A 20‐epi side chain restores growth‐regulatory and transcriptional activities of an A ring‐modified hybrid analog of 1α,25‐dihydroxyvitamin D3 without increasing its affinity to the vitamin D receptor

1α-hydroxymethyl-25-hydroxyvitamin D3 and 1β-hydroxymethyl-3α,25-hydroxyvitamin D3, two analogs with modifications restricted to the A ring, bind poorly to vitamin D receptor (VDR). The effective doses required for 50% of maximal binding activity (ED50) are 7 x 10-7 M for the former and 8 x 10-8 M for the latter, and the ED50 for their growth-inhibitory activities is greater than 10-6 M. Unexpectedly, a hybrid analog with 20-epi configuration at its side chain and a 1β-hydroxymethyl group but not a 1α-hydroxymethyl group inhibits malignant cell growth with an ED50 of 7 x 10-9 M. To determine if the restored biological activity of the hybrid analog is associated with better binding to VDR, we performed competitive binding assays in vitro with calf thymus VDR and in vivo with recombinant human VDR. We found that the 20 epi side chain reduced the affinity of the 1β- and the 1α-hydroxymethyl hybrid analogs for VDR in vitro and in vivo fourfold to tenfold. To determine whether the 1β-hydroxymethyl 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 binding activity of VDR to VDRE in COS-1 cells. We found that the ED50 for transcriptional activity of 1β-hydroxymethyl-3α,25-hydroxyvitamin D3 was greater than 10-6 M, but its 1α diastereomer had barely detectable transcriptional activity. The 20-epi side chain preferentially increased the transcriptional activity of the 1β-hydroxymethyl hybrid analog to an ED50 of 10-8 M, but the 1α-hydroxymethyl hybrid analog remained inactive. To confirm that this transcriptional activity was dependent on the VDR, we repeated the assay in VDR-negative CV-1 cells and compared ligand-dependent expression of the VDRE/growth hormone reporter in the presence of either wild-type or transcriptionally inactive mutant VDR expression vectors. Transcription was induced by the 1β-hydroxymethyl compounds only in the presence 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 transcriptional activities without increasing binding to the VDR of A ring-modified analogs. © 1996 Wiley-Liss, Inc.