Search

Vitamin D3 metabolites inhibit the expression of lipogenic genes by impairing sterol regulatory element-binding protein (SREBP), a master transcription factor of lipogenesis, independent of their canonical activity through a vitamin D receptor (VDR). Herein, we designed and synthesized a series of vitamin D derivatives to search for a drug-like small molecule that suppresses the SREBP-induced lipogenesis without affecting the VDR-controlled calcium homeostasis in vivo. Evaluation of the derivatives in cultured cells and mice led to the discovery of VDR-silent SREBP inhibitors and to the development of KK-052 (50), the first vitamin D-based SREBP inhibitor that has been demonstrated to mitigate hepatic lipid accumulation without calcemic action in mice. KK-052 maintained the ability of 25-hydroxyvitamin D3 to induce the degradation of SREBP but lacked in the VDR-mediated activity. KK-052 serves as a valuable compound for interrogating SREBP/SCAP in vivo and may represent an unprecedented translational opportunity of synthetic vitamin D analogues.

Active vitamin D form 1a,25-dihydroxtvitamin D3 (1,25(OH)2D3) plays pivotal roles in calcium homeostasis and osteogenesis via its transcription regulation effect via binding to vitamin D receptor (VDR). Mutated VDR often causes hereditary vitamin D-dependent rickets (VDDR) type II, and patients with VDDR-II are hardly responsive to physiological doses of 1,25(OH)2D3. Current therapeutic approaches, including high doses of oral calcium and supraphysiologic doses of 1,25(OH)2D3, have limited success and fail to improve the quality of life of affected patients. Thus, various vitamin D analogues have been developed as therapeutic options. In our previous study, we generated genetically modified rats with mutated Vdr(R270L), an ortholog of human VDR(R274L) isolated from the patients with VDDR-II. The significant reduced affinity toward 1,25(OH)2D3 of rat Vdr (R270L) enabled us to evaluate biological activities of exogenous VDR ligand without 1a-hydroxy group such as 25(OH)D3. In this study, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exerted much higher affinity for Vdr(R270L) in in vitro ligand binding assay than both 25(OH)D3 and 1,25(OH)2D3. A robust osteogenic activity of AH-1 was observed in Vdr(R270L) rats. Only a 40-fold lower dose of AH-1 than that of 25(OH)D3 was effective in ameliorating rickets symptoms in Vdr(R270L) rats. Therefore, AH-1 may be promising for the therapy of VDDR-II with VDR(R274L).

The complete understanding of the excretion of surplus 25-hydroxyvitamin Dlt;subgt;3lt;/subgt; [25(OH)Dlt;subgt;3lt;/subgt;] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin Dlt;subgt;3lt;/subgt; [24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt;] 24-glucuronide was identified as a major urinary vitamin Dlt;subgt;3lt;/subgt; metabolite, while the glucuronide of 23,25-dihydroxyvitamin Dlt;subgt;3lt;/subgt; [23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt;] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; liberated in urine by the treatment with β-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; and 24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; was almost as much as that of 24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt;, in contrast to the fact that the plasma concentration of 23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; was much lower than that of 24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt;. These results strongly suggested that 23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt;. Furthermore, the amount ratios of 23,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; to 24,25(OH)lt;subgt;2lt;/subgt;Dlt;subgt;3lt;/subgt; in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin Dlt;subgt;3lt;/subgt; supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)Dlt;subgt;3lt;/subgt;.

The vitamin D3 structure consists of the A-ring, a linker originating from the B-ring, C-ring, D-ring, and side-chain moieties. Each unit has its unique role in expressing the biological activities of vitamin D3. Many efforts have been made to date to assess the possible clinical use of vitamin D. Some organic chemists focused on the D-ring structure of vitamin D and synthesized D-ring-modified vitamin D analogues, and their biological activities were studied. This review summarizes the synthetic methodologies of D-ring-modified vitamin D analogues, except for seco-D, and their preliminary biological profiles.

