Analog Of 1α Research Articles

Vitamin D and its analogs as anticancer and anti-inflammatory agents.

Vitamin D3 is the precursor of the steroid hormone calcitriol (1α,25-dihydroxyvitamin D3), a potent agonist of the transcription factor vitamin D receptor (VDR). Calcitriol has received large appreciation due to the impact not only on mineral homeostasis, but also on metabolic diseases, immunological disorders, and cancer. A supraphysiological dose of calcitriol is required to reduce the proliferation of cancer cells. However, the dose will lead to calcemic side effects such as hypercalcemia and hypercalciuria. Although several analogs of 1α,25(OH)2D3 have shown potent anticancer or anti-inflammatory activity on cell cultures or in animal models, few vitamin D analogs have been successfully applied in the treatment of cancer or inflammatory diseases. This review will present and discuss vitamin D analogs that have the potential to be used as anticancer or anti-inflammatory agents.

Eldecalcitol (ED-71)-induced exosomal miR-6887-5p suppresses squamous cell carcinoma cell growth by targeting heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1).

Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) was purified from A431 cell-conditioned media based on its capacity to bind to fibroblast growth factor 1 and 2 (FGF-1 and FGF-2). HBp17/FGFBP-1 has been observed to induce the tumorigenic potential of epithelial cells and is highly expressed in oral cancer cell lines and tissues. HBp17/FGFBP-1 is also recognized as a pro-angiogenic molecule as a consequence of its interaction with FGF-2. We have previously reported that Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, downregulated the expression of HBp17/FGFBP-1 and inhibited the proliferation of squamous cell carcinoma (SCC) cells in vitro and in vivo through NF-κb inhibition. To explore the possibility of microRNA (miRNA) control of HBp17/FGFBP-1, we analyzed exosomal miRNAs from medium conditioned by A431 cells treated with ED-71. Microarray analysis revealed that 12 exosomal miRNAs were upregulated in ED-71-treated A431 cells. Among them, miR-6887-5p was identified to have a predicted mRNA target matching the 3' untranslated region (3'-UTR) of HBp17/FGFBP-1. The 3'-UTR of HBp17/FGFBP-1 was confirmed to be a direct target of miR-6887-5p in SCC/OSCC cells, as assessed with a luciferase reporter assay. Functional assessment revealed that overexpression of miR-6887-5p in SCC/OSCC cells inhibited cell proliferation and colony formation in vitro, and inhibited tumor growth in vivo compared with control. In conclusion, our present study supports a novel anti-cancer mechanism involving the regulation of HBp17/FGFBP-1 function by exosomal miR-6887-5p in SCC/OSCC cells, which has potential utility as a miRNA-based cancer therapy.

MART-10, a 1α,25(OH)2D3 Analog, Potently Represses Metastasis of ER+ Breast Cancer Cells with VEGF-A Overexpression.

Breast cancer ranks second in the list of cancer-related deaths for women. Even under multidisciplinary treatment, 25-50% of patients with breast cancer still ultimately develop metastasis, leading to poor prognosis. In addition to inducing angiogenesis, vascular endothelial growth factor-A (VEGF-A) is believed to directly increase cancer cell metastatic potential and overexpression of VEGF-A is associated with higher invasiveness of breast cancer. 1α,25(OH)2D3, the active form of vitamin D, and its analogs have been widely applied as anticancer agents in the past. Western blot, migration and invasion assays, enzyme-linked immunosorbent assay, and immunofluorescent stain were applied in this study. VEGF-A increased cell migration and invasion in estrogen receptor-positive (ER+) breast cancer MCF-7 cells. VEGF-A induced an autocrine loop in MCF-7 cells as VEGF-A treatment increased both VEGF-A expression and secretion. The expression of VEGF receptor type 2 (VEGFR2) and neuropilin 1 was also up-regulated by VEGF-A in MCF-7 cells. In addition, F-actin synthesis and LIM domain kinase 1 (LIMK-1) phosphorylation were increased by VEGF-A. VEGF-A also increased β-catenin expression and nuclear translocation of both β-catenin and nuclear factor-ĸB (NF-ĸB), indicating increased β-catenin and NF-ĸB activity. 1α,25(OH)2D3 and MART-10, an analog of 1α,25(OH)2D3, effectively repressed VEGF-A-induced MCF-7 cell migration and invasion and other VEGF-A-induced effects on MCF-7 cells, with MART-10 being more potent than 1α,25(OH)2D3 Conclusion: MART-10 can be deemed as a promising agent for prevention and treatment of metastasis of ER+ breast cancer with VEGF-A overexpression.

Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, inhibits the growth of squamous cell carcinoma (SCC) cells in vitro and in vivo by down-regulating expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) and FGF-2.

Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D3, i.e., 1α,25(OH)2D3 (1,25D3), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D3 inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH)2D3, for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA-HO-1-n-1 and UE-HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D3. Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between -217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the tumor microenvironment.

19-Norvitamin D analogs for breast cancer therapy.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3 or calcitriol), is known to inhibit the proliferation and invasiveness of many types of cancer cells, including breast, colon, pancreatic, prostate, and liver cancer cells. These findings support the use of 1α,25(OH)2D3 for the treatment of these types of cancer. However, 1α,25(OH)2D3 can cause hypercalcemia, so analogs of 1α,25(OH)2D3 that are less calcemic but exhibit more potent anti-tumor activity would be good candidates as therapeutic agents. Therefore, a series of 19-norvitamin D analogs, in which the methylidene group on C19 is replaced with 2 hydrogen atoms, have been synthesized by several laboratories. In our laboratory, we have designed and synthesized a series of 2α-functional group substituted 19-norvitamin D3 analogs and examined their anti-proliferative activity. Among them, 2α- and 2β-(3-hydroxypropyl)-1α,25-dihydroxy-19-norvitamin D3 (MART-10 and MART-11) were found to be the most promising. Here, we review the rationale and approaches for the synthesis of different 19-norvitamin D analogs, and the pre-clinical studies using these analogs in breast cancer cells, in particular, we chose MART-10 for its potential application to the prevention and treatment of breast cancer.

Evaluation of the potential therapeutic role of a new generation of vitamin D analog, MART-10, in human pancreatic cancer cells in vitro and in vivo

Pancreatic cancer is a lethal disease with no known effective chemotherapy and radiotherapy, and most patients are diagnosed in the late stage, making them unsuitable for surgery. Therefore, new therapeutic strategies are urgently needed. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is known to possess antitumor actions in many cancer cells in vitro and in vivo models. However, its clinical use is hampered by hypercalcemia. In this study, we investigated the effectiveness and safety of a new generation, less calcemic analog of 1α,25(OH)2D3, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10), in BxPC-3 human pancreatic carcinoma cells in vitro and in vivo. We demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)2D3 in inhibiting BxPC-3 cell proliferation in a time- and dose-dependent manner, accompanied by a greater upregulation of cyclin-dependent kinase inhibitors p21 and p27 and a greater downregulation of cyclin D3 and cyclin-dependent kinases 4 and 5, leading to a greater increase in the fraction of cells in G0/G1 phase. No induction of apoptosis and no effect on Cdc25 phosphatases A and C were observed in the presence of either MART-10 or 1α,25(OH)2D3. In a xenograft mouse model, treatment with 0.3 µg/kg body weight of MART-10 twice/week for 3 weeks caused a greater suppression of BxPC-3 tumor growth than the same dose of 1α,25(OH)2D3 without inducing hypercalcemia and weight loss. In conclusion, MART-10 is a promising agent against pancreatic cancer growth. Further clinical trial is warranted.

Potent 19-Norvitamin D Analogs For Prostate And Liver Cancer Therapy

The active form of vitamin D(3), 1α,25(OH)(2)D(3) or calcitriol, is known to inhibit the proliferation and invasiveness of many types of cancer cells, including prostate and liver cancer cells. These findings support the use of 1α,25(OH)(2)D(3) for prostate and liver cancer therapy. However, 1α,25(OH)(2)D(3) can cause hypercalcemia, thus, analogs of 1α,25(OH)(2)D(3) that are less calcemic but exhibit potent antiproliferative activity would be attractive as therapeutic agents. We have developed 2α-functional group substituted 19-norvitamin D(3) analogs with and without 14-epimerization. Among them, 2α- and 2β-(3-hydroxypropyl)-1α,25-dihydroxy-19-norvitamin D(3) (MART-10 and -11, respectively) and 14-epi-2α- and 14-epi-2β-(3-hydroxypropyl)-1α,25-dihydroxy-19-norvitamin D(3) (14-epi-MART-10 and 14-epi-MART-11, respectively) were found to be the most promising. In this review, we discuss the synthesis of this unique class of vitamin D analogs, the molecular mechanism of anticancer actions of vitamin D, and the biological evaluation of these analogs for potential application to the prevention and treatment of prostate and liver cancer.

