25S-Adamantyl-23-yne-26,27-dinor-1Î±,25-dihydroxyvitamin D3

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25S-Adamantyl-23-yne-26,27-dinor-1#,25-dihydroxyvitamin D: Synthesis, Tissue Selective Biological Activities and X-ray Crystal Structural Analysis of Its Vitamin D Receptor Complex 3

Rocio Otero, Michiyasu Ishizawa, Nobutaka Numoto, Teikichi Ikura, Nobutoshi Ito, Hiroaki Tokiwa, Antonio Mouriño, Makoto Makishima, and Sachiko Yamada J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00427 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of Medicinal Chemistry

25S-Adamantyl-23-yne-26,27-dinor-1α,25dihydroxyvitamin D3: Synthesis, Tissue Selective Biological Activities and X-ray Crystal Structural Analysis of Its Vitamin D Receptor Complex Rocio Otero,†,# Michiyasu Ishizawa,‡,# Nobutaka Numoto,§ Teikichi Ikura,§ Nobutoshi Ito,§,* Hiroaki Tokiwa,|| Antonio Mourino,†,* Makoto Makishima‡,* and Sachiko Yamada‡ †

Departamento de Química Orgánica, Laboratorio de Investigación Ignacio Ribas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain

‡

Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan

§

Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo113-8510, Japan ||

Department of Chemistry, Faculty of Science, Rikkyo University, Toshima-ku, Tokyo 1718501, Japan

ABSTRACT. Both 25R- and 25S-25-adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D3 (4a and 4b) were stereoselectively synthesized by a Pd(0)-catalyzed ring closure and Suzuki-

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Miyaura coupling between enol-triflate 7 and alkenyl-boronic ester 8. The 25S isomer (4b) showed high vitamin D receptor (VDR) affinity (50% of that of the natural hormone 1α,25dihydroxyvitamin D3, 1) and transactivation potency (kidney HEK293, 90%). In endogenous gene expression, it showed high cell-type selectivity for kidney cells (HEK293, CYP24A1 160% of 1), bone cells (MG63, osteocalcin 64%) and monocytes (U937, CAMP 96%) over intestine (SW480, CYP24A1 8%) and skin (HaCaT, CYP24A1 7%) cells. The X-ray crystal structural analysis of 4b in complex with rat VDR-ligand binding domain (LBD) showed the highest Cα positional shift from the 1/VDR-LBD complex at helix 11. Helix 11 of the 4b and 1 VDR-LBD complexes also showed significant differences in surface properties. These results suggest that 4b should be examined further as another candidate for a mild preventive osteoporosis agent.

INTRODUCTION Osteoporosis encompasses a heterogeneous group of disorders that represents a major risk for bone fractures and a substantial burden on the health care system.1 Therapies for osteoporosis mostly inhibit bone resorption. By acting at this site in the bone remodeling cycle, estrogens, 2 selective estrogen receptor modulators,3 and the bisphosphonates4 increase bone mineral density and reduce the risk of new fractures. Although, a number of antiresorptive agents prevent further bone loss, they do not build bone once lost. Recently, an anabolic bone-building agent, a recombinant human parathyroid hormone (teriparatide)5 has been approved for the treatment of osteoporosis. Teriparatide inhibits the development and activity of osteoclasts decreasing bone resorption and increasing bone density. However, because of the potential risk of osteosarcoma, the duration of teriparatide treatment is limited to 2 years. The human monoclonal antibody denosumab6 to the receptor activator of nuclear factor-κB ligand (RANKL), which blocks its binding to the receptor activator of nuclear factor-κB (RANK), has also been approved for the


