4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure–Function Study

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure–activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4β,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4β,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.

Thus, 4-hydroxylation is a major pathway of 25D3 metabolism.However, the occurrence of the 4-hydroxylation of 1,25D3 by CYP3A4 remains to be demonstrated and the biological significance of 4-hydroxymetabolites remains elusive.The only available biological data concern synthetic 1,4α,25D3 and 1,4β,25D3 ligands (Figure 1B) and the results of luciferase reporter assays in transfected human osteosarcoma cells indicate that both isomers are less active than 1,25D3 [15,16].Interestingly, significant differences in CYP24A1-induced 1,4,25D3 metabolism have been observed between the two isomers, and only the 4α isomer has been shown to be glucuronidated by certain hepatic UGT(s) [16,17].In addition, 4β,25D3 has been shown to have greater metabolic stability and resistance to CYP24A1 than 4α,25D3 [18].To further unveil the structure-activity relationships of 4-hydroxylated-1,25D3-VDR complexes, we describe a detailed synthetic route to the synthesis of 1,4α,25D3 and 1,4β,25D3 as well as some of the biological properties and crystal structures of their complexes with the VDR ligand binding domain (LBD).

Synthesis: General Information
All experiments were conducted under an argon atmosphere unless otherwise mentioned.All solvents and reagents were purified when necessary using standard procedure.Column chromatography was performed on silica gel 60 N (Kanto Chemical Co., Inc., Tokyo, Japan, 100-210 µm), flush column chromatography was performed on silica gel 60 Thus, 4-hydroxylation is a major pathway of 25D 3 metabolism.However, the occurrence of the 4-hydroxylation of 1,25D 3 by CYP3A4 remains to be demonstrated and the biological significance of 4-hydroxymetabolites remains elusive.The only available biological data concern synthetic 1,4α,25D 3 and 1,4β,25D 3 ligands (Figure 1B) and the results of luciferase reporter assays in transfected human osteosarcoma cells indicate that both isomers are less active than 1,25D 3 [15,16].Interestingly, significant differences in CYP24A1-induced 1,4,25D 3 metabolism have been observed between the two isomers, and only the 4α isomer has been shown to be glucuronidated by certain hepatic UGT(s) [16,17].In addition, 4β,25D 3 has been shown to have greater metabolic stability and resistance to CYP24A1 than 4α,25D 3 [18].
To further unveil the structure-activity relationships of 4-hydroxylated-1,25D 3 -VDR complexes, we describe a detailed synthetic route to the synthesis of 1,4α,25D 3 and 1,4β,25D 3 as well as some of the biological properties and crystal structures of their complexes with the VDR ligand binding domain (LBD).

Synthesis: General Information
All experiments were conducted under an argon atmosphere unless otherwise mentioned.All solvents and reagents were purified when necessary using standard procedure.Column chromatography was performed on silica gel 60 N (Kanto Chemical Co., Inc., Tokyo, Japan, 100-210 µm), flush column chromatography was performed on silica gel 60 (Merck, Tokyo, Japan, 0.040-0.063mm), and preparative thin-layer chromatography was performed on silica gel 60 F 254 (Merck, Tokyo, Japan, 0.5 mm).NMR spectra were measured on JEOL AL-400 ( 1 H at 400 MHz) and ECP-600 ( 13 C at 150 MHz) nuclear magnetic resonance spectrometers.Specific optical rotations were measured on a JASCO DIP-370 digital polarimeter.

Thermal Unfolding and Differential Scanning Fluorimetry (nanoDSF)
Fluorescence-based thermal experiments were performed using Prometheus NT.48 (NanoTemper Technologies, Munich, Germany) with capillaries containing 10 µL zVDR LBD at 3.2 mg/mL in the absence and presence of 2 equivalent ligands and/or 4 equivalents of the NCOA1 NR2 peptide (RHKILHRLLQEGSPS).The temperature was increased from 20 to 95 • C at a rate of 1 • C/min and fluorescence was measured at emission wavelengths of 330 nm and 350 nm.NanoTemper PR.Stability Analysis v1.0.2 was used to fit the data and to determine melting temperatures Tm.Triplicates of each sample were made.

