Rita Sigüeiro

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.

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.

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.

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.

Metabolic stability of 3‐Epi‐1α,25‐dihydroxyvitamin D3 over 1α, 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. J. Cell. Biochem. 114: 2293–2305, 2013.

Synthesis and biological evaluation of 6-methyl analog of 1α,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.

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.

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.