Gene Regulatory Potential of Nonsteroidal Vitamin D Receptor Ligands

Mikael Peräkylä, Marjo Malinen, Karl-Heinz Herzig and Carsten Carlberg

Departments of Chemistry (M.P.) and Biochemistry (M.M., C.C.), A. I. Virtanen Institute (K.-H.H.), University of Kuopio, FIN-70211 Kuopio, Finland

Address all correspondence and requests for reprints to: Prof. Carsten Carlberg, Department of Biochemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: carlberg{at}messi.uku.fi.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The seco-steroid 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3] is a promising drug candidate due to its pleiotropic function including the regulation of calcium homeostasis, bone mineralization and cellular proliferation, differentiation, and apoptosis. We report here a novel class of nonsteroidal compounds, represented by the bis-aromatic molecules CD4409, CD4420, and CD4528, as ligands of the 1{alpha},25(OH)2D3 receptor (VDR). Taking the known diphenylmethane derivative LG190178 as a reference, this study provides molecular evaluation of the interaction of nonsteroidal ligands with the VDR. All four nonsteroidal compounds were shown to induce VDR-retinoid X receptor heterodimer complex formation on a 1{alpha},25(OH)2D3 response element, stabilize the agonistic conformation of the VDR ligand-binding domain, enable the interaction of VDR with coactivator proteins and contact with their three hydroxyl groups the same residues within the ligand-binding pocket of the VDR as 1{alpha},25(OH)2D3. Molecular dynamics simulations demonstrated that all four nonsteroidal ligands take a shape within the ligand-binding pocket of the VDR that is very similar to that of the natural ligand. CD4528 is mimicking the natural hormone best and was found to be in vitro at least five times more potent than LG190178. In living cells, CD4528 was only two times less potent than 1{alpha},25(OH)2D3 and induced mRNA expression of the VDR target gene CYP24 in a comparable fashion. At a noncalcemic dose of 150 µg/kg, CD4528 showed in vivo a clear induction of CYP24 expression and therefore may be used as a lead compound for the development of therapeutics against psoriasis, osteoporosis, and cancer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR (NR) for the seco-steroid 1{alpha},25(OH)2D3, VDR, is one of the 11 endocrine members of the NR superfamily, i.e. it binds its ligand with high affinity (dissociation constant value of 1 nM or lower) (1). 1{alpha},25(OH)2D3 is a key player in calcium homeostasis and bone mineralization (2) and also has antiproliferative and differentiation inducing effects on various cell types (3). Like all NRs, VDR has a highly conserved DNA binding domain and a less conserved ligand binding domain (LBD). The LBD is formed by 12 {alpha}-helices, and its overall architecture is similar for all NRs (4). VDR acts preferentially as a heterodimer with the retinoid X receptor (RXR) on specific DNA sequences in promoter regions of 1{alpha},25(OH)2D3 target genes, referred to as 1{alpha},25(OH)2D3 response elements (VDREs) (5).

Due to its growth-inhibitory properties 1{alpha},25(OH)2D3 could act as an anticancer drug, but its calcemic effects limit its application in pharmacological doses. Therefore, more than 3000 synthetic analogs of 1{alpha},25(OH)2D3 have been synthesized with the goal to improve the potency and specificity of the physiological effects of vitamin D (6, 7). Basically all of these analogs interact with the VDR-RXR-VDRE complex. The central element of this molecular switch is the intermolecular interaction of the VDR-LBD (8) with a 1{alpha},25(OH)2D3 analog either in its agonistic or its antagonistic conformation. Most of these ligands are agonists of the VDR and only a few antagonists have been reported (7). The stabilization of the agonistic conformation of the VDR-LBD is achieved by the repositioning of its most carboxy-terminal {alpha}-helix (helix 12). In detail, this is achieved by a hydrogen bond between the C25-hydroxyl group of 1{alpha},25(OH)2D3 and H397 of the receptor (9) and is supported by an additional, less important hydrogen bond with H305 (10). In the presence of agonist, H397 is able to form van der Waals interactions with F422 of helix 12. This keeps helix 12 in a position that allows the binding of the LXXLL NR interaction domain of coactivator (CoA) proteins into a hydrophobic cleft on the surface of the VDR-LBD (11). CoA proteins of the p160 family (12) link the ligand-activated VDR to enzymes displaying histone acetyltransferase activity, that cause chromatin relaxation. This reverses the action of unliganded VDR, which recruits corepressor proteins (13). In a subsequent step, ligand-activated VDR changes rapidly from interacting with the CoAs of the p160 family to those of mediator complexes acting as a bridge to the basal transcriptional machinery (14). In this way, ligand-activated VDR induces two tasks, the modification of chromatin and the activation of transcription.

Most 1{alpha},25(OH)2D3 analogs have been modified at their side chain, which often increases their metabolic stability and the half-life of the VDR-ligand complex (15, 16). Most superagonists identified so far carry only minor modifications compared with the natural hormone and stabilize the same agonistic VDR conformation via the H397-F422 interaction (17). However, there are also semisteroidal analogs of 1{alpha},25(OH)2D3, which either have significant changes in their A, C, or D ring, such as deletion of the CD ring (18), or carry substantial additions, such as the second side chain in Gemini (19, 20). Gemini is an interesting 1{alpha},25(OH)2D3 analog because it is able to act both as a potent agonist (19) as well as a conditional inverse agonist (21). In the presence of CoAs, RXR, and DNA, Gemini is able to stabilize the VDR in its agonistic conformation (19, 22). Molecular dynamics (MD) simulations of the Gemini-VDR complex demonstrated that one of the two side chains of Gemini takes the same location as in the natural hormone, whereas for the second side chain, two approximately equal positions were identified (23).

