Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo.

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize.

Andrographolide derivative as antagonist of vitamin D receptor to induce lipidation of microtubule associate protein 1 light chain 3 (LC3).

Lipidation of microtubule associated protein 1 light chain 3 (LC3) is the critical step in autophagosome formation, numerous efforts have been made to design and develop small molecules that trigger LC3 lipidation to activate autophagy. In this study, we discovered a series of andrographolide derivatives as potent antagonists of vitamin D receptor (VDR) by luciferase reporter assay. Structure-activity-relationship study revealed that andrographolide derivative ZAV-12 specifically inhibited VDR signaling but not NF-κB or STAT3 activation. Western blot analysis indicates that ZAV-12 markedly triggered lipidation of LC3 in MPP+-induced Parkinsonism in vitro in an mTOR-independent manner. The ZAV-12 triggered lipidation was mediated through SREBP2 activation instead of changing expression levels of lipid synthesis genes. Furthermore, ZAV-12 treatment increased the ratio of LC3-II/LC3-I and oligomerization of A53T α-synuclein (SNCA) in SNCA triggered neurotoxicity. Taken together, these results demonstrate the therapeutic potential of VDR antagonist as novel drug candidate for neurodegenerative diseases.

Strategies for developing pregnane X receptor antagonists: Implications from metabolism to cancer.

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor (NR) that was originally identified as a master regulator of xenobiotic detoxification. It regulates the expression of drug-metabolizing enzymes and transporters to control the degradation and excretion of endobiotics and xenobiotics, including therapeutic agents. The metabolism and disposition of drugs might compromise their efficacy and possibly cause drug toxicity and/or drug resistance. Because many drugs can promiscuously bind and activate PXR, PXR antagonists might have therapeutic value in preventing and overcoming drug-induced PXR-mediated drug toxicity and drug resistance. Furthermore, PXR is now known to have broader cellular functions, including the regulation of cell proliferation, and glucose and lipid metabolism. Thus, PXR might be involved in human diseases such as cancer and metabolic diseases. The importance of PXR antagonists is discussed in the context of the role of PXR in xenobiotic sensing and other disease-related pathways. This review focuses on the development of PXR antagonists, which has been hampered by the promiscuity of PXR ligand binding. However, substantial progress has been made in recent years, suggesting that it is feasible to develop selective PXR antagonists. We discuss the current status, challenges, and strategies in developing selective PXR antagonists. The strategies are based on the molecular mechanisms of antagonism in related NRs that can be applied to the design of PXR antagonists, primarily driven by structural information.

Discovery of fused bicyclic derivatives of 1H-pyrrolo[1,2-c]imidazol-1-one as VDR signaling regulators.

The modulation of VDR signaling is important in regulating tumor-related signal transduction and protecting from microorganismal infection. In this study we discovered by luciferase reporter assay that several fused bicyclic derivatives of 1H-pyrrolo[1,2-c]imidazol-1-one with the assistance of calcitriol result in up to three-fold increases of VDR promoter activity. Preliminary SAR results from 20 compounds disclose that ideal VDR signaling regulators of these compounds are built up by the optimal combination of multiple factors. Western blot analysis indicates that compounds of ZD-3, ZD-4 and ZD-5 not only significantly upregulate p62 and LC3-II but also elevate the ratio of LC3-II/LC3-I, which possibly leads to activated autophagy. All of five compounds also significantly downregulate p65 and upregulate p-p65 and ZD-3 is the most active one to NF-κB signaling, suggesting a possible induction of apoptosis through the regulation of NF-κB signal transduction mediated by VDR signaling. Compounds of ZD-3, ZD-4 and ZD-5 significantly counteract the interference by VDR shRNA, in which ZD-3 gets the highest compensation of VDR expression and the highest ratio of LC3-II/LC3-I, indicating that ZD-3 very likely activates VDR-mediated autophagy. Taken together, these 1H-pyrrolo[1,2-c]imidazol-1-one derivatives can modulate VDR signaling, possibly resulting in the regulation of some signal pathways to induce autophagy and apoptosis.

Sulfonyl-containing phenyl-pyrrolyl pentane analogues: Novel non-secosteroidal vitamin D receptor modulators with favorable physicochemical properties, pharmacokinetic properties and anti-tumor activity.

