Hector Deluca

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Organization: University of Wisconsin

Department: Department of Biochemistry

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Chemicals
References

UVB radiation, vitamin D and multiple sclerosis

Co-reporter:H. F. DeLuca;L. Plum

Photochemical & Photobiological Sciences (2002-Present) 2017 vol. 16(Issue 3) pp:411-415

Publication Date(Web):2017/03/16

DOI:10.1039/C6PP00308G

Acheson et al. (1960) observed an inverse relationship between sunlight exposure and the incidence of Multiple Sclerosis (MS). This led to the suggestion that increased levels of vitamin D caused by sunlight in some way suppresses MS. Further, super physiological doses of the metabolically active metabolite of vitamin D, i.e. 1α,25 dihydroxy vitamin D suppresses the animal model of MS i.e. experimental autoimmune encephalomyelitis (EAE). However, this response was accompanied by hypercalcemia. Hypercalcemia itself can suppress EAE. The ability of 1,25(OH)2D3 to suppress EAE in mice is largely eliminated by a low calcium diet until hypercalcemia is induced by high doses of 1,25(OH)2D3 that causes mobilization of calcium from the skeleton. Of great importance is the finding that vitamin D deficiency prevents EAE, a finding dramatically opposite to the original hypothesis. Further, vitamin D receptor knock out animals do not develop EAE supporting the idea that vitamin D does not suppress EAE. Upon revisiting the inverse relationship between light exposure and incidence of MS, a narrow band of light (300–315 nm) was discovered that prevents EAE without a change in serum levels of 25 hydroxy vitamin D (indicator of vitamin D status). Clinical trials are underway to explore the possible use of this narrow band light as a treatment to stop the progression of MS, while biochemical studies are underway to evaluate the mechanism of action of the narrow band light.

Pharmacokinetics of a New Oral Vitamin D Receptor Activator (2-Methylene-19-Nor-(20S)-1α,25-Dihydroxyvitamin D3) in Patients with Chronic Kidney Disease and Secondary Hyperparathyroidism on Hemodialysis

Co-reporter:Richa Pandey;Julia B. Zella;Jinge G. Zhu;Lori A. Plum

Drugs in R&D 2017 Volume 17( Issue 4) pp:597-605

Publication Date(Web):13 September 2017

DOI:10.1007/s40268-017-0210-z

2-Methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (DP001 or 2MD) is a novel, potent 1α-hydroxylated vitamin D analog that binds to the vitamin D receptor and suppresses parathyroid hormone synthesis and secretion with potential for an improved safety profile compared to existing active vitamin D analogs. The purpose of this study was to evaluate the pharmacokinetics of DP001 given orally after hemodialysis.DP001 (550 ng) was given orally to 11 hemodialysis patients with secondary hyperparathyroidism after each dialysis session (3 times/week) for 4 weeks. Pharmacokinetic analyses were performed after the first and final dose.After the first and final dose, the half-life of DP001 was similar (55.8 ± 13.0 and 50.8 ± 8.2 h, respectively). At 4 weeks, the time to maximum plasma concentration was 4.0 ± 0.8 h, with a concentration maximum of 3.4 ± 0.3 pg/mL. The area under the curve (0 to infinity) after the final dose was 204.3 ± 23.9 pg h/mL, and apparent volume of distribution was 2.03 ± 0.22 L/kg. At week 4, mean intact parathyroid hormone was suppressed 33% from the baseline (pre-dose) value (313 ± 52 vs 462 ± 39 pg/mL, respectively). No clinically significant changes from baseline values were found for vital signs, electrocardiogram measurements, or other laboratory parameters, including serum calcium and phosphorus.In hemodialysis patients, DP001 has a longer half-life than existing vitamin D therapies and enables control of parathyroid hormone when administered every 2–3 days on the day of dialysis. It is effective at a lower concentration maximum and area under the curve than other clinically available vitamin D compounds. DP001 may represent a therapeutic improvement over existing compounds due to rapid and extensive distribution to its target and its long half-life enabling sustained parathyroid hormone suppression. These studies support further evaluation of DP001 in longer-term treatment of secondary hyperparathyroidism.

Design, synthesis and biological properties of seco-d-ring modified 1α,25-dihydroxyvitamin D3 analogues

Co-reporter:Marcin Szybinski, Katarzyna Sektas, Rafal R. Sicinski, Lori A. Plum, Jadwiga Frelek, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology 2017 Volume 171(Volume 171) pp:

Publication Date(Web):1 July 2017

DOI:10.1016/j.jsbmb.2017.03.006

•Total synthesis of the CD-ring synthon was achieved.•19-Norvitamins D with the cleaved D ring were synthesized by Wittig-Horner coupling.•Analogue with an exomethylene group at C-2 showed increased biological activity in vitro.As a continuation of our efforts directed to the structure-activity relationship studies of vitamin D compounds, we present in this paper the synthesis of new analogues of 1α,25-(OH)2D3 characterized by numerous structural modifications, especially a cleaved D ring. Total synthesis of the CD fragment required for the construction of the target vitamins was based on the Stork approach. The structure of the key intermediate – bicyclic hydroxy lactone – was established by crystallographic and electronic circular dichroism (ECD) spectral analysis. Following the attachment of the hydroxyalkyl side chain, the formed D-seco Grundmann ketone was subjected to Wittig-Horner coupling with the corresponding A-ring phosphine oxides providing two desired D-seco analogues of 19-nor-1α,25-(OH)2D3, one without a substituent at C-2 and the other possessing a 2-exomethylene group. Both compounds were biologically tested and the latter was found to be more active in in vitro tests. Despite so many structural changes introduced in its structure, the biological activity of the 2-methylene analogue approached that of the natural hormone. The synthesized D-seco vitamins, however, proved to be inactive on bone and intestine in vivo.Download high-res image (91KB)Download full-size image

A Methylene Group on C-2 of 24,24-Difluoro-19-nor-1α,25-dihydroxyvitamin D3 Markedly Increases Bone Calcium Mobilization in Vivo

Co-reporter:Agnieszka Flores; Ilaria Massarelli; James B. Thoden; Lori A. Plum;Hector F. DeLuca

Journal of Medicinal Chemistry 2015 Volume 58(Issue 24) pp:9731-9741

Publication Date(Web):December 2, 2015

DOI:10.1021/acs.jmedchem.5b01564

Four side chain fluorinated analogues of 1α,25-dihydroxy-19-norvitamin D have been prepared in convergent syntheses using the Wittig–Horner reaction as a key step. Structures and absolute configurations of analogues 3 and 5 were confirmed by X-ray crystallography. All analogues showed high potency in HL-60 cell differentiation and vitamin D-24-hydroxylase (24-OHase) transcription as compared to 1α,25-dihydroxyvitamin D3 (1). Most important is that all of the 20S-configured derivatives (4 and 6) had high bone mobilizing activity in vivo. However, in the 20R series, a 2-methylene group was required for high bone mobilizing activity. A change in positioning of the 20R molecule in the vitamin D receptor when the 2-methylene group is present may provide new insight into the molecular basis of bone calcium mobilization induced by vitamin D.

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

Co-reporter:Izabela K. Sibilska; Marcin Szybinski; Rafal R. Sicinski; Lori A. Plum;Hector F. DeLuca

Journal of Medicinal Chemistry 2015 Volume 58(Issue 24) pp:9653-9662

Publication Date(Web):November 17, 2015

DOI:10.1021/acs.jmedchem.5b01295

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

Synthesis and Biological Activity of 25-Hydroxy-2-methylene-vitamin D3 Analogues Monohydroxylated in the A-ring

Co-reporter:Izabela K. Sibilska ; Rafal R. Sicinski ; Justin T. Ochalek ; Lori A. Plum ;Hector F. DeLuca

Journal of Medicinal Chemistry 2014 Volume 57(Issue 20) pp:8319-8331

Publication Date(Web):September 15, 2014

DOI:10.1021/jm500750b

The 20R- and 20S-isomers of 25-hydroxy-2-methylene-vitamin D3 and 3-desoxy-1α,25-dihydroxy-2-methylene-vitamin D3 have been synthesized. Two alternative synthetic routes were devised for preparation of the required A-ring synthons, starting from the chiral compound derived from the (−)-quinic acid and, alternatively, from the commercially available achiral precursor, monoprotected 1,4-cyclohexanedione. The A-ring dienynes were coupled by the Sonogashira process with the respective C,D-ring fragments, the enol triflates derived from the protected (20R)- or (20S)-25-hydroxy Grundmann ketones. All four compounds possessed significant in vivo activity on bone calcium mobilization and intestinal calcium transport. The presence of a 2-methylene group increased intestinal calcium transport activity of all four analogues above that of the native hormone, 1α,25-dihydroxyvitamin D3. In contrast, bone calcium mobilization was equal to that produced by 1α,25-dihydroxyvitamin D3 in compounds having a (20S)-configuration or diminished to one-tenth that of 1α,25-dihydroxyvitamin D3 in compounds with a (20R)-configuration.