Nonsteroidal ligands are known for several members of the NR superfamily, such as flutamine for the androgen receptor (24) or raloxifene for the estrogen receptor (25). However, for the VDR so far only diphenylmethane derivatives, such as LG190178 (26, 27) and its derivatives (28, 29), have been described as pure nonsteroidal ligand. These compounds were shown to mimic various activities of 1{alpha},25(OH)2D3 in vitro and in vivo, such as VDR binding, VDR-dependent transcriptional activation, inhibition of proliferation and differentiation of various cell types (27). Interestingly, LG190178 and its analogs do not bind to the serum vitamin D binding protein and show a reduced calcium mobilization activity.

In this study, three novel nonsteroidal compounds, the bis-aromatic molecules CD4409, CD4420, and CD4528, and the known diphenylmethane derivative LG190178 were compared in different in vitro assays for their ability to act as VDR ligands. Moreover, mutagenesis analysis of the six most critical amino acids of the ligand-binding pocket of the receptor as well as MD simulations indicated that all four compounds take a similar position as the natural hormone and act as VDR agonists. In vitro CD4528 was found to be at least five times more potent than LG190178, in living cells nearly as potent as the natural hormone and in vivo noncalcemic up to a dose of 150 µg/kg. Therefore, CD4528 has the potential to become a therapeutically important drug.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A mammalian one-hybrid screening of more than 50 bis-aromatic compounds (30, 31) was performed using HeLa cells that were transiently transfected with an expression vector for a GAL4DBDVDRLBD-fusion protein together with a reporter gene construct containing a GAL4 binding site-driven luciferase gene (data not shown). The mammalian one-hybrid assay represents a simple reporter gene assay system because it is reduced to the VDR-LBD as a functional unit and functions independently of RXR. The most potent compounds of this screening, CD4409, CD4420, and CD4528 (for structures see Fig. 1Go) were chosen for a more detailed inspection (Fig. 2Go). The chemical structure of the compounds is characterized by two OH-groups, which are separated by 12- to 13-carbon atoms as in 1{alpha},25(OH)2D3 and its CD- or C- or D-ring-deleted analogs. In fact, CD4420 and CD4528 are similar to D-ring-deleted 1{alpha},25(OH)2D3 analogs. The natural hormone 1{alpha},25(OH)2D3 and LG190178 (27) were used as a reference. In the absence of the vitamin D 24-hydroxylase (CYP24) inhibitor ketoconazole (32) the dose-response curves for CD4409, CD4420, and CD4528 provided half-maximal activation (EC50) values of 8, 5, and 1.7 nM, respectively, whereas for 1{alpha},25(OH)2D3 and LG190178 the values of 1 and 10 nM were obtained (Fig. 2AGo). This suggests that, in this ex vivo functional assay, CD4528 is nearly as potent as 1{alpha},25(OH)2D3 and more than five times as sensitive as LG190178. The same experiment was repeated in the presence of 500 nM ketoconazole and resulted in EC50 values of 0.12, 10, 6, 2, and 12 nM for 1{alpha},25(OH)2D3, CD4409, CD4420, CD4528, and LG190178, respectively. This indicates that during the 16-h incubation period in HeLa cells, the natural hormone is metabolized by the CYP24 enzyme, but not the nonsteroidal compounds, i.e. that they are significantly more stable metabolically than 1{alpha},25(OH)2D3.



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Fig. 1. Structures of CD4409, CD4420, and CD4528 in Reference to 1{alpha},25(OH)2D3 and LG190178

 


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Fig. 2. Functional Activity of Nonsteroidal Analogs in Absence and Presence of CYP24 Inhibitor

Luciferase reporter gene assays were performed from extracts of HeLa cells that were transiently transfected with a reporter gene construct driven by three copies of the GAL4 binding site and an expression vector for a GAL4DBDVDRLBD fusion protein (schematically depicted on top). Cells were treated for 16 h with graded concentrations of the indicated ligands in the absence (A) or presence of 500 nM ketoconazole (B). Stimulation of normalized luciferase activity was calculated in comparison to solvent-induced controls. Data points represent the mean of triplicates, and the tips of the bars indicate SD. The EC50 values for the induction of gene activity were determined from the respective dose-response curves.

 
The ligand-dependent gel shifts represent a straightforward, DNA-dependent assay for the analysis of the functionality of VDR-RXR heterodimers (33). In this assay, the complex formation of in vitro-translated VDR-RXR heterodimers on a VDRE is measured as a function of ligand concentration and 1{alpha},25(OH)2D3 is known to provide an EC50 value of 0.1–0.2 nM (15, 19, 34). The EC50 value was found to be independent of sequence variations between natural VDREs and at saturating concentrations an approximately 3-fold increased VDR-RXR heterodimer complex formation can be detected (35). Graded concentrations of CD4409, CD4420, CD4528, and LG190178 were analyzed for their ability to stabilize VDR-RXR heterodimers on the DR3-type VDRE of the rat atrial natriuretic factor (ANF) gene (Fig. 3AGo). Saturating concentrations of all four compounds increased VDR-RXR complex formation on the VDRE by a factor of 2.5 to 3.0 and EC50 values of 6, 4, 4.8, and 29 nM were measured for CD4409, CD4420, CD4528, and LG190178, respectively. This means that in vitro the three best bis-aromatic compounds are at least five times more sensitive than LG190178, but they are still at least 20 times less sensitive than 1{alpha},25(OH)2D3.



