A 20-EPI SIDE-CHAIN RESTORES GROWTH-REGULATORY AND TRANSCRIPTIONAL ACTIVITIES OF AN A RING-MODIFIED HYBRID ANALOG OF 1-ALPHA,25-DIHYDROXYVITAMIN D-3 WITHOUT INCREASING ITS AFFINITY TO THE VITAMIN-D-RECEPTOR

1 alpha-hydroxymethyl-25-hydroxyvitamin D-3 and 1 beta-hydroxymethyl-3 alpha,25-hydroxyvitamin D-3, two analogs with modifications restricte d to the A ring, bind poorly to vitamin D receptor (VDR). The effectiv e doses required for 50% of maximal binding activity (ED(50)) are 7 x 10(-7) M for the former and 8 x 10(-8) M for the latter, and the ED(50 ) for their growth-inhibitory activities is greater than 10(-6) M. Une xpectedly, a hybrid analog with 20-epi configuration at its side chain and a 1 beta-hydroxymethyl group but not a 1 alpha-hydroxymethyl grou p inhibits malignant cell growth with an ED(50) of 7 x 10(-9) M. To de termine if the restored biological activity of the hybrid analog is as sociated with better binding to VDR, we performed competitive binding assays in vitro with calf thymus VDR and in vivo with recombinant huma n VDR. We found that the 20 epi side chain reduced the affinity of the 1 beta- and the 1 alpha-hydroxymethyl hybrid analogs for VDR in vitro and in vivo fourfold to tenfold. To determine whether the 1 beta-hydr oxymethyl analogs induced a VDR-mediated transcription, we tested the induction of reporter gene expression through the osteocalcin vitamin D response element (VDRE) in ROS 17/2.8 cells and the induction of bin ding activity of VDR to VDRE in COS-1 cells. We found that the ED(50) for transcriptional activity of 1 beta-hydroxymethyl-3 alpha,25-hydrox yvitamin D-3 was greater than 10(-6) M, but its 1 alpha diastereomer h ad barely detectable transcriptional activity. The 20-epi side chain p referentially increased the transcriptional activity of the 1 beta-hyd roxymethyl hybrid analog to an ED(50) of 10(-8) M, but the 1 alpha-hyd roxymethyl hybrid analog remained inactive. To confirm that this trans criptional activity was dependent on the VDR, we repeated the assay in VDR-negative CV-1 cells and compared ligand-dependent expression of t he VDRE/growth hormone reporter in the presence of either wild-type or transcriptionally inactive mutant VDR expression vectors. Transcripti on was induced by the 1 beta-hydroxymethyl compounds only in the prese nce of wild-type VDR. Thus, we conclude that it is possible, by adding a 20 epi side chain, to restore growth-inhibitory and VDR-mediated tr anscriptional activities without increasing binding to the VDR of A ri ng-modified analogs. (C) 1996 Wiley-Liss, Inc.