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treatment of osteoporosis. The secreted glycoprotein sclerostin7a has emerged as a key negative regulator of Wnt signaling in bone, and the optimization of anti-sclerostin antibodies has led to a vast array of preclinical studies documenting its ability to enhance bone formation, strength, and density.7b,c 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3 (1)], a hormonally active form of vitamin D3, plays a central role in regulating mineral metabolism via its actions in intestinal and kidney epithelial cells and in specific bone cells.8 This activity is achieved directly by its binding to the vitamin D receptor (VDR) in intestine, kidney, and bone and by the feedback inhibition of parathyroid hormone (PTH) production at the parathyroid glands. Activity at the skeleton is driven primarily by RANKL, a factor produced by stromal cells and osteoblasts.9 Thus 1,25(OH)2D3 (1), which regulates the production of both PTH and RANKL and induces bone anabolic activity,10 is a natural anti-osteoporosis agent. Active vitamin D synthetic analogs (alfacalcidol11 and eldecalcitol12) are being successfully used in the treatment of osteoporosis in Japan but their clinical applications have been restricted in the USA and European countries, primarily because of their hypercalcemic effects and non-definitive bone forming activity. Alfacalcidol, which is recognized as an important drug for the prevention of bone loss and the minimization of traumatic fracture in postmenopausal women and old men, is being reconsidered in Europe for treatment of osteoporosis.13 Our research is focused on the development of vitamin D analogs with selective action to prevent osteoporosis. On this basis, we designed and synthesized vitamin D analogs with an adamantyl group at C25 or C26 of a side chain possessing a double bond14a,b,c or one (2)14d or two triple bonds (3)14e (Chart 1). The two terminal methyl groups of 1,25(OH)2D3 have been shown to be important for binding to the VDR, especially helix 12 residues [human(h) Phe422 and Val418; rat (r) Phe418 and Val414], and for agonistic activity.15 The displacement of one of


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the methyl groups with a large adamantyl group was considered as a way to modify the conformations of helix 11 to 12 region. The introduction of a double or triple bond was thought to increase the side chain rigidity. The 10(19)-methylene group of the vitamin D A-ring was replaced with a 2-methylene group to give 19-nor-2-methylene analogs, which are more stable to oxidation, acids, irradiation, and heat than classic vitamin D analogs bearing the conjugated triene system. The 19-nor-2-methylene A-ring system was first introduced by DeLuca’s group and is also a structural component of the vitamin D super agonist 2MD (1α,25-dihydroxy-2methylene-19-norvitamin D3).16 Both 25- and 26-adamantyl vitamin D derivatives with a double bond at C22 have been shown to induce antagonistic activity,14a-c while ones with a triple bond at C23 induce partial agonism.14d Adamantyl vitamin D analog 2b showed significant VDR affinity and fair cell type selectivity in VDR gene expressions.14d To attain a higher degree of selectivity, we synthesized the vitamin D analogs 4a and 4b, possessing the natural 5,7,10(19)-conjugated triene unit and the same side chain as 2b. Here, we report the biological activity and X-ray crystal structural analysis of 4a and 4b in addition to their stereoselective synthesis.

Chart 1. Structures of the Compounds Discussed in this Article


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RESULTS Stereoselective Synthesis of (25R)- and (25S)-25-Adamantyl-26,27-dinor-23-yne-1α,25dihydroxyvitamin D3 (4a and 4b). Scheme 1 depicts the synthetic plan for the target vitamin D3 analogs 4a and 4b, which possess propargylic and adamantyl units at the side chain. The conjugated triene system of the target vitamin D compounds was assembled by a Pd-catalyzed Heck-cyclization/Suzuki-Miyaura-coupling approach17 between enol-triflate 7 (A-ring precursor) and boronate 8 (CD-ring fragment). A key feature of the whole synthesis is the separation of the diastereomeric alcohols 6 via the carbamates 5 (Pirkle’s method).17,18 Scheme 1. Retrosynthesis of Target Compounds 4a and 4b


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The synthesis of the target compounds 4a and 4b started from the known nitrile 919 (Scheme 2), which was converted in 62% yield to the diastereomeric mixture of propargyl alcohols 13 by a three step sequence via aldehyde 10 and dibromide 11. Thus, the reduction of nitrile 9 with DIBAL-H and the treatment of the resulting aldehyde 1020 with the ylide Ph3P=CBr221 gave the dibromide 11. Deprotonation of 11 with n-butyllithium and trapping of the corresponding lithium acetylide with adamantyl carbaldehyde 1214e furnished the desired mixture of alcohols 13. The benzoylation of 13, desilylation of the resulting benzoate 14 with hydrofluoric acid and subsequent oxidation of the resulting alcohols 15 with pyridinium dichromate gave the ketones 16, which upon treatment with Ph3P=CHBr22 provided (E)-vinyl bromide 17 (69%, four steps from 13). Not surprisingly, all attempts to separate the propargylic diastereoisomers at the benzoate stage by HPLC were unsuccessful. The formation of the upper boronate fragment worked best on the propargylic alcohols 18, which were smoothly prepared by the DIBAL-H reduction of the benzoates 17. The Miyaura borylation23 of 18 with bis(pinacolato)diboron in the presence

