Structure-function study of gemini derivatives with two different side chains at C-20, Gemini-0072 and Gemini-0097

Abstract

Derivatives of vitamin D(3) containing a second side-chain emanating at C-20 are known as gemini and act as vitamin D receptor agonists. Recently, two of these, namely Gemini-0072 and the epimeric Gemini-0097, were selected for further studies in view of their high biological activities and lack of hypercalcemic effects. We now show that the two analogs recruit coactivator SRC-1 better than the parental gemini and act as VDR superagonists. The crystal structures of complexes of zVDR with Gemini-0072 and Gemini-0097 indicate that these ligands induce an extra cavity within the ligand-binding pocket similar to gemini and that their superagonistic activity is due to an increased stabilization of helix H12.

Fig. 1

Chemical structures of 1α,25(OH)₂D₃, gemini, Gemini-0072 (C20S) and Gemini-0097 (C20R).

Fig. 2

Gemini-0072 and Gemini-0097 are highly potent VDR superagonists in vitro. Transient transfections and luciferase reporter gene assays using MCF-7 human breast cancer cells were performed as reported in the experimental procedures. Relative luciferase activities were calculated by dividing the luciferase activity by the respective β-galactosidase activity to correct for differences in transfection efficiency. For every triplicate the mean and S.D. were calculated.

Fig. 3

Conformations and interactions of the bound ligands in the LBP of zVDR at a 4.0 Å cut-off. (A) The Gemini-0072 (left) and Gemini-0097 (right) are shown in their Fo – Fc electron density omit map contoured at 3σ. Ligands are shown in stick representation with carbon, oxygen and fluorine atoms in yellow, red and grey, respectively. (B) Superposition of Gemini-0072 (magenta), Gemini-0097 (orange) and 1α,25(OH)₂D₃ (grey). (C) zVDR LBD–Gemini-0072 and (D) zVDR LBD–Gemini-0097 are shown in magenta and orange, respectively. The gemini ligands are represented in stick with carbon, oxygen and fluorine atoms in yellow, red and light blue, respectively. Superimposition with 1α,25(OH)₂D₃ is shown with 1α,25(OH)₂D₃ in grey and red for the carbon and oxygen atoms, respectively. Additional interactions compared to the parental Gemini are marked in red.

Fig. 4

Additional contacts involving the fluorine side chain in zVDR LBD–Gemini-0072 and zVDR LBD–Gemini-0097 complexes. The zVDR LBD–Gemini-0072 and –Gemini-0097 complexes are shown in magenta and orange, respectively. The ligands are represented in stick with oxygen and fluorine atoms colored in red and light blue, respectively. Hydrophobic residues involved in additional contacts with the ligand are shown in green.