Different Molecular Mechanisms of Vitamin D3 Receptor Antagonists

  1. Andrea Toell1,
  2. Manuel Macias Gonzalez1,2,
  3. Dagmar Ruf1,
  4. Andreas Steinmeyer3,
  5. Seiichi Ishizuka4 and
  6. Carsten Carlberg1,2
  1. 1Institut für Physiologische Chemie I and Biomedizinisches Forschungszentrum, Heinrich-Heine-Universität, Düsseldorf, Germany (A.T., M.M.G., D.R., C.C); 2Department of Biochemistry, University of Kuopio, Kuopio, Finland (M.M.G., C.C.); 3Medicinal Chemistry, Schering AG, Berlin, Germany (A.S.); and 4Department of Bone and Calcium Metabolism, Teijin Institute for Bio-Medical Research, Tokyo, Japan (S.I.)

    Abstract

    Two structurally different antagonists of the nuclear hormone 1α,25-dihydroxyvitamin D3[1α,25(OH)2D3], the 25-carboxylic ester ZK159222 and the 26,23-lactone TEI-9647, have recently been described. In this study, the molecular mechanisms and the efficacy of both antagonists were compared. ZK159222 showed similar potency and sensitivity to 1α,25(OH)2D3 in ligand-dependent gel shift assays using the vitamin D receptor (VDR), the retinoid X receptor, and specific DNA binding sites, whereas TEI-9647 displayed reduced potency and >10-fold lower sensitivity in this assay system. Limited protease digestion and gel shift clipping assays showed that the two antagonists stabilized individual patterns of VDR conformations. Both antagonists prevented the interaction of the VDR with coactivator proteins, as demonstrated by GST-pull-down and supershift assays; like the natural hormone, however, they were able to induce a dissociation of corepressor proteins. Interestingly, ZK159222 demonstrated functional antagonism in reporter gene assays both in HeLa and MCF-7 cells, whereas TEI-9647 functioned as a less sensitive antagonist only in MCF-7 cells. In conclusion, the two 1α,25(OH)2D3 analogs act in part via different molecular mechanisms, which allows us to speculate that ZK159222 is a more complete antagonist and TEI-9647 a more selective antagonist.

    Footnotes

    • Send reprint requests to: Prof. Carsten Carlberg. Department of Biochemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: carlberg{at}messi.uku.fi

    • This work was supported by Deutsche Forschungsgemeinshaft Grant Ca229/1, the Fonds der Chemischen Industrie, and the Wilhelm Sander Foundation (all to C.C.). M.M.G. is a fellow of the Spanish Ministry of Culture and Education.

    • Abbreviations:
      1α,25(OH)2D3
      1α,25-dihydroxyvitamin D3
      VDR
      1α,25-dihydroxyvitamin D3 receptor
      VDRE
      1α,25-dihydroxyvitamin D3response element
      RXR
      retinoid X receptor
      DR3
      direct repeat spaced by three nucleotides
      DBD
      DNA binding domain
      LBD
      ligand binding domain
      AF-2
      trans-activation function-2
      TIF2
      transcriptional intermediary factor 2
      ANF
      atrial natriuretic factor
      GST
      glutathione S-transferase
      DMEM
      Dulbecco's modified Eagle's medium
      FBS
      fetal bovine serum
      DOTAP
      N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
      • Received November 14, 2000.
      • Accepted February 26, 2001.
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    1. Molecular Pharmacology June 1, 2001 vol. 59 no. 6 1478-1485
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