We have developed an in vitro system to easily examine the affinity for vitamin D receptor (VDR) and CYP24A1-mediated metabolism as two methods of assessing vitamin D derivatives. Vitamin D derivatives with high VDR affinity and resistance to CYP24A1-mediated metabolism could be good therapeutic agents. This system can effectively select vitamin D derivatives with these useful properties. We have also developed an in vivo system including a Cyp27b1-gene-deficient rat (a type I rickets model), a Vdr-gene-deficient rat (a type II rickets model), and a rat with a mutant Vdr (R270L) (another type II rickets model) using a genome editing method. For Cyp27b1-gene-deficient and Vdr mutant (R270L) rats, amelioration of rickets symptoms can be used as an index of the efficacy of vitamin D derivatives. Vdr-gene-deficient rats can be used to assess the activities of vitamin D derivatives specialized for actions not mediated by VDR. One of our original vitamin D derivatives, which displays high affinity VDR binding and resistance to CYP24A1-dependent metabolism, has shown good therapeutic effects in Vdr (R270L) rats, although further analysis is needed.

Vitamin D3 metabolites are capable of controlling gene expression in mammalian cells through two independent pathways: vitamin D receptor (VDR) and sterol regulatory element-binding protein (SREBP) pathways. In the present study, we dissect the complex biological activity of vitamin D by designing synthetic vitamin D3 analogs specific for VDR or SREBP pathway, i.e., a VDR activator that lacks SREBP inhibitory activity, or an SREBP inhibitor devoid of VDR activity. These synthetic vitamin D probes permitted identification of one of the vitamin D-responsive genes, Soat1, as an SREBP-suppressed gene. The chemical probes developed in the present study may prove useful in dissecting the intricate interplay of vitamin D actions, thereby providing insights into how vitamin D target genes are regulated.

Two novel 23-fluorinated 25-hydroxyvitamin D3 analogues were synthesized using Inhoffen–Lythgoe diol as a precursor of the CD-ring, efficiently. Introduction of the C23 fluoro group was achieved by the deoxy-fluorination reaction using N,N-diethylaminosulfur trifluoride or 2-pyridinesulfonyl fluoride (PyFluor). Kinetic studies on the CYP24A1-dependent metabolism of these two analogues revealed that (23S)-23-fluoro-25-hydroxyvitamin D3 was more resistant to CYP24A1-dependent metabolism than its 23R isomer.

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.

Vitamin D is recognized to play important roles in the onset of immunological diseases as well as the regulation of the amount of Ca in the blood. Since these physiological actions caused by active vitamin D are triggered by the specific interaction between the vitamin D receptor (VDR) and active vitamin D, many types of compounds have been developed as potent ligands against VDR. It was found that the binding affinity between VDR and its ligand depends significantly on the chirality of the ligand. However, the reason for the dependence has, thus far, not been elucidated. In the present study, we investigated the specific interactions between VDR and some ligands with different chirality, using ab initio fragment molecular orbital (FMO) calculations. The FMO results reveal that two histidine residues of VDR contribute significantly to the binding between VDR and ligand and that their protonation states can affect the specific interactions between VDR and ligand. We therefore considered other possible protonation states of these histidine residues and determined their most stable states, using the ab initio FMO calculations. The results illustrate the possibility that the difference in the chirality of a ligand can induce the change in protonation states of the histidine residues of VDR existing near the ligand. This finding provides an important warning that the protonation states of histidine residues existing near the ligand should be considered more precisely in the molecular simulations for investigating the specific interactions between protein and ligand.

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.

Lactone-vitamin D3 is a major metabolite of vitamin D3, a lipophilic vitamin biosynthesized in numerous life forms by sunlight exposure. Although lactone-vitamin D3 was discovered 40 years ago, its biological role remains largely unknown. Chemical biological analysis of its photoaffinity probe identified the hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), a mitochondrial enzyme that catalyzes β-oxidation of long-chain fatty acids, as its selective binding protein. Intriguingly, the interaction of lactone-vitamin D3 with HADHA does not affect the HADHA enzymatic activity but instead limits biosynthesis of carnitine, an endogenous metabolite required for the transport of fatty acids into the mitochondria for β-oxidation. Lactone-vitamin D3 dissociates the protein-protein interaction of HADHA with trimethyllysine dioxygenase (TMLD), thereby impairing the TMLD enzyme activity essential in carnitine biosynthesis. These findings suggest a heretofore undescribed role of lactone-vitamin D3 in lipid β-oxidation and carnitine biosynthesis, and possibly in sunlight-dependent shifts of lipid metabolism in animals.

