Differential activities of 1α,25-dihydroxy-16-ene-vitamin D3 analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis

Nakagawa, Kimie; Sowa, Yoshiko; Kurobe, Mayuko; Ozono, Keiichi; Siu-Caldera, Mei-Ling; Reddy, G. Satyanarayana; Uskoković, Milan R.; Okano, Toshio

doi:10.1016/s0039-128x(00)00142-2

Cited by 39 publications

(27 citation statements)

References 18 publications

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“…In addition, we established that a trans conformation of the Aring's 1α and 3β hydroxyl groups (1S, 3R) is a key determinant of high-affinity binding and recognition of substrate for both wild-type and S57 mutant rat CYP24A1. This observation may help explain the extended efficacy of 3-epi-vitamin D compounds whose cellular activity has long been attributed to altered target tissue metabolism [40,41]. Our studies with the 3-deoxy analog further clarified the determinants of high affinity binding by showing that the 3-OH group is important but not essential for active-site recognition; our previous work demonstrated a more putative role for the 1α-hydroxyl group in this process [21].…”

Section: Discussionsupporting

confidence: 51%

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Annalora

Bobrovnikov-Marjon

Serda

et al. 2007

Archives of Biochemistry and Biophysics

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Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/ BM-3) homology modeling and affinity labeling studies clarified the role of key domains (Nterminus, A', A, and F-helices, β3a strand, & β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membraneassociated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective binding function and profound metabolic defects for 25-hydroxylated vitamin D 3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH) 2 D 3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α & 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane § Deceased

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Section: Discussionsupporting

confidence: 51%

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Annalora

Bobrovnikov-Marjon

Serda

et al. 2007

Archives of Biochemistry and Biophysics

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“…The 3-epi-1,25(OH) 2 D3 diastereomer stimulated calcium transport in excess of its relative ability to bind to the VDR. Other studies on 3-epi-1,25(OH) 2 D3 indicate that its reduced binding to the VDR does generally correlates with its reduced biological activity [30, 32, 34, 35], but there are notable exceptions such as the maintenance of the ability to suppress parathyroid hormone secretion by cultured parathyroid cells and enhancement of the ability to stimulate HL-60 cell apoptosis, relative to 1,25(OH) 2 D3 [36, 37]. …”

Section: Introductionmentioning

confidence: 99%

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as “biased” agonists on VDR and inverse agonists on RORα and RORγ

Slominski

Kim²,

Hobrath

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

125

143

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The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)2D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)2D3, 20,23(OH)2D3, 17,20,23(OH)3D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,22(OH)3D3, 20,24(OH)2D3, 1,20,24(OH)3D3, 20,25(OH)2D3, 1,20,25(OH)3D3, 20,26(OH)2D3 and 1,20,26(OH)3D3. 20(OH)D3 and 20,23(OH)2D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as “biased” agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)2D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)2D3 have been tested in cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as “biased” agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)2D3 and VDR.

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“…While the transcriptional activity in MG-63 cells using a vitamin D-responsive element (VDRE) from human osteocalcin (−848/+10) and rat CYP24 (−291/+9) gene promoters was lower upon stimulation with 1α,25(OH) 2 -3-epi-D 3 compared to 1α,25(OH) 2 D 3 [47], using 2xVDREs reporter from CYP24 gene promoter in human melanoma G-361 cells comparable transcriptional activity was observed [48]. This response is mainly achieved in cells in which the 1α,25(OH) 2 -3-epi-D 3 metabolite is produced [29].…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2011

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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.

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Differential activities of 1α,25-dihydroxy-16-ene-vitamin D3 analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis

Cited by 39 publications

References 18 publications

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as “biased” agonists on VDR and inverse agonists on RORα and RORγ

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

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