Modulating the vitamin D receptor (VDR) is an effective way to treat for cancer. We previously reported a potent non-secosteroidal VDR modulator (sw-22) with modest anti-tumor activity, which could be due to its undesirable physicochemical and pharmacokinetic properties. In this study, we investigated the structure-activity and structure-property relationships around the 2'-hydroxyl group of sw-22 to improve the physicochemical properties, pharmacokinetic properties and anti-tumor activity. Compounds 19a and 27b, the potent non-secosteroidal VDR modulators, were identified as the most effective molecules in inhibiting the proliferation of three cancer cell lines, particularly breast cancer cells, with a low IC50 via the distribution of cell cycle and induction of apoptosis by stimulating the expression of p21, p27 and Bax. Further investigation revealed that 19a and 27b possessed favorable rat microsomal metabolic stability (2.22 and 2.3 times, respectively, more stable than sw-22), solubility (43.9 and 50.2 times, respectively, more soluble than sw-22) and in vivo pharmacokinetic properties. In addition, 19a and 27b showed excellent in vivo anti-tumor activity without cause hypercalcemia, which is the main side effect of marketed VDR modulators. In summary, the favorable physicochemical properties, pharmacokinetic properties and anti-tumor activity of 19a and 27b highlight their potential therapeutic applications in cancer treatment.

'Psoriasis 1' reduces psoriasis­like skin inflammation by inhibiting the VDR­mediated nuclear NF­κB and STAT signaling pathways.

'Psoriasis 1', a Chinese herbal medicine (CHM) formulation, is extensively used to treat psoriasis in China. Although this CHM formulation yields good therapeutic effect, the underlying mechanism of how this works remains unknown. The present study aimed to test the hypothesis that the CHM formulation 'psoriasis 1' inhibits vitamin D receptor (VDR)­mediated inflammation in psoriasis. To test this, a model of psoriasis was established by stimulating keratinocytes (HaCaT cells) with tumor necrosis factor (TNF)­α; these cells were subsequently transfected with a lentiviral VDR RNA interference expression vector. The expression levels of 25­hydroxyvitamin D3 (25HVD3), TNF­α, interleukin (IL)­4, IL­1, IL­17C, IL­23 and IL­6 were measured using ELISA, and the expression levels of VDR, inhibitor of nuclear factor (NF)­κB (IKK), NF­κB, signal transducer and activator of transcription (STAT) 3 and STAT4 were measured using reverse transcription­quantitative polymerase chain reaction analysis and western blotting. It was observed that 'psoriasis 1' downregulated the concentrations of TNF­α, IFN­Î³, IL­22, IL­17C, IL­1ß and IL­4, and upregulated the concentration of 25HVD3; furthermore, 'psoriasis 1' downregulated the expression levels of NF­κB, phosphorylated (p)­NF­κB, IKK, p­IKK, STAT3, p­STAT3, STAT4 and p­STAT4, and upregulated the expression level of VDR in TNF­α­induced HaCaT cells. These results suggested that 'psoriasis 1' suppressed the inflammatory response and the activation of the NF­κB and STAT signaling pathways. In addition, it was identified that silencing VDR expression decreased the levels of TNF­α, IFN­Î³, IL­22, IL­17C, IL­1ß and IL­4, and increased the level of 25HVD3; silencing VDR expression additionally downregulated the expression levels of NF­ÐºB, p­NF­ÐºB, IKK, p­IKK, STAT3, p­STAT3, STAT4 and p­STAT4, and upregulated the level of VDR in TNF­α­induced HaCaT cells. It was concluded that 'psoriasis 1' exerts inflammation­suppressive effects in psoriasis by suppressing the NF­ÐºB and STAT signaling pathways.

Vitamin D Receptor Is Necessary for Mitochondrial Function and Cell Health.

Vitamin D receptor (VDR) mediates many genomic and non-genomic effects of vitamin D. Recently, the mitochondrial effects of vitamin D have been characterized in many cell types. In this article, we investigated the importance of VDR not only in mitochondrial activity and integrity but also in cell health. The silencing of the receptor in different healthy, non-transformed, and cancer cells initially decreased cell growth and modulated the cell cycle. We demonstrated that, in silenced cells, the increased respiratory activity was associated with elevated reactive oxygen species (ROS) production. In the long run, the absence of the receptor caused impairment of mitochondrial integrity and, finally, cell death. Our data reveal that VDR plays a central role in protecting cells from excessive respiration and production of ROS that leads to cell damage. Because we confirmed our observations in different models of both normal and cancer cells, we conclude that VDR is essential for the health of human tissues.