We report the synthesis of a peptide nucleic acid (PNA) monomer containing preQ

Background 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. Material and methods Western blot, migration and invasion assays, enzyme-linked immunosorbent assay, and immunofluorescent stain were applied in this study. Result 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.

Our previous study revealed that the 2α-substituted vitamin D analog 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exhibited a higher osteocalcin promoter transactivation activity in human osteosarcoma cells and a greater effect on bone mineral density in a rat model of osteoporosis than 1α,25(OH)2D3 without increasing blood calcium concentration. Thus, we hypothesized that AH-1 could be a promising therapeutic agent for osteoporosis without any serious side effects. In this study, we compared the CYP24A1-dependent metabolism of AH-1 with that of 1α,25(OH)2D3. The resistance to CYP24A1-dependent metabolism could be an important property of vitamin D analogs in prolonging their biological effects. A kinetic analysis was performed using a membrane fraction prepared from recombinant E. coli expressing human CYP24A1. The kcat/Km (μM-1 min-1) value for AH-1 was 31% of that for 1α,25(OH)2D3, suggesting that AH-1 is not as resistant to CYP24A1-dependent metabolism as the other C2-substituted vitamin D analogs such as eldecalcitol [2β-hydroxypropoxy-1α,25(OH)2D3]. The major metabolite of AH-1 was the 24R-hydroxylated metabolite, which had high vitamin D receptor (VDR) binding affinity and high HL-60 cell differentiation activity similar to AH-1 itself. In contrast, 1α,25(OH)2D3 was metabolized by multistep monooxygenation reactions, which led to the loss of affinity for VDR. Thus, the greater therapeutic effects of AH-1 than those of 1α,25(OH)2D3 in in vivo studies using osteoporosis rat models may be due to 24R-hydroxy-AH-1 whose VDR affinity was 91% of that of AH-1.

Vitamin D plays an important role in the regulation of the calcium and phosphorus metabolism as well as in bone formation. These physiological actions caused by vitamin D are triggered by the specific binding of vitamin D to its receptor (VDR). Here we investigated the specific interactions and binding affinities between VDR and vitamin D derivatives, using ab initio fragment molecular orbital (FMO) calculations. The FMO results elucidate that relative position of the two hydroxyl groups of the derivatives is essential for the strong binding affinity between the derivative and Arg274 residue of VDR. It is therefore expected that novel potent ligands, which have a great binding affinity for VDR, are developed by adjusting the positions of the hydroxyl groups in the derivatives in such a way as these groups form strong hydrogen bonds with VDR residues. We proposed these novel derivatives and investigated their specific interactions with VDR at atomic and electronic levels to obtain a more potent ligand for VDR.

Among numerous studies on synthetic approaches to and the biological activities of vitamin D analogues, we herein focused on falecalcitriol, an analogue of calcitriol (1α,25-dihydroxyvitamin D3), in which a 26,26,26,27,27,27-hexafluoroisopropanol unit has been introduced into the side chain. Falecalcitriol was designed to escape from the metabolism of CYP24A1 and has been used as a drug to treat secondary hyperparathyroidism since 2001. Its metabolite, the 23-hydroxy form, retains biological activity and resistants to further metabolism. Recent developments in synthetic methodologies for introducing the hexafluoroisopropanol unit into the vitamin D CD-ring side chain were described herein.

Two 24-fluoro-25-hydroxyvitamin D3 analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D3 side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D3 revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D3 (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.

The discovery of a large variety of functions of vitamin D3 and its metabolites has led to the design and synthesis of a vast amount of vitamin D3 analogues in order to increase the potency and reduce toxicity. The introduction of highly electronegative fluorine atom(s) into vitamin D3 skeletons alters their physical and chemical properties. To date, many fluorinated vitamin D3 analogues have been designed and synthesized. This review summarizes the molecular structures of fluoro-containing vitamin D3 analogues and their synthetic methodologies.

