# 1α,25(OH)2-3-Epi-Vitamin D3, a Natural Physiological Metabolite of Vitamin D3: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor

Ferdinand Molnar, Rita Sigueiro, Yoshiteru Sato, Clarisse Araujo, Inge Schuster, Pierre Antony, Jean Peluso, Christian Muller, Antonio Mourino,

Dino Moras, et al.

► To cite this version:

Ferdinand Molnar, Rita Sigueiro, Yoshiteru Sato, Clarisse Araujo, Inge Schuster, et al. 1α,25(OH)2-3-Epi-Vitamin D3, a Natural Physiological Metabolite of Vitamin D3: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor. PLoS ONE, 2011, 6 (3), pp.e18124. 10.1371/journal.pone.0018124. hal-04500358

HAL Id: hal-04500358

https://hal.science/hal-04500358v1

Submitted on 4 Feb 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# 1α,25(OH)₂-3-Epi-Vitamin D₃, a Natural Physiological Metabolite of Vitamin D₃: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor

Ferdinand Molnár¹,², Rita Sigüeiro³, Yoshiteru Sato¹, Clarisse Araujo³, Inge Schuster⁴, Pierre Antony¹, Jean Peluso⁵, Christian Muller⁵, Antonio Mouriño³, Dino Moras¹, Natacha Rochel¹*

¹Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France, ²School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland, ³Departamento de Química Organica, Universidad de Santiago de Compostela and Unidad Asociada al CSIC, Santiago de Compostela, Spain, ⁴Institute of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria, ⁵Faculty of Pharmacy, Institut Gilbert Laustriat, UMR 7175 CNRS, University of Strasbourg, Illkirch, France

## Abstract

**Background:** The 1α,25-dihydroxy-3-epi-vitamin-D₃ (1α,25(OH)₂-3-epi-D₃), a natural metabolite of the seco-steroid vitamin D₃, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. *In vivo* action of 1α,25(OH)₂-3-epi-D₃ is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)₂D₃. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)₂-3-epi-D₃ and its receptor complex, we characterized some of its *in vitro* biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD).

**Methodology/Principal Findings:** In the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)₂D₃. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)₂-3-epi-D₃ in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)₂D₃.

**Conclusions/Significance:** The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)₂D₃ lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)₂D₃.

**Citation:** Molnár F, Sigüeiro R, Sato Y, Araujo C, Schuster I, et al. (2011) 1α,25(OH)₂-3-Epi-Vitamin D₃, a Natural Physiological Metabolite of Vitamin D₃: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor. PLoS ONE 6(3): e18124. doi:10.1371/journal.pone.0018124

**Editor:** Moray Campbell, Roswell Park Cancer Institute, United States of America

**Received** October 24, 2010; **Accepted** February 21, 2011; **Published** March 31, 2011

**Copyright:** © 2011 Molnár et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Funding:** This study received financial support from CNRS, INSERM, ULP, the European Commission as SPINE2-complexes (contract no. LSHG-CT-2006-031220) under the RDT programme 'Quality of Life and Management of Living Resources', the Spanish Ministry of Education and Science (Grant SAF2007-67205) and Xunta de Galicia (Projects INCITE08PXIB-209130PR and ACEUIC-2006/XA050). Dishman-Netherlands provided the gift of vitamin D2. R.S. thanks the Spanish MEC for a predoctoral fellowship. F.M. thanks the Academy of Finland for a postdoctoral fellowship (128226). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

## Introduction

The 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃ or calcitriol), is the most active form of vitamin D₃ and mediates its pleiotropic effects through VDR activation, which heterodimerizes with retinoid X receptor (RXR). VDR-induced genomic action results in growth inhibition of lymphomas, breast or prostate primary tumor cells, renal osteodystrophy, osteoporosis, psoriasis or autoimmune diseases [1,2]. Consequently, VDR is an exquisite therapeutic target to combat human metabolic diseases and uncontrolled cell proliferation in many tissues [3–5]. In addition 1α,25(OH)₂D₃ is a key regulator

of calcium and phosphate homeostasis and bone metabolism but its intrinsic hypercalcemic effect prevents its use in therapeutic applications [6].

1α,25(OH)₂D₃ is subjected to enzymatic inactivation via two major pathways leading to C-24 and C-23 hydroxylated metabolites in various tissues [7–17]. While the side chain oxidation is a general pathway associated to inactivation, another metabolite modified at the A-ring, the 1α,25(OH)₂-3-epi-D₃, has been shown to retain significant biological activity compared to the natural hormone [18,19]. The 1α,25(OH)₂-3-epi-D₃ was initially identified in the culture of human neonatal keratinocytes [20,21]. Further *in vivo* studies have characterized the occurrence of a C-3

<figure><img src="image_2.png" /></figure>

<footnote>* E-mail: rochel@igbmc.fr</footnote>
epimerization pathway [22]. Indeed, this natural vitamin D₃
metabolite was detected in serum of rats treated with pharmaco-
logical doses of 1α,25(OH)₂D₃, and may therefore play an important
physiological role by buffering the level of 1α,25(OH)₂D₃. In
addition, significant accumulation of 1α,25(OH)₂-3-epi-D₃ was
observed in different human adenocarcinoma cell lines such as

colon-derived Caco-2 cells[23] or NCI-H441 pulmonary cells [24].
Moreover, 1α,25(OH)₂-3-epi-D₃ was readily quantified in bovine
parathyroid cells, [25] rat osteoblastic UMR 106 and Ros17/2.8 cells
[26].

The production of 1α,25(OH)₂-3-epi-D₃ is initiated via A-ring C3-epimerization (Figure 1), where the C-3 hydroxyl moiety is

<figure><img src="image_3.png" /><figcaption>**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.</figcaption></figure>

doi:10.1371/journal.pone.0018124.g001

<figure><img src="image_4.png" /></figure>
changed from position β to its diastereomer α. The enzymes responsible for the C3-epimerization have not been identified to present date. It was also proposed by Reddy *et al.* that this pathway might be used for metabolites that resist inactivation through C-24 oxidation [18] a phenomenon well characterized in the bile acid metabolism where the reaction is catalyzed by bile acid hydroxysteroid dehydrogenase [27]. This pathway plays also a major role in the activation and/or inactivation of steroid hormones such as androgens [28].

Despite a lower binding affinity than calcitriol, 1α,25(OH)₂-3-epi-D₃ possess significant biological activity only in specific tissues where it is produced [29]. The transcriptional response of the 1α,25(OH)₂-3-epi-D₃ compound varies for different VDR-regulated genes in different tissues. For instance, it shows lower activation of osteocalcin gene and lower HL60 differentiation [30] but has almost equipotent activity to 1α,25(OH)₂D₃ in inhibiting cellular proliferation in keratinocytes [19] and in suppressing parathyroid secretion in bovine parathyroid cells [25]. These *in vitro* properties associated with its low calcemic activity [31,32] assign potential therapeutic interest to this compound.