26- and 27-Methyl groups of 2-substituted, 19-nor-1α,25-dihydroxylated vitamin D compounds are essential for calcium mobilization in vivo

Co-reporter:Pawel Grzywacz, Lori A. Plum, Margaret Clagett-Dame, Hector F. DeLuca

Bioorganic Chemistry 2013 Volume 47() pp:9-16

Publication Date(Web):April 2013

DOI:10.1016/j.bioorg.2013.01.001

Twelve new analogs of 19-nor-1α,25-dihydroxyvitamin D36–17, were prepared by a multi-step procedure from known alcohols 18 and 19. We have examined the influence of removing two methyl groups located at C-25, as well as the 25-hydroxy group, on the biological in vitro and in vivo biological activity. Surprisingly, removal of the 26- and 27-methyl groups from either the 2α-methyl or 2-methylene-19-nor-1α,25-dihydroxyvitamin D3 reduced vitamin D receptor binding, HL-60 differentiation, and 25-hydroxylase transcription in vitro only slightly to moderately (compounds 6–13). However, these compounds were devoid of in vivo bone mobilization activity and had markedly reduced activity on intestinal calcium transport. The analogs 14–17 with a 2β-methyl substitution had little or no activity in vitro and in vivo as expected from previous work.Graphical abstractHighlights► 26- and 27-Methyl groups of 19-nor-1,25(OH)2D required for Ca mobilization in vivo. ► A β-methyl on C-2 of 19-nor-1,25-(OH)2D eliminates in vitro and in vivo activity. ► 26,27-Methyls of 19-nor-1,25(OH)2D3 are not required for intestinal Ca transport.

CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo

Co-reporter:Glenville Jones;Jinge G. Zhu;Hector F. DeLuca;Martin Kaufmann;Justin T. Ochalek

PNAS 2013 Volume 110 (Issue 39 ) pp:15650-15655

Publication Date(Web):2013-09-24

DOI:10.1073/pnas.1315006110

Bioactivation of vitamin D consists of two sequential hydroxylation steps to produce 1α,25-dihydroxyvitamin D3. It is clear that the second or 1α-hydroxylation step is carried out by a single enzyme, 25-hydroxyvitamin D 1α-hydroxylase CYP27B1. However, it is not certain what enzyme or enzymes are responsible for the initial 25-hydroxylation. An excellent case has been made for vitamin D 25-hydroxylase CYP2R1, but this hypothesis has not yet been tested. We have now produced Cyp2r1 −/− mice. These mice had greater than 50% reduction in serum 25-hydroxyvitamin D3. Curiously, the 1α,25-dihydroxyvitamin D3 level in the serum remained unchanged. These mice presented no health issues. A double knockout of Cyp2r1 and Cyp27a1 maintained a similar circulating level of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Our results support the idea that the CYP2R1 is the major enzyme responsible for 25-hydroxylation of vitamin D, but clearly a second, as-yet unknown, enzyme is another contributor to this important step in vitamin D activation.

A 20S Combined with a 22R Configuration Markedly Increases both in Vivo and in Vitro Biological Activity of 1α,25-Dihydroxy-22-methyl-2-methylene-19-norvitamin D3

Co-reporter:Agnieszka Flores ; Rafal R. Sicinski ; Pawel Grzywacz ; James B. Thoden ; Lori A. Plum ; Margaret Clagett-Dame ;Hector F. DeLuca

Journal of Medicinal Chemistry 2012 Volume 55(Issue 9) pp:4352-4366

Publication Date(Web):April 10, 2012

DOI:10.1021/jm300187x

Six new analogues of 1α,25-dihydroxy-19-norvitamin D3 (3a–4b, 5, and 6) were prepared by a convergent synthesis applying the Wittig–Horner reaction as a key step. The influence of methyl groups at C-22 on their biological activity was examined. It was established that both in vitro and in vivo activity is strongly dependent on the configuration of the stereogenic centers at C-20 and C-22. Introduction of the second methyl group at C-22 (analogues 5 and 6) generates the compounds that are slightly more potent than 1α,25-(OH)2D3 in the in vitro tests but much less potent in vivo. The greatest in vitro and in vivo biological activity was achieved when the C-20 is in the S configuration and the C-22 is in the R configuration. The building blocks for the synthesis, the respective (20R,22R)-, (20R,22S)-, (20S,22R)-, and (20S,22S)-diols, were obtained by fractional crystallization of mixtures of the corresponding diastereomers. Structures and absolute configurations of the diols 21a, 21b, and 22a as well as analogues 3a, 5, and 6 were confirmed by the X-ray crystallography.

Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice

Co-reporter:Atsushi Arai;Yasuhiro Kobayashi;Toshihide Mizoguchi;Josef M. Penninger;Yuko Nakamichi;Hisataka Yasuda;Shigeaki Kato;Masahiro Sato;Tatsuo Suda;Hector F. DeLuca;Nobuyuki Udagawa;Naoyuki Takahashi

PNAS 2012 Volume 109 (Issue 25 ) pp:10006-10011

Publication Date(Web):2012-06-19

DOI:10.1073/pnas.1207361109

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1–mutated CSF-1op/op mice as well as RANKL−/− mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL−/− mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1op/op mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1op/op mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1–induced osteoclastogenesis in CSF-1op/op mice. Osteoclasts appeared in aged CSF-1op/op mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)2D3 (2MD), a potent analog of 1α,25-dihidroxyvitamin D3, into CSF-1op/op mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1op/op mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.

Development of experimental autoimmune encephalomyelitis (EAE) in mice requires vitamin D and the vitamin D receptor

Co-reporter:Yanping Wang;Jinge G. Zhu;Kyle S. Severson;Steven J. Marling;Hector F. DeLuca

PNAS 2012 Volume 109 (Issue 22 ) pp:8501-8504

Publication Date(Web):2012-05-29

DOI:10.1073/pnas.1206054109

The development of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, has been studied in mice that were (i) vitamin D-deficient, (ii) minus the vitamin D receptor, (iii) minus a vitamin D 25-hydroxylase, and (iv) minus the vitamin D 25-hydroxyvitamin D-1α-hydroxylase. EAE development was markedly suppressed in mice lacking the vitamin D receptor and partially suppressed in vitamin D-insufficient mice. However, the absence of either of the two key hydroxylases (i.e., 25-hydroxylase and 1α-hydroxylase) neither inhibits nor enhances the development of EAE. These results indicate that vitamin D and the vitamin D receptor are required for the development of EAE. The results also suggest that 1,25-dihydroxyvitamin D3 may not play a role in this autoimmune response.

Synthesis and Biological Activity of 2-(3′-Hydroxypropylidene)-1α-hydroxy-19-norvitamin D Analogues with Shortened Alkyl Side Chains

Co-reporter:Agnieszka Glebocka ; Rafal R. Sicinski ; Lori A. Plum ;Hector F. DeLuca

Journal of Medicinal Chemistry 2011 Volume 54(Issue 19) pp:6832-6842

Publication Date(Web):September 8, 2011

DOI:10.1021/jm200743p

As a continuation of our efforts directed to vitamin D compounds of promising biological properties, 19-norvitamins 9–13, possessing a 3′-hydroxypropylidene fragment attached to C-2 and shortened 17β-alkyl chains, were synthesized. A new synthetic pathway providing the CD-ring ketones 20–24 is described starting from the epimeric aldehydes 25 and 26. The hydrindanones 20–24 were subjected to the Wittig–Horner reaction with the phosphine oxide 14, and the vitamin D compounds 9–13 were obtained after hydroxyl deprotection. In comparison to 1α,25-(OH)2D3 (1), the prepared analogues, except for the 20R-compound 12, were only ca. 3 times less potent in binding to VDR. Compounds 9–11 and 13 exhibited HL-60 cellular activity 5–20 times lower and transcriptional activity ca. 10 times decreased related to those for the hormone 1. When tested in vivo, all the analogues showed no ability to mobilize calcium from bone, and intestinal calcium transport activity was observed only at high doses of the vitamins 10, 12, and 13.