ChemInform Abstract: An Alternative and Efficient Synthesis of 24,24-Difluoro-1α,25- dihydroxyvitamin D3.

An alternative synthesis of the title compound 2, a highly potent analog of 1α, 25-dihydroxyvitamin D3 (1), is described. Starting with 1α, 3β-bis[(tert-butyldimethylsilyl)oxy]androst-5-ene (8), 2 was obtained in 3.8% total yield through 10 steps. This method provides compound 2 in much higher yield than that reported previously.

MSITE: A new computational tool for comparison of homological proteins in holo form

The mechanism by which nuclear receptors respond differentially to structurally distinct agonists is not a well understood process. However, it is now obvious that transcriptional activity of nuclear receptors is a function of their interactions with co-activators. Recently, we released a new computational tool, CCOMP, for comparing side chain conformations in crystal structures of homologous protein complexes. Application of the CCOMP program revealed that 20- epi-1α,25-(OH) 2D 3 changes the side chain conformation of vitamin D receptor amino acids residing mostly far away from the ligand–receptor contacts. This strongly suggests that the ligand-co-activator signaling pathway involves indirect interactions between amino acids lining the binding pocket and outer surface residues that could attract co-activators. To facilitate identification of amino acids transmitting the subtle receptor changes upon ligand/modulator binding we developed another simple tool, MSITE. The program automatically lists the nearest neighbors of a given amino acid (for example neighbors of residues that are in contact with a ligand or reorient their side chains in the presence of a co-factor) in an arbitrary number of compared complexes. Comparison of seven binary vitamin D receptor complexes holding as ligands the analogs of 1α,25-(OH) 2D 3 with inverted configuration at carbon 14 or 20, or with incorporated oxolane ring bridging carbons 20 and 23, is reported.

Alisol-B, a novel phyto-steroid, suppresses the RANKL-induced osteoclast formation and prevents bone loss in mice

Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1α,25(OH)2D3-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10−8M 1α,25(OH)2D3 caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20S)-1α,25(OH)2D3 (2MD), an analog of 1α,25(OH)2D3, administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.

Synthesis and biological evaluation of 6-methyl analog of 1α,25-dihydroxyvitamin D 3

Among more than 3000 analogs of 1α,25-dihydroxyvitamin D 3 synthesized to date, only a few were characterized by structural modifications in the seco-B-ring. The compounds alkylated at C-6 seemed to be interesting targets for synthetic efforts. Such vitamin D analogs easily undergo thermal conversion to their previtamin forms. The results of molecular modeling indicate that significant deviation from planarity must be present in their molecules associated with the interaction of the 6-alkyl substituent and hydrogens from the C-ring. The synthesis of the analog of 1α,25-(OH) 2D 3, being characterized by the presence of the 6-methyl group, is reported here, together with the results of preliminary testing of its biological potency. This 6-alkylated compound was efficiently prepared using a novel stereoconvergent strategy in which the ring A and the triene unit of the vitamin D skeleton are constructed by a one-pot Pd-catalyzed tandem cyclization-Negishi coupling process.

Removal of C-ring from the CD-ring skeleton of 1α,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly

It is now well established that 1α,25(OH)2D3 is metabolized in its target tissues through the modifications of both side chain and A-ring. The C-24 oxidation pathway is the side chain modification pathway through which 1α,25(OH)2D3 is metabolized into calcitroic acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1α,25(OH)2D3 is metabolized into 1α,25(OH)2-3-epi-D3. During the past two decades, a great number of vitamin D analogs were synthesized by altering the structure of both side chain and A-ring of 1α,25(OH)2D3 with the aim to generate novel vitamin D compounds that inhibit proliferation and induce differentiation of various types of normal and cancer cells without causing significant hypercalcemia. Previously, we used some of these analogs as molecular probes to examine how changes in 1α,25(OH)2D3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1α,25(OH)2D3 with unique biological activity profiles were synthesized. Two of the analogs, SL 117 and WU 515 lack the C-ring of the CD-ring skeleton of 1α,25(OH)2D3. SL 117 contains the same side chain as that of 1α,25(OH)2D3, while WU 515 contains an altered side chain with a 23-yne modification combined with hexafluorination at C-26 and C-27. Presently, it is unknown how the removal of C-ring from the CD-ring skeleton of 1α,25(OH)2D3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1α,25(OH)2D3 in both the isolated perfused rat kidney, which expresses only the C-24 oxidation pathway and rat osteosarcoma cells (UMR 106), which express both the C-24 oxidation and C-3 epimerization pathways. The results of our present study indicate that SL 117 is metabolized like 1α,25(OH)2D3, into polar metabolites via the C-24 oxidation pathway in both rat kidney and UMR 106 cells. As expected, WU 515 with altered side chain structure is not metabolized via the C-24 oxidation pathway. Unlike in rat kidney, both SL 117 and WU 515 are also metabolized into less polar metabolites in UMR 106 cells. These metabolites displayed GC and MS characteristics consistent with A-ring epimerization and were putatively assigned as C-3 epimers of SL 117 and WU 515. In summary, we report that removal of the C-ring from the CD-ring skeleton of 1α,25(OH)2D3 does not alter its target tissue metabolism significantly.

