Antagonistic action of novel 1alpha,25-dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1alpha,25-dihydroxyvitamin D3.

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.     

(23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone function as antagonists of vitamin D receptor-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3)

We synthesized various analogues of 1alpha,25-(OH)(2)D(3)-26,23-lactone and examined the effects of them on HL-60 cell differentiation using the evaluation system of the genomic action of 1alpha,25-(OH)(2)D(3). We found that (23S)- and (23R)-25-dehydro-1alpha-OH-D(3)-26,23-lactone (TEI-9647 and TEI-9648) strongly bound to the VDR, but did not induce HL-60 cell differentiation. Intriguingly, TEI-9647 and TEI-9648 did inhibit that induced by 1alpha,25-(OH)(2)D(3), whereas they did not suppress that caused by retinoic acid or TPA. On the contrary, the similar 25-dehydrated 24-dehydro analogues, TEI-D1807 and TEI-D1808, weakly but significantly induced HL-60 cell differentiation, never showing inhibitory effect on HL-60 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In other experiments, TEI-9647 and TEI-9648 markedly suppressed 25-OH-D(3)-24-hydroxylase gene expression induced by 1alpha,25-(OH)(2)D(3) in HL-60 cells. TEI-9647 also inhibited the heterodimer formation between VDR and RXRalpha, and the VDR interaction with co-activator SRC-1 according to the results obtained from the mammalian two-hybrid system in Saos-2 cells. Taking all these results into consideration, we reached a manifest conclusion that TEI-9647 and TEI-9648 are the specific and first antagonists of 1alpha,25-(OH)(2)D(3) action, specifically VDR-VDRE mediated genomic action.     

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs on 25-hydroxyvitamin-D(3)-24-hydroxylase gene expression induced by 1alpha,25-dihydroxy-vitamin D(3) in human promyelocytic leukemia (HL-60) cells

We have demonstrated that 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs, (23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647, TEI-9648, respectively), inhibit HL-60 cell differentiation induced by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], but not differentiation caused by all-trans retinoic acid (D. Miura et al., 1999, J. Biol. Chem. 274, 16392). To assess whether the antagonistic actions of TEI-9647 and TEI-9648 in HL-60 cells are related to 1alpha,25(OH)(2)D(3) breakdown, we investigated their effects on catabolism of 1alpha,25(OH)(2)D(3). In HL-60 cells, the C-24 but not the C-23 side-chain oxidation pathway of 1alpha,25(OH)(2)D(3) has been reported. Here we demonstrate that 1alpha,25(OH)(2)D(3) was metabolized both to 24,25,26,27-tetranor-1alpha,23-(OH)(2)D(3) and 1alpha,25(OH)(2)D(3)-26,23-lactone; thus HL-60 cells constitutively possess both the 24- and the 23-hydroxylases. Metabolism of 1alpha, 25(OH)(2)D(3) was strongly suppressed by 10(-7) M TEI-9647 or 10(-6) M TEI-9648. 1alpha,25(OH)(2)D(3) alone slightly induced 24-hydroxylase gene expression by 8 h with full enhancement by 24-48 h; this induction was inhibited by 10(-6) M TEI-9647 and 10(-6) M TEI-9648 (86.2 and 31.9%, respectively) 24 h after treatment. However, analogs of TEI-9647 and TEI-9648 without the 25-dehydro functionality induced 24-hydroxylase gene expression. These results indicate that TEI-9647 and TEI-9648 clearly mediate their stereoselective antagonistic actions independent of their actions to block the catabolism of 1alpha,25(OH)(2)D(3). Therefore, TEI-9647 and TEI-9648 appear to be the first antagonists specific for the nuclear 1alpha,25(OH)(2)D(3) receptor-mediated genomic actions of 1alpha,25(OH)(2)D(3) in HL-60 cells.     

Molecular mechanism of the vitamin D antagonistic actions of (23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone depends on the primary structure of the carboxyl-terminal region of the vitamin d receptor

We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.     

Analysis of the molecular mechanism for the antagonistic action of a novel 1alpha,25-dihydroxyvitamin D(3) analogue toward vitamin D receptor function.