Crystallization and Structure Determination
The concentrated protein at 5 mg/mL was incubated with a 2-fold excess of ligand and a 3-fold excess of the coactivator NCOA2 (KHKILHRLLQDSS) peptide.Crystallization experiments were carried out via sitting drop vapor diffusion at 290 K by mixing equal volumes (0.2µL) of the protein-ligand complexes and of the reservoir solution (0.1 M MES pH 6.0,2.5 M NaOAc).The crystals of the complexes were transferred to an artificial mother liquor containing 0.1 M MES at pH 6.0 and 3 M NaOAc and were flash-cooled in liquid nitrogen.Data on the crystals of zVDR-1,4α,25D 3 and of zVDR-1,4β,25D 3 were collected on Proxima2 beamline at Soleil synchrotron.Crystallographic raw data were processed with XDS [19] and scaled with AIMLESS [20].The structures were solved and refined using Phenix [21] and iterative model building using COOT [22].Crystallographic refinement statistics are presented in Supplementary Table S1.

Mice
A cohort of C57BL6J mice aged between 8 and 12 weeks were treated per os with 1 µg/kg/day of 1,25D 3 , 1,4α,25D 3 , or 1,4β,25D 3 in 100 µL of oil daily for 4 days [23].On day 4, blood and kidneys were harvested for analysis.A cohort of mice treated with 100 µL of oil were used as a control.All animal experimental protocols were conducted in compliance with French and EU regulations on the use of laboratory animals for research and approved by the IGBMC Ethical Committee and the French Ministry for National Education, Higher Education, and Research (#10047-2017052615101492).

Serum Calcium Levels
Mouse blood was collected in Microvette ® 500 lithium heparin (SARSTEDT) and centrifuged at 400 g for 10 min at 4 • C. The supernatant corresponding to the serum was retained.Serum calcium levels were determined using a colorimetric assay (MAK022, Sigma Aldrich, St. Quentin Fallavier, France) in accordance with the supplier's instructions.

Synthesis
Compounds 1,4α,25D 3 (1a) and 1,4β,25D 3 (1b) were synthesized as follows: the A-ring precursors enyne 2a and 2b were synthesized from methyl α-D-glucopyranoside by our reported procedures [17], and stereochemistry at the C4-position (4R) of major product 2b was determined by the modified Mosher's method (Scheme 1) [16,24].Triethylamine (1.9 mL) and Pd(PPh 3 ) 4 (21 mg, 190 µmol) were added to a solution of the enyne mixture (without the separation of 2a and 2b, 95 mg, 190 µmol, 2a/2b = 1/3) and bromoolefin 3 (81 mg, 230 µmol) in toluene (1.9 mL) at room temperature.The reaction mixture was stirred at 80 • C overnight.After cooling to room temperature, the mixture was concentrated in vacuo.The residue was purified by flush column chromatography on silica gel (hexane/EtOAc = 20/1) to give the coupling products of silyl-protected 1a and 1b as an inseparable mixture (63 mg, 43% from enynes) as a pale yellow oil.This was used for the next reaction without further purification.TBAF (0.4 mL, 1.0 M solution in THF, 400 µmol) was added to a solution of the mixture of silyl-protected 1a and 1b (30 mg, 39 µmol) in THF (0.8 mL) at 0 • C. The reaction mixture was stirred at room temperature for 3 h, and then at 80 • C for 5 h.After cooling, the reaction was quenched with water at 0 • C. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated.The residue was purified by preparative silica gel TLC plate (hexane/EtOAc = 2/1) to give the mixture of 1a and 1b (17 mg, quant) as a pale yellow oil.The mixture was re-purified and separated by reversed-phase HPLC (YMC-Pack ODS column, 20 × 250 mm, CH 3 OH/H 2 O = 9/1) to give 1a (1.2 mg,) and 1b (6.4 mg), each as a colorless oil [16].