of

[1,1′-bis(diphenyl-phosphino)ferrocene]-dicloro-palladium(II)−dichloromethane

complex as a catalyst and tricyclohexylphosphine as a ligand afforded the boronate 8 in 97% yield. The triene system of 6 was installed by the treatment of an equimolecular mixture of 8 and 724 in H2O/THF with catalytic PdCl2(PPh3)2 in the presence of K3PO4 (75% yield).25 Attempts to


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separate the vitamin D protected diastereoisomers 6 by HPLC were fruitless. Next, efforts were focused on the separation of stereoisomers 6 by Pirkle’s method.17,18 Exposure of the mixture of alcohols 6 to (S)-(+)-1-(1-naphthyl)ethyl isocyanate in the presence of 4-(dimethylamino)pyridine and pyridine followed by a work up and flash column chromatography afforded the carbamates 5a (32% yield) and 5b (43% yield), which were successfully converted to the desired adamantyl vitamin D3 analogs 4a (56% yield) and 4b (47% yield) by hydrolysis with NaOH/THF followed by desilylation with n-Bu4NF/THF. At this stage, the configuration of carbamates 4 at C25 could not be assigned by NMR. Finally, it was possible to establish the configuration of all stereoisomers at C25 on the basis of the crystallographic structures of 4a and 4b in complex with the ligand binding domain (LBD) of the VDR (see below). Scheme 2. Stereoselective Synthesis of Adamantyl Vitamin D Compounds (4a and 4b)


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Reagents and conditions: (a) DIBAL-H, CH2Cl2, -10 ºC, 1 h; 10% HCl/Et2O (1:1), 0 ºC, 2 h. (b) CBr4, Zn, PPh3, CH2Cl2, 23 ºC, 2 h. (c) n-BuLi, THF, -78 ºC; 12, THF, -78 ºC to 23 ºC. (d) nBuLi, THF, -78 ºC; BzCl. (e) 48% HF, CH2Cl2/CH3CN, 23 ºC, 48 h. (f) PDC, CH2Cl2, 23 ºC, 2 h. (g) Ph3P=CHBr, THF/toluene (1:7), -17 ºC (1.5 h) 23 ºC (2 h). (h) DIBAL-H, CH2Cl2, -78 ºC, 2 h. (i) PdCl2(dppf)·CH2Cl2, PCy3, B2Pin2, DMSO, 80 ºC, 3 h. (j) PdCl2(PPh3)2, 2 M K3PO4, THF, 23 ºC, 3 h. (k) 19, py, DMAP, 0 ºC to 23 ºC, 24 h. Flash chromatography: 5a (32%), 5b (43%). (l) NaOH, THF, 50 ºC, 48 h; TBAF, THF, 23 ºC, 8 h; dppf: 1,1´bis(diphenylphosphino)ferrocene; pin: pinacol.

Biological Activities of Adamantyl-23-yne-vitamin D Compounds (4a and 4b). Affinity for hVDR. This property is the most important for compounds targeting the VDR, because without binding to the receptor, no compound can induce expression of the target genes. The VDR affinity of 4a and 4b was evaluated using a solution of recombinant hVDR-LBD14b,d and


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compared with that of the natural hormone 1 (Figure 1). The 25S-isomer (4b) showed significantly higher activity (IC50 0.24 nM, 50% activity of 1) than the 25R isomer 4a (3.13 nM, 4%). The vitamin D type analog 4b was also shown to be less active (56%) than its 2-methylene19-nor analog (2b).14d