13,13-Dimethyl-des-C,D analogues of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): Total synthesis, docking to the VDR, and biological evaluation

Co-reporter:Katarzyna Plonska-Ocypa, Izabela Sibilska, Rafal R. Sicinski, Wanda Sicinska, Lori A. Plum, Hector F. DeLuca

Bioorganic & Medicinal Chemistry 2011 Volume 19(Issue 23) pp:7205-7220

Publication Date(Web):1 December 2011

DOI:10.1016/j.bmc.2011.09.048

As a continuation of our studies focused on the vitamin D compounds lacking the C,D-hydrindane system, 13,13-dimethyl-des-C,D analogues of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2, 2MD) were prepared by total synthesis. The known cyclohexanone 30, a precursor of the desired A-ring phosphine oxide 11, was synthesized starting with the keto acetal 13, whereas the aldehyde 12, constituting an acyclic ‘upper’ building block, was obtained from the isomeric esters 34, prepared previously in our laboratory.The commercial 1,4-cyclohexanedione monoethylene ketal (13) was enantioselectively α-hydroxylated utilizing the α-aminoxylation process catalyzed by l-proline, and the introduced hydroxy group was protected as a TBS, TPDPS, and SEM ether. Then the keto group in the obtained compounds 15–17 was methylenated and the allylic hydroxylation was performed with selenium dioxide and pyridine N-oxide. After separation of the isomers, the newly introduced hydroxy group was protected and the ketal group hydrolyzed to yield the corresponding protected (3R,5R)-3,5-dihydroxycyclohexanones 30–32. The esters 34, starting compounds for the C,D-fragment 12, were first α-methylated, then reduced and the resulted primary alcohols 36 were deoxygenated using the Barton-McCombie protocol. Primary hydroxy group in the obtained diether 38 was deprotected and oxidized to furnish the aldehyde 12. The Wittig–Horner coupling of the latter with the anion of the phosphine oxide 11, followed by hydroxyl deprotection furnished two isomeric 13,13-dimethyl-des-C,D analogues of 2MD (compounds 10 and 42) differing in configuration of their 7,8-double bond. Pure vitamin D analogues were isolated by HPLC and their biological activity was examined. The in vitro tests indicated that, compared to the analogue 7, unsubstituted at C-13, the synthesized vitamin D analogue 10 showed markedly improved VDR binding ability, significantly enhanced HL-60 differentiation activity as well as increased transcriptional potency. Docking simulations provided a rational explanation for the observed binding affinity of these ligands to the VDR. Biological in vivo tests proved that des-C,D compound 10 retained some intestinal activity. Its geometrical isomer 42 was devoid of any biological activity.

Vitamin D, disease and therapeutic opportunities

Co-reporter:Lori A. Plum & Hector F. DeLuca

Nature Reviews Drug Discovery 2010 9(12) pp:941

Publication Date(Web):2010-12-01

DOI:10.1038/nrd3318

The discovery of the vitamin D endocrine system and a receptor for the hormonal form, 1α,25-dihydroxyvitamin D3, has brought a new understanding of the relationship between vitamin D and metabolic bone diseases, and has also established the functions of vitamin D beyond the skeleton. This has ushered in many investigations into the possible roles of vitamin D in autoimmune diseases, cardiovascular disorders, infectious diseases, cancers and granuloma-forming diseases. This article presents an evaluation of the possible roles of vitamin D in these diseases. The potential of vitamin D-based therapies in treating diseases for which the evidence is most compelling is also discussed.

Removal of the 26-Methyl Group from 19-nor-1α,25-Dihydroxyvitamin D3 Markedly Reduces in Vivo Calcemic Activity without Altering in Vitro VDR Binding, HL-60 Cell Differentiation, and Transcription

Co-reporter:Pawel Grzywacz ; Grazia Chiellini ; Lori A. Plum ; Margaret Clagett-Dame ;Hector F. DeLuca

Journal of Medicinal Chemistry 2010 Volume 53(Issue 24) pp:8642-8649

Publication Date(Web):November 24, 2010

DOI:10.1021/jm1010447

Twelve new analogues of 19-nor-1α,25-dihydroxyvitamin D3 (5−16) were prepared by convergent syntheses, employing the Wittig−Horner reaction. The necessary Grundmann type ketones (45−48), possessing fixed configurations of the hydroxyl group at C-25, were obtained by a multistep procedure from commercial vitamin D2 and enantiomers of 1,3-butanediol (23 and 24). We have examined the influence of removal of one of the methyl groups located at C-25 on the biological in vitro and in vivo activity. The in vivo tests showed that the synthesized vitamin D compounds (5−16) exhibit reduced calcemic activity both in bone and in the intestine. However, in vitro potency of 2-methylene and 2α-methyl compounds (5−10, 13, and 14) remained similar or enhanced as compared to that of 1α,25-(OH)2D3.

Screening of Selective Inhibitors of 1α,25-Dihydroxyvitamin D3 24-Hydroxylase Using Recombinant Human Enzyme Expressed in Escherichia coli

Co-reporter:Jinge Zhu, Rafal Barycki, Grazia Chiellini, and Hector F. DeLuca

Biochemistry 2010 Volume 49(Issue 49) pp:

Publication Date(Web):November 8, 2010

DOI:10.1021/bi101488p

High-level heterologous expression of human 1α,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) in Escherichia coli was attained via a fusion construct by appending the mature CYP24A1 without the leader sequence to the maltose binding protein (MBP). Facile purification was achieved efficiently through affinity chromatography and afforded fully functional enzyme of near homogeneity, with a kcat of 0.12 min−1 and a KM of 0.19 μM toward 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. A convenient and reliable cell-free assay was established and used to screen vitamin D analogues with potential inhibitory properties toward CYP24A1. Some of the compounds exhibited potent inhibition with KI values as low as 0.021 μM. Furthermore, TS17 and CPA1 exhibited superior specificity toward CYP24A1 over 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1), with selectivities of 39 and 80, respectively. Addition of TS17 or CPA1 to a mouse osteoblast culture sustained the level of 1,25(OH)2D3 in the medium. Their activities in vitamin D receptor (VDR) binding, CYP24A1 transcription, and HL-60 cell differentiation were evaluated as well.

Reply to Handunnetthi and Ramagopalan: UV radiation, experimental autoimmune encephalomyelitis, and multiple sclerosis

Co-reporter:Bryan R. Becklund;Kyle S. Severson;Souriya V. Vang;Hector F. DeLuca

PNAS 2010 107 (33 ) pp:E131

Publication Date(Web):2010-08-17

DOI:10.1073/pnas.1008059107

UV radiation suppresses experimental autoimmune encephalomyelitis independent of vitamin D production

Co-reporter:Bryan R. Becklund;Kyle S. Severson;Souriya V. Vang;Hector F. DeLuca;

Proceedings of the National Academy of Sciences 2010 107(14) pp:6418-6423

Publication Date(Web):March 22, 2010

DOI:10.1073/pnas.1001119107

Although the exact cause of multiple sclerosis (MS) is unknown, a number of genetic and environmental factors are thought to influence MS susceptibility. One potential environmental factor is sunlight and the subsequent production of vitamin D. A number of studies have correlated decreased exposure to UV radiation (UVR) and low serum 25-hydroxyvitamin D₃ [25(OH)D₃] levels with an increased risk for developing MS. Furthermore, both UVR and the active form of vitamin D, 1α,25-dihydroxyvitamin D₃, suppress disease in the experimental autoimmune encephalomyelitis (EAE) animal model of MS. These observations led to the hypothesis that UVR likely suppresses disease through the increased production of vitamin D. However, UVR can suppress the immune system independent of vitamin D. Therefore, it is unclear whether UVR, vitamin D, or both are necessary for the putative decrease in MS susceptibility. We have probed the ability of UVR to suppress disease in the EAE model of MS and assessed the effect of UVR on serum 25(OH)D₃ and calcium levels. Our results indicate that continuous treatment with UVR dramatically suppresses clinical signs of EAE. Interestingly, disease suppression occurs with only a modest, transient increase in serum 25(OH)D₃ levels. Further analysis demonstrated that the levels of 25(OH)D₃ obtained upon UVR treatment were insufficient to suppress EAE independent of UVR treatment. These results suggest that UVR is likely suppressing disease independent of vitamin D production, and that vitamin D supplementation alone may not replace the ability of sunlight to reduce MS susceptibility.

New 1α,25-Dihydroxy-19-norvitamin D3 Compounds Constrained in a Single A-Ring Conformation: Synthesis of the Analogues by Ring-Closing Metathesis Route and Their Biological Evaluation

Co-reporter:Agnieszka Glebocka ; Katarzyna Sokolowska ; Rafal R. Sicinski ; Lori A. Plum ;Hector F. DeLuca

Journal of Medicinal Chemistry 2009 Volume 52(Issue 11) pp:3496-3504

Publication Date(Web):April 29, 2009

DOI:10.1021/jm9001583

Vitamin D compounds possessing A rings prohibited from flipping to the alternative chair form (i.e., analogues 2 and 26) were synthesized. The bicyclic fragment 22 consisting of the fused cyclohexane and dihydropyran rings was constructed via the ring-closing metathesis route. Also, a homologous synthon 23 with an attached dihydropyran ring was successfully synthesized using this strategy. The carbonyl deprotection in 22 yielded cyclohexanone 5 that was subjected to Julia coupling with the anion of the phenylthiazoline sulfone 25. In the resulting isomeric 19-norvitamins 2 and 26, their A rings can exist only in the α- and β-conformation. The analogue 26 was 300 times more active in binding to the vitamin D receptor protein, 30 times more effective in causing HL-60 differentiation, and 10 times more active in transcription. These results confirm that the β-chair form of the vitamin D ring A is necessary for the binding to the receptor.

