Structural analysis and biological activities of C25-amino and C25-nitro vitamin D analogs.

Intense synthetic efforts have been directed towards the development of noncalcemic analogs of 1,25-dihydroxyvitamin D3. We describe here the structural analysis and biological evaluation of two derivatives of 1,25-dihydroxyvitamin D3 with modifications limited to the replacement of the 25-hydroxyl group by a 25-amino or 25-nitro groups. Both compounds are agonists of the vitamin D receptor. They mediate biological effects similar to 1,25-dihydroxyvitamin D3, the 25-amino derivative being the most potent one while being less calcemic than 1,25-dihydroxyvitamin D3. The in vivo properties of the compounds make them of potential therapeutic value.

Molecular determinants of MED1 interaction with the DNA bound VDR-RXR heterodimer.

The MED1 subunit of the Mediator complex is an essential coactivator of nuclear receptor-mediated transcriptional activation. While structural requirements for ligand-dependent binding of classical coactivator motifs of MED1 to numerous nuclear receptor ligand-binding domains have been fully elucidated, the recognition of the full-length or truncated coactivator by full nuclear receptor complexes remain unknown. Here we present structural details of the interaction between a large part of MED1 comprising its structured N-terminal and the flexible receptor-interacting domains and the mutual heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) bound to their cognate DNA response element. Using a combination of structural and biophysical methods we show that the ligand-dependent interaction between VDR and the second coactivator motif of MED1 is crucial for complex formation and we identify additional, previously unseen, interaction details. In particular, we identified RXR regions involved in the interaction with the structured N-terminal domain of MED1, as well as VDR regions outside the classical coactivator binding cleft affected by coactivator recruitment. These findings highlight important roles of each receptor within the heterodimer in selective recognition of MED1 and contribute to our understanding of the nuclear receptor-coregulator complexes.

Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR-RXR.

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.

Vitamin D Analogs Bearing C-20 Modifications Stabilize the Agonistic Conformation of Non-Responsive Vitamin D Receptor Variants.

The Vitamin D receptor (VDR) plays a key role in calcium homeostasis, as well as in cell proliferation and differentiation. Among the large number of VDR ligands that have been developed, we have previously shown that BXL-62 and Gemini-72, two C-20-modified vitamin D analogs are highly potent VDR agonists. In this study, we show that both VDR ligands restore the transcriptional activities of VDR variants unresponsive to the natural ligand and identified in patients with rickets. The elucidated mechanisms of action underlying the activities of these C-20-modified analogs emphasize the mutual adaptation of the ligand and the VDR ligand-binding pocket.

Design, Synthesis, Evaluation and Structure of Allenic 1α,25-Dihydroxyvitamin D3 Analogs with Locked Mobility at C-17.

Vitamin D receptor ligands have potential for the treatment of hyperproliferative diseases and disorders related to the immune system. However, hypercalcemic effects limit their therapeutical uses and call for the development of tissue-selective new analogs. We have designed and synthesized the first examples of 1α,25-dihydroxyvitamin D3 analogs bearing an allenic unit attached to the D ring to restrict the side-chain conformational mobility. The triene system was constructed by a Pd0 -mediated cyclization/Suzuki-Miyaura cross-coupling process in the presence of an allenic side chain. The allenic moiety was built through an orthoester-Claisen rearrangement of a propargylic alcohol. The biological activity and structure of (22S)-1α,25-dihydroxy-17,20-dien-24-homo-21-nor-vitamin D3 bound to binding domain of the vitamin D receptor, provide information concerning side-chain conformational requirements for biological activity.

Design, synthesis and evaluation of side-chain hydroxylated derivatives of lithocholic acid as potent agonists of the vitamin D receptor (VDR).

A high number of biologically active and low-calcemic secosteroidal ligands of the vitamin D receptor (VDR) have been developed, some of which are already used clinically although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages induce toxicity. We describe here the in silico design, synthesis, structural analysis and biological evaluation of two novel active lithocholic acid derivatives hydroxylated at the side chain as highly potent inhibitors of atopic dermatitis-relevant keratinocyte inflammation of potential therapeutic interest.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists.