Figure 1. Binding of the adamantyl vitamin D (4a and 4b) compared with that of 1,25(OH)2D3 (1) for hVDR-LBD. Glutathione S-transferase-VDR fusion proteins were incubated with [3H]1,25(OH)2D3 in the presence of nonradioactive 1,25(OH)2D3 (1) (red), 4a (green), or 4b (blue) at a range of concentrations. All values represent means ± standard deviations of triplicate assays. Transcriptional Activity. The transcriptional activity of the two compounds (4a and 4b) was evaluated by the luciferase reporter assay in human embryonic kidney-derived HEK293 cells.14b,d This assay system evaluates the activity of a substrate by using a gene expression system in HEK293 cells transfected with VDR and luciferase expression vectors. The 25S-isomer 4b showed activity (EC50 0.03 nM) as high as that of 1,25(OH)2D3 and its maximum activity


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(efficacy) was 87% of that of 1. Meanwhile, the 25R-isomer 4a showed significantly lower activity (EC50 >5.69 nM) (Figure 2), which was consisted with its low VDR affinity.

Figure 2. Transactivation of VDR by adamantyl vitamin D (4a and 4b) compared with that by 1,25(OH)2D3 (1) in HEK293 cells. HEK293 cells were transfected with pCMX-VDR and TKSpp × 3-LUC reporter, and treated with 0 M (vehicle control; white bar) or several varying concentrations of 4a, 4b or 1,25(OH)2D3 (1) (10-14 M to 10-8 M). After 24 h, the cells were harvested in order to assay the luciferase and β-galactosidase activities. Effects on Cofactor Bindings. After binding the ligand 1,25(OH)2D3 (1), the VDR forms a heterodimer with retinoid X receptor (RXR).26 The heterodimer binds to the VDR responsive element on its target genes, whereupon coactivators are recruited, and chromatin remodeling and transactivation follow. Therefore, the VDR/ligand complex needs to bind to RXR and interact with coactivator proteins. When the corepressors instead of coactivators are recruited, the chromatin remodeling and transactivation processes are prohibited. The effects of analogs binding to VDR on heterodimer formation with RXRα (Figure 3A) and on steroid receptor coactivator 1 (SRC-1)27 (Figure 3B) or nuclear receptor corepressor 1 (NCoR)28 protein recruitment (Figure 3C) were evaluated by using mammalian two hybrid assays,14b,d and the


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results were compared with those of the natural hormone 1. In the activation of VDR binding to all of these cofactors, 4b showed similar activity to 1: RXRα (EC50 > 0.14 nM; 0.07 nM for 1), SRC-1 (> 1.2 nM; > 0.90 nM for 1), and NCoR (IC50 0.09 nM; 0.05 nM for 1). The epimer 4a had lower activity than 4b: RXRα (EC50 >1.2 nM), SRC-1 (>3.2 nM), and NCoR (IC50 1.75 nM). The results showed that 4b had suitable properties to affect gene expression in HEK293 cells.

Figure 3. Effect of adamantyl vitamin D 4a and 4b on interaction between VDR and RXRα (A), SRC-1 peptide (B) or NCoR peptide (C). Mammalian two-hybrid assays were performed in HEK293 cells by transfecting CMX-GAL4-RXRα (A), CMX-GAL4-SRC-1 (B) or CMX-GAL4NCoR (C) together with CMX-VP16-VDR and MH100(UAS) × 4-tk-LUC. Cells were treated with 4a, 4b or 1,25(OH)2D3 (1) over a range of concentrations [0 M (vehicle control; white bar) or 10-10 M to 10-8 M].


