TY - JOUR T1 - In Silico and Intuitive Predictions of CYP46A1 Inhibition by Marketed Drugs with Subsequent Enzyme Crystallization in Complex with Fluvoxamine JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.112.080424 SP - mol.112.080424 AU - Natalia Mast AU - Marlin Linger AU - Matthew Clark AU - Jeffrey Wiseman AU - C. David Stout AU - Irina A. Pikuleva Y1 - 2012/01/01 UR - http://molpharm.aspetjournals.org/content/early/2012/08/02/mol.112.080424.abstract N2 - Cytochrome P450 46A1 (CYP46A1), or cholesterol 24-hydroxylase, is an important brain enzyme which may be inhibited by structurally distinct pharmaceuticals both in vitro and in vivo. To identify additional inhibitors of CYP46A1 among FDA-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 Kd values in a low micromolar range. One of the identified tight binders, the widely used antidepressant fluvoxamine, was co-crystallized 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 NH2-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 CF3-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 likely further adjusts its configuration and elicits the movement of the protein side chains. ER -