Interaction between vitamin D receptor and vitamin D ligands: two-dimensional alanine scanning mutational analysis

We present a new method to investigate the details of interaction between vitamin D nuclear receptor (VDR) and various ligands, namely a two-dimensional alanine scanning mutational analysis. In this method, the transactivation of various ligands is studied in conjunction with a series of alanine scanning mutations of the residues lining the ligand binding pocket (LBP) of VDR, and the complete set of results is profiled in a patch table. We investigated examples from four structurally diverse groups of known VDR ligands: the native vitamin D hormone and two compounds with the same side chain configuration; four 20-epi compounds; three 19-nor compounds; and two nonsecosteroids. The patch table of the results indicates characteristics of each group in terms of its interaction with 18 LBP residues. We demonstrate the validity of this approach by application to docking studies of the two nonsecosteroids.     

Synthesis of vitamin D(3) and calcitriol dimers as potential chemical inducers of vitamin D receptor dimerization

The design and synthesis of vitamin D(3) dimers 2 and 3 and 1 alpha, 25-dihydroxyvitamin D(3) (calcitriol) dimers 4 and 5 are described. The dimers were designed with a view to doubly binding the vitamin D receptor (VDR) and inducing the receptor homodimerization. In the dimers the units are linked through the C-11 position in ring C by an alkyl side chain of six or 10 carbon atoms, far from the hydroxy groups responsible for the VDR binding. The linker is formed by olefin metathesis of an olefinic side chain at the C-11 position introduced by stereoselective cuprate addition. The synthesis, which is both short and convergent, uses the Wittig-Horner approach to construct the vitamin D triene system and allows the preparation of dimers with a linker of modulated length with the purpose of optimizing the vitamin D(3)-VDR interaction.     

Design and concise synthesis of novel vitamin D analogues bearing a functionalized aromatic ring on the side chain

Five novel vitamin D analogues (2a, 2b, 3a, 3b and 4) bearing an aromatic side chain have been designed and synthesized in a convergent manner. The requisite CD-ring synthons (10a–c) were prepared from C22 aldehyde (5) using four- or five-step procedures. Using turbo-Grignard reagents allowed aromatic side chains with a polar functional moiety to be installed in a single step with excellent yields. A preliminary biological evaluation using bovine thymus vitamin D receptor (VDR) suggested that incorporating a carboxylic acid instead of the C25-hydroxy group had a positive effect on the VDR affinity compared with the corresponding esters.     

New C15-substituted active vitamin D3

C15-Substituted 1α,25-dihydroxyvitamin D(3) analogs were synthesized for the first time to investigate the effects of the modified CD-ring on biological activity concerning the agonistic positioning of helix-3 and helix-12 of the vitamin D receptor (VDR). X-ray cocrystallographic analysis proved that 0.6 ? shifts of the CD-ring and shrinking of the side chain were necessary to maintain the position of the 25-hydroxy group for proper interaction with helix-12. The 15-hydroxy-16-ene derivative showed higher binding affinity for hVDR than the natural hormone.     

Synthesis of C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation, especially biological effect on bone by modification at the C-2 position

In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 α-substituted vitamin D derivatives were synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We evaluated these compounds by two in vitro assays, affinity to VDR and transactivation assays, using human osteosarcoma (Hos) cells, and demonstrated the SAR of the C-2 position of VD(3). Furthermore, by using the OVX model, we found that compound 5c, which has a hydroxypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in the CD-ring side chain, has a strong activity for enhancing bone growth, same as the reported compound, 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D(3)1d, and this derivative shows a possibility that calcemic activity is less than 1d in vivo.     

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3.

BACKGROUND: The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS: A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS: These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.     

Synthesis and biological evaluation of all A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: possible binding modes of potent A-ring analogues to vitamin D receptor.

BACKGROUND: The secosteroid 1 alpha,25-dihydroxyvitamin D(3) (1) has a wide variety of biological activities, which makes it a promising therapeutic agent for the treatment of cancer, psoriasis and osteoporosis. Insight into the structure-activity relationships of the A-ring of 1 is still needed to assist the development of more potent and selective analogues as candidate chemotherapeutic agents, as well as to define the molecular mode of action. RESULTS: All possible A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) (6a-h) and their 20-epimers (7a-h) were designed and efficiently synthesized. The dependence of the affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP), as well as the HL-60 cell differentiation-inducing activity, upon the stereochemistry of the A-ring and at C20 in the side chain was evaluated. CONCLUSIONS: The binding affinities and potency of the 5,6-trans and 5,6-cis analogues were enhanced by a 2-methyl substituent in a certain orientation. Molecular docking studies based upon the X-ray crystal structure of VDR suggested that the axial 2-methyl group would be accommodated in a pocket surrounded by hydrophobic amino acid residues in the ligand binding domain, resulting in enhanced interaction.     

Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor

Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of beta-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization.     

Total syntheses of (+/-)-anchinopeptolide D and (+/-)-cycloanchinopeptolide D

The first synthesis of (+/-)-anchinopeptolide D (4) has been accomplished in seven steps in 10% overall yield from octopamine hydrochloride (17), N-(Boc)glycine (16), and 5-amino-2-hydroxypentanoic acid (22). The key step is the aldol dimerization and hemiaminal formation of alpha-keto amide 26, which gives primarily protected anchinopeptolide D 27 under kinetically controlled conditions. Cycloanchinopeptolide D (31) has been prepared by the unprecedented head-to-head photodimerization of the two hydroxystyrylamides of 4 using the hydrophobic effect in water to force the two side chains into close proximity so that [2 + 2] cycloaddition is faster than trans to cis double bond isomerization. Coupling of amine 21 with pyroglutamic acid affords the naturally occurring tripeptide 35, which had been assigned glutamic acid structure 34.     