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Fig. 3. In Vitro Evaluation of Nonsteroidal Analogs

Gel shift experiments were performed with in vitro-translated VDR-RXR heterodimers that were preincubated at room temperature with the indicated ligands and the [32P]-labeled DR3-type VDRE from the rat ANF gene promoter (A). Protein-DNA complexes were separated from free probe through 8% nondenaturing polyacrylamide gels. LPD assays were performed by preincubating in vitro-translated [35S]-labeled VDR with unlabeled RXR and the unlabeled DR3-type VDRE from the rat ANF gene promoter in the presence of indicated ligands (B). After digestion with trypsin, samples were electrophoresed through 15% SDS-polyacrylamide gels. For both assays, representative experiments with saturating ligand concentrations (1 µM) are shown. The amount of VDR-RXR-VDRE complexes in relation to free probe (A) and the amount of ligand-stabilized VDR conformations 1 (c1LPD) and 3 (c3LPD) in relation to VDR input (B) were quantified by using a Fuji FLA3000 reader. Data points represent the mean of triplicates, and the tips of the bars indicate standard deviation. The EC50 values for VDR-RXR-VDRE complex formation (A) and for the stabilization of the agonistic conformation c1LPD (B) were determined from the respective dose-response curves. The reference value for 1{alpha},25(OH)2D3 is 0.2 nM (A) and 0.3 nM (B), respectively (15 ).

 
Comparable with the ligand-dependent gel shift assay, the DNA-dependent limited protease digestion (LPD) assay is able to measure the in vitro functionality of VDR-RXR heterodimers and also provides for 1{alpha},25(OH)2D3 an EC50 value of 0.1 to 0.2 nM (15). In addition, the LPD assay monitors via the protease-resistant VDR fragments c1LPD and c3LPD, whether the ligand stabilizes the receptor preferentially in its agonistic or in its nonagonistic conformation, respectively (19, 36, 37). Moreover, the observation of a third VDR fragment, c2LPD, is an indication for a possible antagonistic ligand (34). The natural hormone stabilizes 40–80% of all VDR molecules in a dose-dependent fashion in the agonistic conformation c1LPD, a constant amount of 10% in c3LPD, but no receptors in c2LPD (19, 37). The same was observed with saturating concentrations of more than 50 bis-aromatic compounds of the initial screening (data not shown). CD4409, CD4420, CD4528, and LG190178 stabilized the agonistic conformation with EC50 values of 9, 26, 3.1, and 22 nM, respectively (Fig. 3BGo). Taken together, all tested nonsteroidal compounds stabilized the VDR in its agonistic conformation, i.e. they are VDR agonists and not antagonists or inverse agonists. CD4528 showed to be seven times more sensitive than LG190178, but still at least 10 times less sensitive than the natural hormone. This confirms the results of the ligand-dependent gel shift assays (Fig. 3BGo).

To confirm that the nonsteroidal ligands stabilize the VDR in its agonistic conformation, supershift experiments were performed with the p160 family CoA transcription intermediary factor 2 (TIF2). The natural hormone, LG190178, CD4409, CD4420, and CD4528 were tested at concentrations of 1, 10, and 100 nM for their ability to induce an interaction of in vitro-translated VDR-RXR heterodimers with the NR interaction domain of bacterially expressed TIF2 (Fig. 4Go). In the absence of ligand, no VDR-TIF2 interaction was observed, whereas 1 nM of the natural hormone was sufficient to allow all DNA-bound VDR-RXR heterodimers to interact with TIF2. At the same concentration, LG190178 and CD4409 induced clearly weaker amounts of supershifted complexes than CD4420 and CD4528. At higher concentrations, all four nonsteroidal ligands showed to be equally potent in inducing an interaction between VDR and TIF2. This indicates that the nonsteroidal ligands stabilize the VDR in an agonistic conformation that allows CoA interaction. In summary, in line with the previous assays (Figs. 2Go and 3Go) also with supershifts CD4528 showed to be more sensitive than LG190178 but less than 1{alpha},25(OH)2D3.



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Fig. 4. Nonsteroidal Analogs Induce Interaction of the VDR with CoA Protein

Supershift experiments were performed with in vitro-translated VDR-RXR heterodimers that were preincubated in presence of bacterially expressed GST-TIF2646–926 with the indicated concentrations of ligands and the [32P]-labeled rat ANF DR3-type VDRE. Protein-DNA complexes were separated from free probe through 8% nondenaturing polyacrylamide gels. Representative experiments are shown. The amount of VDR-RXR-VDRE or VDR-RXR-VDRE-TIF2 (supershift) complexes in relation to free probe was quantified by using a Fuji FLA3000 reader. Columns represent the mean of triplicates, and the tips of the bars indicate standard deviation. NS, Nonspecific complex.

 
The crystal structure of the VDR-LBD (9), receptor modeling (38), and biochemical assays (10) indicated that the amino acids Y143, S237, R274, S278, H305, and H397 are the most critical for the receptor-ligand contact. To answer the question, how the nonsteroidal ligands bind into the ligand-binding pocket of the VDR-LBD, the six amino acids were individually mutated to alanines. The effect of the mutagenesis on the functionality of the VDR was tested for 1{alpha},25(OH)2D3 and the four nonsteroidal ligands by conventional reporter gene assays in MCF-7 human breast cancer cells (Fig. 5Go). The residual activities of the individual mutated VDRs varied between 13% (S237A) and 73% (H305A) of wild-type (wt) receptor activity, which was an approximately 100-fold induction of reporter gene activity by ligand. In general, this characteristic mutagenesis pattern appeared to be conserved for the four nonsteroidal ligands, but significant deviations were observed for Y143A (increased activity with CD4409, CD4420, and LG190178), S237A (increased activity with CD4409 and CD4420), H305 (decreased with LG190178) and H397A (increased activity with CD4409 and CD4420). This indicates that the nonsteroidal ligands bind the ligand-binding pocket of the VDR-LBD in the same fashion as the natural ligand with minor variations at a few positions.



