Figure 1.
Proposed pathway of the 1α,25(OH)2-3-epi-D3 production [18].
The reaction is initiated via A-ring C3-epimerization, where the C-3 hydroxyl moiety is changed from β to its diastereomer α. Two distinct pathways may be employed by cells to generate 1α,25(OH)2-3-epi-D3. The first, more likely used pathway, starts with dehydrogenation catalyzed by yet unidentified enzyme leading to a keto-intermediate, which is converted most probably by the same enzyme to the final product 1α,25(OH)2-3-epi-D3. The second one uses dehydration and a subsequent hydroxylation at C-3 α position.
Figure 2.
1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 show similar biological properties.
(A) Coactivator peptide recruitment assay was performed using AlphaScreen method in the presence of increasing concentrations of either 1α,25(OH)2D3 (green circles) or 1α,25(OH)2-3-epi-D3 (blue circles). The data represents two independent measurements in triplicates for which the mean and the S.D. of the mean was calculated. (B) Transactivation assays were performed in human breast cancer cells MCF7 cells with subsequent treatments of the increasing concentrations of either 1α,25(OH)2D3 (green circles) or 1α,25(OH)2-3-epi-D3 (blue circles). For every triplicate the mean and the S.D. were calculated. (C) Metabolism of 3H-25(OH)D3 in human keratinocytes. Kinetics of the primary metabolite 1α,25(OH)2D3 and its 3-epimer, is shown. The time point 5 h, where the 1α,25(OH)2-3-epi-D3 is the major metabolite is highlighted with red arrow. Confluent keratinocytes derived from lid skin were incubated in KGM (0.06 mM calcium) with 20.6 nM 3H[26], [27]-25(OH)D3 for the indicated time periods. CHCl3-extracts of the incubations were analyzed on Zorbax-Sil and individual metabolites identified by matching with authentic reference compounds and quantified as described in Materials and Methods. Data (± SD) was calculated from duplicate experiment. (D) Anti-proliferative cellular effect of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 in human keratinocytes. Keratinocytes in serum-free KGM (0.06 mM calcium) were seeded into 96-well plates and 24 h later the indicated metabolites (range 0.1–100 nM). After further 24 h, 1 µCi 3H-thymidine was applied to each well and its incorporation determined as described in Methods. Data are mean values (± SD) from a representative experiment out of two independent studies, each done in triplicates. For all experiments Student's unpaired t-test was performed and p-values were calculated between values obtained for 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 (* p<0.05, ** p<0.01, *** p<0.001).
Figure 3.
Overall structure of the VDR-1α,25(OH)2-3-epi-D3 and conformation of the bound ligand.
(A) Superimposition of the hVDR LBD– 1α,25(OH)2-3-epi-D3 (blue) and the hVDR LBD–1α,25(OH)2D3 (white). The ligands are shown in stick representation in blue for the 1α,25(OH)2-3-epi-D3 and in green for the 1α,25(OH)2D3. (B) The 1α,25(OH)2-3-epi-D3 is shown in its Fo – Fc electron density omit map contoured at 3 σ. The ligand is shown in stick representation with carbon and oxygen atoms in blue and red, respectively. (C) Stereo view of the ligand 3D conformations of 1α,25(OH)2-3-epi-D3 (blue) and 1α,25(OH)2D3 (green) in their VDR ligand-binding pockets (LBP).
Figure 4.
Adaptability of the hVDR LBP upon 1α,25(OH)2-3-epi-D3 binding.
(A) The adaptation of the LBP is depicted by mesh representation of the superimposed LBP volumes calculated with Voidoo software. The green surface represent the LBP area where the 1α,25(OH)2D3 bound pocket is larger. The blue area represents similar increase but for 1α,25(OH)2-3-epi-D3 and the two main expanded regions are highlighted with red circles. (B) Adaptation of the residues Tyr147 and Glu277 in the LBP of the 1α,25(OH)2-3-epi-D3 hVDR complex. The distances between the ligand-specific positions of the residues are displayed in Å.
Table 1.
Volume of VDR ligands and their resulting LBPs.
Figure 5.
Specific interactions of 1α,25(OH)2-3-epi-D3 in the LBP of the hVDR.
The ligands and residues in the superimposed structures are highlighted in color (1α,25(OH)2-3-epi-D3 in blue and in 1α,25(OH)2D3 green) and the important water molecules are represented with colored dots.