Previously, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 (AH-1) showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect on ovariectomized (OVX) rats for enhancing bone mineral density than those of 1α,25(OH)2D3 without hypercalcemic side effects in vivo. Although CYP24A1 catalyzes multi-step oxidations toward the CD-ring side chain of the active vitamin D3 [1α,25(OH)2D3], the CYP24A1-dependent metabolism of AH-1 tended to stop at the first step hydroxylation at the C24-position of AH-1. Interestingly, the metabolite 24-hydroxy-AH-1 [24(OH)AH-1] showed potent VDR binding affinity, and the new chiral center of the 24-position might be the 24R configuration compared with the process of the natural 1α,25(OH)2D3 catabolism. This time, (24R)-2α-[2-(tetrazol-2-yl)ethyl]-1α,24,25-trihydroxyvitamin D3 [(24R-OH)AH-1] was synthesized as a candidate for the major metabolite of AH-1 using the Trost Pd-mediated coupling reaction between A-ring and CD-ring precursors to identify the metabolite and evaluate its biological activity. We confirmed that the CYP24A1-dependent major metabolite of AH-1 was (24R-OH)AH-1 by HPLC analyses.

According to the binding mode of 14-epi-1α,25-dihydroxy-19-nortachysterol in the ligand binding domain of human vitamin D receptor (hVDR), i.e., 5,6- and 7,8-s-trans configuration that was shown by X-ray co-crystallographic analysis, 7,8-cis-locked 1α,25(OH)2D3 analogs were synthesized. In this paper, the synthesis and biological activity of 2α- and 2β-(3-hydroxypropyl)-7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 are reported. The A-ring and CD-ring precursors for the Julia-Kociensky coupling reaction to create a diene system of the target molecules were prepared using our original methods. hVDR binding affinity and osteocalcin promoter transactivation activity of the new 7,8-cis-14-epi-vitamin D3 analogs were evaluated. Interestingly, the 2β-substituted 7,8-cis-analog was a better binder for hVDR than the 2α-isomeric counterpart.

Here we report the synthesis of new PNA monomers for pseudocomplementary PNA (pcPNA) that are fully compatible with standard Fmoc chemistry. The thiocarbonyl group of the 2-thiouracil (sU) monomer was protected with the 4-methoxy-2-methybenzyl group (MMPM), while the exocyclic amino groups of diaminopurine (D) were protected with Boc groups. The newly synthesized monomers were incorporated into a 10-mer PNA oligomer using standard Fmoc chemistry for solid-phase synthesis. Oligomerization proceeded smoothly and the HPLC and MALDI-TOF MS analyses indicated that there was no remaining MMPM on the sU nucleobase. The new PNA monomers reported here would facilitate a wide range of applications, such as antigene PNAs and DNA nanotechnologies.

Vitamin D is recognized to play important roles not only in the bone metabolism and the regulation of Ca amount in the blood but also in the onset of immunological diseases. These physiological actions caused by vitamin D are triggered by the specific interaction between vitamin D receptor (VDR) and vitamin D. In the present study, we investigated the interactions between VDR and vitamin D derivatives using ab initio molecular simulation, in order to elucidate the reason for the significant difference in their effects on VDR activity. Based on the results simulated, we elucidated which parts of the derivatives and which residues of VDR mainly contribute to the specific binding between VDR and the derivatives at an electronic level. This finding will be helpful for proposing new vitamin D derivatives as a potent modulator or inhibitor against VDR.

Hereditary vitamin D-resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR) gene. Arg274 located in the ligand binding domain (LBD) of VDR is responsible for anchoring 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by forming a hydrogen bond with the 1α-hydroxyl group of 1α,25(OH)2D3. The Arg274Leu (R274L) mutation identified in patients with HVDRR causes a 1000-fold decrease in the affinity for 1α,25(OH)2D3, and dramatically reduces vitamin D- related gene expression. Recently, we successfully constructed fusion proteins consisting of split-luciferase and LBD of the VDR. The chimeric protein LucC-LBD-LucN, which displays the C-terminal domain of luciferase (LucC) at its N-terminus, can detect and discriminate between VDR agonists and antagonists. The LucC-LBD (R274L)-LucN was constructed to screen high-affinity ligands for the mutant VDR (R274L). Of the 33 vitamin D analogs, 5 showed much higher affinities for the mutant VDR (R274L) than 1α,25(OH)2D3, and 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-(OH)2D3 showed the highest affinity. These compounds might be potential therapeutics for HVDRR caused by the mutant VDR (R274L).

