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 Ͼ50%. 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.

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

“…We selected one representative drug from nucleotide derivatives and platinum-containing compounds, all aromatase inhibitors, and other drugs on the list. We also included seven noncancer pharmaceuticals identified as strong inhibitors of cholesterol-metabolizing CYP46A1 and CYP11A1 in our previous studies (Mast et al, 2010(Mast et al, , 2012(Mast et al, , 2013a. Thus, a total of 26 compounds were selected for evaluations in the present work.…”

“…Drug selection was based on approaches developed in our prior studies of CYP46A1 and CYP11A1 (Mast et al, 2012(Mast et al, , 2013a, which like CYP27A1 act on cholesterol as a substrate, but generate different products, 24-hydroxycholesterol and pregnenolone, respectively. We used intuitive predictions and were guided by the knowledge of the CYP27A1 substrate specificities, shape of the active sites of CYP46A1 and CYP11A1, and the previously generated CYP27A1 model based on crystal structure of CYP11A1 (Mast et al, 2006(Mast et al, , 2008(Mast et al, , 2011Charvet et al, 2013).…”

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

“…This is an important reaction for the clearance of excess cholesterol from the brain. Interestingly, the substratefree structure of 46A1 exhibits a much different cavity shape (34), and the 46A1 active site adapts to bind several structurally unrelated inhibitors (35,36). In contrast, structures of microsomal 2R1 (37) indicate that the secosterol moiety of vitamin D 3 and related compounds is bound between helices I and G and the helix B-C loop.…”

Antifungal Azoles: Structural Insights into Undesired Tight Binding to Cholesterol-Metabolizing CYP46A1

“…Unexpectedly, CYP46A1 was found to have submicromolar K d values for clotrimazole and voriconazole in vitro Shafaati et al, 2010), and to be inhibited by voriconazole in vivo in mice (Shafaati et al, 2010). In subsequent studies, CYP46A1 was also found to tightly bind another azole posaconazole (Mast et al, 2012); hence, in the present work we cocrystallized CYP46A1 with this azole to gain insight into the features of the enzyme active site that enable binding of the three structurally very …”

“…POS was identified as a tight binder of CYP46A1 based on the interaction with the full-length enzyme (Mast et al, 2012). However, for crystallization we used the truncated Δ(2-50) variant; we determined the apparent K d of POS as well as its K i for Δ(2-50) CYP46A1.…”