20-epi Analogues Research Articles

Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3

Analogs of 1α,25-dihydroxyvitamin D3 (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D3 (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs. For example, 1α,25-dihydroxy-16-ene-20-epi-vitamin D3 (S4), but not 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR-106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3-epi-S4, a metabolite of S4 produced through C-3 epimerization pathway. Using GC/MS, ESI-MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25-hydroxy-16-ene-20-epi-vitamin D3-1-stearate and 25-hydroxy-16-ene-20-epi-vitamin D3-1-oleate and their respective C-3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR-106 cells in which both 1α,25(OH)2-16-ene-20-epi-D3 and 1α,25(OH)2-16-ene-20-epi-3-epi-D3 undergo C-1 esterification into stearic and oleic acid esters.

Partial prevention of active Heymann nephritis by 1 alpha, 25 dihydroxyvitamin D3.

The hormone 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunosuppressive effects in vitro. Recent publications also described a protective effect of the hormone in various animal models of immune-mediated diseases. To test its in vivo activity we induced active Heymann nephritis in Lewis rats that were either untreated or treated with 1,25(OH)2D3 or its synthetic 20-epi analogue, KH1060. Treatment with cyclosporine A (CsA) was used as an immunosuppressive control. In this nephrotic model the administration of 1,25(OH)2D3 (0.5 microgram/kg body weight) given on alternate days during the first 13 days after active immunization significantly reduced the proteinuria as measured by weeks 7-9. This reduction was comparable to the reduction observed in rats treated with CsA (20 mg/kg) on alternate days. A second series of experiments with 1,25(OH)2D3 confirmed these findings. The level of autoantibodies was found to be significantly suppressed during the treatment time in the CsA (20 mg/kg) group, whereas the limit of significance (P = 0.06) was reached in the 1,25(OH)2D3 (0.5 microgram/kg) group. The size of the immune deposits also was found to be substantially smaller in the groups that developed less proteinuria. The administration of 1,25(OH)2D3 transiently increased the mean serum calcium concentration with 2.5 mg/dl above the pretreatment values, and the urinary calcium excretion by a factor of 3-5 during the short treatment time. Treatment with the analogue KH1060 did not reduce the proteinuria significantly. Our experiments add evidence to the hypothesis that 1,25(OH)2D3 in pharmacological doses has immunosuppressive potency.

Synthesis and Conformational Analysis of 17α,21-Cyclo-22-Unsaturated Analogues of Calcitriol

Six new calcitriol analogues, conformationally restricted at their side chain by the introduction of both a cyclopropane ring at C17-C20 and a double or triple bond at C22, were synthesized using the Wittig-Horner approach to construct the triene system. The six CD-ring and side-chain bearing fragments were prepared from ketone 14 by a divergent route to generate both series of epimers at C20, followed by stereoselective cyclopropanation. The (E)-alkenyl side chain was synthesized by means of a Wittig reaction. The alkynyl side chain was prepared by Corey-Fuchs homologation, followed by alkylation. The (Z)-alkenyl side chain was prepared from the previous alkyne by partial hydrogenation. The 20-epi analogues bind more strongly to VDR than the corresponding analogues with the C20 natural stereochemistry. These results can be reasoned by conformational analysis and hydrophobic interactions with the VDR ligand-binding domain.

Design and Synthesis of Novel 20-epiAnalogues of Calcitriol with Restricted Side Chain Conformation

The design and efficient preparation of two 20-epi analogues of calcitriol with restricted side chain conformation is described. The formation of the tetrahydropyran ring was achieved via zinc chloride mediated etherification of alcohols. Docking experiments show that these novel analogues could be interesting synthetic targets.

