Pharmacologic stimulation of cytochrome P450 46A1 and cerebral cholesterol turnover in mice

Abstract

Cytochrome P450 46A1 (CYP46A1) is a brain-specific cholesterol 24-hydroxylase responsible for the majority of cholesterol elimination from the brain. Genetically increased CYP46A1 expression in mice leads to improved cognition and decreases manifestations of Alzheimer disease. We found that four pharmaceuticals (efavirenz (EFV), acetaminophen, mirtazapine, and galantamine) prescribed for indications unrelated to cholesterol maintenance increased CYP46A1 activity in vitro. We then evaluated the anti-HIV medication EFV for the mode of interaction with CYP46A1 and the effect on mice. We propose a model for CYP46A1 activation by EFV and show that EFV enhanced CYP46A1 activity and cerebral cholesterol turnover in animals with no effect on the levels of brain cholesterol. The doses of EFV administered to mice and required for the stimulation of their cerebral cholesterol turnover are a hundred times lower than those prescribed to HIV patients. At such small doses, EFV may be devoid of adverse effects elicited by high drug concentrations. CYP46A1 could be a novel therapeutic target and a tool to further investigate the physiological and medical significance of cerebral cholesterol turnover.

Keywords: Alzheimer Disease; Brain Metabolism; Cholesterol; Cholesterol Metabolism; Cytochrome P450.

Figures

FIGURE 1.

EFV activation of CYP46A1Δ(2–50). Under identical assay conditions (see “Experimental Procedures” for optimized enzyme assay), full-length CYP46A1 and the truncated mutant (CYP46A1Δ(2–50)) showed similar activation of cholesterol 24-hydroxylation by EFV. The results of these and all other in vitro experiments represent the means ± S.D. of triplicate measurements. Statistical analysis is described under “Experimental Procedures”. ***, p < 0.001.

FIGURE 2.

Chemical structures of the drugs tested in this study.

FIGURE 3.

Effect of different drugs on CYP46A1 activity in the screening enzyme assay (A), assay with isolated brain microsomes (B), and optimized enzyme assay at three fixed cholesterol concentrations (C).Bars and lines of the same color pertain to the same drug. Conditions of the assays and statistical analysis are described under “Experimental Procedures.” *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Effect of EFV on the enzymatic and spectral properties of CYP46A1.A and B, rates of cholesterol 24-hydroxylation versus increasing concentrations of cholesterol and OR, respectively, in the absence (black lines) and presence of 20 μm (gray lines) and 100 μm (red lines) EFV. The data were fit to the Michaelis-Menten equation. C and E, the amplitude (ΔA) of cholesterol- and EFV-induced changes, respectively, in the difference spectrum of CYP46A1. D, cholesterol-induced changes in the CYP46A1 ΔA in the presence of 20 μm EFV. F, EFV-induced changes in the CYP46A1 ΔA in the presence of 20 μm cholesterol. The spectral data were fit to a hyperbolic equation (when the apparent spectral Kd was higher than the enzyme concentration), to a quadratic equation (when the apparent spectral Kd was lower than the enzyme concentration assuming a 1:1 stoichiometry), or to the Hill equation (when cooperativity of binding was observed). Insets show ligand-induced P450 difference spectra indicating the positions of the spectral peaks and troughs. The schematic representation of ligand binding (green rectangles for cholesterol and magenta triangles for EFV) to CYP46A1 (open circles) is also shown and is discussed in detail in Fig. 7.

FIGURE 5.

The extent of CYP46A1 activation in the in vitro enzyme assay depends on the order in which the components of the reconstitution system are mixed. The solid line shows CYP46A1 activation when the enzyme is first mixed with EFV, followed by the addition of 20 μm cholesterol. The dashed line shows CYP46A1 activation when the P450 is first mixed with 20 μm cholesterol, followed by the addition of EFV. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Computational analysis of EFV binding to the CYP46A1 surface. The CYP46A1 crystal structure is shown in light gray, cholesterol sulfate in the enzyme active site is shown in green, and EFV bound to each (A-C) of the three putative allosteric sites is shown in magenta. The heme group is shown in salmon, and amino acid residues are shown in dark gray. The black horizontal lines separate the cytosol (above) and the lipid bilayer (below). The proposed membrane orientation is taken from Ref. . The lowest energy EFV-binding site is near Lys-216 (A), whereas the sites near Phe-468 (B) and Pro-429 (C) have ∼3-fold higher relative free energies of EFV binding. The Lys-216-containing site is the most likely allosteric site for EFV.