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Fig. 5. Single Point Mutagenesis Analysis of Critical Amino Acid Residues within the VDR Ligand Binding Pocket

Luciferase reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected with a reporter gene construct driven by four copies of the rat ANF DR3-type VDRE and expression vectors for RXR and wt or mutant VDR proteins (schematically depicted on top). The cells were treated for 16 h with the indicated ligands (each 100 nM). Stimulation of luciferase activity was calculated in comparison to solvent-induced controls and normalized for each ligand to the activity of wt VDR. Columns represent the mean of three experiments, and the tips of the bars indicate SD. Statistical analysis was performed by two-tailed, paired Student’s t test, and P values were calculated in reference to the relative activity obtained with 1{alpha},25(OH)2D3 (*, P < 0.05).

 
MD simulations were performed to analyze in more detail the interactions between the four nonsteroidal ligands and the ligand-binding pocket of the VDR-LBD. By molecular modeling the crystal structure of the VDR-LBD (9) was first completed with the amino acid residues 118, 119, 375–377, and 424–427. The four nonsteroidal VDR ligands were then docked to the ligand-binding pocket so that the three hydroxyl groups of the ligands were positioned close to the hydroxyl groups of 1{alpha},25(OH)2D3 at C1, C3, and C25. After energy minimizing of the water molecules and the moving part of the protein-ligand complex, MD simulations of 1 nsec were performed. The distance between the 15 closest amino acid residues of the ligand-binding pocket and the ligands are shown (published as supplemental Table 1 on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), whereas supplemental Table 2 lists the energies derived from their van der Waals and electrostatic interactions. A comparison between the distances obtained from the crystal structure and from a reference MD simulation of 1{alpha},25(OH)2D3 demonstrates the reliability of the method (supplemental Table 1). The four nonsteroidal compounds fit nicely in the ligand-binding pocket and formed stable complexes with the VDR-LBD. In general, the amino acids Y143, S237, R274, S278, H305, and H397 were found to be the closest contact points for the hydroxyl groups of the natural ligand as well as for those of the four nonsteroidal compounds. The interaction energies (supplemental Table 2) of the hydrogen bond forming residues are in line with the corresponding distances (supplemental Table 1). The total interaction energy is in accordance with the experimental data, i.e. the natural hormone showed the strongest interactions (–100.6 kcal/mol) with all residues of the LBD [Sum(all)] followed by CD4528 (–93.7 kcal/mol), CD4420 (–93.0 kcal/mol), and CD4409 (–92.9 kcal/mol). This order is retained for the interactions of the 15 closest residues [Sum (15)], except for CD4409, which in that comparison showed second strongest interactions. Interestingly, 1{alpha},25(OH)2D3 showed higher total interaction energies only due to increased van der Waals interactions. Concerning the latter aspect LG190178 is as potent as the natural hormone, but it shows significantly lower electrostatic interactions with the amino acids of the ligand binding pocket, so that its total interaction energy was with –91.3 kcal/mol the lowest of all tested compounds.

The distances of the six most critical amino acids with their respective contact atom of the ligand (Fig. 6Go) increased for CD4528 only in one case (a slight increase in the O7-R274 distance). This fits with the result of the mutagenesis analysis (Fig. 5Go), in which CD4528 showed only with R274 a significant deviation from the pattern of the natural ligand. In contrast, CD4420 and LG190178 showed each in two cases and CD4409 in three cases a significantly increased distance. Also, these simulation results nicely fit with mutagenesis results (Fig. 5Go). Taken together, the MD simulations suggest that CD4528 mimics the structure of the natural ligand better than the three other nonsteroidal VDR ligands.



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Fig. 6. MD Simulations of Ligand-VDR-LBD Complexes

Detailed view of the VDR-LBD complexed with 1{alpha},25(OH)2D3, CD4409, CD4420, CD4528, and LG190178. Only those six amino acids are shown that are closest to the ligand (compare with supplemental Table 1). All structures are averages from the last 200 psec of 1-nsec MD simulations.

 
Previous studies (15, 16) have suggested that the stabilization of the VDR-agonist complex over time, i.e. the half-life of the complex, may be a more meaningful parameter for evaluation of superagonists than EC50 values in different in vitro and ex vivo assays. Therefore, the kinetics of VDR-ligand dissociation within VDR-RXR-VDRE complexes was analyzed by DNA-dependent LPD assays (compare Fig. 3Go), which were performed with in vitro-translated VDR-RXR heterodimers bound to the rat ANF DR3-type VDRE and saturating concentrations of 1{alpha},25(OH)2D3, CD4528, and LG190178 (Fig. 7AGo). The incubation time with trypsin varied between 2 and 24 h. It is important to note that trypsin was found to be still active even at the end of the incubation (15). The amount of the agonistic VDR conformation c1LPD was quantified and plotted over time, which allows the determination of the half-life of the VDR-ligand complex. With 1{alpha},25(OH)2D3 we obtained a half-life of the agonistic VDR conformation of 452 min, which reproduced the value of our earlier report (15). In contrast, with CD4528 and LG190178 we measured only half-lives of 165 and 97 min, respectively. This significantly lower half-lives reflect the lower affinities of both nonsteroidal ligands compared with the natural hormone. However, the values also indicate that CD4528 stabilized the agonistic conformation of the VDR 1.7 times longer than LG190178.



