Journal of Cellular Biochemistry

Metabolic stability of 3-Epi-1α,25-dihydroxyvitamin D3 over 1α, 25-dihydroxyvitamin D3: Metabolism and molecular docking studies using rat CYP24A1

Authors

  • Steve Y. Rhieu,

    1. Epimer LLC, North Smithfield, Rhode Island
    2. Division of Biology and Medicine, Brown University, Providence, Rhode Island
    3. School of Engineering, Brown University, Providence, Rhode Island
    Current affiliation:
    1. National Institute of Standards and Technology, Gaithersburg, Maryland
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  • Andrew J. Annalora,

    1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California
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  • Guochun Wang,

    1. Epimer LLC, North Smithfield, Rhode Island
    2. Division of Biology and Medicine, Brown University, Providence, Rhode Island
    3. School of Engineering, Brown University, Providence, Rhode Island
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  • Caroline C. Flarakos,

    1. Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts
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  • Rose M. Gathungu,

    1. Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts
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  • Paul Vouros,

    1. Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts
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  • Rita Sigüeiro,

    1. Departamento de Química Orgánica, Laboratorio de Investigación Ignacio Ribas, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
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  • Antonio Mouriño,

    1. Departamento de Química Orgánica, Laboratorio de Investigación Ignacio Ribas, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
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  • Inge Schuster,

    1. Institute for Theoretical Chemistry, University of Vienna, Vienna, Austria
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  • G. Tayhas R. Palmore,

    1. Division of Biology and Medicine, Brown University, Providence, Rhode Island
    2. School of Engineering, Brown University, Providence, Rhode Island
    3. Department of Chemistry, Brown University, Providence, Rhode Island
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  • G. Satyanarayana Reddy

    Corresponding author
    • Epimer LLC, North Smithfield, Rhode Island
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  • Steve Y. Rhieu and Andrew J. Annalora contributed equally to this work.
  • Conflict of interest: The authors have no conflict of interest to declare.

Correspondence to: Dr. G. Satyanarayana Reddy, M.D., Epimer LLC, 1 Valley View Drive, North Smithfield, RI 02896.

E-mail: satyareddy125@gmail.com

ABSTRACT

3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), a natural metabolite of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), exhibits potent vitamin D receptor (VDR)-mediated actions such as inhibition of keratinocyte growth or suppression of parathyroid hormone secretion. These VDR-mediated actions of 3-epi-1α,25(OH)2D3 needed an explanation as 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, exhibits low affinity towards VDR. Metabolic stability of 3-epi-1α,25(OH)2D3 over 1α,25(OH)2D3 has been hypothesized as a possible explanation. To provide further support for this hypothesis, we now performed comparative metabolism studies between 3-epi-1α,25(OH)2D3 and 1α,25(OH)2D3 using both the technique of isolated rat kidney perfusion and purified rat CYP24A1 in a cell-free reconstituted system. For the first time, these studies resulted in the isolation and identification of 3-epi-calcitroic acid as the final inactive metabolite of 3-epi-1α,25(OH)2D3 produced by rat CYP24A1. Furthermore, under identical experimental conditions, it was noted that the amount of 3-epi-calcitroic acid produced from 3-epi-1α,25(OH)2D3 is threefold less than that of calcitroic acid, the analogous final inactive metabolite produced from 1α,25(OH)2D3. This key observation finally led us to conclude that the rate of overall side-chain oxidation of 3-epi-1α,25(OH)2D3 by rat CYP24A1 leading to its final inactivation is slower than that of 1α,25(OH)2D3. To elucidate the mechanism responsible for this important finding, we performed a molecular docking analysis using the crystal structure of rat CYP24A1. Docking results suggest that 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, binds to CYP24A1 in an alternate configuration that destabilizes the formation of the enzyme-substrate complex sufficiently to slow the rate at which 3-epi-1α,25(OH)2D3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid. J. Cell. Biochem. 114: 2293–2305, 2013. © 2013 Wiley Periodicals, Inc.

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