Assessment of endocrine disruption potential of essential oils of culinary herbs and spices involving glucocorticoid, androgen and vitamin D receptors.

Essential oils (EOs) of culinary herbs and spices are consumed on a daily basis. They are multicomponent mixtures of compounds with already demonstrated biological activities. Taking into account regular dietary intake and the chemical composition of EOs, they may be considered as candidates for endocrine-disrupting entities. Therefore, we examined the effects of 31 EOs of culinary herbs and spices on transcriptional activities of glucocorticoid receptor (GR), androgen receptor (AR) and vitamin D receptor (VDR). Using reporter gene assays in stably transfected cell lines, weak anti-androgen and anti-glucocorticoid activity was observed for EO of vanilla and nutmeg, respectively. Moderate augmentation of calcitriol-dependent VDR activity was caused by EOs of ginger, thyme, coriander and lemongrass. Mixed anti-glucocorticoid and VDR-stimulatory activities were displayed by EOs of turmeric, oregano, dill, caraway, verveine and spearmint. The remaining 19 EOs were inactive against all receptors under investigation. Analyses of GR, AR and VDR target genes by means of RT-PCR confirmed the VDR-stimulatory effects, but could not confirm the anti-glucocorticoid and anti-androgen effects of EOs. In conclusion, although we observed minor effects of several EOs on transcriptional activities of GR, AR and VDR, the toxicological significance of these effects is very low. Hence, 31 EOs of culinary herbs and spices may be considered safe, in terms of endocrine disruption involving receptors GR, AR and VDR.

Vitamin D Receptor: A Novel Therapeutic Target for Kidney Diseases.

BACKGROUND: Kidney disease is a serious problem that adversely affects human health, but critical knowledge is lacking on how to effectively treat established chronic kidney disease. Mounting evidence from animal and clinical studies has suggested that Vitamin D Receptor (VDR) activation has beneficial effects on various renal diseases. METHODS: A structured search of published research literature regarding VDR structure and function, VDR in various renal diseases (e.g., IgA nephropathy, idiopathic nephrotic syndrome, renal cell carcinoma, diabetic nephropathy, lupus nephritis) and therapies targeting VDR was performed for several databases. RESULT: Included in this study are the results from 177 published research articles. Evidence from these papers indicates that VDR activation is involved in the protection against renal injury in kidney diseases by a variety of mechanisms, including suppression of RAS activation, anti-inflammation, inhibiting renal fibrogenesis, restoring mitochondrial function, suppression of autoimmunity and renal cell apoptosis. CONCLUSION: VDR offers an attractive druggable target for renal diseases. Increasing our understanding of VDR in the kidney is a fertile area of research and may provide effective weapons in the fight against kidney diseases.

Development of a highly sensitive in vitro system to detect and discriminate between vitamin D receptor agonists and antagonists based on split-luciferase technique.

Split-luciferase techniques are widely used to detect protein-protein interaction and bioactive small molecules including some hormones and vitamins. Previously, we successfully expressed chimeric proteins of luciferase and the ligand binding domain (LBD) of the vitamin D receptor (VDR), LucC-LBD-LucN in COS-7 cells. The LucC-LBD-LucN biosensor was named split-luciferase vitamin D biosensor (SLDB). This biosensor can detect and discriminate between VDR agonists and antagonists in mammalian cells. In this study, we established an in vitro screening system for VDR ligands using the SLDB proteins expressed in Escherichia coli (E. coli) cells. Our in vitro screening system using cell lysate of recombinant E. coli cells could be completed within 30min, and its activity was unchanged after 10 freeze-thaw cycles. This highly sensitive and convenient system would be quite useful to screen VDR ligands with therapeutic potential for various bone-related diseases, age-related cognitive disorders, cancer, and immune disorders. In addition, our system might be applicable to diagnostic measurement of serum concentrations of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3.

Creation of Potent Vitamin D Receptor Agonists and Antagonists with 2α-(ω-Hydroxyalkylation) Concept to the seco-Steroid Skeleton.