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.

Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

Structural Basis for α-Helix Mimicry and Inhibition of Protein-Protein Interactions with Oligourea Foldamers.

Efficient optimization of a peptide lead into a drug candidate frequently needs further transformation to augment properties such as bioavailability. Among the different options, foldamers, which are sequence-based oligomers with precise folded conformation, have emerged as a promising technology. We introduce oligourea foldamers to reduce the peptide character of inhibitors of protein-protein interactions (PPI). However, the precise design of such mimics is currently limited by the lack of structural information on how these foldamers adapt to protein surfaces. We report a collection of X-ray structures of peptide-oligourea hybrids in complex with ubiquitin ligase MDM2 and vitamin D receptor and show how such hybrid oligomers can be designed to bind with high affinity to protein targets. This work should enable the generation of more effective foldamer-based disruptors of PPIs in the context of peptide lead optimization.

9-cis-13,14-Dihydroretinoic Acid Is an Endogenous Retinoid Acting as RXR Ligand in Mice.

The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.

Solution Behavior of the Intrinsically Disordered N-Terminal Domain of Retinoid X Receptor α in the Context of the Full-Length Protein.

Retinoid X receptors (RXRs) are transcription factors with important functions in embryonic development, metabolic processes, differentiation, and apoptosis. A particular feature of RXRs is their ability to act as obligatory heterodimerization partners of class II nuclear receptors. At the same time, these receptors are also able to form homodimers that bind to direct repeat separated by one nucleotide hormone response elements. Since the discovery of RXRs, most of the studies focused on its ligand binding and DNA binding domains, while its N-terminal domain (NTD) harboring a ligand-independent activation function remained poorly characterized. Here, we investigated the solution properties of the NTD of RXRα alone and in the context of the full-length receptor using small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. We report the solution structure of the full-length homodimeric RXRα on DNA and show that the NTD remains highly flexible within this complex.

Structure of the full human RXR/VDR nuclear receptor heterodimer complex with its DR3 target DNA.

Transcription regulation by steroid hormones and other metabolites is mediated by nuclear receptors (NRs) such as the vitamin D and retinoid X receptors (VDR and RXR). Here, we present the cryo electron microscopy (cryo-EM) structure of the heterodimeric complex of the liganded human RXR and VDR bound to a consensus DNA response element forming a direct repeat (DR3). The cryo-EM map of the 100-kDa complex allows positioning the individual crystal structures of ligand- and DNA-binding domains (LBDs and DBDs). The LBDs are arranged perpendicular to the DNA and are located asymmetrically at the DNA 5'-end of the response element. The structure reveals that the VDR N-terminal A/B domain is located close to the DNA. The hinges of both VDR and RXR are fully visible and hold the complex in an open conformation in which co-regulators can bind. The asymmetric topology of the complex provides the structural basis for RXR being an adaptive partner within NR heterodimers, while the specific helical structure of VDR's hinge connects the 3'-bound DBD with the 5'-bound LBD and thereby serves as a conserved linker of defined length sensitive to mutational deletion.

Architecture of DNA Bound RAR heterodimers.

Nuclear Retinoic Acid receptors (RARs) consist of three subtypes, α, ß, and γ, encoded by separate genes. They function as ligand-dependent transcriptional regulators, forming heterodimers with Retinoid X receptors (RXRs). RARs mediate the effects of retinoic acid (RA), the active metabolite of Vitamin A, and regulate many biological functions such as embryonic development, organogenesis, homeostasis, vision, immune functions, and reproduction. During the two last decades, a number of in-depth structure-function relationship studies have been performed, in particular with drug design perspectives in the therapeutics for cancer, dermatology, metabolic disease, and other human diseases. Recent structural results concerning integral receptors in diverse functional states, obtained using a combination of different methods, allow a better understanding of the mechanisms involved in molecular regulation. The structural data highlight the importance of DNA sequences for binding selectivity and the role of promoter response elements in the spatial organization of the protein domains into functional complexes.

Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors.