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Effects on Endogenous VDR Target Gene Expression in Various Tissue Cells. We examined endogenous gene expression to determine the tissue-selective behavior of adamantyl vitamin D 4a and 4b. The CYP24A1 gene encodes vitamin D 24-hydroxylase and is the VDR target that is induced in many VDR-expressing cells.8c,14,29 We first examined the expression of the CYP24A1 gene in various cells: kidney epithelium-derived HEK293, intestinal mucosa-derived SW480, osteoblast-derived MG63, myeloid-derived U937, and skin-keratinocyte-derived HaCaT cells. We next examined the effect of the analogues on the expression of other genes, such as the epithelial calcium channel transient receptor potential vanilloid 6 (TRPV6),30 the colon cancerrelated E-cadherin31 in SW480 cells, osteocalcin (OCN)32 in MG63 cells, and the innate cathelicidin antimicrobial peptides (CAMP)33 in U937 and HaCaT cells. TRPV6 plays a role in vitamin D-induced intestinal calcium absorption,8c,30 E-cadherin is involved in the maintenance of the adhesive and polarized phenotype of epithelial cells,8c,31 OCN is an osteoblast-secreted protein that regulates bone homeostasis,8c,32 and CAMP plays a role in innate immunity in myeloid cells, keratinocytes and epithelial cells.8c,33 In all the experiments, we used an equal concentration (100 nM) of the ligands (4a, 4b and 1), and the percentage of expression relative to the natural hormone (1) was used to compare the activities (Figure 4). The 25S-isomer 4b was more active than its 25R counterpart 4a in the expression of all genes in all cell types examined (Figure 4). In CYP24A1 expression, 4b showed the highest activity, 160% of that of natural hormone 1, in kidney HEK293 cells, followed by that in monocyte U937 (54%) and bone MG63 cells (44%). In these cells, 4b also activated the expression of other genes, such as OCN (64%) in MG63 and CAMP (96%) in U937. Compound 4b showed weaker effects on the expression of CYP24A1 in intestinal SW480 (8%) and skin HaCaT (7%) cells. It also did not show considerable effects on the expression of other genes such as TRPV6 (22%) and E-cadherin


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(12%) in SW480 and CAMP (24%) in HaCaT cells. Thus, it should be noted that the adamantyl vitamin D 4b showed high cell-type selectivity in gene expression. Although its potency was weaker than that of 4b, the selectivity of 4a was similar to that of 4b (Figure 4).


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Figure 4. Effects of adamantyl vitamin D 4a and 4b compared with those of 1,25(OH)2D3 (1) on VDR target gene expression in different cell types. We evaluated mRNA levels of VDR target genes by reverse transcription and quantitative real-time PCR analysis: CYP24A1 in kidneyderived HEK293 cells (A), intestinal mucosa-derived SW480 cells (B), keratinocyte-derived HaCaT cells (C), bone-derived MG63 cells (D), monocyte-derived U937 cells (E); TRPV6 (F) and E-cadherin (G) in SW480 cells; CAMP in HaCaT cells (H); OCN in MG63 cells (I); CAMP in U937 cells (J). Cells were treated with each sample (100 nM) for 24 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle control. Cellular Uptake and Metabolic Stability of Compounds 4a and 4b. We investigated the cellular uptake and metabolic stability of 4a and 4b compared with those of the natural hormone 1 and the 19-nor-2-methylene analog 2b. MG63 cells were cultured with 4a, 4b, 2b or 1 (1 µM) in the presence of 10% fetal bovine serum (FBS) for 5 and 10 min, and the concentrations of these compounds in the cells and culture medium were determined by HPLC analysis.14e The cellular uptakes of 4a, 4b, 2b and 1 at 5 min were 6.9%, 3.1%, 6.1% and 3.4%, respectively, and at 10 min were 9.5%, 6.0%, 9.7% and 2.3%, respectively (Supporting information, Figure S1A). At 5