To further unveil, the structural mechanism and structure-activity relationships of 1α,25(OH)₂-3-epi-D₃/hVDR-LBD complex, we describe a more effective synthetic route to the synthesis of 1α,25(OH)₂-3-epi-D₃, some of its *in vitro* biological properties and the crystal structure of its complex with hVDR LBD.

## Results and Discussion

### Synthesis of the 1α,25(OH)₂-3-epi-D₃

The synthesis of the target 1α,25(OH)₂-3-epi-D₃ (1, Scheme S1) was first described by Okamura's group at Riverside from (R)-carvone using the dienyne approach (13 steps, 8.5%) [33]. We describe here an efficient and alternative convergent synthesis of 1 from (S)-carvone (9 steps, 13%) that features a palladium catalyzed tandem process that produces the vitamin D triene unit stereoselectively in one pot by coupling enol triflate 3 (A-ring fragment) with an alkenyl metal intermediate 2 (CD-side chain fragment) [34]. For reproducibility reasons we employed Indium intermediates (M = InR₂) instead of Zinc intermediates [35–37].

### Synthesis of the A-ring fragment 3

Our synthesis starts with commercial (S)-carvone (4, Scheme S2), which was reduced under Luche conditions [38] to alcohol 5a and its epimer 5b (9:1 ratio as determined by ¹H-NMR). The mixture of alcohols 5 was subjected to Sharpless epoxidation [39] to provide the desired epoxyalcohol 6a (58% yield, two steps) and the starting ketone 4 (28%). The formation of 4 can be explained by oxidation of 5a through the corresponding chair-like equatorially oriented vanadium ester intermediate. *Tert*-butyldimethylsilyl protection of 6a gave 6b in 96% yield. Side-chain degradation on 6b by Daniewski's method [40] afforded alcohol 7a (71%), which was protected to 7b in the usual way (91%). Epoxide 7b was converted in 77% yield to dibromide 8b by the two-step sequence: 1) oxidative cleavage with periodic acid; 2) Corey-Fuchs side-chain extension [41]. Finally, consecutive treatment of 8b with lithium diisopropylamide and *n*-butyllithium followed by trapping of the resulting enolate with N-(5-Cl-2-pyridil)bis(triflate) gave the desired enol triflate 3 in 76% yield [42].

### Synthesis of the upper fragment 2 and 1α,25(OH)₂-3-epi-D₃ (1b)

Alkenyl bromide 10 was prepared from ketone 9 by a modified [43] Trost procedure [44]. Treatment of a mixture of bromide 10 and indium trichloride with *tert*-butyllithium, and coupling of the

resulting indium intermediate 2a with enol triflate 3 in the presence of catalytic amounts of (Ph₃P)₄Pd and (dppf)PdCl₂, gave, after desilylation, the desired metabolite 1b in 58% yield (Scheme S3). The detailed synthesis is described in the Methods S1.

### 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ show similar properties in coactivator peptide recruitment

The human transcriptional intermediary factor TIF2 coactivator (NCOA2) has been shown to interact with VDR [45]. The induced recruitment of TIF2 coactivator peptide bearing the 3rd LXXLL motif to the hVDR LBD was monitored in the presence of increasing concentrations of 1α,25(OH)₂D₃ or 1α,25(OH)₂-3-epi-D₃ using the luminescent oxygen channeling assays [46]. Our results show that EC₅₀ value for both metabolites are in the lower nanomolar range, 1.2 and 2.5 nM for 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃, respectively (Figure 2A).

### 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ induce expression of vitamin D target genes in human breast cancer (MCF-7) cells with similar potency

The transactivation potency of 1α,25(OH)₂-3-epi-D₃ has been reported for several VDR target genes in different model cell lines such as MG-63 or ROS17/2.8 osteosarcoma cells [24,30]. While the transcriptional activity in MG-63 cells using a vitamin D-responsive element (VDRE) from human *osteocalcin* (−848/+10) and rat *CIP24* (−291/+9) gene promoters was lower upon stimulation with 1α,25(OH)₂-3-epi-D₃ compared to 1α,25(OH)₂D₃ [47], using 2xVDREs reporter from *CIP24* gene promoter in human melanoma G-361 cells comparable transcriptional activity was observed [48]. This response is mainly achieved in cells in which the 1α,25(OH)₂-3-epi-D₃ metabolite is produced [29]. We monitored the dose-dependent VDR induced transcriptional activity in human breast cancer cells (MCF-7) cells transfected with human *CIP24* promoter (−414 to −64) containing VDRE fused to reporter *luciferase* gene (Figure 2B). Here, we show that 1α,25(OH)₂-3-epi-D₃ is slightly less potent than 1α,25(OH)₂D₃ in directing transactivation assay as the EC₅₀ induced by 1α,25(OH)₂-3-epi-D₃ is twice higher than that of 1α,25(OH)₂D₃ (5.9 nM vs 2.9 nM). This difference is in agreement with our results obtained from cell free coactivator peptide recruitment assays. Our transactivation assays show that the dose-dependent comparison between the 1α,25(OH)₂-3-epi-D₃ and 1α,25(OH)₂D₃ reveals that at 50% of the dose-response, the transcriptional activity of the 3-epimer is 65% of that obtained with 1α,25(OH)₂D₃. Statistical analysis revealed a significant correlation between both the induced-coactivator recruitment and transactivation assays (Pearson *r* = 0.961** and *r* = 0.986**, respectively), indicating the similarity in the course of the dose response curves for both 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃. The reason for the discrepancy from the previously reported lower transactivation potential of 1α,25(OH)₂-3-epi-D₃ may have its origin in different *CIP24* promoter fragment used in our experiments. Although, the EMSA assays with nuclear extracts and *in vitro* translated full length VDR and RXR reported by Nakagawa *et al.* [47] showed decreased DNA complex formation of VDR-RXR heterodimer in the presence of 1α,25(OH)₂-3-epi-D₃ compared to 1α,25(OH)₂D₃, the same authors showed using two-hybrid system that the strength of VDR-RXR heterodimerization in presence of 10nM of the 3-epimer is 40% compared to that observed for 1α,25(OH)₂D₃.