2α-modification on the vitamin D skeleton with a 2α-(ω-hydroxyalkyl) or 2α-(ω-hydroxyalkoxy) group improves vitamin D receptor (VDR) binding affinity, lengthens the half-life in target cells because of increased resistance to CYP24A1 metabolism, and enhances biological activity. The introduced terminal hydroxy group forms an additional hydrogen bond to Arg274, which is the most important amino acid residue for recognizing the ligand hormone 1α,25-dihydroxyvitamin D3 of human VDR. According to our 2α-functionalization concept, we synthesized several hundred vitamin D analogs, and some had selective potent biological activity, such as bone formation (by AH-1) or anticancer activity (by MART-10), without the side-effects of vitamin D such as hypercalcemia. A potent hVDR antagonist NS-74c and stable 14-epi-tachysterol derivatives are also described in this short review.

Regulation of uridine diphosphate-glucuronosyltransferase 2B15 expression during ovulation in the rat.

Uridine diphosphate-glucuronosyltransferase 2B15 (UGT2B15) conjugates 5α-androstane-3α, 17ß-diol (3α-diol) to 3α-diol glucuronide (3α-diol G) in steroid target tissues. The present study investigated the regulation of UGT2B15 expression during the ovulatory process in the rat. Real-time PCR analysis revealed that treatment of immature rats with equine chorionic gonadotropin followed by human chorionic gonadotropin transiently stimulated UGT2B15 gene expression in granulosa cells of preovulatory follicles within 6 h. The progesterone receptor antagonist RU486 suppressed the gonadotropin-induced UGT2B15 expression. The expression of UGT2B15 and the levels of 3α-diol G were transiently increased by luteinizing hormone (LH) treatment in cultured preovulatory follicles. The LH-stimulated UGT2B15 mRNA level in cultured preovulatory follicles was inhibited by inhibitors of adenylyl cyclase, phosphoinositide 3-kinase and mitogen-activated protein kinase. Furthermore, a vitamin D receptor agonist (calcitriol) suppressed the LH-stimulated UGT2B15 expression in a dose-dependent manner. Taken together, these results indicate that gonadotropins transiently stimulate UGT2B15 expression and activity in preovulatory follicles, and UGT2B15 mRNA levels are regulated by the progesterone receptor and vitamin D receptor.

Vitamin D inhibits lymphangiogenesis through VDR-dependent mechanisms.

Excessive lymphangiogenesis is associated with cancer progression and renal disease. Attenuation of lymphangiogenesis might represent a novel strategy to target disease progression although clinically approved anti-lymphangiogenic drugs are not available yet. VitaminD(VitD)-deficiency is associated with increased cancer risk and chronic kidney disease. Presently, effects of VitD on lymphangiogenesis are unknown. Given the apparently protective effects of VitD and the deleterious associations of lymphangiogenesis with renal disease, we here tested the hypothesis that VitD has direct anti-lymphangiogenic effects in vitro and is able to attenuate lymphangiogenesis in vivo. In vitro cultured mouse lymphatic endothelial cells (LECs) expressed VitD Receptor (VDR), both on mRNA and protein levels. Active VitD (calcitriol) blocked LEC tube formation, reduced LEC proliferation, and induced LEC apoptosis. siRNA-mediated VDR knock-down reversed the inhibitory effect of calcitriol on LEC tube formation, demonstrating how such inhibition is VDR-dependent. In vivo, proteinuric rats were treated with vehicle or paricalcitol for 6 consecutive weeks. Compared with vehicle-treated proteinuric rats, paricalcitol showed markedly reduced renal lymphangiogenesis. In conclusion, our data show that VitD is anti-lymphangiogenic through VDR-dependent anti-proliferative and pro-apoptotic mechanisms. Our findings highlight an important novel function of VitD demonstrating how it may have therapeutic value in diseases accompanied by pathological lymphangiogenesis.

SRC2-3 binds to vitamin D receptor with high sensitivity and strong affinity.

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates the expression of target genes through ligand binding. To express the target gene, coactivator binding to the VDR/ligand complex is essential. Although there are many coactivators in living cells, precise interactions between coactivators and VDR have not been clarified. Here, we synthesized two coactivator peptides, DRIP205-2 and SRC2-3, evaluated their affinity for the ligand-binding domain (LBD) of VDR using 1α,25-dihydroxyvitamin D3, partial agonist 1, and antagonist 2 by surface plasmon resonance (SPR), and assessed their interaction modes with VDR-LBD using X-ray crystallographic analysis. This study showed that the SRC2-3 peptide is more sensitive to the ligands (agonist, partial agonist, and antagonist) and shows more intimate interactions with VDR-LBD than DRIP205-2 peptide.

