1 are in red and salmon, respectively. Coloring of atoms is the same as in Fig. 4.

Fig. 7.

Possible conformations of the FLV E-isomer in solution as suggested by computational modeling. Dashed black lines indicate the maximal width of the conformers. RPE, the relative potential energy; SA, surface area.