### Cell specific effects of 1α,25(OH)₂-3-epi-D₃

The magnitude of 1α,25(OH)₂-3-epi-D₃-mediated specific biological outcomes versus that induced by 1α,25(OH)₂D₃ is cell line specific. As such, it is established based only on CD11b

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

**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)₂D₃ 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).

doi:10.1371/journal.pone.0018124.g002

antigen positive cell numbers that 1α,25(OH)₂-3-epi-D₃ is biologically less potent than 1α,25(OH)₂D₃ in the human leukemia anti-proliferation and pro-differentiation cellular model (HL60), compared to 1α,25(OH)₂D₃ [47]. We monitor the precise dose-dependent study of 1α,25(OH)₂-3-epi-D₃ - directed HL60 cell anti-proliferation and differentiation by live cell enumeration and flow cytometry based on the expression of both CD11c and CD14 cell surface markers. In our experiments for both, 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃, only the saturating 100 nM concentration of ligand reduced the numbers of HL60 cells (Figure S1 and Methods S2). Although for 1α,25(OH)₂-3-epi-D₃ the related percentage of single positive or double CD11c/CD14 sub-populations was higher compared to that observed in control incubations, it was markedly reduced compared to that induced with 100 nM 1α,25(OH)₂D₃, consistent with the previous study [47].

Further, we hypothesized about the absence of the 1α,25(OH)₂-3-epi-D₃ signaling in HL60 cellular model and thus turned to characterize some of the biological properties of 1α,25(OH)₂-3-epi-D₃ in cells where it is produced [20,21]. We first determined the kinetics of *CIP27B1*- and *CIP24A1*-catalyzed oxidation by monitoring the major lipophilic metabolites arising from a single pulse of ³H[26,27]-25(OH)D₃ at physiological concentration (20.6 nM). During the first two hours, we observed a rapid appearance of 1α,25(OH)₂D₃, from which at a slower rate the 3-epimer was irreversibly formed. In total, some 60 independent incubation experiments were performed on the kinetics of ³H[26,27]-25(OH)D₃ using primary keratinocytes from various donors and skin sites. In all experiments, highly comparable time course of 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ were recorded with 3-epimer exceeding 1α,25(OH)₂D₃ after longer incubation as shown in Figure 2C and in the detailed HPLC analysis in Figure

<figure><img src="image_10.png" /></figure>
S2 and Methods S2. Since the 1α,25(OH)₂-3-epi-D₃ is present steadily up to 5 h in rather high concentration in this tissue and the fact that the primary genomic effects of hVDR ligands are exerted in first hours suggested that primary keratinocytes may be a good cellular model to investigate the anti-proliferative actions of this metabolite. Therefore we determined the dose-dependent anti-proliferative effects of 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ using ³H-thymidine incorporation assay (Figure 2D), and found that the IC₅₀ values for 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ were highly similar (41.4 and 66.1 nM, respectively) with no significant statistical difference (using unpaired t-test *p* = 0.074). In addition, we correlated the course of the anti-proliferation data between the two epimers and find a strong correlation (Pearson *r* = 0.940**) between them indicating the similar anti-proliferative activity for 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃. The anti-proliferative effects of the two metabolites are comparable and they are in close agreement with our coactivator peptide recruitment (Figure 2A) and reporter gene assays (Figure 2B). Although in this assay we cannot totally exclude the possibility that the potential cell type specific difference in the function of the two natural ligands may be partly due to the accumulation of 1α,25(OH)₂-3-epi-D₃ in 1α,25(OH)₂D₃ treated cells with C3-epimerization ability leading to additive effect, we consider this accumulation process as a naturally occurring *in vivo* physiological event when 1α,25(OH)₂D₃ is present in these cells.

## Overall structure of the hVDR complexed to 1α,25(OH)₂-3-epi-D₃

The mechanistic action of analogues of 1α,25(OH)₂D₃ is unveiled by the determination at high resolution of the crystal structure of their complexes with the VDR LBD [49–53]. We solved the crystal structure of the complex formed by 1α,25(OH)₂-3-epi-D₃ with the hVDR LBD mutant previously used to solve the structures of hVDR LBD in complexes with 1α,25(OH)₂D₃ or several synthetic agonists [49–54]. The crystal was isomorphous and the structure of hVDR LBD bound to 1α,25(OH)₂-3-epi-D₃

determined at a resolution of 1.9 Å (PDB ID: 3A78). The crystallographic data are summarized in Table S1. After refinement of the protein alone, the map showed an unambiguous electron density where to fit the ligand (Figure 3B). The complex formed by the hVDR LBD bound to 1α,25(OH)₂-3-epi-D₃ adopts the canonical active conformation as described in all previously reported agonist-bound nuclear receptor LBDs (Figure 3A). The conformation of the activation helix 12 is strictly maintained. When compared to the structure of hVDR LBD-1α,25(OH)₂D₃ complex, the atomic coordinates of hVDR LBD bound to 1α,25(OH)₂-3-epi-D₃ show very small root-mean-square deviation (RMSD) of 0.17 Å for all 255 Cα atoms, reflecting its high structural homology.

## Conformation of the 3-epimer in the ligand-binding pocket of hVDR

The 1α,25(OH)₂-3-epi-D₃, is buried in the predominantly hydrophobic ligand-binding pocket (LBP) of the VDR. The conformation of the 3-epi-hydroxyl group does not modify the A-ring chair conformation of the ligand. Furthermore the secα B-, C-, D- rings, and the aliphatic side chain present conformations similar to those observed with 1α,25(OH)₂D₃ (Figure 3B and C).

In the complexes of hVDR LBD bound to 1α,25(OH)₂D₃ versus 1α,25(OH)₂-3-epi-D₃, the distance between the C1-OH and the C25-OH groups varies from 13.1 Å to 12.7 Å and between the C3-OH and the C25-OH groups from 15.3 Å to 16.0 Å, respectively. The adaptation of the hVDR's LBP to different ligands can be described with the differential changes in the volumes of LBPs and bound ligands. In addition the parameter representing the % of LBP filling with ligand can provide useful information about the activity of ligand [55]. All these parameters are summarized in Table 1. Although the two diastereomer have the same molecular weight and differ only in the position of the C3-OH group, the 1α,25(OH)₂-3-epi-D₃ takes a slightly more compact conformation in the LBP. The graphical 0.2 Å mesh representation of the superimposed LPBs presented in Figure 4A

<figure><img src="image_11.png" /><figcaption>**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). doi:10.1371/journal.pone.0018124.g003</figcaption></figure>

<figure><img src="image_12.png" /></figure>
<table><caption>**Table 1.** Volume of VDR ligands and their resulting LBPs.</caption><thead><tr><th>Ligand</th><th>Ligand volume [Å³] *</th><th>Ligand volume [%]*</th><th>LBP volume [Å³]**</th><th>LBP volume [%]**</th><th>Filling of the LBP with ligand [%]</th></tr></thead><tbody><tr><td>1α,25(OH)₂D₃</td><td>416.56</td><td>100.00</td><td>667.13</td><td>100.00</td><td>62.44</td></tr><tr><td>1α,25(OH)₂-3-epi-D₃</td><td>407.65</td><td>97.86</td><td>633.75</td><td>95.00</td><td>64.32</td></tr></tbody></table>

The absolute values in Å³ as well as relative values in reference to those of 1α,25(OH)₂D₃ (100%) are indicated. From these values the percent filling of the LBP with ligands was also calculated.