Design and Efficient Synthesis of 2α-(ω-Hydroxyalkoxy)-1α,25-dihydroxyvitamin D3Analogues, Including 2-epi-ED-71 and Their 20-Epimers with HL-60 Cell Differentiation Activity

A concise and efficient synthetic approach to 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D(3) (4a-c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the construction of the 2 alpha-modified A-ring precursors (11a-c). It was found that the best ligand for the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in the induction of HL-60 cell differentiation for 4a-c was almost the same or weaker than that of 1 despite the strong binding affinity for the VDR. Next, we were interested in the "double modification"of 1 based on 4a-c with C20-epimerization, affording 2 alpha-(omega-hydroxyalkoxy)-20-epi-1 alpha,25-dihydroxyvitamin D(3) (20-epi-4a-c). All three 2 alpha-substituted 20-epi analogues of 1 (20-epi-4a-c) exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a-c showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher than that of the natural hormone 1.

Novel Gemini analogs of 1α,25-dihydroxyvitamin D 3 with enhanced transcriptional activity

The active form of vitamin D, 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3), exerts its effects through regulation of target gene transcription. Configuration at C-20 of 1,25(OH) 2D 3 is important in determining potency, as shown by the high potency of analogs with inverted configuration at C-20 (20-epi compounds). Gemini analogs of 1,25(OH) 2D 3 contain two side chains, combining a C-20-normal with a C20-epi side chain. We studied the potency of analogs combining double (Gemini) side chains with a 23-triple bond and a C-26,27-hexafluoro substitution in either the 20-epi (analog 20R) or 20-normal (analog 20S) side chain. These novel Gemini analogs were 8–50-fold more potent than 1,25(OH) 2D 3 in inducing U937, HL-60G, and THP-1 differentiation and 5–50-fold more potent in inducing transcription from the osteocalcin vitamin D response element or the 25-hydroxyvitamin D 3-24-hydroxylase (24OHase) promoter. In vivo, following i.p. injection in vitamin D-deficient mice, the 20S analog induced significantly higher levels of calbindin-D 9K mRNA in intestine, and 24OHase and calbindin-D 28K in kidney than 1,25(OH) 2D 3 or analog 20R. Increased potency did not correlate with ligand-receptor binding affinity. In GST-pull down assays using in vitro translated VDR, Gemini analogs showed equivalent (or even attenuated) potency to 1,25(OH) 2D 3 in recruiting cofactors DRIP205 and GRIP-1 to VDR. However, Gemini analogs were up to 15-fold more potent than 1,25(OH) 2D 3 in recruiting the same cofactors to VDR in GST-pull down assays using equal amounts of VDR from nuclear extracts of VDR transfected and hormone treated (24 hr) COS-7 cells. Deletion of C-19 in either 20S or 20R Gemini analogs resulted overall in slightly less potent analogs compared to Gemini itself. We conclude that enhanced potency of the novel Gemini analogs is at least partly due to increased metabolic stability of the analogs, resulting in more cofactor binding and elevated levels of transcription.

Inhibition of MG-63 cell cycle progression by synthetic vitamin D 3 analogs mediated by p27, Cdk2, cyclin E, and the retinoblastoma protein

Progression through eukaryotic cell division cycle is regulated by synergistic activities of both positive and negative regulatory factors. The active form of vitamin D 3 (1α,25(OH) 2D 3, 1,25D) and a number of its synthetic analogs have been shown to arrest cells in the G 1 phase of the cell cycle. In the present study, 1α,25(OH) 2D 3 and the analogs KH1060, EB1089, and CB1093 were used to study the mechanism of the cell cycle arrest and to compare the effectiveness of these compounds in human MG-63 osteosarcoma cells. The 20-epi analogs KH1060 and CB1093, as well as the 20-normal analog EB1089, were found to be more potent than 1α,25(OH) 2D 3 in inhibiting cell proliferation and arresting the MG-63 cells in the G 1 phase. These analogs were more active than 1α,25(OH) 2D 3 in increasing the cyclin dependent kinase inhibitor p27 protein levels (approximately 2.3–2.5-fold compared to 1α,25(OH) 2D 3) by both increasing its formation and decreasing its degradation rate. The increased p27 formation was accompanied by stabilization of binding of nuclear proteins to the Sp1+NF-Y responsive promoter region of the p27 gene. The increase in p27 protein levels and the simultaneous decrease in cyclin E protein levels was accompanied by decreased Cdk2 kinase activity, retinoblastoma (Rb) protein hypophosphorylation and, finally, cell cycle arrest in the G 1 phase. In summary, the analogs KH1060, EB1089, and CB1093 keep Rb protein in its growth-suppressing, hypophosphorylated form and prevent cell cycle progression through the restriction point. Therefore, these synthetic vitamin D 3 analogs may be potential candidates for treating diseases, where cell cycle regulation is needed.