Rational design of vitamin D3 analogues which selectively restore activity to a vitamin D receptor mutant associated with rickets

[formula: see text] Vitamin D3-resistant rickets (VDRR) is associated with mutations to the Vitamin D receptor (VDR) which effect ligand-dependent transactivation. Some VDRR associated mutants directly disrupt ligand binding. Using the reported VDR-1,25-dihydroxy vitamin D3 (1,25(OH)2D3) cocrystal structure, three 1,25(OH)2D3 analogues were designed to uniquely complement the rickets associated mutant VDR(Arg274-->Leu). The three analogues were 17 to 286 times more potent than 1,25(OH)2D3 with the mutant in cell-based assays and did not substantially activate cellular calcium influx.     

Synthesis and conformational analysis of 17alpha,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.     

A structural basis for the species-specific antagonism of 26,23-lactones on vitamin D signaling

The 26,23-lactone derivative of 1alpha,25-dihydroxyvitamin D3, TEI-9647, is a partial antagonist of the of human vitamin D receptor (VDR). However, we found that TEI-9647 in rat cells behaves as a weak VDR agonist. This behavior could be mimicked in human cells by the double mutagenesis of human VDR (specifically C403S and C410N). The increased agonistic action of TEI-9647 correlates to a gain in the interaction of the VDR with coactivator protein and a decreased stabilization of the antagonistic conformation of the receptor. Molecular dynamics simulations indicated that TEI-9647 acts as antagonist of human VDR by reducing the stability of helix 12 of the ligand binding domain. In contrast, N410 of the rat VDR stabilized, via backbone contacts, the interaction between helices 11 and 12. This results in TEI-9647 becoming a weak agonist in this organism.     

Modulation effects of zinc on the formation of vitamin D receptor and retinoid X receptor alpha-DNA transcription complexes: analysis by microelectrospray mass spectrometry

The vitamin D receptor (VDR) binds zinc, and the activity of vitamin D dependent genes in cells is influenced by intracellular zinc concentrations. To determine whether zinc influences vitamin D action in cells by modulating the formation of VDR and retinoid x receptor alpha (RXR alpha) heterodimer-DNA complexes, we used microelectrospray ionization mass spectrometry (microESI-MS) to assess receptor-DNA interactions in the presence of varying amounts of zinc. In the absence of DNA, VDR and RXR alpha proteins were primarily monomeric with small amounts of protein homodimers also observed. Zn(2+) (up to 300 microM) did not change VDR or RXR alpha monomer/homodimer ratios. Mass spectra of VDR combined with RXR alpha were a sum of individual protein spectral data. Zn(2+) had no effect on the interactions of receptors. With increasing amounts of Zn(2+), additional Zn(2+) ions were detected bound to VDR and RXR alpha. microESI-MS analyses of RXR alpha in the presence of an osteopontin vitamin D DNA response element (OP-VDRE) showed RXR alpha homodimer/OP-VDRE complexes. DNA-protein complex formation increased on addition of Zn(2+) up to 200 microM; at 300 microM, Zn(2+) dissociation of the RXR alpha homodimer/OP-VDRE complexes occurred, coincident with the appearance of RXR alpha monomeric protein. When microESI-MS analyses were carried out with VDR and OP-VDRE, VDR homodimer/OP-VDRE complexes were not detected. Addition of Zn(2+) did not result in VDR/OP-VDRE complex formation. Heterodimeric VDR/RXR alpha complexes with OP-VDRE were detected by microESI-MS. Addition of 300 microM Zn(2+) resulted in dissociation of the heterodimeric VDR/RXR alpha/OP-VDRE complex. Addition of Mg(2+) in place of Zn(2+) did not alter protein/OP-VDRE complexes. Our results show that zinc modulates steroid hormone receptor-DNA interactions.     

Design, synthesis, evaluation, and structure of vitamin D analogues with furan side chains

Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.     

Vitamin D side chain triazole analogs via cycloaddition `click' chemistry

Cycloaddition of a vitamin D side chain terminal acetylene with phenyl azide and separately with a vitamin D side chain terminal azide produced the corresponding 1,2,3-triazole monomeric and dimeric analogs of 1α-hydroxyvitamin D₃ in good yields.     

Vitamin D receptor gene polymorphisms in breast cancer

Breast cancer is the leading cause of cancer death among women around the world and its incidence is annually increasing. The vitamin D receptor (VDR) gene is a member of the nuclear receptor superfamily, which is expressed in breast tissue and known to modulate the rate of cell proliferation. Association between the VDR gene polymorphisms and cancer development has been suggested by several studies. However, the relationship between VDR polymorphisms and breast cancer is controversial and has not been confirmed by all studies. The purpose of this study was to investigate the genotype frequencies and association of the VDR Bsm I and Taq I polymorphisms with breast cancer in Turkish patients. In this study, 78 patients with breast cancer and 27 healthy individuals were enrolled. The prevalence of the VDR Taq I and Bsm I alleles and the genotype frequencies in patients with breast cancer was similar to that in the normal population. Our data indicate that no significant differences exist between the patients and control subjects.