*and ** Connolly solvent accessible surfaces calculated by GRASP and Voidoo respectively The quality of the cubic grid spacing for the surface for both ligands and LBP = 0.5 Å.

doi:10.1371/journal.pone.0018124.t001

and B show the surface area which is enlarged in case of 1α,25(OH)₂D₃ (in green) and the one enlarged in case of 1α,25(OH)₂-3-epi-D₃ bound hVDR LBP (in blue). This suggests that the hydrophobic residues lining the LBP are closer to the 3-epimer and may compensate for the canonical hydrogen bonds. We observed a notable adaptation with the displacement of the side chain of the residue Tyr147 by 2.0 Å compared to the 1α,25(OH)₂D₃ bound complex and the reorientation of the Glu277 side chain away from the 1α,25(OH)₂-3-epi-D₃ due to the α-position of the C3-OH group (Figure 4B). These specific rearrangements lead to a more compact conformation resulting in a 5% decrease in the volume of the LBP compared to 1α,25(OH)₂D₃.

## Specific interactions of the 1α,25(OH)₂-3-epi-D₃

The hydrophobic and electrostatic interactions between the receptor and the ligand are similar between the two structures except around the C3-OH group. While the C1-OH and C25-OH display the canonical hydrogen bonds, the 3-epi-OH of 1α,25(OH)₂-3-epi-D₃ interacts through hydrogen bonding only with Tyr143 instead of interacting with both Tyr143 and Ser278 (Figure 5). A significant feature of the 1α,25(OH)₂-3-epi-D₃ is the

compensation of the loss of interaction with Ser278 by a water-mediated hydrogen bond with the water molecule H₂O1 (W1 in [50]). As such, the position of water H₂O1 is moved 0.7 Å towards 1α,25(OH)₂-3-epi-D₃, thereby facilitating the specific water-mediated contacts. This water molecule is part of the network connecting another water molecule H₂O2 to Arg274. All these water molecules are also present in the 1α,25(OH)₂D₃-hVDR complex [50]. The C3-OH hydrogen bonds have longer distances in the 3-epimer (3.0 Å instead of 2.8 Å with Tyr143 and 3.1 Å with the water molecule instead of 2.9 Å with Ser278). A study on the mutations of the residues forming the hydrogen bonds with the hydroxyl groups of 1α,25(OH)₂-3-epi-D₃ revealed that mutated residues contacting the 3-hydroxyl group are the less affected in term of activity. Mutation of Ser278 in Ala may result in a lower binding affinity for 1α,25(OH)₂D₃ [56] while showing a similar potency to activate the transcription [57,58]. Due to the shift of the side chain of Tyr147, a hydrophobic interaction with this residue is lost in the 3-epimer structure. These structural data agree well with the lower binding affinity of this compound for VDR and to its induced biological activity.

In conclusion, we described a more effective synthesis of the highly stable 1α,25(OH)₂-3-epi-D₃, a natural metabolite. We have

<figure><img src="image_13.png" /><figcaption>**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.</figcaption></figure>

doi:10.1371/journal.pone.0018124.g004

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>**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. doi:10.1371/journal.pone.0018124.g005</figcaption></figure>

solved the crystal structure of hVDR LBD in complex with 1α,25(OH)₂-3-epi-D₃, which provides a mechanistic insight for the specific recognition of the two naturally occurring 3-epimers by hVDR. Indeed, the crystal structure reveals that the 3-epimer metabolite maintains the number of H-bonds by an alternative water-mediated interaction. In MCF-7 cells, the 1α,25(OH)₂-3-epi-D₃ on *CYP24* gene promoter retains significant transcriptional activity. In addition, the anti-proliferative action of 1α,25(OH)₂-3-epi-D₃ is cell specific and the IC₅₀ values of 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ in primary keratinocytes are in the same nanomolar range. Therefore, we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)₂D₃. The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear and needs further investigation. However, its high metabolic stability together with its significant biological activity makes this natural metabolite an interesting ligand for clinical applications. Further study on its target specificity and selectivity is required to the design of selective analogues. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)₂D₃ lead to significant activity in biological systems.

# Materials and Methods

## Ligands

1α,25(OH)₂D₃ was purchased from Cayman Chemical (Tallinn, Estonia) and the synthesis of 1α,25(OH)₂-3-epi-D₃ is described in more details in the Methods S1. Additional ligands and reference compounds are described in Methods S2. IUPAC rules were used

for the name of the compounds. In addition to NMR spectra (summarized in Methods S1), HPLC analysis was used to determine the purity (>95%) of the vitamin D analogues.

## Protein expression vectors for transactivation assays

Full-length cDNAs for human VDR [58] was subcloned into the T7/SV40 promoter-driven pSG5 expression vector (Stratagene, Heidelberg, Germany) and full-length cDNAs for green fluorescent protein (GFP) [59] was subcloned into parent vector resulting the pEGFP-C2 mammalian expression vector (Clontech Laboratories, Inc., USA).

## Luciferase reporter gene construct

The fragment of the proximal promoter region (–414 to –64) of the human *CYP24A1* gene was fused with the *thymidine kinase* promoter driving the firefly *luciferase* reporter gene [60].

## Coactivator peptide recruitment assays

Biochemical interaction between human VDR-LBD and the coactivator peptide in the presence of 1α,25(OH)₂D₃ or 1α,25(OH)₂-3-epi-D₃ was assayed using the AlphaScreen technology. The assay was performed in white opaque 384-well microplate (OptiPlate-384 Perkin Elmer) using a final volume of 15 µl containing final concentrations of 100 nM *E. coli*-expressed hexahistidine (6xHis)-tagged VDR-LBD protein, 20 nM of the human TIF2-3 biotinylated peptide ( Button-QEPVSPKKKENALL-RYLLDKDDTKD), and 10 µg/ml of both AlphaLISA Ni²⁺-chelate acceptor beads and (AL108C) and AlphaScreen streptavidin coated donor beads (6760002S) in an assay buffer containing

<figure><img src="image_16.png" /></figure>
50 mM MOPS pH = 7.4, 50 mM NaF, 50 mM CHAPS, and 100 µg/ml bovine serum albumin. Different concentrations of 1α,25(OH)₂D₃ or 1α,25(OH)₂-3-epi-D₃ dissolved in DMSO (maintained at a final concentration of 1%) were added as indicated. The experiment represents two independent measurements in triplicates for, which the mean and the S.D. of the mean was calculated.