Transcription regulation by steroid hormones, vitamin derivatives, and metabolites is mediated by nuclear receptors (NRs), which play an important role in ligand-dependent gene expression and human health. NRs function as homodimers or heterodimers and are involved in a combinatorial, coordinated and sequentially orchestrated exchange between coregulators (corepressors, coactivators). The architecture of DNA-bound functional dimers positions the coregulators proteins. We previously demonstrated that retinoic acid (RAR-RXR) and vitamin D3 receptors (VDR-RXR) heterodimers recruit only one coactivator molecule asymmetrically without steric hindrance for the binding of a second cofactor. We now address the problem of homodimers for which the presence of two identical targets enhances the functional importance of the mode of binding. Using structural and biophysical methods and RAR as a model, we could dissect the molecular mechanism of coactivator recruitment to homodimers. Our study reveals an allosteric mechanism whereby binding of a coactivator promotes formation of nonsymmetrical RAR homodimers with a 21 stoichiometry. Ligand conformation and the cofactor binding site of the unbound receptor are affected through the dimer interface. A similar control mechanism is observed with estrogen receptor (ER) thus validating the negative cooperativity model for an established functional homodimer. Correlation with published data on other NRs confirms the general character of this regulatory pathway.

Phosphorylation of the retinoic acid receptor alpha induces a mechanical allosteric regulation and changes in internal dynamics.

Nuclear receptor proteins constitute a superfamily of proteins that function as ligand dependent transcription factors. They are implicated in the transcriptional cascades underlying many physiological phenomena, such as embryogenesis, cell growth and differentiation, and apoptosis, making them one of the major signal transduction paradigms in metazoans. Regulation of these receptors occurs through the binding of hormones, and in the case of the retinoic acid receptor (RAR), through the binding of retinoic acid (RA). In addition to this canonical scenario of RAR activity, recent discoveries have shown that RAR regulation also occurs as a result of phosphorylation. In fact, RA induces non-genomic effects, such as the activation of kinase signaling pathways, resulting in the phosphorylation of several targets including RARs themselves. In the case of RARα, phosphorylation of Ser369 located in loop L9-10 of the ligand-binding domain leads to an increase in the affinity for the protein cyclin H, which is part of the Cdk-activating kinase complex of the general transcription factor TFIIH. The cyclin H binding site in RARα is situated more than 40 Å from the phosphorylated serine. Using molecular dynamics simulations of the unphosphorylated and phosphorylated forms of the receptor RARα, we analyzed the structural implications of receptor phosphorylation, which led to the identification of a structural mechanism for the allosteric coupling between the two remote sites of interest. The results show that phosphorylation leads to a reorganization of a local salt bridge network, which induces changes in helix extension and orientation that affects the cyclin H binding site. This results in changes in conformation and flexibility of the latter. The high conservation of the residues implicated in this signal transduction suggests a mechanism that could be applied to other nuclear receptor proteins.

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells.

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM-FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells.

4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure-Function Study.

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4ß,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4ß,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.

Design, synthesis, and biological activity of D-bishomo-1α,25-dihydroxyvitamin D3 analogs and their crystal structures with the vitamin D nuclear receptor.

The biologically active metabolite of vitamin D3 - calcitriol - is a hormone involved in the regulation of calcium-phosphate homeostasis, immunological processes and cell differentiation, being therefore essential for the proper functioning of the human body. This suggests many applications of this steroid in the treatment of diseases such as rickets, psoriasis and some cancers. Unfortunately, using therapeutic doses of calcitriol is associated with high concentrations of this compound which causes hypercalcemia. For this reason, new calcitriol analogs are constantly sought, devoid of calcemic effects but maintaining its beneficial properties. In this study, we present the synthesis of vitamin D derivatives characterized by an enlarged (seven-membered) ring D. Preparation of the designed vitamin D compounds required separate syntheses of crucial building blocks (C/D-rings fragments with side chain and rings A) which were combined by different methods, including Wittig-Horner reaction and Suzuki coupling. Biological activities of the target vitamin D analogs were assessed both in vitro and in vivo, demonstrating their significant potency compared to the natural hormone. Furthermore, the successful crystallization of these compounds with the vitamin D receptor (VDR) enabled us to investigate additional molecular interactions with this protein.