Synthesis of novel hapten derivatives of 1alpha,25-dihydroxy-vitamin D3 and its 20-epi analogue.

Hapten derivatives of 1alpha,25-dihydroxyvitamin D(3) and its 20-epimer were synthesized and conjugated to a carrier protein for raising polyclonal antibodies. The haptens were linked through spacers at C-16, thereby exposing both the A-ring and the side chain of the molecules, to maximize antibody specificity. The spacers were introduced via stereoselective hydroboration of 16-ene intermediates as the key step. In immunoassays, the antibodies raised toward the natural hormone were selective to this compound over derivatives with modifications in the A-ring or the side chain. The antibodies toward the 20-epimer, however, were unable to recognize modifications in the side chain.

Asymmetric total synthesis of (-)-callystatin A and (-)-20-epi-callystatin A employing chemical and biological methods.

An efficient asymmetric total synthesis of the potent cytotoxic marine natural product (-)-callystatin A and its 20-epi analogue has been achieved. The synthetic pathway involved the preparation of three fragments to be coupled with each other at the end of the route. The first fragment 3 was obtained using a biocatalytic enantioselective reduction of a 3,5-dioxocarboxylate as the key step. For the second intermediate 4 the asymmetric alpha-alkylation of an O-protected derivative of 4-hydroxybutanal was performed exploiting the SAMP/RAMP hydrazone alkylation methodology, and followed by a highly Z-selective Horner-Wadsworth-Emmons reaction under modified conditions. For the synthesis of the polypropionate fragment 5 a diastereoselective syn-aldol reaction was employed between a chiral ethyl ketone and an alpha-substituted chiral aldehyde, both prepared in enantiopure form again by means of the asymmetric alkylation of their corresponding RAMP hydrazones. Finally, these three building blocks were coupled using highly E-selective Wittig reactions via allyltributylphosphonium ylides to afford the target compounds after a final oxidation/deprotection sequence.

Vitamin D analogue-specific recruitment of vitamin D receptor coactivators.

Synthetic ligands for the vitamin D receptor (VDR) are potential therapeutic agents for metabolic, neoplastic, and autoimmune disorders. Some of these ligands have similar or more potent antiproliferative, yet reduced hypercalcemic actions, than calcitriol. However, the mechanisms for these differential actions have not been clearly defined. We hypothesized that these gene- and tissue-specific effects may relate to ligand-directed selective recruitment of transcriptional coactivators. To identify key elements in ligand structure that facilitate VDR-coactivator interactions, the current studies assessed the ability of the VDR to recruit the coactivators GRIP1 and RAC3 following activation by a series of 20-R- and 20-S (20-epi)-modified analogues. The strength of VDR-coactivator interactions was ligand-specific and did not always correlate with ligand-receptor binding affinity. In general, the 20-epi analogues enhanced these interactions, whereas the 20-R-modified analogues were less effective than calcitriol. The 16-ene,23-yne modification and fluorinated substituents to the side-chain attenuated interaction with coactivators. The enhanced ability of the VDR to recruit GRIP1 following activation by the 20-epi analogues was consistent with potentiation of 20-epi analogue-induced transactivation of the osteocalcin gene promoter by GRIP1. Overall, the structure of the ligand side-chain as well as its orientation seemed to affect the avidity of coactivator binding. These results suggest that selective recruitment of coactivators may contribute to gene- and tissue-specific effects of vitamin D analogues.

A vitamin D analogue (MC 1288) has immunomodulatory properties and suppresses collagen-induced arthritis (CIA) without causing hypercalcaemia.