Transient transfections and luciferase reporter gene
assays

MCF-7 cells were seeded into 24-well plates (100,000 cells/well) and grown overnight in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% charcoal-stripped fetal bovine serum (FCS) and 0.6 µg/ml insulin. Plasmid DNA containing liposomes were formed by incubating 40 ng of an expression vector for hVDR, 100 ng of reporter plasmid and 10 ng pEGF-C2 with Fugene 6 (Roche Diagnostics, Switzerland) transfection reagent according to the recommendation of the manufacturer for 15 min at room temperature. After dilution with 500 µl of phenol red-free DMEM, the liposomes were added to the cells. Phenol red-free DMEM supplemented with 500 µl of 20% charcoal-stripped FCS was added 4 h after transfection, in the presence of ligands or solvent. The cells were lysed 16 h after the onset of stimulation using reporter gene lysis buffer (Roche Diagnostics, Switzerland). The lysates were assayed for luciferase activity as recommended by the supplier (Perkin-Elmer, The Netherlands). The luciferase activities were normalized to GFP expression. Data represent one triplicate for which the mean and the S.D. of the mean was calculated.

Data analysis for dose response curves

A non-linear curve fit was performed for the AlphaScreen and reporter gene assay experimental dose response data and from sigmoidal dose response curve then the EC₅₀ values for the respective ligands were calculated using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA). The Student's unpaired t-test and Pearson correlation were performed with the SPSS software (SPSS Inc., version 14.0, Chicago, IL, USA).

Keratinocyte cell cultures

Normal human keratinocytes were isolated from fresh adult skin obtained from surgery and immediately transported to the laboratory under sterile conditions. Isolation and culture under serum-free conditions and without a feeder layer followed a modified protocol as used by Bikle *et al* [11]. The isolated epidermis was incubated in a 0.25% trypsin solution for 45 min at 37°C. Thereafter, the cells were scraped off and put in 50 ml Hank's balanced salt solution (HBSS) containing 10% FCS to block further trypsin digestion and centrifuged at 2000 rpm/2 min. The resulting cell pellet was suspended in Keratinocyte Growth Medium (KGM, Clonetics Corp., San Diego), a defined serum-free medium at low (0.06 mM) calcium containing 0.1 ng/ml epidermal growth factor, 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, bovine pituitary extract, antibiotics (gentamycin, amphothericin) gave the primary culture. After 24 h, the cells were incubated at 37°C in 95% air/5% CO₂ and the attached cells were washed and provided with fresh KGM medium. The culture medium was changed every other day and the cells were passaged when they reached 80–90% confluency (usually 6–10 days after plating).

Incubations of primary keratinocytes with ³H-25(OH)D₃

Confluent human keratinocytes in 1 ml KGM and in 6-well plates were incubated in duplicates at 37°C with 20.6 nM ³H-

25(OH)D₃ (around 600 000 dpm/ml) for 1–23 h. Incubations were stopped with 1 ml methanol/well, the cells were scraped off, transferred to a test tube together with the supernatant and two washings (with 1 ml methanol and 0.8 ml water). Unmodified ³H-25(OH)D₃ and most of the products were totally extracted from the combined solutions plus cell pellet according to the method of Bligh and Dyer [61] by three subsequent extractions with 2, 1 and 1 ml volumes of CHCl₃ at room temperature. ³H-activity in the CHCl₃-phase, in the water and total ³H-yield were determined. The combined CHCl₃ extracts were then evaporated under argon at 35°C, the residues dissolved in 0.4 ml ethanol and an aliquot (containing around 250 000 dpm ³H-activity) subjected to HPLC-analysis (see Methods S2).

³H-Thymidine incorporation (anti-proliferation assay in primary keratinocytes)

Keratinocytes (second passage) in 200 µl KGM (low calcium) were plated in 96-well plates at an initial density of 10⁴ cells/well, kept 24 h at 37°C in an incubator with 95% air/5% CO₂. Thereafter, the test compounds 1,25(OH)₂D₃ or its 3-epimer were added in 1 µl ethanol to give final concentrations ranging from 0 to 100 nM, each condition in triplicates. After further 24 h, 50 µl ³H-thymidine (1 µCi) were added and incubation continued for additional 7 h. Then, incubations were stopped by cell harvesting (Filtermate 196 Harvester, Packard-Canberra) and lysis: After removing the supernatant (see below), the adherent cells were released by 5 min treatment with 100 ml 0.125% trypsin in PBS at 37°C, harvested on a filterplate and washed 3 x with redistilled water. After drying the plates, their bottoms were sealed with a film and 50 µl scintillation cocktail (MicroScint O, Packard) were added. The whole plates were sealed with Packard Cover Film and ³H-activity counted on a Microplate Scintillation Counter (TopCount, Packard Canberra). To check whether proliferative (³H-thymidine incorporating) cells could have been shed off, the supernatants were soaked through a 96-well filterplate (Unifilter Plate GF/C) and 3 x washed with redistilled water: in all conditions, ³H-activity was undetectable on these filterplates (in order to roughly assess cell numbers and check for substance related morphological changes/toxic effects, photographs were taken prior to compound addition and immediately before harvesting.) Data - used as means ± SD – were normalized (incorporated ³H-activity sample vs. blank) and analyzed using the GRAFIT Erithacus 4.0.19 IC₅₀ software.

Protein purification and Crystallization

Purification and crystallization of the hVDR LBD complexed with 1α,25(OH)₂-3-epi-D₃ were performed as previously described [49]. The LBD of the hVDR (residues 118-427 Δ166-216) was cloned in pET-28b expression vector (Novagen) to obtain an N-terminal 6xHis fusion protein and was overproduced in E. coli BL21 (DE3) strain. Cells were grown in Luria Bertani medium and subsequently incubated for 6 h at 20°C with 1 mM isopropyl thio-β-D-galactoside. The protein purification included a metal affinity chromatography step on a Co²⁺-chelating resin (Clontech). The 6xHis tag was removed by thrombin digestion overnight at 4°C, and the protein was further purified by gel filtration on a Superdex S200 16/60. The sample buffer prior to protein concentration contained 10 mM Tris, pH = 7.5, 100 mM NaCl, and 10 mM dithiothreitol. The protein was concentrated to 3.5 mg/ml and incubated in the presence of a 1.5-fold molar excess of ligand. The purity and homogeneity of the protein were assessed by SDS-PAGE. The protein crystals were obtained at 4°C by vapor diffusion method using crystals of hVDR LBD-1α,25(OH)₂D₃ as

<figure><img src="image_17.png" /></figure>
microseeds. The reservoir solution contained 0.1 M MES and 1.4 M ammonium sulfate pH = 6.0.