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Fig. 7. Time Courses of in Vitro VDR Stabilization and ex Vivo CYP24 Expression

LPD assays were performed by preincubating in vitro-translated [35S]-labeled VDR with unlabeled RXR and the unlabeled DR3-type VDRE from the rat ANF gene promoter in the presence of saturating concentrations (1 µM) of 1{alpha},25(OH)2D3, CD4528 and L190178 (A). The samples were incubated for indicated times with trypsin and were then electrophoresed through 15% SDS polyacrylamide gels. The amount of the agonistic VDR conformation c1LPD in relation to VDR input was quantified by using a Fuji FLA3000 reader. Representative experiments are shown. The half-lives (t1/2) were determined from the respective time-course curves. MCF-7 cells were treated for 1, 2, and 4 h with 1{alpha},25(OH)2D3, CD4528 and L190178 (100 nM each) and were compared with nontreated cells (0 h, B). Total RNA was isolated and RT-PCR was performed with primer pairs that were specific for the CYP24 gene. The mRNA expression was normalized for ß2-microglobulin control gene mRNA expression. Data points (A) and columns (B) represent mean values of three experiments, and the tips of the bars indicate SD. Statistical analysis was performed by two-tailed, paired Student’s t test, and P values were calculated in reference to nonstimulated cells (*, P < 0.05; **, P < 0.005).

 
Finally, we compared the performance of CD4528 and LG190178 with that of the natural hormone concerning the induction of CYP24 mRNA expression in living cells (MCF-7 breast cancer) and in vivo (kidney of Balb/c mice). The MCF-7 cells were treated with 100 nM of the three VDR agonists for 1, 2, and 4 h, total RNA was extracted and CYP24 mRNA expression in relation to ß2-microglobuline control gene was determined by RT-PCR (Fig. 7BGo). All three ligands steadily induced CYP24 expression over time and showed at none of the three time points a significant difference, i.e. the induction profile was indistinguishable. This result indicates that, at least at a pharmacological dose, the two nonsteroidal VDR ligands induce primary VDR target genes as potently as the natural hormone.

For the in vivo experiments, 8-wk-old male Balb/c mice were fed orally every day for 3 and 5 d with 1{alpha},25(OH)2D3, CD4409, CD4420, CD4528, and LG190178. Serum calcium concentrations were measured and the induction of renal CYP24 mRNA expression in reference to control gene was determined by real-time PCR (Table 1Go). In this primarily, limited in vivo study, at the high dose of 500 µg/kg CD4409 and CD4528 showed an increase of serum calcium concentrations by 21 to 64%, whereas no significant increase was observed with the other VDR ligands and doses, i.e. at doses of 50 and 150 µg/kg CD4528 was not calcemic. It has to be noted that at a dose of 500 µg/kg three of five mice died after being treated with LG190178, whereas the two surviving mice showed after 5 d an 81% increased serum calcium level. Therefore, the dose for this compound was reduced to 50 µg/kg. Surprisingly, at a dose of 500 µg/kg CD4420 showed neither a calcemic effect nor any induction of CYP24 expression. Probably, this compound is either not resorbed properly or metabolically very unstable. In contrast, the relative induction of CYP24 expression of a noncalcemic dose of CD4528 (150 µg/kg) is higher than that of a noncalcemic dose of 1{alpha},25(OH)2D3 (1 µg/kg). At a dose of 50 µg/kg, LG190178 also gave a clear induction of CYP24 expression, but the toxicity at higher doses is limiting its application. It has to be emphasized that the mice were treated orally, which may cause some differences in intestinal absorption and availability of the tested VDR ligands.


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Table 1. Limited in Vivo Profile of CD4409, CD4420, CD4528, and LG190178

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study represents a molecular evaluation of the interaction of nonsteroidal ligands with the VDR. The bis-aromatic compounds CD4409, CD4420, and CD4528 showed to be the most potent representatives of a collection of more than 50 closely related molecules (30, 31), whereas the diphenylmethane derivative LG190178 is the most active compound of a previous study (27). All four nonsteroidal compounds were analyzed in detail, and each showed the essential criteria of VDR agonists. These are 1) the induction of VDR-RXR heterodimer complex formation on a VDRE, 2) the stabilization of the agonistic conformation of the VDR-LBD, 3) enabling the interaction of VDR with CoA molecules, and 4) contact between their third hydroxyl group and H397, so that the H397-F422 interaction locks helix 12 in its agonistic position. Quantification of these criteria via the determination of 1) EC50 values from dose response curves, 2) the deviation from the mutagenesis pattern, and 3) measuring atomic distances between ligand and receptor allowed a ranking of the four compounds. We could demonstrate that in each of these points CD4528 was more potent than LG190178 because it showed at least five times lower EC50 values in the induction of VDR-RXR heterodimer complex formation (Fig. 3AGo) and in the stabilization of the agonistic VDR conformation c1LPD (Fig. 3BGo), a more potent induction of a VDR-CoA interaction at low ligand concentrations (Fig. 4Go), only one deviation from the mutagenesis pattern of the natural ligand (Fig. 5Go), only one significantly increased distance to the critical contact amino acids of the ligand-binding pocket of the VDR (Fig. 6Go and supplemental Table 1), higher total interaction energy (supplemental Table 2), and a longer stabilization of the agonistic VDR conformation (Fig. 7AGo). However, in all these assay systems CD4528 was not as perfect as the natural hormone and showed at least 10 times higher EC50 values, but at pharmacologic doses (100 nM) it induced CYP24 mRNA expression as potently as 1{alpha},25(OH)2D3 (Fig. 7BGo). In a limited in vivo study, CD4528 was noncalcemic at a dose of 150 µg/kg, but showed a significant induction of CYP24 expression (Table 1Go). LG190178 resulted at noncalcemic doses of 50 µg/kg in an even higher stimulation of CYP24 mRNA production but showed in contrast to CD4528 severe toxic effects at 500 µg/kg. The latter observation may be the reason that Boehm et al. (27) did no present any in vivo data with LG190178.