Biological Assays
Nano differential scanning fluorimetry analysis confirmed the binding of the 1,4α,25 (OH) 3 D 3 and 1,4β,25(OH) 3 D 3 ligands to the zebrafish (z)VDR LBD, which has been shown to bind ligands similarly to the human VDR [25].The two 4-hydroxylated ligands increased the stabilization of the zVDR LBD, which was further increased in the presence of a NCOA1 coactivator peptide, which encompasses one nuclear receptor LXXLL interacting motif (Figure 2A).However, the stabilization of the complexes was weaker compared to the effect of 1,25D 3 in this assay.The interaction of the LBD of zVDR with the NCOA1 coactivator peptide was next quantified by fluorescence polarization in the presence of saturating ligand concentrations of 1,25D3, 1,4α,25D 3 , or 1,4β,25D 3 (Figure 2B).Whereas the zVDR LBD in its apo form did not bind the NCOA1 peptide [26], the NCOA1 peptide bound to zVDR LBD with a similar affinity in the presence of the tested ligands (Kd = 0.54 µM, 0.50 µM, and 0.48 µM for the 1,25D 3 , 1,4α,25D 3 , and 1,4β,25D 3 complexes, respectively).The effects of these 4-hydroxylated ligands on VDR activities were previously studied using reporter gene assays [15,16].To determine their potency to induce endogenous VDR target genes in intestinal cells, a key vitamin D target tissue, we evaluated the expression of the transcript levels of several VDR target genes (Cyp24a1, Trpv6, and S100g) in rat intestinal epithelial (IEC-18) cells treated for 24 h with vehicle or 100 nM of 1,25D3, 1,4α,25D3, and 1,4β,25D3 by RT-qPCR.The transcript levels of these VDR target genes were induced at least twofold in the presence of the tested compounds compared to vehicle-treated cells (Figure 3).Consequently, 4-hydroxylated compounds are potent VDR agonist ligands with a potency comparable to that of 1,25D3.Note that Cyp24a1 transcripts The effects of these 4-hydroxylated ligands on VDR activities were previously studied using reporter gene assays [15,16].To determine their potency to induce endogenous VDR target genes in intestinal cells, a key vitamin D target tissue, we evaluated the expression of the transcript levels of several VDR target genes (Cyp24a1, Trpv6, and S100g) in rat intestinal epithelial (IEC-18) cells treated for 24 h with vehicle or 100 nM of 1,25D 3 , 1,4α,25D 3 , and 1,4β,25D 3 by RT-qPCR.The transcript levels of these VDR target genes were induced at least twofold in the presence of the tested compounds compared to vehicle-treated cells (Figure 3).Consequently, 4-hydroxylated compounds are potent VDR agonist ligands with a potency comparable to that of 1,25D 3 .Note that Cyp24a1 transcripts were higher in 1,4β,25D 3treated cells than in 1,25D 3 -and 1,4α,25D 3 -treated cells, indicating that 1,4β,25D 3 is more potent.Thus, in contrast to the previous study using reporter assays in VDR-transfected cells, the two metabolites enhanced endogenous VDR activities in IEC-18 cells.
The effects of these 4-hydroxylated ligands on VDR activities were previously studied using reporter gene assays [15,16].To determine their potency to induce endogenous VDR target genes in intestinal cells, a key vitamin D target tissue, we evaluated the expression of the transcript levels of several VDR target genes (Cyp24a1, Trpv6, and S100g) in rat intestinal epithelial (IEC-18) cells treated for 24 h with vehicle or 100 nM of 1,25D3, 1,4α,25D3, and 1,4β,25D3 by RT-qPCR.The transcript levels of these VDR target genes were induced at least twofold in the presence of the tested compounds compared to vehicle-treated cells (Figure 3).Consequently, 4-hydroxylated compounds are potent VDR agonist ligands with a potency comparable to that of 1,25D3.Note that Cyp24a1 transcripts were higher in 1,4β,25D3-treated cells than in 1,25D3-and 1,4α,25D3-treated cells, indicating that 1,4β,25D3 is more potent.Thus, in contrast to the previous study using reporter assays in VDR-transfected cells, the two metabolites enhanced endogenous VDR activities in IEC-18 cells.The results in IEC18 cells prompted us to investigate the in vivo activities of 1,4α,25D3 and 1,4β,25D3.We determined the effects of 1,4α,25D3 and 1,4β,25D3 administration on the expression of the VDR target genes Cyp24a1 and Cyp27b1, two genes encoding proteins The results in IEC18 cells prompted us to investigate the in vivo activities of 1,4α,25D 3 and 1,4β,25D 3 .We determined the effects of 1,4α,25D 3 and 1,4β,25D 3 administration on the expression of the VDR target genes Cyp24a1 and Cyp27b1, two genes encoding proteins involved in the metabolic pathway of vitamin D and known to be upregulated and downregulated by 1,25D 3 , respectively.After treatment with the 4-hydroxylated metabolites, Cyp24a1 transcript levels were induced, whereas the expression of Cyp27b1 decreased (Figure 4A,B).The expression of Trpv5, a VDR target gene encoding for a channel involved in kidney calcium reabsorption, was induced by 1,4α,25D 3 or 1,4β,25D 3 with a similar potency to that of 1,25D 3 (Figure 4C).Then, we determined the pro-calcemic activities of the 4-hydroxylated metabolites.In accordance with previous results, mice treated with 1 µg/kg/day of 1,25D 3 for 4 days were hypercalcemic [23].Interestingly, serum calcium levels after 1,4α,25D 3 or 1,4β,25D 3 intoxication were similar to those in 1,25D 3 -treated mice.All together, these results indicate that 1,4α,25D 3 or 1,4β,25D 3 have comparable in vivo activities to those of 1,25D 3 (Figure 4D).

