Abstract

A 25-carboxylic ester analog of 1α,25-dihydroxyvitamin D₃[1α,25(OH)₂D₃], ZK159222 (compound 1), was recently described as a novel type of antagonist of 1α,25(OH)₂D₃ signaling. In this study five derivatives of compound 1 (compounds 2–6) were selected because of their sensitivity in facilitating complex formation between the 1α,25(OH)₂D₃ receptor (VDR) and the retinoid X receptor on a 1α,25(OH)₂D₃ response element that was comparable to that of the natural hormone (0.2–0.9 nM). Most derivatives of compound 1 reacted as typical agonists, because they were able to promote ligand-dependent interaction of the VDR with the coactivator TIF2, stabilized the VDR preferentially in its agonistic conformation c1_LPD, and stimulated VDR-dependent gene activity with a potency similar to 1α,25(OH)₂D₃. In contrast, only compound 2 showed the antagonistic profile of compound 1, which includes the incompetence to induce a VDR-TIF2 contact, the stabilization of the antagonistic conformation c2_LPD, and only a very weak and insensitive functional activity. Accordingly, only compounds 1 and 2, but not compounds 3 to 6, showed prominent antagonistic effects in cellular systems. The comparison of the structures of the compounds indicates that the essential requirements for an antagonistic function are a cyclopropyl ring at carbon 25, a hydroxy group at carbon 24, and at least a butylester. Interestingly, compound 2 was an approximately 3 times more sensitive antagonist than compound 1 and even displayed a lower residual agonistic activity. In conclusion, only a very limited number of structural variations of compound 1 are possible to keep its antagonistic profile, but the tools presented here for their in vitro evaluation allow an accurate prediction of the effects and are suited to screening for even more potent 1α,25(OH)₂D₃ antagonists.