ED-71, a novel vitamin D analog, promotes bone formation and angiogenesis and inhibits bone resorption after bone marrow ablation

ED-71, a novel analog of 1α,25-(OH) 2 D 3, increases bone mass to a greater extent than alfacalcidol, an 1α,25-(OH) 2 D 3 prodrug. In this study, we used a murine bone marrow ablation model to compare the effect of ED-71 on bone formation and resorption in vivo with that of 1α,25-(OH) 2 D 3. We discovered that bone matrix remodeling occurring within the first week after bone marrow ablation was enhanced by a single injection of ED-71, but not by 1α,25-(OH) 2 D 3. This enhancement was associated with an increase in bone surface. Trabecular bone resorption occurring from 1 to 2 weeks after the procedure was suppressed by a single injection of ED-71, but not 1α,25-(OH) 2 D 3, with treated mice exhibiting a reduction in osteoclast numbers, despite increases in osteoblast surface. As seen with the single injection, daily administration of ED-71 also enhanced bone modeling. Bone marrow osteoblast differentiation was also augmented by ED-71 pretreatment. Furthermore, ED-71 treatment immediately after bone marrow ablation enhanced angiogenesis within the bone marrow cavity via enhancement of VEGF 120 expression. In this paper, we clearly demonstrate that ED-71 is an orally administered small molecular weight compound with an anabolic effect on bone metabolism.

1alpha,25-dihydroxyvitamin D3 induces vascular smooth muscle cell migration via activation of phosphatidylinositol 3-kinase.

The steroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] promotes vascular smooth muscle cell (VSMC) growth and calcification, but the precise mechanism by which 1alpha, 25-(OH)2D3 regulates VSMC migration is unknown. In rat aortic SMCs, we found that 1alpha, 25-(OH)2D3 (0.1 to 100 nmol/L) induced a dose-dependent increase in VSMC migration. This response required the activation of phosphatidylinositol 3-kinase (PI3 kinase) because 1alpha, 25-(OH)2D3-induced migration was completely abolished by the PI3 kinase inhibitors, LY294002 (10 micromol/L) or wortmannin (30 nmol/L). Furthermore, the RNA polymerase inhibitor, 5,6-dichlorobenzimidazole riboside (50 micromol/L), did not affect 1alpha, 25-(OH)2D3-induced VSMC migration, suggesting that gene transcription is not involved in this rapid response. Using analogs of 1alpha, 25-(OH)2D3, which have been characterized for their abilities to induce either transcriptional or nontranscriptional responses of 1alpha, 25-(OH)2D3, we found that 1alpha,25-dihydroxylumisterol, which is a potent agonist of the rapid, nongenomic responses, was equipotent with 1alpha, 25-(OH)2D3 in inducing PI3 kinase activity and VSMC migration. Moreover, 1beta, 25-(OH)2D3, which specifically antagonizes the nongenomic actions of 1alpha, 25-(OH)2D3, abolished 1alpha, 25-(OH)2D3-induced PI3 kinase activity and VSMC migration, whereas the inhibitor of the genomic actions of vitamin D, (23S)-25-dehydro-1alpha-OH-D3-26,23-lactone, did not affect these responses. These results indicate that 1alpha, 25-(OH)2D3 induces VSMC migration independent of gene transcription via PI3 kinase pathway, and suggest a possible mechanism by which 1alpha, 25-(OH)2D3 may contribute to neointima formation in atherosclerosis and vascular remodeling.