FIGURE 7.

Model for CYP46A1 activation and inhibition by EFV. Molecules of the enzyme, drug, and substrate are shown as open circles (CYP46A1), magenta triangles (EFV), and green rectangles (cholesterol), respectively. A diagram for the lipid bilayer is also shown. We suggest that only one ligand, either cholesterol or EFV, can bind to the CYP46A1 active site, with ligand binding altering the shape of the active site cavity. Our previous crystallographic studies showed that when the substrate (cholesterol sulfate) is present, there is no space in the CYP46A1 active site for a second molecule the size of EFV (14). Also, only one drug molecule was found in the substrate-binding cavity in all seven CYP46A1-drug complexes crystallized so far (15, 16, 20, 50). Furthermore, in all ligand-bound CYP46A1 crystal structures, the P450 active site undergoes conformational changes to better fit the ligand. Our earlier studies indicated that the entrance to the CYP46A1 active site is embedded in the lipid bilayer, i.e. cholesterol enters CYP46A1 through the membrane (49). We envision that when EFV is at low concentrations (e.g. 20 μm) in vitro or in vivo, it binds to the CYP46A1 allosteric site, as this site is more accessible than the P450 active site embedded in the lipid bilayer. EFV binding to the allosteric site does not change the shape of the CYP46A1 active site when cholesterol is there, as this shape is rigidified by substrate-protein interactions. However, EFV alters the shape of the active site of cholesterol-free CYP46A1, making cholesterol binding tighter and enzyme catalysis more efficient. This would explain the enzyme's activation at low doses of EFV. The dependence of CYP46A1 activation on the addition of EFV prior to the addition of cholesterol in Fig. 5 supports this notion. However, when the concentration of EFV is high (e.g. 100 μm), not only does the drug bind to the allosteric site, but it also begins to bind to the active site and either prevents cholesterol from binding or displaces cholesterol from the active site. This causes CYP46A1 inhibition, which does not depend on whether EFV binds to substrate-free or substrate-bound CYP46A1. Our model is based on the assumption that despite its abundance in the ER, cholesterol may not always be present in the CYP46A1 active site when EFV binds to the allosteric site. This could be due to the low catalytic efficiency of CYP46A1 (its in vitro kcat for cholesterol is only ∼0.1 min−1 (14)), suggesting that the time between the CYP46A1 catalytic cycles may be sufficient for a drug (EFV) to occupy the CYP46A1 allosteric site. Alternatively, EFV could bind to the allosteric site when cholesterol still occupies the active site and elicit conformational changes in CYP46A1 after the substrate is metabolized to the product, and the product has left the active site.

FIGURE 8.

Effect of different doses of EFV on the cerebral sterol profile in mice (n = 3 per dose and time point) when the drug is delivered by gavage.Thin gray lines correspond to the 2-week treatment (2-wk Tx), whereas thick black lines reflect the 4-week treatment (4-wk Tx). The percentages of the sterol change relative to the untreated control are also shown, as is a scheme of the cholesterol biosynthesis pathway. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 9.

Effect of different doses of EFV on sterol content in mouse brain and serum (n = 3 per dose and time point) when the drug is delivered in drinking water.Dashed lines, untreated controls; thin lines, 0.09 mg/kg/day dose; thick lines, 0.22 mg/kg/day dose. Lines of the same color pertain to the same sterol. The percentages of the sterol increase relative to the untreated control are also shown. Tx, treatment. Please note that sterol normalizations are different compared with those in Fig. 8 and that one 1 mg of wet brain contains ∼115 μg of total protein. *, p < 0.05; **, p < 0.01; ***, p < 0.001.