We have recently reported that 23(S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) efficiently blocks the differentiation of HL-60 cells induced by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (Miura, D., Manabe, K., Ozono, K., Saito, M., Gao, Q., Norman, A. W., and Ishizuka, S. (1999) J. Biol. Chem. 274, 16392-16399). To clarify the molecular mechanisms of this antagonism, we examined whether TEI-9647 antagonizes the genomic effects of 1alpha,25(OH)(2)D(3). 10(-7) to 10(-9) M TEI-9647 inhibited the transactivation effect of 10(-8) M 1alpha,25(OH)(2)D(3) in a dose-dependent manner, while TEI-9647 alone did not activate the reporter activity driven by SV40 promoter containing two vitamin D response elements in Saos-2 cells. The antagonistic effect of TEI-9647 was also observed using the rat 24-hydroxylase gene promoter, but the effect was weaker in HeLa and COS-7 cells than in Saos-2 cells. TEI-9647 also exhibited antagonism in an assay system where the VDR fused to the GAL4 DNA-binding domain and the reporter plasmid containing the GAL4 binding site were used in Saos-2 cells, but did not in HeLa cells. TEI-9647 reduced the interaction between VDR and RXRalpha according to the results obtained from the mammalian two-hybrid system in Saos-2 cells, but did not in HeLa cells. The two-hybrid system also revealed that the interaction between VDR and SRC-1 was reduced by TEI-9647 in Saos-2 cells. These results demonstrate that the novel 1alpha,25(OH)(2)D(3) analogue, TEI-9647, is the first synthetic ligand for the VDR that efficiently antagonizes the action of 1alpha, 25(OH)(2)D(3), although the extent of its antagonism depends on cell type.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 from pagetic bone marrow cells

(23S)-25-Dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) functions an antagonist of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1alpha,25-(OH)(2)D(3) from bone marrow cells of patients with Paget's disease. TEI-9647 itself never induced osteoclast formation even at 10(-6)M, but dose-dependently (10(-10) to 10(-6)M) inhibited osteoclast formation induced by physiologic concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10)M) from bone marrow cells of patients with Paget's disease. At the same time, 10(-8)M of TEI-9647 alone did not cause 1alpha,25-(OH)(2)D(3) dependent gene expression, but almost completely suppressed TAF(II)-17, a potential coactivator of VDR and 25-hydroxyvitamin D(3)-24-hydroxylase (25-OH-D(3)-24-hydroxylase) gene expression induced by 10(-10)M 1alpha,25-(OH)(2)D(3) in bone marrow cells of patients with Paget's disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10(-9)M 1alpha,25-(OH)(2)D(3) by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget's disease.     

Culture serum-induced conversion from agonist to antagonist of a Vitamin D analog, TEI-9647

The nuclear receptor for Vitamin D (VDR) mediates many of the effects of Vitamin D in target tissues by regulating gene expression. The transactivation function of ligand-bound VDR in target tissues is thought to depend on the tissue-type and the cellular-environment, but the molecular basis for these differences has not been fully understood. In this study, during characterization of TEI-9647 as a synthetic ligand for the VDR, we found that depletion of serum from the culture medium converted TEI-9647 from an antagonist to an agonist of VDR-mediated transactivation, whereas it retained antagonistic activity in the presence of serum. Consistent with these results, using a mammalian two-hybrid system, we found that TEI-9647 recruited different coactivators to the VDR in the presence and absence of serum. These findings suggest that an unknown serum factor modulates the transactivation function of the VDR.     

Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.     

Ligand-mediated conformational changes of the VDR are required for gene transactivation

The central element of the molecular switch of nuclear 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) signaling is the ligand-binding domain (LBD) of the Vitamin D receptor (VDR), which can be stabilized by 1alpha,25(OH)(2)D(3) or its analogues in to agonistic, antagonistic or inverse agonistic conformations. The positioning of helix 12 of the LBD is of most critical importance for these conformations, because it determines the distance between the charge clamp amino acids K246 and E420 that are essential for VDR-coactivator (CoA) interaction. Most VDR ligands have been identified as agonists and only a few (e.g., ZK168281 and TEI-9647) as pure or partial antagonists. Antagonists induce corepressor (CoR) dissociation from the VDR but prevent completely or partially CoA interaction and thus transactivation. Gemini is a 1alpha,25(OH)(2)D(3) analogue with two identical side chains that despite its significantly increased volume binds to the VDR and acts under most conditions as an agonist. Interestingly, supramolar CoR concentrations shift Gemini from an agonist to an inverse agonist, which actively recruits CoR to the VDR and thus mediates repression of 1alpha,25(OH)(2)D(3) target genes. Gemini is the first described (conditional) inverse agonist to an endocrine nuclear receptor (NR) and may function as a sensor for cell-specific CoA/CoR ratios.     

Design and synthesis of potent vitamin D receptor antagonists with A-ring modifications: remarkable effects of 2alpha-methyl introduction on antagonistic activity

Novel A-ring analogues of the vitamin D receptor (VDR) antagonist (3a), ZK-159222, and its 24-epimer (3b) were convergently synthesized. Preparation of the CD-ring portions with the side chains of 3a,b, followed by palladium-catalyzed cross-coupling with the A-ring enyne precursors (15a,b), (3S,4S,5R)- and (3S,4S,5S)-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded the 2alpha-methyl-introduced analogues (4a,b) and their 3-epimers (5a,b). The biological profiles of the hybrid analogues were assessed in terms of affinity for VDR, and antagonistic activity to inhibit HL-60 cell differentiation induced by the natural hormone, 1alpha,25-dihydroxyvitamin D(3). The analogue 4a showed an approximately fivefold higher antagonistic activity compared with 3a. The 2alpha-methyl introduction into 3a increased the receptor affinity, thereby enhancing VDR antagonism. This approach to design potent antagonists based on hybridization of structural motifs in the A-ring and in the side chain may prove to be valuable.     