Effect of 1,25-(OH)2D3 and lipopolysaccharide on mononuclear cell inflammation in type 2 diabetes mellitus and diabetic nephropathy uremia.

The prevention and treatment of type-2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), which are disorders with high incidence rates, is of primary importance. In this study, we analyzed the effect of 1,25-(OH)2D3 and lipopolysaccharide (LPS) in combination with interleukin (IL)-15 on the inflammatory immune response and expression of vitamin D receptor (VDR) in mononuclear cells of T2DM and DN uremia (DNU) patients. The human acute monocytic leukemia cell line THP-1 was treated with peripheral blood serum isolated from 30 healthy controls and T2DM and DNU patients each, cultured in the presence or absence of 1,25-(OH)2D3, and subsequently treated with LPS and IL-15. The VDR mRNA and protein expression in THP-1 cells was detected by real-time polymerase chain reaction and western blot (and immunofluorescence assay), respectively, and IL-6 and IL-10 concentrations in the culture supernatant were detected by enzyme-linked immunosorbent assay. LPS treatment induced a significant decrease in VDR mRNA expression in T2DM and DNU serum-treated THP-1 cells compared to the control cells (P < 0.05). The VDR protein expression in DNU serum-treated THP-1 cells was also significantly down-regulated (P < 0.05). LPS treatment induced IL-6 secretion in serum-treated THP-1 cells (P < 0.05), while 1,25-(OH)2D3 treatment inhibited IL-6 secretion to some extent. These findings suggested that LPS down-regulates the expression of VDR in mononuclear cells of T2DM and DNU patients and induces an imbalance in the pro-inflammatory and anti-inflammatory cytokine response, while 1,25-(OH)2D3 partially reversed the effect of LPS and protected patients with T2DM and DNU.

1,25(OH)2D3 and VDR Signaling Pathways Regulate the Inhibition of Dectin-1 Caused by Cyclosporine A in Response to Aspergillus Fumigatus in Human Corneal Epithelial Cells.

BACKGROUND: The objective of this study is to observe whether cyclosporine A (CsA) inhibits the expression of dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (A. fumigatus) and to investigate the molecular mechanisms of the inhibition. METHODS: Immortalized human corneal epithelial cells (HCECs) were pretreated with 1,25(OH)2D3 and VDR inhibitor for 1 h, and then they were pretreated with CsA for 12h. After these pretreatments, the HCECs were stimulated with A. fumigatus and curdlan respectively, and the expression of dectin-1 and proinflammatory cytokines (IL-1ß and TNF-α) were detected by RT-PCR, western blot and ELISA. RESULTS: Dectin-1 mRNA and dectin-1 protein expression increased when HCECs were stimulated with A. fumigatus or curdlan, and CsA inhibited the dectin-1 expression both in mRNA and protein levels specifically. Dectin-1 and proinflammatory cytokine expression levels were higher when HCECs were pretreated with VDR inhibitor and CsA compared to pretreatment with CsA alone, while dectin-1 and proinflammatory cytokine levels were lower when HCECs were pretreated with 1,25(OH)2D3 and CsA compared to pretreatment with CsA alone. CONCLUSIONS: These data provide evidence that CsA can inhibit the expression of dectin-1 and proinflammatory cytokines through dectin-1 when HCECs are stimulated by A. fumigatus or curdlan. The active form of vitamin D, 1,25(OH)2D3, and VDR signaling pathway regulate the inhibition of CsA. The inhibition is enhanced by 1,25(OH)2D3, and the VDR inhibitor suppresses the inhibition.

The UVB1 Vitamin D analogue inhibits colorectal carcinoma progression.