A non-calcemic analog of 1α,25 dihydroxy vitamin D 3 (JKF) upregulates the induction of creatine kinase B by 17β estradiol in osteoblast-like ROS 17/2.8 cells and in rat diaphysis

We have reported that multiple treatments with so-called ‘non-hypercalcemic’ analogs of 1α,25(OH) 2 vitamin D 3 (1,25(OH) 2D 3) stimulate the specific activity of creatine kinase BB (CK) in ROS 17/2.8 osteoblast-like cells, and that pretreatment with these analogs upregulates responsiveness and sensitivity to 17β estradiol (E 2) for the induction of CK. However, since the analogs showed toxicity in vivo, we have now studied the action of a demonstrably non-calcemic hybrid analog of vitamin D in ROS 17/2.8 cells, and prepubertal rats. The analog JKF was designed to separate its calcemic activity from other biological activities by combining a calcemic-lowering 1-hydroxymethyl group with a potentiating C, D-ring side chain modification including 24 difluoronation. Treatment with 1 pM JKF alone significantly stimulated CK specific activity at 4 h by 30±10%. However after three daily pretreatments, JKF upregulated the extent of induction by 30 nM E 2 by 33% at 1 pM and by 97% at 1 nM; the E 2 dose needed for a significant stimulation of CK activity was lowered to 30 pM. The action of the SERMS tamoxifen, tamoxifen methiodide and raloxifene, at 3 μM, was also upregulated by three daily pretreatments with 1 nM JKF; unexpectedly, this pretreatment prevented the inhibition of E 2 stimulation by the SERMS. Upregulation of E 2 action by 1 nM JKF was inhibited by 1 nM ZK159222, an inhibitor of the nuclear action of 1,25(OH) 2D 3. In vivo, three daily injections of 0.05 ng/g body weight of JKF augmented the response of prepubertal female rat diaphysis and epiphysis to E 2. Therefore, demonstrably non-calcemic analogs of 1,25(OH) 2D 3 may have potential for use in combination with estrogens or SERMS in the prevention and/or treatment of metabolic bone diseases such as postmenopausal osteoporosis.

Ligands for the vitamin D endocrine system: different shapes function as agonists and antagonists for genomic and rapid response receptors or as a ligand for the plasma vitamin D binding protein

The integrated operation of the vitamin D endocrine system which produces the steroid hormone 1α,25(OH) 2-vitamin D 3 (1α,25(OH) 2D 3) is dependent on four classes of proteins each of which have inherent in their secondary and tertiary structure a ligand binding domain (LBD) that allows the stereospecific binding of 1α,25(OH) 2D 3 or related analogs as a substrate or ligand. These LBDs include: (a) the cytochrome P450 enzymes in the liver, kidney, and other tissues which metabolize vitamin D 3 into biologically active metabolites; (b) the plasma vitamin D binding protein (DBP) which selectively transports these hydrophobic molecules to the various target organs of the vitamin D endocrine system; (c) the nuclear receptor VDR nuc that is involved in regulation of gene transcription in over 30 cell types which possess this receptor; and (d) a plasma membrane receptor, VDR mem, that is involved in initiation of signal transduction pathways which generate rapid biological responses. This article reviews the evidence that supports the conclusions that the LBD of the DBP, VDR mem and VDR nuc each select as their preferred ligand a unique shape of the conformationally flexible 1α,25(OH) 2D 3. Two critical aspects of the conformationally flexible 1α,25(OH) 2D 3 molecule which defines the optimum ligand shape are (a) the orientation and relative rigidity of the flexible 8 carbon side chain and (b) the position of the A ring in relation to the C/D rings as determined by the extent of rotation around the 6,7 single carbon bond of the seco B ring. These conclusions are based on consideration of structure-function studies of over 300 analogs of 1α,25(OH) 2D 3, of these, 22 analogs are highlighted in this presentation.