## X-Ray data collection and structure determination

The crystal was mounted in fiber loop and flash cooled in liquid nitrogen after cryoprotection with a solution containing the reservoir plus 30% glycerol and 2% polyethylene glycol 400. Data collection from a single frozen crystal was performed at 100 K on the beamline ID29 of the European Synchrotron Radiation Facility (Grenoble, France). The crystal belongs to the orthorhombic space group P2₁2₁2₁, with one monomer per asymmetric unit. Data were integrated and scaled using MOSFLM [62] (see statistics in Table S1). A rigid body refinement was used with the structure of the hVDR LBD complexed to 1α,25(OH)₂D₃ as a starting model. Refinement involved iterative cycles of manual building and refinement calculations. The programs Refmac [63] and COOT [64] were used throughout structure determination and refinement. The omit map from the refined atomic model of hVDR LBD was used to fit the ligand to its electron density, shown in Figure 2A. Individual B-atomic factors were refined isotropically. Solvent molecules were then placed according to unassigned peaks in the difference Fourier map. In the hVDR/1α,25(OH)₂-3-epi-D₃ complex, refined at 1.9 Å with no σ cutoff, the final model consists of residues 118-423 (Δ166-216), the ligand, two sulphate ions and 372 water molecules. According to PROCHECK [65] 92.6% of peptide lies in most favored regions and 7.4% in additional allowed regions. Data are summarized in Table S1. The volumes of the ligand-binding pockets and ligands were calculated as previously reported [49].

## Supporting Information

**Figure S1 Biological properties of 1α,25(OH)₂D₃ and 1α,25(OH)₂-3-epi-D₃ in HL60 cellular model.** (A) 1α,25(OH)₂-3-epi-D₃-mediated HL60 cell growth. 1α,25(OH)₂D₃ or 1α,25(OH)₂-3-epi-D₃-treated HL60 at 1 nM and 100 nM concentrations are counted. Data are presented as mean±S.D. of the mean (*, p<0.05; **, p<0.01; ***, p<0.001). (B) 1α,25(OH)₂-3-epi-D₃-mediated HL60 cell differentiation into monocyte-like cells. HL60 cells were treated with either ethanol or 1 nM and 100"nM concentration of 1α,25(OH)₂D₃ or 1α,25(OH)₂-3-epi-D₃. Cells were labeled with PElabeled anti-human CD11c and FITC-labeled anti-human CD14, and HL60 cell differentiation was estimated by the double-positive CD11c/CD14 population. Data are representative of three distinct experiments.
(PDF)

**Figure S2 Dominant production of the 1α,25(OH)₂-3-epi-D₃ in keratinocytes after 5 h.** HPLC profile of the CHCl₃-extract from keratinocytes after 5 h incubation is shown.

## References

1. Adorini L, Penna G (2008) Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol 4: 404–412.

2. Bouillon R, Eelen G, Verlinden L, Mathieu C, Carmeliet G, et al. (2006) Vitamin D and cancer. J Steroid Biochem Mol Biol 102: 156–162.

3. Pinette KV, Yee YK, Amegadzie BY, Nagpal S (2003) Vitamin D receptor as a drug discovery target. Mini Rev Med Chem 3: 193–204.

4. Campbell MJ, Adorini L (2006) The vitamin D receptor as a therapeutic target. Expert Opin Ther Targets 10: 735–748.

5. DeLuca HF (2008) Evolution of our understanding of vitamin D. Nutr Rev 66: S73–S87.

6. Nagpal S, Na S, Rathnachalam R (2005) Noncalcemic actions of vitamin D receptor ligands. Endocr Rev 26: 662–687.

7. Garabedian M, Lieberherr M, N'Guyen TM, Corvol MT, Du Bois MB, et al. (1978) The in vitro production and activity of 24, 25-dihydroxycholecalciferol in cartilage and calvarium. Clin Orthop Relat Res 135: 241–248.

The amount of 1α,25(OH)₂-3-epi-D₃ (blue star) is the highest from all the metabolites detected with HPLC. The peak of 1α,25(OH)₂D₃ is highlighted with green star.
(PDF)

**Table S1 Data collection and refinement statistics.**

(PDF)

### Methods S1 Synthesis.

(PDF)

### Methods S2

(DOCX)

### Scheme S1 Retrosynthesis of 1b.

(PDF)

**Scheme S2 Synthesis of enol triflate 3.** Si=TBS = Si(t-Bu)(CH₃)₂.TBHP = t-BuOOH (a) NaBH₄, CeCl₃·7H₂O, MeOH, 0°C, 30 min. (b) TBHP, VO(acac)₂, PhH, reflux, 30 min. (c) TBSCl, Im, DMF, rt, 12 h. (d) O₃, MeOH-CH₂Cl₂, -78°C; Ac₂O, Et₃N, DMAP, -35°C to -8°C, 2 h; NaOAc, MeOH, 37°C, 12 h, (e) H₅IO₆, Et₂O, rt, 2 h. (f) CBr₄, Zn, Ph₃P, CH₂Cl₂, rt, 40 min. (g) LDA, THF, -78°C, 1 h; n-BuLi, 15 min; 5-Cl-Py-₂NTf₂, -78°C to rt, 12 h.
(PDF)

**Scheme S3 Synthesis of metabolite 1.** TES = Si(CH₂CH₃)₃. (a) (Ph₃PCH₂Br)Br, KOt-Bu, toluene, -5°C to rt, 1 h, 80%. (b) TESCl, Im, DMAP, DMF, rt, 3 h, 91%. (c) InCl₃, t-BuLi, THF, -78°C to 0°C, 2 h. (d) 3, (Ph₃P)₄Pd, Et₃N, THF, (dppf)PdCl₂, 0°C to rt, 12 h. (e) HF·Py, Et₃N, CH₂Cl₂, CH₃CN, rt, 4 h, 58%.
(PDF)

## Acknowledgments

We thank the beam-line staff at the ESRF (Grenoble, France) for help during data collection and T. Huet for critical reading. We thank N. Rouleau and J. Hyvärinen from Perkin Elmer for support in AlphaScreen Assays and V. Prantner for help with statistical analysis.

## Author Contributions

Conceived and designed the experiments: AM DM NR. Performed the experiments: FM RS YS CA IS PA, JP. Analyzed the data: FM IS AM NR. Contributed reagents/materials/analysis tools: CM. Wrote the paper: FM AM NR.