MD simulations demonstrated that all four nonsteroidal ligands take a shape within the ligand binding pocket of the VDR that is very similar to that of the natural ligand. Most importantly, each of the three hydroxyl groups of the ligands is closely located to the S237/R274, Y143/S278, and H305/H397 contact amino acid pairs, like the hydroxyl groups at C1, C3, and C25 of the natural ligand. The fact that all four nonsteroidal ligands have a significant effect on the activity of the VDR, indicates that the seco-steroid backbone of 1{alpha},25(OH)2D3 is of less importance than the positioning of the three hydroxyl groups. The more exactly the nonsteroidal compounds place their hydroxyl groups, the lower seem to be their respective EC50 values in the different in vitro assays. Results from previous studies (15, 34) have indicated that these values are proportional to the affinity of the ligand to the receptor.

The total electrostatic energy values of CD4409, CD4420, and CD4428 are similar to that of 1{alpha},25(OH)2D3, but the natural hormone’s higher affinity for the VDR is due to stronger van der Waals interactions. LG190178 forms slightly more favorable van der Waals interactions with the LBD than 1{alpha},25(OH)2D3, but clearly less favorable electrostatic interactions. This is also seen in the nonoptimal hydrogen bonds of LG190178. Interestingly, the nonsteroidal analogs make stronger interactions with H309 and H397 than 1{alpha},25(OH)2D3 (13.7 to 14.4 vs. 11.8 kcal/mol). In addition, the fluorinated CD4420 and CD4528 form more favorable interactions with H305 than the other compounds, i.e. the relatively high affinity of CD4528 for the VDR is in part based on the high electronegative six fluor atoms. More traditional 1{alpha},25(OH)2D3 analogs (23) as well as nonsteroidal ligands to other NRs, such as GW3965 and T0901317 of the liver X receptor (39), were shown to acquire prolonged stability of the receptor-ligand complex through fluor substitutions. In conclusion of all assays, we suggest the ranking 1{alpha},25(OH)2D3 > CD4528 > CD4409 > CD4420 = LG190178.

This study has indicated that the in vitro and ex vivo profile of a VDR ligand allows an extrapolation to the compound’s in vivo profile. In ex vivo assays, such as the mammalian one-hybrid assay in HeLa cells (Fig. 2Go), CD4528 was found to be nearly as sensitive as 1{alpha},25(OH)2D3. In fact, the EC50 value of CD4528 in this assay system stayed in the same low nanomolar range as in the in vitro assays, but the natural hormone showed to be less sensitive in living cells than in vitro. This phenomenon was already reported previously (15) and is explained through the binding of 1{alpha},25(OH)2D3 to the serum vitamin D binding protein and through metabolism of the hormone. This lowers the effective concentration of the natural ligand but leaves synthetic compounds largely unaffected because binding proteins and metabolic enzymes do not recognize them (Fig. 2BGo). However, the complete failure of CD4420 in vivo and the toxicity of LG190178 at higher doses can still not be predicted on the basis of in vitro and ex vivo assays. In conclusion, at an appropriate noncalcemic dose (150 µg/kg in mice) CD4528 acts very much like 1{alpha},25(OH)2D3 both in vitro and in vivo. Further chemical modification may convert this lead compound to a powerful VDR ligand that has the potential to be used in the therapy of various disorders, such as psoriasis, osteoporosis, and cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Compounds
The natural VDR ligand 1{alpha},25(OH)2D3 was a gift of Dr. L. Binderup (LEO Pharma, Ballerup, Denmark), LG190178 (2'-[4-(2-hydroxy-3,3-dimethylbutoxy)-3-methylphenyl]-2'-[4-(2,3-dihydroxypropoxy)-3-methylphenyl]pentane) (27) was synthesized by Dr. A. Steinmeyer (Schering AG, Berlin, Germany) and CD4409 ((4E,6E)-7-[5-(3,4-Bis-hydroxymethyl-phenoxymethyl)-thiophen-2-yl]-3-ethyl-nona-4,6-dien-3-ol), CD4420 ((3E,5E)-6-[3-(3,4-Bis-hydroxymethyl-benzyloxy)-phenyl]-1,1,1-trifluoro-2-trifluoromethyl-octa-3,5-dien-2-ol) and CD4528 ((3E,5E)-6-{(E)-3-[2-(3,4-Bis-hydroxymethyl-phenyl)-ethyl]-phenyl}-1,1,1-trifluoro-2-trifluoromethyl-octa-3,5-dien-2-ol) (30, 31) were kindly provided by Dr. U. Reichert (Galderma R&D, Sophia Antipolis, France). The structures of all five compounds are shown in Fig. 1Go. 1{alpha},25(OH)2D3 was dissolved in 2-propanol, whereas the nonsteroidal ligands were dissolved in dimethylsulfoxide; further dilutions were made either in dimethylsulfoxide (for in vitro experiments) or in ethanol (for cell culture experiments). The CYP24 inhibitor ketoconazole was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in dimethylsulfoxide.

DNA Constructs
The full-length cDNAs for human VDR (40) and human RXR{alpha} (41) were subcloned into the simian virus 40 promoter-driven pSG5 expression vector (Stratagene, La Jolla, CA). These constructs are suitable for T7 RNA polymerase-driven in vitro transcription/translation of the respective cDNAs. All point mutants to the pSG5-VDR construct were generated using the QuickChange point mutagenesis kit (Stratagene) and confirmed by sequencing. The DBD of the yeast transcription factor GAL4 (amino acids 1–147) was fused with the cDNA of the human VDR-LBD (amino acids 109–427). For the mammalian one-hybrid assay the luciferase reporter gene was driven by three copies of the GAL4 binding site fused to the thymidine kinase promoter (8) and for the conventional reporter gene assay in MCF-7 cells the luciferase gene was driven by four copies of the rat ANF DR3-type VDRE (core sequence AGAGGTCATGAAGGACA) (42) fused to the thymidine kinase promoter. The NR interaction domain of the human CoA TIF2 (spanning from amino acids 646–926) (43) was subcloned into the glutathione-S-transferase (GST) fusion vector pGEX (Amersham Pharmacia, Uppsala, Sweden).