Ligand Binding Mode to VDR
To decipher the ligand binding mode of the two 4-hydroxy metabolites to VDR, we solved the crystal structures of their complexes with the zVDR LBD in the presence of a NCOA2 coactivator peptide.The structures of the zVDR LBD bound to 1,4α,25D 3 and to 1,4β,25D 3 were determined at a resolution of 1.95 and 1.8 Å, respectively.The crystallographic data are summarized in Supplementary Table S1.After the refinement of the protein alone, the map showed an unambiguous electron density in which the ligands fit (Figure 5A).The complexes formed by the zVDR LBD bound to the two 4-OH compounds adopt the canonical active conformation, as described in all previously reported agonist-bound VDRs (Figure 5B).The conformation of the activation helix 12 is strictly maintained and the coactivator peptide forms similar interactions as in the complex with 1,25D 3 .When compared to the structure of the zVDR LBD-1,25D 3 complex, the atomic coordinates of zVDR LBD bound to 1,4α,25D 3 and 1,4β,25D 3 show a very small root mean square deviation of 0.3 Å over 235 Cα atoms, reflecting their high structural homology.

Ligand Binding Mode to VDR
To decipher the ligand binding mode of the two 4-hydroxy metabolites to VDR, we solved the crystal structures of their complexes with the zVDR LBD in the presence of a NCOA2 coactivator peptide.The structures of the zVDR LBD bound to 1,4α,25D3 and to 1,4β,25D3 were determined at a resolution of 1.95 and 1.8 Å, respectively.The crystallographic data are summarized in Supplementary Table S1.After the refinement of the protein alone, the map showed an unambiguous electron density in which the ligands fit (Figure 5A).The complexes formed by the zVDR LBD bound to the two 4-OH compounds adopt the canonical active conformation, as described in all previously reported agonistbound VDRs (Figure 5B).The conformation of the activation helix 12 is strictly maintained and the coactivator peptide forms similar interactions as in the complex with 1,25D3.When compared to the structure of the zVDR LBD-1,25D3 complex, the atomic coordinates   The addition of an additional hydroxyl group on C4 does not modify the A-ring conformation of the ligand, and the seco-B and C/D-rings and aliphatic side chain have similar conformations to those of 1,25D 3 (Figure 6A).The distances between C1-OH and C25-OH are 13.12Å, 13.11Å, and 12.95Å, and between the C3-OH and C25-OH they are 15.36 Å, 15.38 Å, and 15.28 Å for 1,25D 3 , 1,4α,25D 3 , and 1,4β,25D 3 , respectively.The interactions with the seco-B, C/D-rings and the side chain of the two analogs are similar to those formed by 1,25D 3 as well as the hydrogen bonds formed with 1-OH, 3-OH, and 25-OH (Figure 6B,C).Differences are observed around the 4-OH group; the 4-OH group of the 4β hydroxylated compound forms hydrogen bonds with Ser306 and Cys316, whereas its diastereomer 4α acts through hydrogen bonding only with Cys316 but forms stronger van der Waals interactions with Phe182 and Leu261 (Figure 6D,E).To adapt the additional hydroxyl group, some side chain amino acid residues, notably Tyr179, Phe182, and Cys316, are shifted slightly, by 0.4-0.6Å.These structural data agree well with the induced biological activity of the two compounds, which are similar to the natural hormone.