During our ongoing studies of vitamin D, we focused on the vitamin D

There is no suitable medicine for rickets caused by mutation of vitamin D receptor (VDR). In the present study, we found the promising new compounds for this disease using bioluminescence assay.

We report the synthesis of a peptide nucleic acid (PNA) monomer containing preQ1, a positively charged guanine analogue. The new monomer was incorporated into PNA oligomers using standard Fmoc-chemistry-based solid-phase synthesis. The preQ1 unit-containing PNA oligomers exhibited improved affinity for their complementary DNA through electrostatic attraction, and their sequence specificity was not compromised. It could be beneficial to incorporate preQ1 into PNA oligomers instead of guanine when creating antisense/antigene agents or research tools.

The specific binding of active vitamin-D to the vitamin-D receptor (VDR) is closely related to the onset of immunological diseases. To inhibit the binding, various compounds have been developed as potent inhibitors against VDR. Among them, a compound NS-54c, which was developed based on the first VDR antagonist TEI-9647 (25-dehydro-1α-hydroxyvitamin D3-26,23-lactone), was revealed to posse almost 1000-fold improved antagonistic activity over the original TEI-9647. However, the reason for this significant improvement has not been elucidated. In the present study, we investigated the specific interactions between VDR and these inhibitors, using molecular simulations based on molecular docking, molecular mechanics and ab initio fragment molecular orbital calculations. Based on the results simulated, we furthermore proposed novel inhibitors and investigated their binding properties to VDR. The results elucidate that the replacement of propyl group at the 24th site of NS-54c by a phenethyl group can enhance the binding affinity of the inhibitor to VDR. This finding provides useful information for developing novel potent inhibitors against VDR.

Recently, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect in ovariectomized (OVX) rats in vivo than those of active vitamin D3, 1α,25(OH)2D3. We were interested in introducing a heterocyclic ring to the C2 position of the seco-steroidal structure via an alkyl linker, and four novel C2-(3-tetrazolylpropyl) substituted 1α,25-dihydroxy-19-norvitamin D3 analogs, 2α-[3-(tetrazol-1-yl)propyl]-, 2β-[3-(tetrazol-1-yl)propyl]-, 2α-[3-(tetrazol-2-yl)propyl]-, and 2β-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 were synthesized. Among them, 2α-[3-(tetrazol-1-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak binding affinity for human vitamin D receptor (hVDR) (2.6% of 1α,25(OH)2D3 and ca. 15% of 19-nor-1α,25(OH)2D3) and weak VDR transactivation activity in HOS cells (EC50 7.3nM, when 1α,25(OH)2D30.23nM). Although the other three compounds could not act as VDR binders by evaluation of the competition assays, 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak transactivation activity (EC50 12.5nM). Metabolic stability of the 2α-substituted compounds 2α-[3-(tetrazol-1-yl)propyl]- and 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 was higher than that of the 2β-substituted counterparts 2β-[3-(tetrazol-1-yl)propyl]- and 2β-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 against human CYP24A1. Introduction of a tetrazole ring to the C2-position of the 19-norvitamin D3 skeleton with the propyl linker led to weak VDR agonistic activity with stability against CYP24A1 metabolism.

Four 7,8-cis-1α,25-dihydroxyvitamin D3 derivatives, 7,8-cis- and 7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 as well as 7,8-cis- and 7,8-cis-14-epi-1α,25-dihydroxyvitamin D3 were synthesized, and their chemical stability was characterized. In our previous work, we disclosed that 14-epi-19-nortachysterol showed the unprecedented binding configuration in human vitamin D receptor (hVDR), that is, 5,6- and 7,8-s-trans configuration. However, this configuration is variable because of the rotation at the single bond between C7 and C8. For the precise discussion of the 7,8-s-trans configuration, we designed and synthesized the 7,8-cis-locked skeleton of vitamin D3 derivatives. Among four analogs, the 19-nor derivatives were stable at ambient temperature, and their hVDR binding affinity and co-crystallographic analysis of their hVDR complexes were studied. The other derivatives with the triene system were isomerized to corresponding previtamin D3 and vitamin D3.