The effects of 1α,24(S)-dihydroxyvitamin D 2 analog on cancer cell proliferation and cytokine expression

It is well established that 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2D 3), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1α,25(OH) 2D 3 in regulating the immune system. However, therapeutic application of 1α,25(OH) 2D 3 to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1α,25(OH) 2D 3 have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1α,24(S)-dihydroxyvitamin D 2 (1α,24(S)(OH) 2D 2), a vitamin D 2 analog, in restraining cell proliferation was compared to that of 1α,25(OH) 2D 3. In parallel studies, cancer cell lines were grown in increased concentrations (10 − 10–10 − 7 M) of each compound for various incubation periods (1–4 days). Growth was assessed by measuring [ 3H]thymidine incorporation. The results revealed that 1α,24(S)(OH) 2D 2 significantly inhibits proliferation to an extent similar to that observed for 1α,25(OH) 2D 3. Moreover, incubating the human leukemia cell line, HL-60, with 1α,24(S)(OH) 2D 2 resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1α,25(OH) 2D 3. Using a Western procedure, it was also shown that 1α,24(S)(OH) 2D 2 like 1α,25(OH) 2D 3 enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-α) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be regulated by 1α,25(OH) 2D 3 as well as by 1α,24(S)(OH) 2D 2. Incubations of HPM with 1α,25(OH) 2D 3 or 1α,24(S)(OH) 2D 2, have inhibited the expression of TNF-α on both mRNA and protein levels. These results suggest that 1α,25(OH) 2D 3 has a role in controlling the rate of inflammation in the peritoneal cavity of CAPD treated patients. Since 1α,24(S)(OH) 2D 2 does not cause hypercalcemia, the present results encourage the possible use of this vitamin D 2 analog in the treatment of cancer and hyper-inflammatory diseases.

Metabolites and analogs of 1α,25-dihydroxyvitamin D 3: evaluation of actions in bone

Analogs of 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] activate both genomic mechanisms via the nuclear vitamin D 3 receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D 3 receptor (pmVDR). Both of these pathways are normally activated by 1α,25(OH) 2D 3, but as a result of synthesis of numerous analogs of 1α,25(OH) 2D 3 these pathways can be distinguished. We used increasing doses of vitamin D 3 analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D 3 (R) and 1α,25(OH) 2-16,23E-diene-D 3 (A) are 10-fold more potent than 1α,25(OH) 2D 3 for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1α-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1α,25(OH) 2D 3 to increase osteocalcin mRNA or protein release. 1α,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D 3, (E) is an agent that is 10 fold more potent than 1α,25(OH) 2D 3 at increasing osteocalcin mRNA and protein release, whereas 1α,25(OH) 2-3-epi-D 3 increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1α,25(OH) 2D 3. These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F 6 on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1α-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.

Highly active analogs of 1α,25-dihydroxyvitamin D 3 that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1α,25(OH) 2-3-epi-D 3. A rational design for the synthesis of potent analogs of 1α,25(OH) 2D 3 is developed based on the knowledge of the various metabolic pathways of 1α,25(OH) 2D 3. Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D 3 analogs 1α,25(OH) 2-16,23-E-diene-26,27-hexafluoro-19-nor-D 3 (Ro 25–9022), 1α,25(OH) 2-16,23-Z-diene-26,27-hexafluoro-19-nor-D 3 (Ro 26–2198), and 1α,25(OH) 2-16-ene-23-yne-26,27-hexafluoro-19-nor-D 3 (Ro 25–6760), as indicated by their antiproliferative activities.

Why do we need a three-dimensional architecture of the ligand-binding domain of the nuclear 1α,25-dihydroxyvitamin D 3 receptor?☆

Highly specific binding of 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2D 3) by vitamin D receptor (VDR), a nuclear transcriptional factor, activates a genomic mechanism that is manifested in the multiple biologic properties of 1α,25(OH) 2D 3. Numerous synthetic analogs of 1α,25(OH) 2D 3 have been employed to study the interaction between 1α,25(OH) 2D 3 and VDR, and to identify structural markers in 1α,25(OH) 2D 3 that are important for VDR-binding. On the other hand the three-dimensional structure of VDR remained elusive till very recently. In the present study we employed affinity labeling (by 1α,25-dihydroxyvitamin D 3-3-bromoacetate, 1α,25(OH) 2D 3-3-BE) of VDR to identify C 288 as the anchoring residue for the 3-hydroxyl group of 1α,25(OH) 2D 3 inside the ligand-binding domain of VDR (VDR-LBD). In addition we carried out mutation/hormone-binding analyses to determine the importance of M 284 and W 286 toward hormone binding. We incorporated this information with the three-dimensional structure of the LBD of progesterone receptor to develop a homology-extension model of VDR-LBD. This model identified several amino acid residues as ligand-contact points inside the LBD. Mutational and hormone-binding analyses of these residues verified the structure-functional authenticity of this model, in comparison with the crystal structure of VDR, bound to 1α,25(OH) 2D 3.