8. Howard GA, Turner RT, Sherrard DJ, Baylink DJ (1981) Human bone cells in culture metabolize 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃. J Biol Chem 256: 7738–7740.

9. Jones G, Vriezen D, Lohnes D, Palda V, Edwards NS (1987) Side-chain hydroxylation of vitamin D₃ and its physiological implications. Steroids 49: 29–53.

10. Ishizuka S, Norman AW (1987) Metabolic pathways from 1α,25-dihydroxyvitamin D₃ to 1α,25-dihydroxyvitamin D₃-26,23-lactone. Stereo-retained and stereo-selective lactonization. J Biol Chem 262: 7165–7170.

11. Bikle DD, Nemanic MK, Whitney JO, Elias PW (1986) Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D₃. Biochemistry 25: 1545–1548.

12. Horst RL (1979) 25-OHD₃-26,23-lactone: a metabolite of vitamin D₃ that is 5 times more potent than 25-OHD₃ in the rat plasma competitive protein binding radioassay. Biochem Biophys Res Commun 89: 286–293.

<figure><img src="image_18.png" /></figure>
13. Wichmann JK, DeLuca HF, Schnoes HK, Horst RL, Shepard RM, et al. (1979) 25-Hydroxyvitamin D₃ 26,23-lactone: a new in vivo metabolite of vitamin D. Biochemistry 18: 4775-4780.

14. DeLuca HF, Suda T, Schnoes HK, Tanaka Y, Holick MF (1970) 25,26-dihydroxycholecalciferol, a metabolite of vitamin D₃ with intestinal calcium transport activity. Biochemistry 9: 4776-4780.

15. Reinhardt TA, Napoli JL, Praminik B, Littledike ET, Beitz DC, et al. (1981) 1α-25,26-trihydroxyvitamin D₃: an in vivo and in vitro metabolite of vitamin D₃. Biochemistry 20: 6230-6235.

16. Holick MF, Schnoes HK, DeLuca HF, Gray RW, Boyle IT, et al. (1972) Isolation and identification of 24,25-dihydroxycholecalciferol, a metabolite of vitamin D made in the kidney. Biochemistry 11: 4251-4255.

17. Kumar R, Schnoes HK, DeLuca HF (1978) Rat intestinal 25-hydroxyvitamin D₃- and 1α,25-dihydroxyvitamin D₃-24-hydroxylase. J Biol Chem 253: 3804-3809.

18. Reddy GS, Rao DS, Siu-Caldera ML, Astecker N, Weiskopf A, et al. (2000) 1α,25-dihydroxy-16-ene-23-yne-vitamin D₃ and 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D₃: analogs of 1α,25-dihydroxyvitamin D₃ that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway. Arch Biochem Biophys 383: 197-205.

19. Norman AW, Bouillon R, Farach-Carson MC, Bishop JE, Zhou LX, et al. (1993) Demonstration that 1 beta,25-dihydroxyvitamin D₃ is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1α,25-dihydroxyvitamin D₃. J Biol Chem 268: 20022-20030.

20. Reddy GS, Muralidharan KR, Okamura WH, Tserng K-Y, McLane JA (1994) Metabolism of 1α,25-dihydroxyvitamin D₃ and one of its A-ring diastereomer 1α,25-dihydroxy-3-epivitamin D₃ in neonatal human keratinocytes. In: Norman AW, Bouillon R, Thomasset M, eds. Vitamin D a pluripotent steroid hormone: Structural studies, molecular endocrinology and clinical applications, Walter de Gruyter, NY, USA. Walter de Gruyter, NY, USA. pp 172-173.

21. Reddy GS, Siu-Caldera M-L, Schuster I, Astecker N, Tserng K-Y, et al. (1997) Target tissue specific metabolism of 1α,25-dihydroxyvitamin D₃ through A-ring modification. In: Norman WA, Bouillon R, Thomasset M, eds. Vitamin D, chemistry, biology and clinical applications of the steroid hormone, Riverside, CA, USA. Riverside, CA, USA. pp 139-146.

22. Sekimoto H, Siu-Caldera ML, Weiskopf A, Vouros P, Muralidharan KR, et al. (1999) 1α,25-dihydroxy-3-epi-vitamin D₃: in vivo metabolite of 1α,25-dihydroxyvitamin D₃ in rats. FEBS Lett 448: 278-282.

23. Bischof MG, Siu-Caldera ML, Weiskopf A, Vouros P, Cross HS, et al. (1998) Differentiation-related pathways of 1α,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: production of 1α,25-dihydroxy-3-epi-cholecalciferol. Exp Cell Res 241: 194-201.

24. Rehan VK, Torday JS, Peleg S, Gennaro L, Vouros P, et al. (2002) 1α,25-dihydroxy-3-epi-vitamin D₃, a natural metabolite of 1α,25-dihydroxy vitamin D₃: production and biological activity studies in pulmonary alveolar type II cells. Mol Genet Metab 76: 46-56.

25. Brown AJ, Ritter C, Slatopolsky E, Muralidharan KR, Okamura WH, et al. (1999) 1α,25-dihydroxy-3-epi-vitamin D₃, a natural metabolite of 1α,25-dihydroxyvitamin D₃, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem 73: 106-113.

26. Siu-Caldera ML, Sekimoto H, Weiskopf A, Vouros P, Muralidharan KR, et al. (1999) Production of 1α,25-dihydroxy-3-epi-vitamin D₃ in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed. Bone 24: 457-463.

27. Hylemon PB, Sjövall HDAJ (1985) Chapter 12 Metabolism of bile acids in intestinal microflora. In: New Comprehensive Biochemistry Sterols and Bile Acids Elsevier. pp 331-343.

28. Penning TM, Bennett MJ, Smith-Hoog S, Schlegel BP, Jez JM, et al. (1997) Structure and function of 3α-hydroxysteroid dehydrogenase. Steroids 62: 101-111.

29. Reddy GS, Rao DS, Siu-Caldera ML (2000) Natural metabolites of 1α,25-dihydroxy-vitamin D₃ and its analogs. In: Norman AW, Bouillon R, Thomasset, eds. Vitamin D Endocrine system, Structural, Biological, Genetic and Clinical Aspects, University of California, Printing and Reprographics, Riverside, CA, USA. pp 139-146.

30. Kamao M, Tatematsu S, Hatakeyama S, Sakaki T, Sawada N, et al. (2004) C-3 epimerization of vitamin D₃ metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D₃ is metabolized to 3-epi-25-hydroxyvitamin D₃ and subsequently metabolized through C-1α or C-24 hydroxylation. J Biol Chem 279: 15897-15907.