Discussion
In the present work, we show that 4-hydroxylated 1,25D3 ligands are potent VDR agonists.
The most active form of vitamin D3 is 1,25D3, produced by two sequential hydroxylations of vitamin D3, which can be obtained from dietary sources or synthesized endogenously in the skin after the photolytic conversion of 7-dehydrocholesterol [4,5].But 25D3 is not only a metabolic precursor of 1,25D3: it also acts as a VDR agonist itself, with gene regulatory and anti-proliferative properties [27][28][29].Indeed, 25D3, when present at high concentrations, induces VDR activities, notably in cancer cells when 1,25D3 production is abolished [28][29][30].However, 25D3 binds to VDR less efficiently than 1,25D3 [31], indicating that the hydroxyl group at C1 has a critical role in achieving high affinity.
The recent identification of alternative vitamin D3 pathways and of the major enzymes involved has revealed the existence of other natural vitamin D3 metabolites, notably with modifications of the A-ring [7,8].The 3-epimer of 1,25D3 has been shown to retain significant biological activity compared to the natural hormone, although its activity is lower than that of 1,25D3 [7,32].The crystal structural analysis of VDR-1,25-3-epiD3 showed that 1,25-3-epi-D3 takes a slightly more compact conformation in the VDR ligand binding pocket and compensates for the loss of interaction of the 3-OH group with hSer278 by a water-mediated hydrogen bond [32].Here, we show that the 4-hydroxylated 1,25D3 compounds maintain all anchoring H-bonds of 1,25D3 and form additional interactions.The 4-OH group of the 4β-hydroxylated compound forms hydrogen bonds with zSer306 and zCys316, whereas its diastereomer 4α acts through hydrogen bonding only with zCys316, but forms stronger van der Waals interactions with zPhe182 and zLeu261.

