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Research ArticleArticle

In Silico and Intuitive Predictions of CYP46A1 Inhibition by Marketed Drugs with Subsequent Enzyme Crystallization in Complex with Fluvoxamine

Natalia Mast, Marlin Linger, Matthew Clark, Jeffrey Wiseman, C. David Stout and Irina A. Pikuleva
Molecular Pharmacology November 2012, 82 (5) 824-834; DOI: https://doi.org/10.1124/mol.112.080424
Natalia Mast
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio (N.M., M.L., I.A.P.); Pharmatrope, Ltd., Wayne, Pennsylvania (M.C., J.W.); Drug Discovery Center, University of Cincinnati, Cincinnati, Ohio (J.W.); and Department of Molecular Biology, the Scripps Research Institute, La Jolla, California (C.D.S.)
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Marlin Linger
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio (N.M., M.L., I.A.P.); Pharmatrope, Ltd., Wayne, Pennsylvania (M.C., J.W.); Drug Discovery Center, University of Cincinnati, Cincinnati, Ohio (J.W.); and Department of Molecular Biology, the Scripps Research Institute, La Jolla, California (C.D.S.)
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Matthew Clark
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio (N.M., M.L., I.A.P.); Pharmatrope, Ltd., Wayne, Pennsylvania (M.C., J.W.); Drug Discovery Center, University of Cincinnati, Cincinnati, Ohio (J.W.); and Department of Molecular Biology, the Scripps Research Institute, La Jolla, California (C.D.S.)
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Jeffrey Wiseman
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio (N.M., M.L., I.A.P.); Pharmatrope, Ltd., Wayne, Pennsylvania (M.C., J.W.); Drug Discovery Center, University of Cincinnati, Cincinnati, Ohio (J.W.); and Department of Molecular Biology, the Scripps Research Institute, La Jolla, California (C.D.S.)
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C. David Stout
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio (N.M., M.L., I.A.P.); Pharmatrope, Ltd., Wayne, Pennsylvania (M.C., J.W.); Drug Discovery Center, University of Cincinnati, Cincinnati, Ohio (J.W.); and Department of Molecular Biology, the Scripps Research Institute, La Jolla, California (C.D.S.)
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Irina A. Pikuleva
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio (N.M., M.L., I.A.P.); Pharmatrope, Ltd., Wayne, Pennsylvania (M.C., J.W.); Drug Discovery Center, University of Cincinnati, Cincinnati, Ohio (J.W.); and Department of Molecular Biology, the Scripps Research Institute, La Jolla, California (C.D.S.)
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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 Kd 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 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 probably further adjusts its configuration and elicits the movement of the protein side chains.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM62882]. I.A.P. is a recipient of the Jules and Doris Stein Professorship from Research to Prevent Blindness. Portions of this research were carried out at SSRL, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research and by the National Institutes of Health National Institute of General Medical Sciences [Grant P41-GM103393] and National Institutes of Health National Center for Research Resources [Grant P41-RR001209].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.080424.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    TCP
    tranylcypromine
    FLV
    fluvoxamine
    KPi
    potassium phosphate
    NTA
    nitrilotriacetic
    BME
    β-mercaptoethanol
    DTT
    dithiothreitol
    SSRL
    the Stanford Synchrotron Radiation Lightsource
    PDB
    Protein Data Bank.

  • Received June 6, 2012.
  • Accepted August 2, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (5)
Molecular Pharmacology
Vol. 82, Issue 5
1 Nov 2012
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Research ArticleArticle

CYP46A1-Drug Interactions

Natalia Mast, Marlin Linger, Matthew Clark, Jeffrey Wiseman, C. David Stout and Irina A. Pikuleva
Molecular Pharmacology November 1, 2012, 82 (5) 824-834; DOI: https://doi.org/10.1124/mol.112.080424

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Research ArticleArticle

CYP46A1-Drug Interactions

Natalia Mast, Marlin Linger, Matthew Clark, Jeffrey Wiseman, C. David Stout and Irina A. Pikuleva
Molecular Pharmacology November 1, 2012, 82 (5) 824-834; DOI: https://doi.org/10.1124/mol.112.080424
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