The immunomodulatory properties of the vitamin D analogue MC 1288 (20-epi-1alpha,25-dihydroxycholecalciferol) were investigated in CIA in rats. The analogue was administered systemically at three different time points; (i) for 10 consecutive days before collagen (CII) immunization; (ii) for 10 consecutive days after CII immunization; or (iii) for 7 consecutive days from disease onset. Treatment initiated either 10 days before CII immunization or at the day of collagen immunization effectively suppressed the development of arthritis. Treatment initiated at the day of the onset of arthritis reduced the severity of joint inflammation. Significantly, doses which did not induce hypercalcaemia decreased the incidence and severity of arthritis. In vivo treatment with the 20-epi analogue of 1alpha,25-dihydroxycholecalciferol diminished the serum levels of antibodies to rat CII. Similarly, mitogen-induced proliferation of lymph node cells from rat CII-immunized animals was reduced. The experiments demonstrate that the vitamin D analogue MC 1288 has the ability to prevent, and furthermore to suppress, already established CIA by its immunomodulatory properties without inducing hypercalcaemia.

Erratum to ‘Differential regulation of heterodimerization by 1α,25-dihydroxyvitamin D3 and its 20-epi analog’

Erratum to ‘Differential regulation of heterodimerization by 1α,25-dihydroxyvitamin D3 and its 20-epi analog’

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands.

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1alpha,25(OH)(2)D(3) and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17beta-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Vitamin D 3 analogs (MC 1288, KH 1060, EB 1089, GS 1558, and CB 1093): studies on their mechanism of action

Selected 20-epi and 20-normal vitamin D 3 analogs were studied. First, point mutations were introduced into human vitamin D receptor (VDR) to identify residues important for ligand binding. In helices three, four and five, His229, Asp232, Ser237 and Arg274 seem to have an important role in the binding of calcitriol. Surprisingly, the 20-epi analog MC 1288 did not bind to Ser237. Second, the effects of analogs on VDR degradation were studied. The transcriptionally active 20-epi analogs protected VDR against degradation more efficiently than the 20-normal analogs and calcitriol. With proteasome inhibitor MG-132 formation of Sug-1-RXRβ-VDR-VDRE complex was detected. The 20-epi analogs effectively prevented its formation. Thus, the 20-epi analogs induce a VDR conformation, which prevents binding of factors mediating VDR degradation. Third, the analogs were found to be powerful regulators of cell cycle progression in MG-63 cells. They arrested cell cycle in the G0/G1 phase at lower concentrations and earlier time points than calcitriol. This was accompanied by hypophosphorylation of Rb followed by strong inhibition of Cdk2 activity. This correlated with increased levels of p27. Cdk2 and cyclin E levels were downregulated but those of p21 and cyclin D1 were not affected. Thus, a similar sequence of events with calcitriol and the analogs in inhibiting MG-63 cell growth was detected but the analogs had much longer lasting and stronger effects than calcitriol. A unifying scheme for the varying effects of vitamin D 3 analogs is presented.

Differential regulation of heterodimerization by 1α,25-dihydroxyvitamin D 3 and its 20-epi analog

Twenty-epi analogs of 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2D 3) are 100–1000 times more potent transcriptionally than the natural hormone. To determine to what extent this enhanced activity is mediated through modulation of the dimerization process, we performed quantitative dimerization assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of VDR (GST-VDR) or retinoid X receptor (RXR)α (GST-RXR). We found that VDR did not form homodimers in either the presence or absence of ligand, but heterodimerization of the ligand-binding domains of RXRα and VDR was primarily deltanoid-dependent. The ED 50 for induction of heterodimerization was 1–2 × 10 − 9 M for 1α,25(OH) 2D 3 and 0.5 × 10 − 11 M for 20-epi 1α,25(OH) 2D 3. Mutations in VDR’s activation function 2 domain (AF-2) diminished the abilities of 1α,25(OH) 2D 3 to induce a protease-resistant conformation and heterodimerization. These mutations changed neither the potency of 20-epi-1α,25(OH) 2D 3 to induce protease-resistant conformation nor its potency to induce dimerization. Mutations in heptad 9/helix 10 abolished the ability of both 1α,25(OH) 2D 3 and the 20-epi analog to induce dimerization, but not their potency to fold VDR into a protease-resistant conformation. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR’s dimerization interface, and that interfaces exposed by these ligands are probably not significantly different. However, the mechanisms by which the two ligands expose the dimerization interface are different with respect to participation of the AF-2 domain.

