Abstract
BackgroundThe 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 FindingsIn 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/SignificanceThe 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.
Highlights
The 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3 or calcitriol), is the most active form of vitamin D3 and mediates its pleiotropic effects through vitamin D nuclear receptor (VDR) activation, which heterodimerizes with retinoid X receptor (RXR)
In addition 1a,25(OH)2D3 is a key regulator of calcium and phosphate homeostasis and bone metabolism but its intrinsic hypercalcemic effect prevents its use in therapeutical applications [6]
While the side chain oxidation is a general pathway associated to inactivation, another metabolite modified at the A-ring, the 1a,25(OH)2-3-epi-D3, has been shown to retain significant biological activity compared to the natural hormone [18,19]
Summary
The 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3 or calcitriol), is the most active form of vitamin D3 and mediates its pleiotropic effects through VDR activation, which heterodimerizes with retinoid X receptor (RXR). The enzymes responsible for the C3-epimerization have not been identified to present date It was proposed by Reddy et al that this pathway might be used for metabolites that resist inactivation through C-24 oxidation [18] a phenomenon well characterized in the bile acid metabolism where the reaction is catalyzed by bile acid hydroxysteroid dehydrogenase [27]. The transcriptional response of the 1a,25(OH)2-3-epi-D3 compound varies for different VDR-regulated genes in different tissues It shows lower activation of osteocalcin gene and lower HL60 differentiation [30] but has almost equipotent activity to 1a,25(OH)2D3 in inhibiting cellular proliferation in keratinocytes [19] and in suppressing parathyroid secretion in bovine parathyroid cells [25]. The structural mechanism and structureactivity relationships of 1a,25(OH)2-3-epi-D3/hVDR-LBD complex, we describe a more effective synthetic route to the synthesis of 1a,25(OH)2-3-epi-D3, some of its in vitro biological properties and the crystal structure of its complex with hVDR LBD
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