31. Morrison NA, Eisman JA (1991) Nonhypercalcemic 1,25-(OH)₂D₃ analogs potently induce the human osteocalcin gene promoter stably transfected into rat osteosarcoma cells (ROSCO-2). J Bone Miner Res 6: 893-899.

32. Fleet JC, Bradley J, Reddy GS, Ray R, Wood RJ (1996) 1α,25-(OH)₂-2-vitamin D₃ analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch Biochem Biophys 329: 228-234.

33. Muralidharan KR, De Lera AR, Isaeff SD, Norman AW, Okamura WH (1993) Studies of vitamin D (calciferol) and its analogs. 45. Studies on the A-ring diastereomers of 1α,25-dihydroxyvitamin D₃. J Org Chem 58: 1895-1899.

34. Gómez-Reino C, Vitale C, Maestro M, Mouriño A (2005) Pd-catalyzed carbocyclization-Negishi cross-coupling cascade: a novel approach to 1α,25-dihydroxyvitamin D₃ and analogues. Org Lett 7: 5885-5887.

35. Zhu G, Okamura WH (1995) Synthesis of Vitamin D (Calciferol). Chem Rev 95: 1877-1952.

36. Krause S, Schmalz HG (2000) Palladium-Catalyzed Synthesis of Vitamin D-Active Compounds. In: Organic Synthesis Highlights IV. Germany: Wiley and VCH. pp 212-217.

37. Posner GH, Kahraman M (2003) Organic chemistry of vitamin D analogues (deltanooids). Eur J Org Chem 2003. pp 3889-3895.

38. Luche JL (1978) Lanthanides in organic chemistry. 1. Selective 1, 2 reductions of conjugated ketones. J Am Chem Soc 100: 2226-2227.

39. Sharpless KB, Michaelson RC (1973) High stereo- and regioselectivities in the transition metal catalyzed epoxidations of olefinic alcohols by tert-butyl hydroperoxide. J Am Chem Soc 95: 6136-6137.

40. Daniewski AR, Garofalo LM, Hutchings SD, Kabat MM, Liu W, et al. (2002) Efficient synthesis of the A-ring phosphine oxide building block useful for 1α,25-dihydroxy-vitamin D and analogues. J Org Chem 67: 1580-1587.

41. Corey EJ, Fuchs PL (1972) A synthetic method for formyl-> ethynyl conversion (RCHO--> RCCH or RCCR'). Tetrahedron Lett 13: 3769-3772.

42. Mouriño A, Torneiro M, Vitale C, Fernández S, Pérez-Sestelo J, et al. (1997) Efficient and versatile synthesis of A-ring precursors of 1α,25-dihydroxy-vitamin D₃ and analogues. Applications to the synthesis of Lythgoe-Roche phosphine oxide. Tetrahedron letters 38: 4713-4716.

43. Gogoi P, Sigüeiro R, Eduardo S, Mouriño A (2010) An expeditious route to 1α,25-dihydroxyvitamin D(3) and its analogues by an aqueous tandem palladium-catalyzed a-ring closure and suzuki coupling to the C/D unit. Eur J Chem 16: 1432-1435.

44. Trost BM, Dumas J, Villa M (1992) New Strategies for the synthesis of vitamin D metabolites via Pd-catalyzed reactions. J Am Chem Soc 114: 9836-9845.

45. Takeyama K, Masuhiro Y, Fuse H, Endoh H, Murayama A, et al. (1999) Selective synthesis of vitamin D receptor with transcriptional coactivators by a vitamin D analog. Mol Cell Biol 19: 1049-1055.

46. Ullman EF, Kirakossian H, Singh S, Wu ZP, Irvin BR, et al. (1994) Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci U S A 91: 5426-5430.

47. Nakagawa K, Sowa Y, Kurobe M, Osono K, Siu-Caldera ML, et al. (2001) Differential activities of 1α,25-dihydroxy-16-ene-vitamin D(3) analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis. Steroids 66: 327-337.

48. Harant H, Spinner D, Reddy GS, Lindley IJ (2000) Natural metabolites of 1α,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor. J Cell Biochem 78: 112-120.

49. Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D (2000) The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5: 173-179.

50. Hourai S, Fujishima T, Kittaka A, Suhara Y, Takayama H, et al. (2006) Probing a water channel near the A-ring of receptor-bound 1α,25-dihydroxyvitamin D₃ with selected 2α-substituted analogues. J Med Chem 49: 5199-5205.

51. Eelen G, Valle N, Sato Y, Rochel N, Verlinden L, et al. (2008) Superagonistic fluorinated vitamin D₃ analogs stabilize helix 12 of the vitamin D receptor. Chem Biol 15: 1029-1034.

52. Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D (2001) Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci U S A 98: 5491-5496.

53. Hourai S, Rodrigues LC, Antony P, Reina-San-Martin B, Ciesielski F, et al. (2008) Structure-based design of a superagonist ligand for the vitamin D nuclear receptor. Chem Biol 15: 383-392.

54. Rochel N, Tocchini-Valentini G, Egea PF, Juntunen K, Garnier JM, et al. (2001) Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor. Eur J Biochem 268: 971-979.

55. Molnár F, Peräkylä M, Carlberg C (2006) Vitamin D receptor agonists specifically modulate the volume of the ligand-binding pocket. J Biol Chem 281: 10516-10526.

56. Reddy MD, Stoynova L, Acevedo A, Collins ED (2007) Residues of the human nuclear vitamin D receptor that form hydrogen bonding interactions with the three hydroxyl groups of 1α,25-dihydroxyvitamin D₃. J Steroid Biochem Mol Biol 103: 347-351.

57. Choi M, Yamamoto K, Masuno H, Nakashima K, Taga T, et al. (2001) Ligand recognition by the vitamin D receptor. Bioorg Med Chem 9: 1721-1730.

58. Carlberg C, Bendik I, Wyss A, Meier E, Sturzenbecker LJ, et al. (1993) Two nuclear signalling pathways for vitamin D. Nature 361: 657-660.

59. Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173: 33-38.

60. Väisänen S, Dunlop TW, Sinkkonen L, Frank C, Carlberg C (2005) Spatiotemporal activation of chromatin on the human CYP24 gene promoter in the presence of 1α,25-Dihydroxyvitamin D₃. J Mol Biol 350: 65-77.

61. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917.

62. Leslie AGW (1992) Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26.

<figure><img src="image_19.png" /></figure>
63. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. *Acta Crystallogr D Biol Crystallogr* 53: 240–255.

64. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. *Acta Crystallogr D Biol Crystallogr* 60: 2126–2132.

65. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: A program to check the stereochemical quality of protein structures. *J Appl Crystallogr* 26: 283–291.

<figure><img src="image_20.png" /></figure>