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min, the uptakes of 4b and 1 were approximately twice as slow as those of 4a and 2b. At 10 min, the uptakes of 4a, 4b and 2b were increased, but that of 1 was decreased slightly, probably because the catabolism of 1 had begun. When the culture was continued for 24 h, the stability of each compound in MG63 cells could be observed. Compounds 4b and 2b were the least stable (49% and 43% remained, respectively), 4a was the most stable (90%), and the natural hormone 1 was intermediate (70%). (Supporting information, Figure S1B). The results suggested that 30% of 1 was decomposed by catabolism, 4b and 2b were decomposed faster than 1 by a similar catabolic mechanism because of the rapid uptakes of the substrates by the cells, and 4a was stably present in the cells, probably because of its weak VDR affinity and the resulting smaller production of catabolic enzymes. X-ray Crystallographic Analysis of the rVDR-LBD Complexes of 25-Adamantyl-23-yne Vitamin D (4a and 4b). The crystal structures of the ternary complexes of the new adamantyl vitamin D compounds (4a and 4b) with rVDR-LBD34 and an oligopeptide of DRIP205 (MED1) were determined at resolutions of 2.0 and 2.1 Å, respectively (Supporting information, Table S1). The stereochemistry at C25 of the two isomers (4a and 4b) was determined as R and S, respectively, by these crystal structures. The overall structures of these complexes (4a and 4b) are quite similar, with rmsd values of 0.23 and 0.21 Å for all atoms, respectively, to a ternary VDR complex of 1,25(OH)2D3 (2ZLC)34b (4b complex, Figure 5A; 4a complex, Supporting information, Figure S2). The structure of 4b complex is also quite similar (rmsd for all atoms, 0.26 Å) to the VDR complex (3VTB) of 19-nor-2-methylene analog (2b).14e The 25S isomer 4b binds tightly to the VDR, forming three pairs of hydrogen bonds via its three hydroxyl groups (magenta stick representation, Figure 5B). The hydrogen bonds at the 1α- and 3β-hydroxyl groups in the A-ring are nearly the same as those in the 1,25(OH)2D3 1 complex34b (green stick,


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Figure 5B), as the four residues are nearly superimposable, but those at the 25-hydroxyl group are significantly different. The ligand 4b interacts with 19 residues within 4 Å (Figure 5C) in addition to the 6 hydrogen-bonding residues (Figure 5B). Since 1,25(OH)2D3 (1) in the rVDR complex (2ZLC) interacts with only 12 residues within 4 Å, except for 6 hydrogen-bonding residues, the adamantane ring induces interactions with 7 residues. The 25R isomer 4a forms hydrogen bonds similarly to 4b except that its 25-hydroxyl group forms only one hydrogen bond with His393, while His301 is slightly more distant from it (4.76 Å) (Figure 5D). The lack of one hydrogen-bonding interaction comparing with 4b might be a reason why 4a shows less than 1/10 weaker VDR affinity than 4b does. Also, the number of van der Waals interaction of 4a with VDR residue is smaller (15 residues) than those of 4b (19 residues) (Supporting information, Figure S3). Because of the special characteristic of the ligands 4a and 4b, a large adamantane ring and a 23-triple bond, the residues around the adamantane ring changed the Cα position significantly. In the 4b complex, Cα of His393 shifted significantly (0.57 Å) and a line of residues at the helix 11 region, Ser394 to Gln403, changed not only their Cα positions (0.42-1.78 Å, Figures 5E)14c but also their conformations significantly (Figure 5F). Another His301 forming hydrogen-bond with the 25-hydroxyl group changed the Cα position significantly (0.54 Å) but the residues around it (Ala299-Pro308) changed the Cα position (0.34-0.74 Å) and side chain conformations less than in the helix 11 region (Figures 5E and G). In the 4a complex, two regions with similarly large positional shift were also observed (Figure 5H). In the helix 11 region (H393-Q403) of the 4a complex, the residues showed lower Cα shifts (0.17-1.17 Å) than those in 4b complex but their side chain conformations were significantly changed (Figure 5I). The helix 7 region (K298-E307) of the 4a complex showed Cα shifts of 0.2-0.89 Å and the side chain conformation changed similarly to that of the 4b complex (Figure 5J).


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Figure 5. X-ray crystal structures of ternary rVDR-LBD complexes of adamantyl vitamin D compounds 4a and 4b. (A) Overall structure of ternary rVDR-LBD complex with 25S-isomer (4b) and DRIP205 co-activator peptide. The protein and peptide are drawn in ribbon representation with rainbow colors (blue to red) for helixes 1 to 12 and the co-activator peptide. The ligand 4b is shown in stick representation with atom-type colors. (B) Hydrogen bonding interactions of the ligand 4b with the VDR residues (the figure colors showing distances are the same as those of the structures): overlay of the complexes of 4b (atom-type colors with magenta carbon) and 1 (atom-type colors with green carbon). (C) Van der Waals interactions (

25S-Adamantyl-23-yne-26,27-dinor-1Î±,25-dihydroxyvitamin D3

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