Vitamin D has been shown to display a wide variety of antitumour effects, but their therapeutic use is limited by its severe side effects. We have designed and synthesized a Gemini vitamin D analogue of calcitriol (UVB1) which has shown to display antineoplastic effects on different cancer cell lines without causing hypercalcemia. The aim of this work has been to investigate, by employing in silico, in vitro, and in vivo assays, whether UVB1 inhibits human colorectal carcinoma progression. We demonstrated that UVB1 induces apoptotic cell death and retards cellular migration and invasion of HCT116 colorectal carcinoma cells. Moreover, the analogue reduced the tumour volume in vivo, and modulated the expression of Bax, E-cadherin and nuclear ß-catenin in tumour animal tissues without producing toxic effects. In silico analysis showed that UVB1 exhibits greater affinity for the ligand binding domain of vitamin D receptor than calcitriol, and that several characteristics in the three-dimensional conformation of VDR may influence the biological effects. These results demonstrate that the Gemini vitamin D analogue affects the growth of the colorectal cancer and suggest that UVB1 is a potential chemotherapeutic agent for treatment of this disease.

Synthesis and Biological Activity of 2-Methylene Analogues of Calcitriol and Related Compounds.

In an attempt to prepare vitamin D analogues that are superagonists, (20R)- and (20S)-isomers of 1α-hydroxy-2-methylenevitamin D3 and 1α,25-dihydroxy-2-methylenevitamin D3 have been synthesized. To prepare the desired A-ring dienyne fragment, two different approaches were used, both starting from the (-)-quinic acid. The obtained derivative was subsequently coupled with the C,D-ring enol triflates derived from the corresponding Grundmann ketones, using the Sonogashira reaction. Moreover, (20R)- and (20S)-1α,25-dihydroxy-2-methylenevitamin D3 compounds with an (5E)-configuration were prepared by iodine catalyzed isomerization. All four 2-methylene analogues of the native hormone were characterized by high in vitro activity. As expected, the 25-desoxy analogues were much less potent. Among the synthesized compounds, two of them, 1α,25-dihydroxy-2-methylenevitamin D3 and its C-20 epimer, were found to be almost as active as 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) on bone but more active in intestine.

Synthesis, Biological Activities, and X-ray Crystal Structural Analysis of 25-Hydroxy-25(or 26)-adamantyl-17-[20(22),23-diynyl]-21-norvitamin D Compounds.

Novel 19-norvitamin D analogues (ADYW1-4, 5a-d) in which an adamantyl diyne side chain is attached directly to the 17-position of the D ring are designed and stereoselectively synthesized. The adamantane ring of these analogues was expected to interfere with helix 12 (H12, activation function 2) of the vitamin D receptor (VDR) to modulate its activities. The analogue 5b binds to the VDR (7% of the natural hormone) and shows significant partial agonistic activity in transactivation assay. Compound 5b showed considerable selectivity in VDR target genes expressions in vitro, it was taken up by target cells 2-3 times more readily, and its lifetime was three times longer than the natural hormone. The X-ray crystal structure of 5b in complex with VDR reveals that the ligand binds similarly to the natural hormone, but the diyne moiety is slightly bent (angles around the diyne 5° to 8°) with respect to the original diyne vitamin D compound 6 in VDR (<1°) due to steric hindrance with helix 12.

Development of Novel Bioluminescent Sensor to Detect and Discriminate between Vitamin D Receptor Agonists and Antagonists in Living Cells.

Active forms of vitamin D regulate the expression of multiple genes that play essential roles in calcium and phosphate homeostasis, cell differentiation, and the immune system via the vitamin D receptor (VDR). Many vitamin D analogs have been synthesized for clinical use in the treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer. We have constructed two fusion proteins containing split-luciferase and the ligand binding domain (LBD) of the VDR designated as LucN-LBD-LucC and LucC-LBD-LucN. Remarkably, the LucC-LBD-LucN, which has the C-terminal domain of luciferase at the N-terminus of the fusion protein, was a significantly better biosensor than LucN-LBD-LucC. Addition of the VDR agonists to COS-7 cells expressing LucC-LBD-LucN dramatically reduced luciferase activity. In contrast, the VDR antagonist significantly increased the chimeric luciferase activity in a dose- and time-dependent manner. Our results on chimeric luciferases containing the LBDs of mutant VDRs derived from patients with vitamin D-dependent type II rickets indicated that our system could detect a conformational change of the LBD of the VDR likely based on a positional change of the helix 12, which occurs upon ligand binding. This novel system to detect and discriminate between VDR agonists and antagonists could be useful for the screening and identification of chemical compounds that bind to normal or mutant VDRs with high affinity.