13-Methyl-substituted des-C,D analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): Synthesis and biological evaluation

Co-reporter:Katarzyna Plonska-Ocypa, Rafal R. Sicinski, Lori A. Plum, Pawel Grzywacz, Jadwiga Frelek, Margaret Clagett-Dame, Hector F. DeLuca

Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 4) pp:1747-1763

Publication Date(Web):15 February 2009

DOI:10.1016/j.bmc.2008.11.082

Analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2, 2MD), substituted at C-13 but lacking both C and D rings, were prepared in convergent syntheses, starting with the chiral ester 14 and the phosphine oxide 29. Two of the synthesized vitamins (11 and 32) were analogs in which the 13-methyl group constituted a substituent of an unsaturated fragment, that is, C(13)–C(17) double bond, whereas in the two other cases (12 and 13), the methyl group belonged to a ternary carbon stereogenic center. The aim of these studies was to further explore extensive modifications in the ‘upper’ part of the vitamin D skeleton in the hope of finding biologically active analogs of potential therapeutic value.The commercial (R)-(−)-methyl-3-hydroxy-2-methylpropionate (14) was converted in six steps to alcohol 18, the vitamin D side chain fragment. Its subsequent three-step transformation led to aldehyde 20 which was subjected to the Still–Gennari HWE reaction with anion derived from ester 21. The obtained α,β-unsaturated esters 22 and 23 served as convenient starting compounds to the syntheses of the corresponding chiral acyclic aldehydes, β,γ-unsaturated (28) and saturated (39 and 40), required for the final Wittig–Horner coupling with the anion of the phosphine oxide 29. After hydroxyl deprotection, the synthesized vitamin D analogs 11–13 and 32 were purified and biologically tested. Only the (13R,20S)-analog 12 retained substantial, although 30 times lower than 1α,25-(OH)2D3, binding ability to the full-length rat recombinant vitamin D receptor (VDR). This analog was also very effective in differentiation of HL-60 cells, and it exerted significant transcriptional activity (2 times and 15 times less potent, respectively, as compared to the native hormone). The in vivo tests showed that all synthesized vitamin D analogs were devoid of calcemic activity.

Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) selectively eliminates bone calcium mobilization activity

Co-reporter:Rafal Barycki, Rafal R. Sicinski, Lori A. Plum, Pawel Grzywacz, Margaret Clagett-Dame, Hector F. DeLuca

Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 22) pp:7658-7669

Publication Date(Web):15 November 2009

DOI:10.1016/j.bmc.2009.09.047

The 18-nor (7), 21-nor (8) and 18,21-dinor (9) analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (6, 2MD) were prepared by convergent syntheses. The known phosphine oxide 10 was coupled by the Wittig–Horner process with the corresponding C,D-fragments (13–15), obtained by a multi-step procedure from commercial vitamin D2. The goal of our studies was to examine the influence of removal of the methyl groups located at carbons 13 and 20 on the biological potency of 2MD in the hope of finding analogs with improved therapeutic profiles.Replacement of the 20-methyl with hydrogen in 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) did not affect binding to the rat vitamin D receptor and had little effect on transcription activity and on HL-60 differentiation. However, the mobilization of calcium from bone was largely eliminated while intestinal calcium transport remained strong. Curiously, removal of both the C-13-methyl and 20-methyl restored slightly the bone calcium mobilizing activity. Thus, the 21-nor analog of 2MD may provide a potent analog with a greater margin of safety than 2MD.

The Functional Metabolism and Molecular Biology of Vitamin D Action

Co-reporter:Lori A. Plum;Hector F. DeLuca

Clinical Reviews in Bone and Mineral Metabolism 2009 Volume 7( Issue 1) pp:20-41

Publication Date(Web):2009 March

DOI:10.1007/s12018-009-9040-z

The evolution of our understanding of the biological impact of vitamin D is briefly reviewed, with a focus on the physiology and endocrinology of the vitamin D system. This chapter attempts to bring the molecular discoveries in vitamin D metabolism and mechanisms of action into focus on known physiology and endocrinology. The latest developments on metabolism of vitamin D, the enzymes involved, and the genes responsible are presented. The impact of the molecular discoveries on current views of the importance of vitamin D in public health is also presented.

Enhancement of 1,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis by calcitonin

Co-reporter:Donald W. Hansen, Jr.;Bryan R. Becklund;Hector F. DeLuca

PNAS 2009 Volume 106 (Issue 13 ) pp:5276-5281

Publication Date(Web):2009-03-31

DOI:10.1073/pnas.0813312106

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], suppresses disease development in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). However, complete disease prevention only occurs with doses that dramatically elevate serum calcium levels, thus limiting the usefulness of 1,25(OH)2D3 as a potential MS therapeutic agent. Because calcitonin (CT) is believed to be released by hypercalcemia and has been shown to be anti-inflammatory, we examined whether suppression of EAE by 1,25(OH)2D3 could be mediated either in part or entirely by CT. Continuous administration of pharmacological doses of CT did not prevent EAE. However, a combination of CT and a subtherapeutic dose of 1,25(OH)2D3 additively suppressed EAE without causing hypercalcemia. Moreover, CT decreased the dose of 1,25(OH)2D3 required for disease suppression. Our results suggest that CT may be a significant factor but cannot account entirely for 1,25(OH)2D3-mediated suppression of EAE.

TRPV6 is not required for 1α,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo

Co-reporter:Galina D. Kutuzova;Bulli Padmaja Tadi;Flora Sundersingh;Jennifer Vaughan;Susan E. Ansay;Sylvia Christakos;Hector F. DeLuca

PNAS 2008 Volume 105 (Issue 50 ) pp:19655-19659

Publication Date(Web):2008-12-16

DOI:10.1073/pnas.0810761105

The requirement for TRPV6 for vitamin D-dependent intestinal calcium absorption in vivo has been examined by using vitamin D-deficient TRPV6 null mice and littermate wild-type mice. Each of the vitamin D-deficient animals received each day for 4 days 50 ng of 1,25-dihydroyvitamin D3 in 0.1 ml of 95% propylene glycol:5% ethanol vehicle or vehicle only. Both the wild-type and TRPV6 null mice responded equally well to 1,25-dihydroxyvitamin D3 in increasing intestinal calcium absorption. These results, along with our microarray data, demonstrate that TRPV6 is not required for vitamin D-induced intestinal calcium absorption and may not carry out a significant role in this process. These and previous results using calbindin D9k null mutant mice illustrate that molecular events in the intestinal calcium absorption process in response to the active form of vitamin D remain to be defined.

Lithocholic acid can carry out in vivo functions of vitamin D

Co-reporter:Claudia Zierold;Jamie A. Nehring;Hector F. DeLuca

PNAS 2007 Volume 104 (Issue 24 ) pp:10006-10009

Publication Date(Web):2007-06-12

DOI:10.1073/pnas.0703512104

The physiological ligand for the vitamin D receptor (VDR) is 1,25-dihydroxyvitamin D3. Lithocholic acid (LCA), a bile acid implicated in the progression of colon cancer, was recently shown to bind to VDR with low affinity and increase expression of the xenobiotic enzymes of the CYP3A family. Thus, LCA can induce its own catabolism through the VDR. We have now found that LCA can substitute for vitamin D in the elevation of serum calcium in vitamin D-deficient rats. Further, LCA in the diet will also replace vitamin D in the mobilization of calcium from bone. Further, LCA induces CYP24-hydroxylase mRNA gene expression in the kidney of vitamin D-deficient rats. It is clear, therefore, that LCA can be absorbed into the circulation to bind to the VDR at extra-intestinal sites. These findings lend support for the idea that the VDR may have evolved from an original role in detoxification.

CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1α-hydroxylase promoter activity in the skin

Co-reporter:Janeen L. Vanhooke;Jean M. Prahl;Christine Kimmel-Jehan;Monica Mendelsohn;Eric W. Danielson;Kevin D. Healy;Hector F. DeLuca;

Proceedings of the National Academy of Sciences 2006 103(1) pp:75-80

Publication Date(Web):December 21, 2005

DOI:10.1073/pnas.0509734103

The hormonally active form of vitamin D₃,1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is synthesized in the kidney through a tightly regulated reaction catalyzed by 25-hydroxyvitamin D₃-1α-hydroxylase (1α-hydroxylase), the product of the CYP27B1 gene. Through gene targeting in embryonic stem cells, we engineered a mouse strain in which the coding region of the 1α-hydroxylase gene is replaced by the genes for β-galactosidase (lacZ) and neomycin resistance. Null mice produced no detectable 1α-hydroxylase transcript. The mice grew normally when maintained on a balanced diet containing 1,25(OH)₂D₃ but rapidly developed rickets when phosphorus and 1,25(OH)₂D₃ were restricted. Rickets was curable through administration of 1,25(OH)₂D₃ but not its biological precursor, 25-hydroxyvitamin D₃. Upon administration of a diet low in calcium and devoid of any form of vitamin D₃, β-galactosidase activity was detected in the kidneys of the –/– and +/– mice and in placentas harvested from –/– females bred with –/– males. No β-galactosidase activity was detected in skin sections or in primary keratinocyte cultures from –/– animals. Our results demonstrate we have generated 1α-hydroxylase null mice that display phenotypes characteristic of vitamin D-dependency rickets type I. From the histochemical analysis of reporter gene expression in these mice, we conclude that acute 1,25(OH)₂D₃ deficiency in otherwise healthy animals does not stimulate local production of 1,25(OH)₂D₃ in the skin. These findings stand in contrast to previously published reports of 1,25(OH)₂D₃ production in keratinocytes.

Calbindin D9k knockout mice are indistinguishable from wild-type mice in phenotype and serum calcium level

Co-reporter:Galina D. Kutuzova;Shirin Akhter;Sylvia Christakos;Janeen Vanhooke;Christine Kimmel-Jehan;Hector F. DeLuca

PNAS 2006 Volume 103 (Issue 33 ) pp:12377-12381

Publication Date(Web):2006-08-15

DOI:10.1073/pnas.0605252103

Since the discovery of calbindin D9k, its role in intestinal calcium absorption has remained unsettled. Further, a wide distribution of calbindin D9k among tissues has argued for its biological importance. We discovered a frameshift deletion in the calbindin D9k gene in an ES cell line, E14.1, that originated from 129/OlaHsd mice. We produced mice with the mutant calbindin D9k gene by injecting the E14.1 ES cell subline into the C57BL/6 host blastocysts and proved that these mice lack calbindin D9k protein. Calbindin D9k knockout mice were indistinguishable from wild-type mice in phenotype, were able to reproduce, and had normal serum calcium levels. Thus, calbindin D9k is not required for viability, reproduction, or calcium homeostasis.

Parathyroid hormone decreases renal vitamin D receptor expression in vivo

Co-reporter:Kevin D. Healy;Janeen L. Vanhooke;Jean M. Prahl;Hector F. DeLuca

PNAS 2005 Volume 102 (Issue 13 ) pp:4724-4728

Publication Date(Web):2005-03-29

DOI:10.1073/pnas.0501312102

The vitamin D receptor (VDR) is a nuclear transcription factor responsible for mediating the biological activities of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Renal and parathyroid gland VDR content is an important factor in calcium homeostasis, vitamin D metabolism, and the treatment of secondary hyperparathyroidism and renal osteodystrophy. In these tissues, VDR expression is highly regulated by the calcium and vitamin D status. Although 1,25(OH)2D3 up-regulates VDR expression, hypocalcemia and vitamin D deficiency result in drastically reduced expression of the receptor. The generation of 25-hydroxyvitamin D3-1α-hydroxylase-null mice, which are incapable of endogenously producing 1,25(OH)2D3, has allowed us to investigate the influence of parathyroid hormone (PTH) on VDR expression independent of PTH-mediated increases in 1,25(OH)2D3. Administration of human PTH (1-34) (110 μg/kg per day) for 48 h reduced renal VDR levels from 515 to 435 fmol/mg protein (15%, P < 0.03) in wild-type mice. In the 25-hydroxyvitamin D3-1α-hydroxylase-null mice, PTH administration strongly reduced renal VDR levels, from 555 to 394 fmol/mg protein (29%, P < 0.001). These results demonstrate that PTH is a potent down-regulator of VDR expression in vivo.

Biologically active noncalcemic analogs of 1α,25-dihydroxyvitamin D with an abbreviated side chain containing no hydroxyl

Co-reporter:Lori A. Plum;Jean M. Prahl;Xiaohong Ma;Rafal R. Sicinski;Sumithra Gowlugari;Margaret Clagett-Dame;Hector F. DeLuca;

Proceedings of the National Academy of Sciences 2004 101(18) pp:6900-6904

Publication Date(Web):April 26, 2004

DOI:10.1073/pnas.0401656101

Since the discovery of the active metabolite of vitamin D, i.e., 1α,25-dihydroxyvitamin D₃, there has been a continuous effort to synthesize analogs able to carry out many of the functions of the native hormone without raising serum calcium concentration. The present report provides a series of previously undescribed analogs wherein this goal is realized. We have prepared 2-methylene-19-nor-1α-hydroxyvitamin D analogs of 1,25-(OH)₂D₃ that possess only two to four carbons of the side chain without a hydroxyl thereon. Compared to 1,25-(OH)₂D₃, these analogs are slightly less active in binding to the vitamin D receptor, in causing HL-60 differentiation, and are slightly less active in in vitro transcription assays using the 24-hydroxylase promoter attached to a luciferase reporter gene. Of considerable importance is that these analogs, given to rats at daily doses of up to 70 μg/kg of body weight per day, are either unable or only slightly able to raise serum calcium concentration but are nevertheless able to suppress parathyroid hormone levels in plasma up to 100% and induce 24-hydroxylase mRNA in skin. Because of their ability to act in vivo without raising serum calcium levels, they may be of considerable interest for the systemic treatment of diseases such as psoriasis, cancer, and secondary hyperparathyroidism of renal failure, where a rise in serum calcium is undesirable.

Vitamin D and autoimmune diabetes

Co-reporter:Julia B. Zella;Hector F. DeLuca

Journal of Cellular Biochemistry 2003 Volume 88(Issue 2) pp:216-222

Publication Date(Web):31 OCT 2002

DOI:10.1002/jcb.10347

The biologically active form of vitamin D, 1,25(OH)₂D₃, is a potent modulator of the immune system as well as a regulator of bone and mineral metabolism. Vitamin D-deficiency in infancy and vitamin D receptor gene polymorphisms may be risk factors for insulin-dependent Diabetes mellitus (IDDM). 1,25(OH)₂D₃ and its analogs significantly repress the development of insulitis and diabetes in the non-obese diabetic (NOD) mouse, a model of human IDDM. 1,25(OH)₂D₃ may modulate IDDM disease pathogenesis by repression of type I cytokines, inhibition of dendritic cell maturation, and upregulation of regulatory T cells. The function of vitamin D as a genetic and environmental determining factor for IDDM, the protective role of 1,25(OH)₂D₃ and its analogs in a mouse model of IDDM, and the possible mechanisms by which this protection occurs will be reviewed. J. Cell. Biochem. 88: 216–222, 2003. © 2002 Wiley-Liss, Inc.

Regulation of 25-hydroxyvitamin D3-24-hydroxylase mRNA by 1,25-dihydroxyvitamin D3 and parathyroid hormone

Co-reporter:Claudia Zierold;Jamie A. Mings;Hector F. DeLuca

Journal of Cellular Biochemistry 2003 Volume 88(Issue 2) pp:234-237

Publication Date(Web):31 OCT 2002

DOI:10.1002/jcb.10341

The 25-hydroxyvitamin D₃-24-hydroxylase mRNA is tightly and reciprocally regulated by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and parathyroid hormone (PTH). The upregulation of the 24-hydroxylase by 1,25(OH)₂D₃ is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, this induction is blocked by the protein synthesis inhibitor cycloheximide (CHX) indicating a protein component in the regulation pathway. CHX treatment reduced total vitamin D receptor (VDR) protein levels in cells, but reintroduction of VDR and/or retinoid X receptor protein into cells by transfection did not reduce the inhibition by CHX. This indicates that production of another transcription factor or mRNA-stabilizing protein synthesized in response to 1,25(OH)₂D₃ is required for optimal accumulation of 24-hydroxylase mRNA. PTH downregulates the 24-hydroxylase mRNA by affecting its stability. The half-life of 24-hydroxylase mRNA is reduced 4.2-fold in AOK-B50 cells by PTH. Untranslated regions of the 24-hydroxylase mRNA in reporter gene assays did not confer PTH responsiveness. Further analysis of the coding region of the rat 24-hydroxylase may reveal sites of action of PTH. J. Cell. Biochem. 88: 234–237, 2003. © 2002 Wiley-Liss, Inc.