Discussion
In the present work, we show that 4-hydroxylated 1,25D 3 ligands are potent VDR agonists.The most active form of vitamin D 3 is 1,25D 3 , produced by two sequential hydroxylations of vitamin D 3 , which can be obtained from dietary sources or synthesized endogenously in the skin after the photolytic conversion of 7-dehydrocholesterol [4,5].But 25D 3 is not only a metabolic precursor of 1,25D 3 : it also acts as a VDR agonist itself, with gene regulatory and anti-proliferative properties [27][28][29].Indeed, 25D 3 , when present at high concentrations, induces VDR activities, notably in cancer cells when 1,25D 3 production is abolished [28][29][30].However, 25D 3 binds to VDR less efficiently than 1,25D 3 [31], indicating that the hydroxyl group at C1 has a critical role in achieving high affinity.
The recent identification of alternative vitamin D 3 pathways and of the major enzymes involved has revealed the existence of other natural vitamin D 3 metabolites, notably with modifications of the A-ring [7,8].The 3-epimer of 1,25D 3 has been shown to retain significant biological activity compared to the natural hormone, although its activity is lower than that of 1,25D 3 [7,32].The crystal structural analysis of VDR-1,25-3-epiD 3 showed that 1,25-3-epi-D 3 takes a slightly more compact conformation in the VDR ligand binding pocket and compensates for the loss of interaction of the 3-OH group with hSer278 by a water-mediated hydrogen bond [32].Here, we show that the 4-hydroxylated 1,25D 3 compounds maintain all anchoring H-bonds of 1,25D 3 and form additional interactions .The 4-OH group of the 4β-hydroxylated compound forms hydrogen bonds with zSer306 and zCys316, whereas its diastereomer 4α acts through hydrogen bonding only with zCys316, but forms stronger van der Waals interactions with zPhe182 and zLeu261.These interactions explain why those compounds are as active as 1,25D 3 .In IEC-18 cells, the two compounds are potent VDR agonist ligands, with similar activities to 1,25D 3 .In addition, they regulate the expression of renal VDR target genes and increase serum calcium levels, demonstrating that these 4-hydroxylated metabolites enhance VDR activities in vivo.While 4,25D 3 are endogenous metabolites catalyzed by CYP3A4 [11,12], the occurrence of 1,25D 3 C4hydroxylation or 4,25D 3 C1-hydroxylation remains to be demonstrated.The enzymatic activity of CYP3A4 was associated with 1,25D 3 inactivation via C-23 hydroxylation [11,14].However, other presumed hydroxylated "degradation" products have demonstrated transcriptional activity [33].
Their high and differential metabolic stability compared to that of 1,25D 3 , together with their significant biologic activity, makes these synthetic 4-hydroxylated 1,25D 3 analogs promising candidate ligands for clinical applications.However, their target specificity needs further investigation in future studies.In addition, this study provides information for developing novel VDR agonists.Thousands of 1,25D 3 analogs have already been synthetized, and some modifications, such as C20 epimer, C-2 substitutions, or side chain rigidification, have been shown to improve the stability of VDR complexes and ligandinduced activities [34].The incorporation of a 4-hydroxyl group into secosteroidal ligands could provide new potent VDR agonists.

Conclusions
In this study, two 4-hydroxylated analogs of 1,25D 3 were chemically synthetized.1α,4α,25D 3 and 1α,4β,25D 3 ligands showed VDR gene regulatory activities similar to 1,25D 3 .The crystal structures of zVDR LBD in complex with the two epimers provide a mechanistic insight for the specific recognition of 4-hydroxylated metabolites of 1,25D 3 .Therefore, we conclude that the C4-hydroxylation pathway produces active metabolites with similar biochemical and biological properties to those of 1,25D 3 .

Figure 1 .
Figure 1.(A) Pathway of the production of 4-hydroxy metabolites of D3. (B) Chemical structures of the ligands.

Figure 1 .
Figure 1.(A) Pathway of the production of 4-hydroxy metabolites of D 3 .(B) Chemical structures of the ligands.

Figure 5 .
Figure 5. Crystal structures of the zVDR complexes with 1α,4a,25(OH)3D3 and 1a,4b,25(OH)3D3.(A) Ligands modeled into omit Polder maps contoured at 3σ. (B) Superimposed protein structures of 1,4a,25D3 (blue) and 1,4b,25D3 (green) compared to the 1α,25D3 zVDR complex (red).The addition of an additional hydroxyl group on C4 does not modify the A-ring conformation of the ligand, and the seco-B and C/D-rings and aliphatic side chain have similar conformations to those of 1,25D3 (Figure 6A).The distances between C1-OH and C25-OH are 13.12Å, 13.11Å, and 12.95Å, and between the C3-OH and C25-OH they are 15.36 Å, 15.38 Å, and 15.28 Å for 1,25D3, 1,4α,25D3, and 1,4β,25D3, respectively.The interactions with the seco-B, C/D-rings and the side chain of the two analogs are similar to those formed by 1,25D3 as well as the hydrogen bonds formed with 1-OH, 3-OH, and 25-OH (Figure 6B,C).Differences are observed around the 4-OH group; the 4-OH group of the 4β