Advances in Vitamin D Receptor Function and Evolution Based on the 3D Structure of the Lamprey Ligand-Binding Domain.

1α,25-dihydroxyvitamin D3 (1,25D3) regulates many physiological processes in vertebrates by binding to the vitamin D receptor (VDR). Phylogenetic analysis indicates that jawless fishes are the most basal vertebrates exhibiting a VDR gene. To elucidate the mechanism driving VDR activation during evolution, we determined the crystal structure of the VDR ligand-binding domain (LBD) complex from the basal vertebratePetromyzon marinus, sea lamprey (lVDR). Comparison of three-dimensional crystal structures of the lVDR-1,25D3 complex with higher vertebrate VDR-1,25D3 structures suggests that 1,25D3 binds to lVDR similarly to human VDR, but with unique features for lVDR around linker regions between H11 and H12 and between H9 and H10. These structural differences may contribute to the marked species differences in transcriptional responses. Furthermore, residue co-evolution analysis of VDR across vertebrates identifies amino acid positions in H9 and the large insertion domain VDR LBD specific as correlated.

Synthesis of 28,28,28-trideutero-25-hydroxydihydrotachysterol2.

As part of our program on synthesis of labeled vitamin D metabolites and analogs, we describe here an efficient and versatile synthetic approach to 28,28,28-trideutero- 25-hydroxydihydrotachysterol2 where isotopic labeling was incorporated stereoselectively in the last step of the synthesis. This deuterated compound will allow the study this analog in vitro or in vivo and to measure AT10-like compounds in serum by LC-MS/MS.

Aromatic-Based Design of Highly Active and Noncalcemic Vitamin D Receptor Agonists.

We report the design, synthesis, biological evaluation, and structural analysis of a new class of vitamin D analogues that possess an aromatic m-phenylene D-ring and an alkyl chain replacing the C-ring. A key feature of the synthetic strategy is a stereoselective Pd-catalyzed construction of the triene system in aqueous medium that allows the rapid preparation of small amounts of VDR ligands for biological screening. Analogues with the shorter (2a) and longer (2d, 2e) side chains attached to the triene system have no calcemic activity. Compound 2a binds to VDR with the same order of magnitude than calcipotriol and oxacalcitriol. It also reduces proliferation in normal and tumor cells similarly to the natural hormone 1α,25-dihydroxyvitamin D3, calcipotriol, and oxacalcitriol, suggesting preclinical studies related to hyperproliferative disorders such as psoriasis and cancer.

Synthesis of Side-Chain Locked Analogs of 1α,25-Dihydroxyvitamin D3 Bearing a C17 Methyl Group.

A convergent synthesis of side-chain locked vitamin D analogs 3 and 4, which bind strongly in silico to the vitamin D receptor (VDR), is described. The synthetic approach features an SN2'- syn displacement of carbamates by cuprates to set the challenging quaternary stereogenic center at C17 and a Pd-catalyzed construction of the triene system in the presence of a diyne moiety.

Stereoselective Palladium-Catalyzed Approach to Vitamin D3 Derivatives in Protic Medium.

We describe an efficient convergent synthesis of vitamin D3 metabolites and analogues. The synthetic strategy relies on a tandem Pd-catalyzed A-ring closure and Suzuki-Miyaura coupling to the CD-side chain component to set directly the vitamin D triene system under protic conditions. This strategy enables rapid access to vitamin D3 and 3-epi-vitamin D3 metabolites and analogues modified at the side chain for biological evaluation and structural and metabolic studies.

Stereoselective synthesis of 25S,26-dihydroxyvitamin D3.

A convergent approach to 25S,26-dihydroxyvitamin D3 (1) has been developed in our laboratories. The A-ring and the CD-fragment are constructed from ergocalciferol and Inhoffen-Lythgoe diol, respectively. The triene system is assembled by a Wittig-Horner coupling. With this convergent synthesis, a novel hydroxylated vitamin D metabolite in our laboratory is available for biological testing.

A new approach to 19-nor-A-ring phosphine oxide for the convergent synthesis of 19-nor-calcitriol.

A new approach to 19-nor-A-ring phosphine oxide 5 together with a convergent synthesis of the vitamin D3 analogue 1α,25-dihydroxy-19-norvitamin D3 (3) have been developed. The 19-nor-A-ring is constructed from (S)-carvone. The triene system is assembled by a Wittig-Horner coupling.

A convergent approach to side-chain homologated derivatives of 1α,25-dihydroxyergocalciferol.

As part of our program on search for vitamin D analogs with selective biological properties, such as low or negligible calcemic action, we describe here an efficient and versatile synthetic approach to derivatives of 1α,25-dihydroxyvitamin D2 with homologated side-chains and substitution at C24 for biological evaluation.

Stereoselective synthesis of 1ß,25-Dihydroxyvitamin D3 and its 26,27-hexadeuterated derivative.