N-Acyldopamines are endogenous analogs of capsaicin that exhibit cannabinoid-like activities and were identified from brain extracts. Among them, N-arachidonoyldopamine (AADA) and N-oleoyldopamine (ODA) were characterized as transient receptor potential vanilloid type V1 channel (TRPV1) ligands. Recently, it was shown that N-acyldopamines may possess diverse physiological roles in addition to their ligand activities. To study the multiple functions and action mechanisms of endogenous N-acyldopamines, a simple and efficient method of N-acyldopamine synthesis was investigated. The eighteen potentially endogenous N-acyldopamines and two deuterated ones, N-palmitoyl dopamine-d5 and N-stearoyl dopamine-d5, were efficiently synthesized without protective groups in CH2Cl2 under optimized conditions using propylphosphoric acid cyclic anhydride (PPACA) as a condensation agent.

Vitamin D receptor (VDR) ligands, such as 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and its analogs, have been investigated for their potential clinical use in the treatment of various diseases such as type I rickets, osteoporosis, psoriasis, leukemia, and cancer. Previously, we reported a split-luciferase-based biosensor that can detect VDR ligands and assess their affinity for the ligand binding domain (LBD) of the VDR in a short time. However, a further increase in its sensitivity was required to detect plasma levels of 1α,25(OH)2D3 and its analogs. In this study, a novel type of biosensor called LXXLL + LBD was successfully developed. Here, the split luciferase forms a functional complex based on the intermolecular interaction between the LXXLL motif and the ligand-bound form of the LBD. This biosensor has an approximately 10-fold increase in the light intensity compared to the previous versions. Additionally, the binding affinity of the vitamin D analogs for the wild-type and the rickets-associated mutant R274L of VDR was evaluated.

Three 23-hydroxylated vitamin D

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)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)

Both 2α- and 2β-hydroxypropyl substituted 14-epi-1α,25-dihydroxy-19-nortachysterols were synthesized to study the human vitamin D receptor (hVDR) binding affinity, binding configurations, and interactions with amino acid residues in the ligand binding domain of hVDR by X-ray co-crystallographic analysis. In conjunction with our previous results on 14-epi-19-nortachysterol, 2-methylidene-, 2α-methyl-, 2β-methyl, and 2α-hydroxypropoxy-14-epi-19-nortachysterol, we propose a variety of effects of substitution at the C2 position in the 14-epi-19-nortachysterol skeleton on biological activities.

The survival rate of anaplastic thyroid cancer (ATC) is still very poor due to its fast growth and high metastatic potential. Currently, no effective treatment is available. The active form of vitamin D3, 1α,25(OH)2D3, has been shown to have a anti-metastatic effect in pre-clinical studies, however induction of hypercalcemia hampered its clinical application. The new class of less-calcemic vitamin D analog, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10), is much more potent than 1α,25(OH)2D3 to repress cancer growth and metastasis in a variety of cancers. In this study, we demonstrated that both 1α,25(OH)2D3 and MART-10 could effectively inhibit the migration and invasion of ATC cells, 8305C and 8505C, with MART-10 much more potent than 1α,25(OH)2D3. The anti-metastatic effect of 1α,25(OH)2D3 and MART-10 on ATC cells is mediated by reversal of cadherin switch (upregulation of E-cadherin and downregulation of N-cadherin), which led to the attenuation of EMT process, and decrease of F-actin formation. We further showed that the expressions of Slug, the EMT-related transcriptional factor, and MMP-9 were inhibited by 1α,25(OH)2D3 and MART-10 in 8505C cells, but not in 8303C cells. Since metastasis is the important cause of ATC-related death, our results strongly encourage the further in vivo study of MART-10 application against ATC.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3or 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)2D3for the treatment of these types of cancer. However, 1α,25(OH)2D3can cause hypercalcemia, so analogs of 1α,25(OH)2D3that 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 D3analogs 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.