Structural variants of the vitamin D analogue EB1089 reduce its ligand sensitivity and promoter selectivity

The nuclear hormone 1α,25-dihydroxyvitamin D₃ (VD) has important cell-regulatory functions but also a strong calcemic effect. Therefore, various VD analogues have been synthesized and screened for their biological profile. In order to gain more insight into the molecular basis of the high antiproliferative but low calcemic action of the VD analogue EB1089, we characterized this compound in comparison to five structurally related VD analogues. The activities of the six VD analogues in in vitro assays (limited protease digestion assays for determining interaction with monomeric vitamin D receptor (VDR), ligand-dependent gel shift assays for showing the increase of DNA binding of VDR-retinoid X receptor (RXR) heterodimers, and reporter gene assays on different types of VD response elements for demonstrating the efficacy in nuclear VD signalling) were found to represent their biological potency (antiproliferative effect on different malignant cell lines). In this series, EB1089 proved to be the most potent VD analogue; that is, every structural modification (20-epi configuration, cis-configuration at position C24, or changes at the ethyl groups at position C25) appeared to reduce the determined activities mediated through the VDR of these analogues. Moreover, the modifications of EB1089 resulted in a loss of VD response element selectivity, suggesting that this parameter is very critical for the biological profile of this VD analogue. J. Cell. Biochem. 71:340–350, 1998. © 1998 Wiley-Liss, Inc.     

Ligands with a 3,3-diphenylpentane skeleton for nuclear vitamin D and androgen receptors: Dual activities and metabolic activation

Ligands possessing dual vitamin D(3) (VD(3))-agonistic and androgen-antagonistic activities with various activity spectra were prepared based on a substituted 3,3-diphenylpentane (DPP) skeleton. Among the compounds, (R,S)-DPP-1023 [(R,S)-7b] and (S,S)-DPP-0123 [(S,S)-7c] showed the most potent vitamin D(3)-agonistic activity [with potency comparable to that of 1alpha,25-dihydroxyvitamin D(3) (1,25-VD(3))] and nuclear androgen receptor (AR)-binding activity (with higher affinity than that of hydroxyflutamide), respectively. Metabolic activation (reduction of the carbonyl group) of pivaloyl analogs [DPP-1113 (3a), DPP-1013 (3b), DPP-0113 (3c), and DPP-0013 (3d)] in HL-60 cells was found to be necessary for binding to nuclear vitamin D(3) receptor (VDR).     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.     

Concise synthesis and biological activities of 2alpha-alkyl- and 2alpha-(omega-hydroxyalkyl)-20-epi-1alpha,25-dihydroxyvitamin D3

We found a concise route to the Trost A-ring precursor enyne for synthesizing 2alpha-alkylated 1alpha,25-dihydroxyvitamin D(3) (1) from D-glucose. The enynes were coupled with the 20-epi-CD ring part to study the effect of the double modification of 2alpha-substitution and 20-epimerization upon biological activities of 1. The novel three analogues of 2alpha-alkyl- and four analogues of 2alpha-(omega-hydroxyalkyl)-20-epi-1alpha,25-dihydroxyvitamin D(3) (5b-d and 6a-d) showed higher binding affinity for vitamin D receptor (VDR) and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone 1.     

Antagonistic action of a 25-carboxylic ester analogue of 1alpha, 25-dihydroxyvitamin D3 is mediated by a lack of ligand-induced vitamin D receptor interaction with coactivators

A 25-carboxylic ester analogue of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)), ZK159222, was described as a novel type of antagonist of 1alpha,25-(OH)(2)D(3) signaling. The ligand sensitivity of ZK159222, in facilitating complex formation between 1alpha,25-(OH)(2)D(3) receptor (VDR) and the retinoid X receptor (RXR) on a 1alpha,25-(OH)(2)D(3) response element (VDRE), was approximately 7-fold lower when compared with 1alpha,25-(OH)(2)D(3). However, ZK159222 was not able to promote a ligand-dependent interaction of the VDR with the coactivator proteins SRC-1, TIF2, and RAC3, neither in solution nor in a complex with RXR on DNA. Functional analysis in HeLa and COS-7 cells demonstrated a 10-100-fold lower ligand sensitivity for ZK159222 than for 1alpha, 25-(OH)(2)D(3) and, most interestingly, a potency that was drastically reduced compared with 1alpha,25-(OH)(2)D(3). A cotreatment of 1alpha,25-(OH)(2)D(3) with a 100-fold higher concentration of ZK159222 resulted in a prominent antagonistic effect both in functional in vivo and in in vitro assays. These data suggest that the antagonistic action of ZK159222 is due to a lack of ligand-induced interaction of the VDR with coactivators with a parallel ligand sensitivity, which is sufficient for competition with the natural hormone for VDR binding.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.