A mild convergent synthesis of 1ß,25-dihydroxyvitamin D3 (3a), a metabolite of vitamin D3, and its C26,27-hexadeuterated derivative (3b) are described. The A-ring and the CD-fragments are constructed from (R)-carvone and Inhoffen-Lythgoe diol, respectively. The triene system is assembled by a Pd(0)-catalyzed process, which involves an enol-triflate (A-ring fragment) and an alkenyl boronate (CD-side chain fragment). Deuterium labeling is introduced at the last step of the synthesis.

Carborane-based design of a potent vitamin D receptor agonist.

The vitamin D nuclear receptor (VDR) is a potential target for cancer therapy. It is expressed in many tumors and its ligand shows anticancer actions. To combine these properties with the application of boron neutron capture therapy (BNCT), we design and synthesize a potent VDR agonist based on the skeleton of the hormone 1α,25-dihydroxyvitamin D3 (1,25D) and an o-carborane (dicarba-o-closo-1,2-dodecaborane) at the end of its side chain. The present ligand is the first secosteroidal analog with the carborane unit that efficiently binds to VDR and functions as an agonist with 1,25D-like potency in transcriptional assay on vitamin D target genes. Moreover it exhibits similar antiproliferative and pro-differentiating activities but is significantly less hypercalcemic than 1,25D. The crystal structure of its complex with VDR ligand binding domain reveals its binding mechanism involving boron-mediated dihydrogen bonds that mimic vitamin D hydroxyl interactions. In addition to the therapeutic interest, this study establishes the basis for the design of new unconventional vitamin D analogs containing carborane moieties for specific molecular recognition, and drug research and development.

Pit-1 inhibits BRCA1 and sensitizes human breast tumors to cisplatin and vitamin D treatment.

The POU class 1 homeobox 1 (POU1F1, also known as Pit-1), pertaining to the Pit-Oct-Unc (POU) family of transcription factors, has been related to tumor growth and metastasis in breast. However, its role in response to breast cancer therapy is unknown. We found that Pit-1 down-regulated DNA-damage and repair genes, and specifically inhibited BRCA1 gene expression, sensitizing breast cancer cells to DNA-damage agents. Administration of 1α, 25-dihydroxy-3-epi-vitamin D3 (3-Epi, an endogenous low calcemic vitamin D metabolite) reduced Pit-1 expression, and synergized with cisplatin, thus, decreasing cell proliferation and apoptosis in vitro, and reducing tumor growth in vivo. In addition, fifteen primary cultures of human breast tumors showed significantly decreased proliferation when treated with 3-Epi+cisplatin, compared to cisplatin alone. This response positively correlated with Pit-1 levels. Our findings demonstrate that high levels of Pit-1 and reduced BRCA1 levels increase breast cancer cell susceptibility to 3-Epi+cisplatin therapy.

An efficient convergent synthesis of 1α,25-dihydroxy-3-epi-vitamin D2.

The first synthesis of 1α,25-dihydroxy-3-epi-vitamin D2 is described. Key steps of the synthesis entail the construction of the triene unit by a Pd-catalyzed ring closure of an enol-triflate (A-ring fragment) followed by a Suzuki-Miyaura coupling with a boronate (upper fragment), and the installation of the methyl group at C-24 by an SN2'-syn displacement of an allylic carbamate. This article is part of a Special Issue entitled 'SI:17th Vitamin D Workshop'.

Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.

CONTEXT: The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D]. OBJECTIVE: We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 µL of serum with a 5-minute run time. DESIGN: The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter. SETTING: We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models. RESULTS: The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia. CONCLUSIONS: The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

Metabolic stability of 3-epi-1α,25-dihydroxyvitamin D3 over 1 α 25-dihydroxyvitamin D3: metabolism and molecular docking studies using rat CYP24A1.

3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), a natural metabolite of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), exhibits potent vitamin D receptor (VDR)-mediated actions such as inhibition of keratinocyte growth or suppression of parathyroid hormone secretion. These VDR-mediated actions of 3-epi-1α,25(OH)2D3 needed an explanation as 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, exhibits low affinity towards VDR. Metabolic stability of 3-epi-1α,25(OH)2D3 over 1α,25(OH)2D3 has been hypothesized as a possible explanation. To provide further support for this hypothesis, we now performed comparative metabolism studies between 3-epi-1α,25(OH)2D3 and 1α,25(OH)2D3 using both the technique of isolated rat kidney perfusion and purified rat CYP24A1 in a cell-free reconstituted system. For the first time, these studies resulted in the isolation and identification of 3-epi-calcitroic acid as the final inactive metabolite of 3-epi-1α,25(OH)2D3 produced by rat CYP24A1. Furthermore, under identical experimental conditions, it was noted that the amount of 3-epi-calcitroic acid produced from 3-epi-1α,25(OH)2D3 is threefold less than that of calcitroic acid, the analogous final inactive metabolite produced from 1α,25(OH)2D3 . This key observation finally led us to conclude that the rate of overall side-chain oxidation of 3-epi-1α,25(OH)2D3 by rat CYP24A1 leading to its final inactivation is slower than that of 1α,25(OH)2D3. To elucidate the mechanism responsible for this important finding, we performed a molecular docking analysis using the crystal structure of rat CYP24A1. Docking results suggest that 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, binds to CYP24A1 in an alternate configuration that destabilizes the formation of the enzyme-substrate complex sufficiently to slow the rate at which 3-epi-1α,25(OH)2D3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid.

