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ORIGINAL ARTICLE

Superagonistic Action of 14-epi-Analogs of 1,25-Dihydroxyvitamin D Explained by Vitamin D Receptor-Coactivator Interaction

Laboratorium voor Experimentele Geneeskunde en Endocrinologie (G.E., L.V., R.B., A.V.) and Afdeling Biochemie, Gasthuisberg (F.C.) Katholieke Universiteit Leuven, Belgium; Département de Biologie et de Génomique Structurales, Institut de Génétique et Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Illkirch, France (N.R., G.T.-V., D.M.); and Vakgroep voor Organische Chemie, Universiteit Gent, Gent, Belgium (P.D.C., M.V.)

Abstract

Two 14-epi-analogs of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], 19-nor-14-epi-23-yne-1,25-(OH)₂D₃ (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)₂D₃ (TX527), show enhanced antiproliferative (at least 10-fold) and markedly lower calcemic effects both in vitro and in vivo, compared with 1,25-(OH)₂D₃. This study aimed to evaluate their superagonistic effect at the level of interaction between the Vitamin D receptor (VDR) and coactivators. Mammalian two-hybrid assays with VP16-fused VDR and GAL4-DNA-binding-domain-fused steroid receptor coactivator 1 (SRC-1), transcriptional intermediary factor 2 (Tif2), or DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)₂D₃ (up to 16- and 20-fold stronger induction of VDR-SRC-1 interaction for TX522 and TX527 at 10^–10 M). Similar assays in which metabolism of 1,25-(OH)₂D₃ was blocked with VID400, a selective inhibitor of the 1,25-(OH)₂D₃-metabolizing enzyme CYP24, showed that the enhanced potency of these analogs in establishing VDR-coactivator interactions can only partially be accounted for by their increased resistance to metabolic degradation. Crystallization of TX522 complexed to the ligand binding domain of the human VDR demonstrated that the epi-configuration of C14 caused the CD ring of the ligand to shift by 0.5 Å, thereby bringing the C12 atom into closer contact with Val300. Moreover, C22 of TX522 made an additional contact with the CD1 atom of Ile268 because of the rigidity of the triple bond-containing side chain. The position and conformation of the activation helix H12 of VDR was strictly maintained. In conclusion, this study provides deeper insight into the docking of TX522 in the LBP and shows that stronger VDR-coactivator interactions underlie the superagonistic activity of the two 14-epi-analogs.

Address correspondence to: Dr. Roger Bouillon, Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Onderwijs en Navorsing, Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: roger.bouillon{at}med.kuleuven.ac.be

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