Characterization of a Novel Analogue of 1α,25(OH)₂-Vitamin D₃ with Two Side Chains:  Interaction with Its Nuclear Receptor and Cellular Actions

The hormone 1α,25(OH)₂-vitamin D₃ (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1α,25(OH)₂D₃ (20E-125D) increases transcription 200−5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1α,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D₃ (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays:  binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells:  ED₅₀ 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED₅₀(125D)/ED₅₀(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.