Transient Transfections and Reporter Gene Assay
HeLa human cervix carcinoma cells and MCF-7 human breast cancer cells were seeded into six-well plates (105 cells/ml) and grown overnight in phenol red-free DMEM supplemented with 5% charcoal-treated fetal bovine serum. Liposomes were formed by incubating each 1 µg of a luciferase reporter gene plasmid driven by three copies of a GAL4 binding and an expression vector for a GAL4DBDVDRLBD-fusion protein (for mammalian one-hybrid assays in HeLa cells) or 1 µg of each a luciferase reporter gene plasmid driven by four copies the rat ANF DR3-type VDRE and pSG5-based receptor expression vectors for wt or mutant VDR and RXR (for conventional reporter gene assays in MCF-7 cells) with 10 µg N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, Roth, Karlsruhe, Germany) for 15 min at room temperature in a total volume of 100 µl. After dilution with 900 µl phenol red-free DMEM, the liposomes were added to the cells. Phenol red-free DMEM supplemented with 30% charcoal-treated fetal bovine serum (500 µl) was added 4 h after transfection. At this time, 1{alpha},25(OH)2D3, nonsteroidal ligands or solvent (ethanol) were also added. For both type of assays the cells were lysed 16 h after onset of stimulation using the reporter gene lysis buffer (Roche Diagnostics, Mannheim, Germany) and the constant light signal luciferase reporter gene assay was performed as recommended by the supplier (Canberra-Packard, Groningen, The Netherlands). The luciferase activities were normalized with respect to protein concentration and relative activities were calculated.

In Vitro Protein Translation and Bacterial Fusion Protein Overexpression
In vitro-translated VDR and RXR proteins were generated by transcribing their respective pSG5-based cDNA expression vector with T7 RNA polymerase and translating these RNAs in vitro using rabbit reticulocyte lysate as recommended by the supplier (Promega, Madison, WI). [35S]-labeled VDR was generated by translation in the presence of [35S]-methionine. Bacterial overexpression of GST-TIF2646–926 was facilitated in the Escherichia coli BL21(DE3)pLysS strain (Stratagene) by induction with isopropyl-ß-D-thio-galactopyranoside (0.25 mM) for 3 h at 37 C.

Ligand-Dependent Gel Shift and Supershift Assay
Heterodimers formed by in vitro-translated VDR and RXR were incubated with graded or saturating concentrations of 1{alpha},25(OH)2D3 or its analogs for 15 min at room temperature in a total volume of 20 µl binding buffer [10 mM HEPES (pH 7.9), 1 mM dithiothretiol, 0.2 µg/µl poly(deoxyinosine-deoxycytosine) and 5% glycerol]. The buffer had been adjusted to 150 mM by addition of KCl. For the supershift assay, 3 µg of bacterially expressed GST-TIF2646–926 fusion protein were included in the incubation. Approximately 1 ng of the [32P]-labeled DR3-type VDRE from the rat ANF gene was then added to the protein-ligand mixture and incubation was continued for 20 min. Protein-DNA complexes were resolved through 8% nondenaturing polyacrylamide gels in 0.5x TBE [45 mM Tris, 45 mM boric acid, 1 mM EDTA (pH 8.3)] and were quantified on a Fuji FLA3000 reader (Tokyo, Japan) using Image Gauge software (Fuji).

LPD Assay
In vitro-translated, [35S]-labeled VDR protein (2.5 µl) together with unprogrammed lysate (2.5 µl) or in vitro-translated RXR (2.5 µl) and 1 ng of unlabeled rat ANF DR3-type VDRE were incubated with graded or saturating concentrations of ligand for indicated time periods (15 min regular assay, up to 24 h in time courses) at room temperature in 20 µl binding buffer. The buffer was adjusted to 150 mM of monovalent cations by addition of KCl. Trypsin (Promega; final concentration 8.3 ng/µl) was then added and the mixtures were further incubated for 15 min at room temperature. The digestion reactions were stopped by adding 25 µl protein gel loading buffer [0.25 M Tris (pH 6.8), 20% glycerol, 5% mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 0.025% bromophenol blue]. The samples were denatured at 85 C for 3 min and electrophoresed through a 15% SDS-polyacrylamide gel. The individual protease-sensitive VDR fragments were quantified on a Fuji FLA3000 reader.