Regulation of ligand-induced heterodimerization and coactivator interaction by the activation function-2 domain of the vitamin D receptor.

Twenty-epi analogs of 1alpha,25-dihydroxyvitamin D3 (1,25D3) are 100-1000 times more potent transcriptionally than the natural hormone. To determine whether this enhanced activity is mediated through modulation of the dimerization process or through interaction with coactivators, we performed quantitative protein-protein interaction assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of retinoid X receptor (RXRalpha), or the nuclear receptor-interacting domain of the steroid receptor coactivator 1 (SRC-1), or the glucocorticoid receptor-interacting protein 1 (GRIP-1). We found that heterodimerization of the ligand-binding domains of RXRalpha and VDR was primarily deltanoid dependent as was the interaction of VDR with the SRC-1 or with GRIP-1. The ED50 for induction of heterodimerization was 2 nM for 1,25D3 and 0.05 nM for 20-epi-1,25D3. However, the ED50 for induction of VDR interaction with SRC-1 was similar for both 1,25D3 and the 20-epi analog (ED50 = 0.7-1.0 nM) as was the ED50 for ligand-mediated interaction of VDR with GRIP-1 (ED50 = 0.1-0.3 nM). Mutations in heptad 9 diminished both 1,25D3 and the 20-epi analog-mediated dimerization, without changing binding of these ligands to VDR. Mutations in VDR's activation function 2 (AF-2) domain/helix 12 residues diminished the ability of 1,25D3 to induce heterodimerization and interaction with SRC-1. These mutations did not change the ability of 20-epi-1,25D3 to induce dimerization but did diminish its ability to induce interaction with SRC-1. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's functional interfaces. The mechanisms by which the two ligands expose these functional interfaces differ with respect to participation of the AF-2 domain.

Mechanism of action of superactive vitamin D analogs through regulated receptor degradation

We and others have previously shown that selected vitamin D analogs potentiate the vitamin D receptor (VDR) mediated transcription much more efficiently than the natural hormone itself. Here we show that the transcriptionally active 20-epi analogs, namely KH 1060 and MC 1288, protect VDR against degradation more efficiently than calcitriol at 10(-10) M concentration (VDR t(1/2) > 48 h, 17 h, and 10 h, respectively). The conformationally epi-like analog EB 1089 did not significantly alter the half-life of VDR (10.3 h), but retained the VDR levels over longer periods of time than calcitriol. The transcriptionally weak analog GS 1558, on the other hand, enhanced VDR degradation even more than what was observed with the unliganded receptor (t(1/2) 4.5 h and 5 h, respectively). Inhibition of proteasome activity by the inhibitor MG-132 resulted in a marked increase in the VDR levels in cells treated with the vehicle or GS 1558 (2.5-fold and 2.7-fold, respectively), more than twice the levels observed in the presence of calcitriol or EB 1089 (1.2-fold and 1.1-fold, respectively). MG-132 treatment did not increase the VDR levels in cells treated with KH 1060 or MC 1288. The electrophoretic mobility shift assay (EMSA) with nuclear extracts from MG-132-treated cells revealed formation of a high-molecular-weight RXRbeta-VDR-VDRE complex, which also contained Sug1. In the presence of calcitriol, 34% of total VDR in its DNA binding state was present in this complex. The 20-epi analogs effectively prevented the formation of this complex, since, in this case, only 16% of total VDR was found in this complex. These results suggest that KH 1060 and MC 1288 induce a VDR conformation, which prevents binding of proteins mediating receptor degradation. As a result, the regulation of VDR degradation differs from that found with the calcitriol-VDR complex resulting in superactive transcriptional action of the analogs.

Mechanism of action of superactive vitamin D analogs through regulated receptor degradation.