CD8+ T cells are not necessary for 1α,25-dihydroxyvitamin D3 to suppress experimental autoimmune encephalomyelitis in mice

Co-reporter:Hector F. DeLuca;Terrence F. Meehan

PNAS 2002 Volume 99 (Issue 8 ) pp:5557-5560

Publication Date(Web):2002-04-16

DOI:10.1073/pnas.082100699

In addition to its role in calcium and phosphorous homeostasis, 1α,25-dihydroxyvitamin D3 [1,25-(OH)2D3] appears to be a modulator of the immune system. Administration of 1,25-(OH)2D3 prevents disease in several autoimmune animal models, including experimental autoimmune encephalomyelitis (EAE). The vitamin D receptor is believed to mediate this activity. Among cells of the immune system, CD8+ T cells have the highest levels of the vitamin D receptor. Because CD8+ T cells have been implicated as both suppressors and effectors of the inflammation associated with multiple sclerosis and EAE, we examined the question of whether the 1,25-(OH)2D3 suppression of EAE occurs through a CD8+ T cell-dependent mechanism. To test this hypothesis, mice that are homozygous knockouts for the α chain of the CD8 receptor and have been characterized as lacking functional CD8+ T cells (CD8+ −/−) were provided 1,25-(OH)2D3 in their diet before EAE induction. Although CD8+ −/− mice fed the same diet lacking 1,25-(OH)2D3 have a high incidence of EAE, EAE did not occur in CD8+ −/− mice fed the diet containing 1,25-(OH)2D3. We conclude that CD8+ T cells neither are needed nor do they play a role in the prevention of EAE by 1,25-(OH)2D3.

Parathyroid hormone regulates 25-hydroxyvitamin D3-24-hydroxylase mRNA by altering its stability

Co-reporter:Claudia Zierold;Hector F. DeLuca;Jamie A. Mings

PNAS 2001 Volume 98 (Issue 24 ) pp:13572-13576

Publication Date(Web):2001-11-20

DOI:10.1073/pnas.241516798

The up-regulation of the 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, the mechanism of down-regulation of the 24-hydroxylase by parathyroid hormone (PTH) has not yet been elucidated. To study the mechanism of PTH action, we used AOK-B50 cells, a porcine kidney-cell line with stably transfected opossum PTH receptor in which both the 24-hydroxylase mRNA and activity are down-regulated by PTH. Cells dosed with 1,25(OH)2D3 at 0 h, and subsequently at 0, 1, 2, or 4 h with 100 nM of PTH, showed levels of 24-hydroxylase mRNA equivalent to 72.6, 65.3, 57.2, and 37.1%, respectively, of the levels found in cells dosed with 1,25(OH)2D3 only. All cells were collected 7 h after the initial 1,25(OH)2D3 dose. This pattern of expression indicated that PTH does not act by repressing transcription but rather by making the mRNA for 24-hydroxylase susceptible to degradation. At least 1 h is required for PTH to act. Further RNA and protein syntheses are required for PTH to act. However, the sites and mechanism whereby PTH causes 24-hydroxylase mRNA degradation are unknown. Because the untranslated regions of genes can determine the stability of its transcripts, we studied the 5′ untranslated region and the 3′ untranslated region of the rat 24-hydroxylase gene by using reporter-gene strategy to identify possible PTH sites of action. None was found, suggesting that the destabilization site is elsewhere in the coding region.

Isolation and characterization of the chicken vitamin D receptor gene and its promoter

Co-reporter:Zhongjian Lu;Frederic Jehan;Claudia Zierold;Hector F. DeLuca

Journal of Cellular Biochemistry 2000 Volume 77(Issue 1) pp:92-102

Publication Date(Web):16 FEB 2000

DOI:10.1002/(SICI)1097-4644(20000401)77:1<92::AID-JCB10>3.0.CO;2-N

The sequences from several independent cDNA clones encoding the chicken vitamin D receptor as well as primer extension assay have clearly delineated the 5′ terminus and the transcriptional start site. Screening a chicken genomic library produced genomic clones containing vitamin D receptor (VDR) gene fragments. Restriction map of clone 8 showed that the 18.6-kb chicken VDR fragment has exons 1 and 2, intron 1, part of intron 2, and 7-kb 5′ flanking region. Exons 1, 2 , and 3 found in the chicken VDR gene shares low homology with its mammalian counterparts (i.e., E1A, E1B, and E1C in human). By contrast, the fourth exon and following exons for the coding region of VDR gene are highly conserved between avian and mammalian species. While the fourth exon bears the ATG sites for translation initiation in mammals, the third exon in birds has two extra ATG sites for leaky translation as determined previously. Thus, the avian VDR has more N-terminal sequence than the mammalian VDR and is found in two distinct forms. The 5′ flanking region from genomic clone 8 shares considerable homology in several regions with the human and mouse VDR promoters. Moreover, the 5′ flanking region of chicken VDR gene possesses promoter activity, as shown by its ability to drive the luciferase reporter gene in cell transfection assays. Like other steroid receptor promoters, the chicken VDR promoter contains no TATA box but possesses several GC boxes or SP1 sites. A series of deletional promoter constructs established that the proximal GC boxes are the major drivers of gene transcription, while the more upstream sequences have repressive elements. J. Cell. Biochem. 77:92–102, 2000. © 2000 Wiley-Liss, Inc.

DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements

Co-reporter:Hisham M. Darwish;Steve A. Strugnell;Frédéric Jehan;Bridgette Wiefling;Hector F. DeLuca;Christine Kimmel-Jehan;Hisham M. Darwish;Steve A. Strugnell;Frédéric Jehan;Bridgette Wiefling;Christine Kimmel-Jehan;Hector F. DeLuca

Journal of Cellular Biochemistry 1999 Volume 74(Issue 2) pp:220-228

Publication Date(Web):18 JUN 1999

DOI:10.1002/(SICI)1097-4644(19990801)74:2<220::AID-JCB8>3.0.CO;2-T

The ability of vitamin D receptor-retinoid X receptor (VDR-RXR) heterodimers to induce a DNA bend upon binding to various vitamin D response elements (VDRE) has been investigated by circular permutation and phasing analysis. Recombinant rat VDR expressed in the baculovirus system and purified recombinant human RXRβ have been used. The VDREs were from 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂D₃) enhanced genes (rat osteocalcin, rOC; mouse osteopontin, mOP, and rat 1,25-dihydroxyvitamin D₃-24-hydroxylase, r24-OHase), and a 1,25-(OH)₂D₃ repressed gene (human parathyroid hormone, hPTH). As shown by circular permutation analysis, VDR-RXR induced a distortion in DNA fragments containing various VDREs. Calculated distortion angles were similar in magnitude (57°, 56°, 61°, and 59°, respectively for rOC, mOP, r24-Ohase, and hPTH). The distortions took place with or without a 1,25-(OH)₂D₃ ligand. The centers of the apparent bend were found in the vicinity of the midpoint of all VDREs, except for rOC VDRE which was found 4 bp upstream. Phasing analysis was performed with DNA fragments containing mOP VDRE and revealed that VDR-RXR heterodimers induced a directed bend of 26°, not influenced by the presence of hormone. In this study we report that similar to other members of the steroid and thyroid nuclear receptor superfamily, VDR-RXR heterodimers induce DNA bending. J. Cell. Biochem. 74:220–228, 1999. © 1999 Wiley-Liss, Inc.

Additional protein factors play a role in the formation of VDR/RXR complexes on vitamin D response elements

Co-reporter:Claudia Zierold;H. F. DeLuca

Journal of Cellular Biochemistry 1998 Volume 71(Issue 4) pp:515-523

Publication Date(Web):12 DEC 1998

DOI:10.1002/(SICI)1097-4644(19981215)71:4<515::AID-JCB6>3.0.CO;2-C

The vitamin D receptor (VDR) elicits a transcriptional response to 1,25-dihydroxyvitamin D₃ by binding to specific response elements (VDRE) in the promoter of target genes. Retinoic X receptor (RXR) is required for formation of the VDR-VDRE complex when VDR is supplied at physiologic concentrations. When porcine intestinal nuclear extract is used as a source of VDR, two distinct complexes are always observed with native gel electrophoresis. Both complexes contain VDR and RXR. We now show that the faster-migrating complex requires another heretofore unknown nuclear factor for its formation. In addition, we provide evidence that the formation of the slower-migrating complex is enhanced by transcription factor IIB (TFIIB). Using ligand binding assays, we determined that both complexes contain the same ratio of VDR to VDRE. Using RXR subtype-specific antibodies in gel shift assays, we show that the complexes contain more than one RXR subtype. Therefore, the present results demonstrate VDR-RXR-VDRE complexes formed with pig intestinal nuclear extracts contain other proteins and that the complexes formed between VDR and VDRE are not simply heterodimers of VDR and RXR. J. Cell. Biochem. 71:515–523, 1998. © 1998 Wiley-Liss, Inc.

