25-dehydro-1 alpha-hydroxyvitamin D-3-,26,23S-lactone antagonizes the nuclear vitamin D receptor by mediating a unique noncovalent conformational change

(23S)-25-dehydro-1 alpha -Dihydroxyvitamin D-3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1 alpha ,25-dihydroxyvitamin D-3 nucle ar receptor (VDR)- mediated increase in transcriptional activity. Using a t ransient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nM MK antagonizes VDR-me diated transcription by 50% when driven by 1 nM 1 alpha ,25(OH)(2)D-3. Four analogs of la,25(OH)(2)D-3, also at 1 nM, were antagonized 25 to 39% by 20 nM MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1 alpha ,25(OH)(2) D-3, were antagonized by 20 nM MK only at 100 pM or 10 pM, respectively. On e possible mechanism for antagonism is that the 25-dehydroalkene of MK migh t covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK boun d to VDR is freely exchanged with 1 alpha ,25(OH)(2)D-3 in vitro. These dat a support the apparent correlation between VDR transcriptional activation b y agonists and the effective range of MK antagonism by competition. Further more, protease sensitivity analysis of MK bound to VDR indicates the presen ce of a unique conformational change in the VDR ligand-binding domain, show ing a novel doublet of VDR fragments centered at 34 kDa, whereas 1 alpha ,2 5(OH)(2)D-3 as a ligand produces only a single 34-kDa fragment. In comparis on, the natural metabolite 1 alpha ,25-dihydroxyvitamin D-3-26,23-lactone y ields only the 30-kDa fragment that is produced by all ligands to varying d egrees. Collectively, these results support that MK is a potent partial ant agonist of the VDR for 1 alpha ,25(OH)(2)D-3 and its analogs when in approp riate excess of the agonist.