We and others have previously shown that selected vitamin D analogs potentiate the vitamin D receptor (VDR) mediated transcription much more efficiently than the natural hormone itself. Here we show that the transcriptionally active 20-epi analogs, namely KH 1060 and MC 1288, protect VDR against degradation more efficiently than calcitriol at 10(-10) M concentration (VDR t(1/2) > 48 h, 17 h, and 10 h, respectively). The conformationally epi-like analog EB 1089 did not significantly alter the half-life of VDR (10.3 h), but retained the VDR levels over longer periods of time than calcitriol. The transcriptionally weak analog GS 1558, on the other hand, enhanced VDR degradation even more than what was observed with the unliganded receptor (t(1/2) 4.5 h and 5 h, respectively). Inhibition of proteasome activity by the inhibitor MG-132 resulted in a marked increase in the VDR levels in cells treated with the vehicle or GS 1558 (2.5-fold and 2.7-fold, respectively), more than twice the levels observed in the presence of calcitriol or EB 1089 (1.2-fold and 1.1-fold, respectively). MG-132 treatment did not increase the VDR levels in cells treated with KH 1060 or MC 1288. The electrophoretic mobility shift assay (EMSA) with nuclear extracts from MG-132-treated cells revealed formation of a high-molecular-weight RXRbeta-VDR-VDRE complex, which also contained Sug1. In the presence of calcitriol, 34% of total VDR in its DNA binding state was present in this complex. The 20-epi analogs effectively prevented the formation of this complex, since, in this case, only 16% of total VDR was found in this complex. These results suggest that KH 1060 and MC 1288 induce a VDR conformation, which prevents binding of proteins mediating receptor degradation. As a result, the regulation of VDR degradation differs from that found with the calcitriol-VDR complex resulting in superactive transcriptional action of the analogs.

The Importance of the Putative Helices 4 and 5 of Human Vitamin D3 Receptor for Conformation and Ligand Binding

The 3-D structure of the human vitamin D3 receptor has not been solved to date. To study the conformation of the ligand binding pocket and the amino acid residues important for binding of calcitriol and its synthetic 20-epi analog MC1288, we have introduced several point mutations into putative helices 4 and 5 of human vitamin D3 receptor by site-directed mutagenesis. The amino acid residues Ser256, Glu257, Asp258, Gln259, Lys264, Ser265, Ser266, Glu269, Arg274, Ser278, and Phe279 were substituted by alanine. Our results suggest that Arg274 is important for the binding of calcitriol and probably also for the binding of the synthetic vitamin D analog MC1288, whereas Asp258, Gln259, Glu269, and Phe279 may have an important role in stabilizing the conformation of hVDR after ligand binding.

20-Epi Analogues of 1,25-Dihydroxyvitamin D3 Are Highly Potent Inducers of DRIP Coactivator Complex Binding to the Vitamin D3 Receptor

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in the stimulation of bone growth, mineralization, and intestinal calcium and phosphate absorption; it also acts as a general inhibitor of cellular proliferation. Several new, clinically relevant compounds dissociate antiproliferative and calcemic activities of 1,25(OH)2D3, but the molecular basis for this has not been clearly elucidated. Here, we tested whether the potency of one class of compounds, 20-epi analogues, to induce myeloid cell differentiation, is because of direct molecular effects on vitamin D receptor (VDR). We report that two 20-epi analogues, MC1627 and MC1288, induced differentiation and transcription of p21(Waf1,Cip1), a key VDR target gene involved in growth inhibition, at a concentration 100-fold lower than that of 1,25(OH)2D3. We compared this sensitivity to analogue effects on VDR interacting proteins: RXR, GRIP-1, and DRIP205, a subunit of the DRIP coactivator complex. Compared with the interaction of VDR with RXR or GRIP-1, the differentiation dose-response most closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to VDR and to the ability of the receptor to activate transcription in a cell-free system. These results provide compelling links between the efficiency of the 20-epi analogue in inducing VDR/DRIP interactions, transactivation in vitro, and its enhanced ability to induce cellular differentiation.