The absence of 25-hydroxyvitamin D3-1α-hydroxylase potentiates the suppression of EAE in mice by ultraviolet light

Co-reporter:Yanping Wang, Steve J. Marling, Victoria M. Martino, Jean M. Prahl, Hector F. Deluca

The Journal of Steroid Biochemistry and Molecular Biology (October 2016) Volume 163() pp:98-102

Publication Date(Web):1 October 2016

DOI:10.1016/j.jsbmb.2016.04.010

•UV light completely blocks EAE in CYP27B1 KO mice.•UV light suppresses expression of cytokines IFN-γ, IL-10 and CCL-5 in spinal cord in EAE susceptible mice.•1,25-OH-D3 reduces effectiveness of UV light in suppressing EAE.Ultraviolet B (UVB) light suppresses the development of multiple sclerosis (MS) in patients and experimental autoimmune encephalomyelitis (EAE) in mice. Although vitamin D3 is produced by ultraviolet light, the suppression of EAE by narrow band UVB (NBUVB) is independent of vitamin D3. However, it is possible that the NBUVB suppression of EAE can be further influenced by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3).We used NBUVB lamps (10 KJ/m2) to irradiate both wild type (WT) and 1α-hydroxylase knockout mice (CYP27B1 KO) that were then induced to develop EAE. There was a complete elimination of EAE development by NBUVB in the KO mice. On the other hand, the NBUVB treatment of WT mice reduced but did not eliminate the severity or incidence of EAE. This suggests that the presence of 1,25-dihydroxyvitamin D3 actually counteracts the suppressive effect of NBUVB. In support of this concept, cytokines (IFN-γ, IL-10) and chemokine (CCL-5) mRNA in spinal cord were reduced in wild type or eliminated in the KO mice by the NBUVB. Cytokine mRNA levels in the spinal cord correlated with clinical scores in both WT and KO mice.

Supplementation by vitamin D compounds does not affect colonic tumor development in vitamin D sufficient murine models

Co-reporter:Amy A. Irving, Richard B. Halberg, Dawn M. Albrecht, Lori A. Plum, Kathleen J. Krentz, Linda Clipson, Norman Drinkwater, James M. Amos-Landgraf, William F. Dove, Hector F. DeLuca

Archives of Biochemistry and Biophysics (November 2011) Volume 515(Issues 1–2) pp:

Publication Date(Web):November 2011

DOI:10.1016/j.abb.2011.08.011

Epidemiological studies indicate that sunlight exposure and vitamin D are each associated with a lower risk of colon cancer. The few controlled supplementation trials testing vitamin D in humans reported to date show conflicting results. We have used two genetic models of familial colon cancer, the ApcPirc/+ (Pirc) rat and the ApcMin/+ (Min) mouse, to investigate the effect of 25-hydroxyvitamin D3 [25(OH)D3] and two analogs of vitamin D hormone on colonic tumors. Longitudinal endoscopic monitoring allowed us to test the efficacy of these compounds in preventing newly arising colonic tumors and in affecting established colonic tumors. 25(OH)D3 and two analogs of vitamin D hormone each failed to reduce tumor multiplicities or alter the growth patterns of colonic tumors in the Pirc rat or the Min mouse.Highlights► The effect of vitamin D compounds on colon tumors has been studied in the Pirc rat and Min mouse. ► Supplementation with 25(OH)D3 neither prevents nor slows the growth of tumors. ► A highly potent analog and a non-calcemic analog of 1,25(OH)2D3 each failed to affect tumor emergence or growth.

2-Methylene-19-nor-20(S)-1α-hydroxy-bishomopregnacalciferol [20(S)-2MbisP], an analog of vitamin D3 [1,25(OH)2D3], does not stimulate intestinal phosphate absorption at levels previously shown to suppress parathyroid hormone

Co-reporter:Katie B. Williams, Hector F. DeLuca

Steroids (November 2008) Volume 73(Issue 12) pp:1277-1284

Publication Date(Web):1 November 2008

DOI:10.1016/j.steroids.2008.06.003

Chronic kidney disease results in a reduction in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) synthesis and an accumulation of phosphorus in the blood, leading to secondary hyperparathyroidism and renal osteodystrophy. Vitamin D analogs that retain the ability to suppress PTH but that are less calcemic and phosphatemic than the native hormone are preferred therapies for secondary hyperparathyroidism. However, even the most favored analog currently approved for the treatment of chronic kidney disease patients, i.e. 1,25-dihydroxy-19-nor-vitamin D2 (19-nor-D2, Zemplar®), still retains some ability to stimulate intestinal absorption of calcium and phosphate. A recently described analog of vitamin D3, 2-methylene-19-nor-20(S)-1α-hydroxy-bishomopregnacalciferol [20(S)-2MbisP], suppresses PTH levels, but is unable to stimulate intestinal calcium absorption or bone resorption in rats. The present study shows that 20(S)-2MbisP is unable to stimulate intestinal phosphate absorption at levels known to suppress PTH secretion. Further, 19-nor-vitamin D2 under the same circumstances does stimulate phosphate absorption. Thus, 2MbisP has significant potential in the management of secondary hyperparathyroidism of renal failure.

26-Desmethyl-2-methylene-22-ene-19-nor-1α,25-dihydroxyvitamin D3 compounds selectively active on intestine

Co-reporter:Grazia Chiellini, Pawel Grzywacz, Lori A. Plum, Margaret Clagett-Dame, Hector F. DeLuca

Steroids (May 2014) Volume 83() pp:27-38

Publication Date(Web):1 May 2014

DOI:10.1016/j.steroids.2014.01.012

•Six 1,25(OH)2D3 analogs containing a double bond at C-22 are efficiently prepared.•2-Methylene-Δ22-19-nor-1,25(OH)2D3 compounds are active in vitro and in vivo.•C-26 methyl is required for bone restoration of 19-nor-Δ22-1,25(OH)2D3.•Des-26-methyl, Δ22 analogs of 1,25(OH)2D3 retain intestinal Ca transport activity.Six new analogs of 2-methylene-19-nor-1α,25-dihydroxyvitamin D3, 6–7 and 8a,b–9a,b, have been synthesized. All compounds are characterized by a trans double bond located in the side chain between C-22 and C-23. While compounds 6 and 7 possess C-26 and C-27 methyls, compounds 8a,b and 9a,b lack one of these groups. A Lythgoe-based synthesis, employing the Wittig–Horner reaction was used for these preparations. Two different types of Δ22E-25-hydroxy Grundmann’s ketone, having either only one stereogenic center located at position C-20 (20 and 21), or two stereogenic centers located at 20- and 25-positions (24a,b–25a,b) were obtained by a multi-step procedure from commercial vitamin D2. The introduction of a double bond at C-22 appeared to lower biological activity in vitro and in vivo. Further removal of a 26-methyl in these analogs had little effect on receptor binding, HL-60 differentiation and CYP24A expression but markedly diminished or eliminated in vivo activity on bone calcium mobilization while retaining activity on intestinal calcium transport.Download full-size image

Selective analogs of 1α,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

Co-reporter:Hector F. DeLuca, Lori A. Plum, Margaret Clagett-Dame

The Journal of Steroid Biochemistry and Molecular Biology (March 2007) Volume 103(Issues 3–5) pp:263-268

Publication Date(Web):1 March 2007

DOI:10.1016/j.jsbmb.2006.12.005

New analogs of 1α,25-dihydroxyvitamin D3 synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3′-propylidene-19-nor-(20S or 20R))-1α,25-dihydroxyvitamin D3 and 2-methylene-19-21-dinor-1α,25-dihydroxyvitamin D3. Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD), the 2α-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1α-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1α,25-dihydroxyvitamin D3. These latter compounds do not elevate serum calcium until doses of the order of >300 μg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.

2-(3′-Hydroxypropylidene)-1α-hydroxy-19-norvitamin D compounds with truncated side chains

Co-reporter:Agnieszka Glebocka, Rafal R. Sicinski, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (March 2007) Volume 103(Issues 3–5) pp:310-315

Publication Date(Web):1 March 2007

DOI:10.1016/j.jsbmb.2006.12.017

Our recent studies with 2-(3′-hydroxypropylidene) analogs of 1α,25-dihydroxy-19-norvitamin D3 showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1α-hydroxy-19-norvitamin D analogs characterized by the presence of a 3′-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8β-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig–Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7–11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1α,25-(OH)2D3 (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.