Synthesis of 9-alkylated calcitriol and two 1α,25-dihydroxy-9-methylene-10,19-dihydrovitamin D3 analogues with a non-natural triene system by thermal sigmatropic rearrangements.

1α,25-(OH)(2)-9α-Methylvitamin D(3) (4), the first known analogue of the natural hormone 1α,25-(OH)(2)D(3) (3) with an alkyl substituent at C-9, and two 1α,25-(OH)(2)-9-methylene-10,19-dihydrovitamin D(3) analogues (7 and 8) with an unprecedented non-natural triene system were synthesized by thermal isomerization of 1α,25-(OH)(2)-9-methylprevitamin D(3) (6). Three alternative approaches (Sonogashira, Stille, or stereoselective dehydration of a tertiary propargyl alcohol) have been successfully used to construct the dienyne precursors of previtamin 6 possessing two methyl groups capable of participating in the [1,7]-sigmatropic hydrogen shift.

1α,25(OH)2-3-epi-vitamin D3, a natural physiological metabolite of vitamin D3: its synthesis, biological activity and crystal structure with its receptor.

BACKGROUND: The 1α,25-dihydroxy-3-epi-vitamin-D3 (1α,25(OH)2-3-epi-D3), a natural metabolite of the seco-steroid vitamin D3, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1α,25(OH)2-3-epi-D3 is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)2D3. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD). METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)2D3. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)2-3-epi-D3 in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)2D3. CONCLUSIONS/SIGNIFICANCE: The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3.

Design and synthesis of active vitamin D analogs.

A review of the design and synthesis of structural analogs of the vitamin D hormone recently investigated in our laboratories, and the first report on a new class of vitamin D analogs characterized by an aromatic D-ring, is described.

Synthesis and biological evaluation of 6-methyl analog of 1alpha,25-dihydroxyvitamin D3.

Among more than 3000 analogs of 1alpha,25-dihydroxyvitamin D3 synthesized to date, only a few were characterized by structural modifications in the seco-B-ring. The compounds alkylated at C-6 seemed to be interesting targets for synthetic efforts. Such vitamin D analogs easily undergo thermal conversion to their previtamin forms. The results of molecular modeling indicate that significant deviation from planarity must be present in their molecules associated with the interaction of the 6-alkyl substituent and hydrogens from the C-ring. The synthesis of the analog of 1alpha,25-(OH)2D3, being characterized by the presence of the 6-methyl group, is reported here, together with the results of preliminary testing of its biological potency. This 6-alkylated compound was efficiently prepared using a novel stereoconvergent strategy in which the ring A and the triene unit of the vitamin D skeleton are constructed by a one-pot Pd-catalyzed tandem cyclization-Negishi coupling process.

Structure-function relationships and crystal structures of the vitamin D receptor bound 2 alpha-methyl-(20S,23S)- and 2 alpha-methyl-(20S,23R)-epoxymethano-1 alpha,25-dihydroxyvitamin D3.

The vitamin D nuclear receptor is a ligand-dependent transcription factor that controls multiple biological responses such as cell proliferation, immune responses, and bone mineralization. Numerous 1 alpha,25(OH)(2)D(3) analogues, which exhibit low calcemic side effects and/or antitumoral properties, have been synthesized. We recently showed that the synthetic analogue (20S,23S)-epoxymethano-1 alpha,25-dihydroxyvitamin D(3) (2a) acts as a 1 alpha,25(OH)(2)D(3) superagonist and exhibits both antiproliferative and prodifferentiating properties in vitro. Using this information and on the basis of the crystal structures of human VDR ligand binding domain (hVDR LBD) bound to 1 alpha,25(OH)(2)D(3), 2 alpha-methyl-1 alpha,25(OH)(2)D(3), or 2a, we designed a novel analogue, 2 alpha-methyl-(20S,23S)-epoxymethano-1 alpha,25-dihydroxyvitamin D(3) (4a), in order to increase its transactivation potency. Here, we solved the crystal structures of the hVDR LBD in complex with the 4a (C23S) and its epimer 4b (C23R) and determined their correlation with specific biological outcomes.