MD Simulations
The initial coordinates for the MD simulations were obtained from the x-ray crystal structure of the VDR-LBD-1{alpha},25(OH)2D3 complex (Brookhaven Protein Data Bank code 1DB1) determined to 1.8 Å resolution (9). The missing amino acid residues of the x-ray structure (residues 118, 119, 375–377, and 424–427) were built using the Quanta98 molecular modeling package (Molecular Simulations Inc., San Diego, CA). The four residues missing from the C terminus (424–427) were built in an {alpha}-helical conformation ({phi} = – 57°, {psi} = – 47°). Crystallographic water molecules were included in the simulation systems. The four nonsteroidal VDR ligands were docked to the ligand-binding pocket on the basis of 1{alpha},25(OH)2D3 of the crystal structure. The three hydroxyl groups of the ligands, which presumably correspond to the hydroxyl groups at C1, C3, and C25 of 1{alpha},25(OH)2D3, were used as anchor points in the docking. The protein-ligand complexes were solvated by adding a sphere of TIP3P water molecules with a 25 Å radius from the mass-center of the ligand. In total, there were 1042 water molecules in the simulation systems. In the simulations, the water sphere of 25 Å and residues having atoms within 16 Å from the atoms of the ligand were allowed to move. The water molecules of the complexes were first energy-minimized for 2000 steps, heated to 300 K in 10 psec and equilibrated by 40 psec at a constant temperature of 300 K. After that, the moving part of the protein-ligand complexes were minimized for 2000 steps, the temperature of the system was increased to 300 K in 10 psec and equilibrated for 300 psec. Then production simulations of 1 nsec were started. In the simulations, a time step of 2 fsec was used, and all bonds were constrained to their equilibrium lengths using the SHAKE algorithm. The simulations were done using the AMBER7.0 simulation package (University of California, San Francisco, CA) and the Cornell et al. force field (44). The parameters of the ligands were generated with the Antechamber suite of AMBER7.0 in conjunction with the general amber force field. The atomic point charges of the ligands were calculated with the two-stage RESP (45) fit at the HF/6–31G* level using ligand geometries optimized with the semiempirical PM3 method using the Gaussian98 program (Gaussian Inc., Pittsburgh, PA). Gas-phase interaction energies [E(Int)] between the ligands and individual protein residues of the LBD were calculated for the 125 structures collected from the last 250 psec of the production simulations. Dielectric constant was set to 1 in these calculations and E(Int) was divided in van der Waals [E(vdW)] and electrostatic [E(Ele)] contributions.

RNA Extraction and Real-Time Quantitative PCR
Total RNA was extracted using Tri-reagent (Sigma-Aldrich, St. Louis, MO) and was used as a template in cDNA synthesis. Real-time quantitative PCR was performed in an IQ-cycler (Bio-Rad, Hercules, CA) using the dye SybrGreen (Molecular Probes, Leiden, The Netherlands). In PCRs, 3 mM MgCl2 was used for all primers. The PCR cycling conditions used were: 40 cycles of 30 sec at 95 C, 30 sec at 58 C (62 C for CYP24), and 40 sec at 72 C. Fold inductions were calculated using the formula 2–({Delta}{Delta}Ct), where {Delta}{Delta}Ct is the {Delta}Ct(1{alpha},25(OH)2D3) {Delta}Ct(Ethanol), {Delta}Ct is Ct(CYP24) – Ct(control gene), and Ct is the cycle at which the threshold is crossed. The gene-specific primer pairs for the gene analyzed here were as follows: human CYP24 gene forward 5'-CTGCTGCAGATTCTCTGGAA-3' and reverse 5'-ATGATGAAGTTCACAGCTTC-3', human ß2-microglobulin gene forward 5'-CCCCCACTGAAAAAGATGAGTATGCCTG-3' and reverse 5'-CCTGTGGAGCAACCTGCTCAGATACATC-3', mouse CYP24 gene forward 5'-CAAACCCTGGAAAGCCTATC-3' and reverse 5'-TCCTGTCCTTCCAGGATCAT-3', mouse acidic riboprotein P0 (ARP0, also known as 36B4) control gene forward 5'-AGATGCAGCAGATCCGCAT-3' and reverse 5'-GTGGTGATGCCCAAAGCCTG-3'. PCR product quality was monitored using post-PCR melt curve analysis.

In Vivo Gene Regulation and Calcium Potential
Eight-week-old male BALB/c x DAB2 mice (National Laboratory Animal Center, Kuopio, Finland) were housed in stainless steel metabolic cages under controlled temperature (21–23 C) and light conditions (lights on 0700–1900 h). Mice had free access to water and received a vitamin D-deficient, calcium-deplete diet ad libitum (Altromin, Lage, Germany) for 14 d before initiation of treatment. All experiments were approved by the Committee for the Welfare of Laboratory Animals at the University of Kuopio and conducted in accordance with the guidelines of the European Community Council directives 86/609/EEC. VDR ligands were administered in saline by oral gavage (dose volume 5 µl/g body weight). The animals were gavaged everyday between 0800 and 1000 h for 3–5 d (five animals per group). Twenty-four hours after the last gavage, blood was drawn from the tail vein under ketamine (75 mg/kg) and xylazine hydrochloride (10 mg/kg) anesthesia for serum calcium level determinations. Afterward, the animals were killed and the kidneys removed and shock frozen in liquid nitrogen. Total RNA was extracted from kidney for real-time PCR quantification of CYP24 expression in reference to ARP0 expression. Calcium concentrations in serum were measured using a colorimetric assay (Roche).


    ACKNOWLEDGMENTS
 
We thank Dr. U. Reichert for discussions and Drs. L. Binderup, A. Steinmeyer, and U. Reichert for providing VDR ligands.


    FOOTNOTES
 
The Academy of Finland (Grant 205694 to C.C. and Grant 74097 to M.P.) and the Finnish Cancer Organisation supported this work.

First Published Online April 28, 2005

Abbreviations: ANF, Atrial natriuretic factor; CYP24, vitamin D 24-hydroxylase; CoA, coactivator; 1{alpha},25(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; GST, glutathione-S-transferase; LBD, ligand binding domain; LPD, limited protease digestion; MD, molecular dynamics; NR, nuclear receptor; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; TIF2, transcription intermediary factor 2; VDR, 1{alpha},25(OH)2D3 receptor; VDRE, 1{alpha},25(OH)2D3 response element; wt, wild type.

Received for publication October 18, 2004. Accepted for publication April 21, 2005.


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