Antonio Mouriño

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.

Palladium-catalysed coupling of vinyl triflates with enynes and its application to the synthesis of 1α-25-dihydroxyvitamin D3

Abstract We describe a general approach, based on the palladium-catalysed coupling of enynes with vinyl triflates, for the construction of dienynes related to vitamin D metabolites and analogues. As an application of this method, an efficient convergent synthesis of 1α,25-dihydroxyvitamin D 3 starting from the Inhoffen-Lythgoe diol ( 6a ) and natural carvones has been carried out (11 steps, 28% overall yield from 6a ). This strategy allows labelling of the side chain in the final steps of the synthesis

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.

Palladium-catalyzed synthesis of dienynes related to vitamin D from enol triflates

Abstract This note describes a simple synthesis of Lythgoe-type dienynes based on palladium catalyzed coupling of kinetic Grundmanns ketone enol triflate and acetylenic compounds containing the vitamin D A-ring fragment.

Structure-Based Design of a Superagonist Ligand for the Vitamin D Nuclear Receptor

Vitamin D nuclear receptor (VDR), a ligand-dependent transcriptional regulator, is an important target for multiple clinical applications, such as osteoporosis and cancer. Since exacerbated increase of calcium serum level is currently associated with VDR ligands action, superagonists with low calcium serum levels have been developed. Based on the crystal structures of human VDR (hVDR) bound to 1alpha,25-dihydroxyvitamin D(3) and superagonists-notably, KH1060-we designed a superagonist ligand. In order to optimize the aliphatic side chain conformation with a subsequent entropy benefit, we incorporated an oxolane ring and generated two stereo diasteromers, AMCR277A and AMCR277B. Only AMCR277A exhibits superagonist activity in vitro, but is as calcemic in vivo as the natural ligand. The crystal structures of the complexes between the ligand binding domain of hVDR and these ligands provide a rational approach to the design of more potent superagonist ligands for potential clinical application.

An improved synthesis of 1α, 25-dihydroxyvitamin D A synthons

Abstract A new improved preparation of key intermediates ( 9b, 11 ) for the synthesis of 1α, 25-dihydroxyvitamin D is described.

New vitamin D receptor ligands

More than 3000 synthetic analogues of the biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2-D3), are presently known. All analogues interact with the molecular switch controlling nuclear 1α,25-(OH)2-D3 signalling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR) and a 1α,25-(OH)2-D3 response element (VDRE). The central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilised by a 1α,25-(OH)2-D3 analogue in either its agonistic or antagonistic conformation. In this article, the patent literature on 1α,25-(OH)2-D3 analogues described in the last 4 years will be reviewed, and several new VDR ligands with promising selective profiles discussed. Although most of these ligands are agonists of the VDR, a few antagonists have been reported. Non-steroidal VDR ligands will be highlighted, as well as 1α,25-(OH)2-D3 analogues with two side chains.

Vitamin D receptor ligands: the impact of crystal structures.

Introduction: In the past years, the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), has received large appreciation due to the broad physiological impact of the hormone and its nuclear receptor, the transcription factor vitamin D receptor (VDR). Recently, the understanding of VDR actions has progressed greatly, due to VDR crystal structures with various ligands. Areas covered: This review will present and discuss new synthetic agonistic and antagonistic 1α,25(OH)2D3 analogs in the context of the recent insights provided by VDR crystal structures. Expert opinion: During the last 5 years, a large number of new 1α,25(OH)2D3 analogs, many of which have an interesting functional profile, have been patented. Moreover, for a surprisingly high number of 1α,25(OH)2D3 analogs, the crystal structure data of their complex with the VDR is available. This structural information provides important insight into the functional potential of the VDR ligands and explains their agonistic and antagonistic action. However, so far, only for a few VDR ligands, a rational design, based on crystal structure information, has been applied. The design of future analogs may also take the specificity of co-factor interaction into account, in order to create selective VDR modulators.

An Expeditious Route to 1α,25-Dihydroxyvitamin D3 and Its Analogues by an Aqueous Tandem Palladium-Catalyzed A-Ring Closure and Suzuki Coupling to the C/D Unit

1a,25-Dihydroxyvitamin D3 (1), the hormonally active metabolite of the seco-steroid vitamin D3, interacts with the vitamin D nuclear receptor (VDR) to initiate a cascade of events that ultimately controls mineral homeostasis and a multitude of cellular processes including differentiation, antiproliferation, growth, apoptosis, angiogenesis, and immunomodulation. Unfortunately, the therapeutic applications of 1 in pharmacological doses to correct dysfunction of one or more of these processes are severely limited by its potent calcemic effects. Efforts to develop analogues with selectively reduced calcemic effects for treatment of, for example, cancer and skin diseases or with selective activity on bone formation have led to more than 3000 synthetic analogues being tested, although only a few have reached the pharmaceutical market or advanced clinical trials. The most useful convergent methods to synthesize the triene moiety in vitamin D analogues include the Wittig– Horner approach devised by Lythgoe and developed by the Hoffmann La Roche group (Scheme 1, route A) and the palladium-catalyzed route introduced by Trost and co-workers (Scheme 1, route B). These methods have practical drawbacks in that they either require an excess of the lower (A ring) fragment (for small-scale work) or elevated temper-

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.