Synthesis and biological evaluation of a des-C,D-analog of 2-methylene-19-nor-1α,25-(OH)2D3

Co-reporter:Katarzyna Plonska-Ocypa, Pawel Grzywacz, Rafal R. Sicinski, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (March 2007) Volume 103(Issues 3–5) pp:298-304

Publication Date(Web):1 March 2007

DOI:10.1016/j.jsbmb.2006.12.015

Structure–activity studies directed at novel Vitamin D compounds of potential therapeutic value has long attracted an attention of numerous research groups. To date more than 3000 analogs of the natural hormone 1α,25-dihydroxyvitamin D3 (1) have been synthesized and tested. As a continuation of our studies on 19-norvitamin D compounds we have obtained (20S)-des-C,D-2-methylene-1α,25-dihydroxy-19-norvitamin D3 (6) and examined its biological activity. This Vitamin D compound was efficiently prepared in a convergent synthesis. The “upper” fragment was synthesized starting with methyl (R)-(−)-3-hydroxy-2-methylpropionate (9). The key synthetic step involved Lythgoe type Wittig–Horner coupling of the protected hydroxy aldehyde 19 with the phosphine oxide 20. The prepared Vitamin D analog 6 exhibited limited binding ability to the rat intestinal Vitamin D receptor being ca. 80 times less potent when compared to the natural hormone 1. Although the analog 6 did retain some cell differentiating and transcriptional activities, preliminary in vivo tests do not show any activity of this analog in animals.

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

Co-reporter:Rafal R. Sicinski, Agnieszka Glebocka, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (March 2007) Volume 103(Issues 3–5) pp:293-297

Publication Date(Web):1 March 2007

DOI:10.1016/j.jsbmb.2006.12.064

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D3 (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.

Highly potent 2-methylene analogs of 1α,25-dihydroxyvitamin D3: Synthesis and biological evaluation

Co-reporter:Izabela K. Sibilska, Marcin Szybinski, Rafal R. Sicinski, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (July 2013) Volume 136() pp:9-13

Publication Date(Web):1 July 2013

DOI:10.1016/j.jsbmb.2013.02.001

As a continuation of our studies on structure–activity relationship of vitamin D compounds we synthesized new calcitriol analogs characterized by the presence of an exomethylene substituent at C-2. The A-ring dienyne synthon was prepared from commercially available quinic acid by two different synthetic routes, and it was then coupled with the triflate enol derived from the corresponding (20R)- and (20S)-Grundmann ketone by palladium catalyzed Sonogashira reaction.The obtained 1α,25-dihydroxy-2-methylene-vitamin D3 analogs, epimeric at C-20, were biologically evaluated by in vitro and in vivo studies. Both isomers exhibited unique activity profiles and greater biological potency than 1α,25-(OH)2D3. It was established that the biological profiles of the newly obtained vitamin D compounds depend on the configuration at C-20. Thus, introduction of 2-methylene substituent to the calcitriol molecule together with alteration of stereochemistry of its side chain induces remarkable changes in a VDR-mediated signaling response and enhances biological activity.This article is part of a Special Issue entitled ‘Vitamin D Workshop’.Highlights▸ Starting from quinic acid two syntheses of the A-ring synthon were achieved. ▸ 2-Methylene calcitriol analogs were synthesized by Pd-catalyzed cross-coupling. ▸ Presence of 2-methylene group at C-2 enhances biological activity of calcitriol.

Ring-A-seco analogs of 1α,25-dihydroxy-19-norvitamin D3

Co-reporter:Agnieszka Glebocka, Rafal R. Sicinski, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (July 2013) Volume 136() pp:39-43

Publication Date(Web):1 July 2013

DOI:10.1016/j.jsbmb.2012.09.030

The steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] is the most active metabolite of vitamin D3 which exerts its control over a multitude of biological processes related to calcium and phosphorus homeostasis, cell proliferation and differentiation, and immune regulation. Unfortunately, the therapeutic application of 1α,25-(OH)2D3 is limited by induction of hypercalcemia. The need for vitamin D compounds with selective biological profiles has stimulated the synthesis of more than three thousand analogs of 1α,25-(OH)2D3. Most of these compounds have structural modifications in the side chain and A-ring; there is also an increasing number of modifications in the CD-rings and limited number in the triene system (seco-B ring). Herein, we report the synthesis and biological evaluation of seco-A-19-nor analogs of 1α,25-dihydroxyvitamin D3, developed to study the role of ring A in the biological activity of 1α,25-(OH)2D3. Interestingly, compounds 2 and 4 show substantial ability to bind the vitamin D receptor and the former is also characterized by selective intestinal calcium transport activity.This article is part of a Special Issue entitled ‘Vitamin D Workshop’.Highlights▸ Seco-A-19-nor analogs of 1α,25-dihydroxyvitamin D3 were developed to study the role of ring A in the biological activity of 1α,25-(OH)2D3. ▸ Two of the seco-A-19-nor analogs show substantial ability to bind the vitamin D receptor and one of them is also characterized by selective intestinal calcium transport activity. ▸ Opening of the ring A in vitamin D compounds causes large decrease in both (in vitro and in vivo) biological activities of the analogs.

Synthesis and biological evaluation of novel 6-substituted analogs of 1α,25-dihydroxy-19-norvitamin D3

Co-reporter:Katarzyna Sokolowska, Rafal R. Sicinski, Antonio Mouriño, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (July 2013) Volume 136() pp:30-33

Publication Date(Web):1 July 2013

DOI:10.1016/j.jsbmb.2012.10.016

We have recently described the synthesis and biological evaluation of 1α,25-dihydroxy-6-methylvitamin D3 which binds the vitamin D receptor (VDR) very effectively. Unfortunately, this compound has a strong tendency to rearrange to its previtamin form. Taking this into consideration, we decided to synthesize a series of 6-substituted analogs of 1α,25-dihydroxy-19-norvitamin D3 which lack the exomethylene moiety at C-10 and thus are unable to undergo a conversion to their respective previtamin forms. The synthesis of analogs bearing different substituents at C-6 was accomplished by Suzuki–Miyaura cross-coupling reaction of a bicyclic organoboron derivative (CD-ring fragment) with the respective alkenyl halides (A-ring synthons). The biological in vitro studies of the affinity of synthesized analogs to the full-length recombinant rat VDR indicate that presence of a bulky, polar substituent at C-6 results in decrease in binding ability. Moreover, introduction of a 6-methyl substituent into the 1α,25-dihydroxy-19-norvitamin D3 molecule results in the 9 times lower affinity of the homolog to the VDR while the same modification of calcitriol has not influenced binding activity.This article is part of a Special Issue entitled ‘Vitamin D Workshop’.Highlights▸ 6-Substituted 19-norcalcitriol analogs were synthesized by Suzuki–Miyaura coupling. ▸ Biological activity of 6-substituted 19-nor-1α,25-(OH)2D3 analogs was investigated. ▸ Presence of substituent at C-6 impairs binding affinity of 19-norvitamin D to VDR.

Synthesis and biological activity of 25-hydroxy-2-methylene-vitamin D3 compounds

Co-reporter:Izabela Sibilska, Rafal R. Sicinski, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (July 2013) Volume 136() pp:17-22

Publication Date(Web):1 July 2013

DOI:10.1016/j.jsbmb.2012.12.012

We have recently obtained 1-desoxy and 3-desoxy analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD), a compound exerting significantly enhanced calcemic activity and currently being evaluated as a potential drug for osteoporosis. In order to further explore this class of pharmacologically important vitamin D compounds we have decided to synthesize analogs characterized by the presence of two A-ring exocyclic methylene groups attached to C-2 and C-10. The Sonogashira coupling of a triflate enol of the protected (20R)- or (20S)-25-hydroxy Grundmann ketone and the corresponding dienyne A-ring fragment provided the target compounds. A new synthetic path was elaborated, leading to the desired A-ring synthon, that started from commercially available 1,4-cyclohexanedione monoethylene acetal. Biological in vitro and in vivo activities of the synthesized 25-hydroxy-2-methylene-vitamin D3 compounds, belonging to 20R- and 20S-series, were evaluated and discussed.This article is part of a Special Issue entitled ‘Vitamin D Workshop’.Highlights▸ Starting from 1,4-cyclohexanedione monoethylene acetal synthesis of the A-ring synthon was achieved ▸ 2-Methylene vitamin D analogs were synthesized by Pd-catalyzed cross-coupling. ▸ Presence of 2-methylene group at C-2 affected biological activity of vitamin D.

1-Desoxy analog of 2MD: Synthesis and biological activity of (20S)-25-hydroxy-2-methylene-19-norvitamin D3

Co-reporter:Izabela Sibilska, Katarzyna M. Barycka, Rafal R. Sicinski, Lori A. Plum, Hector F. DeLuca

The Journal of Steroid Biochemistry and Molecular Biology (July 2010) Volume 121(Issues 1–2) pp:51-55