Carboxylic ester antagonists of 1 alpha,25-dihydroxyvitamin D-3 show cell-specific actions

Background: The nuclear hormone 1 alpha ,25-dihydroxyvitamin D-3 (1 alpha , 25(OH)(2)D-3) acts through the transcription factor vitamin D receptor (1 a lpha ,25(OH)(2)D-3 receptor, VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins and specific DNA binding sites (1 alph a ,25(OH)(2)D-3 response elements, VDREs). Ligand-mediated conformational c hanges of the VDR are the basis of the molecular mechanisms of nuclear 1 al pha ,25(OH)(2)D-3 signaling. Cell-specific VDR antagonists would allow to d issect and fine regulate the pleiotropic 1 alpha ,25(OH)(2)D-3 endocrine sy stem affecting the regulation of calcium homeostasis, bone mineralization a nd other cellular functions. Results: Two carboxylic ester analogues of 1 alpha ,25(OH)(2)D-3, ZK159222 and ZK168281, which have additional cyclopropyl rings and allylic alcohol s ubstructures in their side chain, were characterized in different 1 alpha , 25(OH)(2)D-3 target tissues as functional antagonists of 1 alpha ,25(OH)(2) D-3 signaling. In all tested systems, ZK168281 showed lower residual agonis tic activity and higher antagonistic effects than ZK159222, but the strengt h of these effects was cell-specific. Both antagonists were shown to act vi a the same mechanisms: they selectively stabilize an antagonistic conformat ion of the ligand-binding domain of the VDR within VDR-RXR-VDRE complexes, which then inhibits the interaction of the VDR with coactivator proteins an d an induction of transactivation. Interestingly, cells that have been trea ted with antagonists were found to contain VDR-RXR heterodimers in a differ ent conformation than cells that were stimulated with an agonist. Moreover, the strength of the functional antagonism of ZK159222 and ZK168281 appears to depend on the VDR/RXR expression ratio and high RXR levels were found t o reduce the antagonistic effect of both compounds. In support of this obse rvation, the overexpression of an transactivation function 2 (AF-2) deletio n mutant of RXR resulted for both ZK159222 and ZK168281 in a reduced agonis tic activity and an increased antagonistic effect. Conclusions: A novel, more potent VDR antagonist, ZK168281, was identified, which stabilizes VDR-RXR heterodimers in living cells in a different confo rmation than agonists. In addition, the VDR/RXR ratio was found as the majo r discriminating factor for understanding cell-specific effects of VDR anta gonists.