Design, Synthesis, and Biological Evaluation of New Type of Gemini Analogues with a Cyclopropane Moiety in Their Side Chain.

We synthesized two new gemini analogues, UG-480 and UG-481, that incorporate a modified longer side chain containing a cyclopropane group. The evaluation of the bioactivities of the two gemini analogues indicated that the 17,20 threo (20S) compound, UG-480, is the most active one and is as active as 1,25(OH)2D3. Docking and molecular dynamics (MD) data showed that the compounds bind efficiently to vitamin D receptor (VDR) with UG-480 to form an energetically more favorable interaction with His397. Structural analysis indicated that whereas the UG-480 compound efficiently stabilizes the active VDR conformation, it induces conformational changes in the H6-H7 VDR region that are greater than those induced by the parental Gemini and that this is due to the occupancy of the secondary channel by its modified side chain.

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.

Further Studies on the Highly Active Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3 (Calcitriol): Refinement of the Side Chain.

Current efforts in the vitamin D field are directed toward the development of highly antiproliferative yet noncalcemic analogues of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3). We have recently reported the design, synthesis, biological evaluation, and crystal structures of a series of novel analogues that both lack the steroidal C-ring and have an m-phenylene ring replacing the steroidal cyclopentane D-ring. We have now investigated the potentiating effects of incorporating selected modifications (hexafluorination and/or an internal triple bond) within the steroidal side chain in our series. An alternative synthetic strategy (Wittig-Horner approach instead of our previously used Pd-catalyzed tandem cyclization/cross-coupling) for the construction of the vitamin D triene system was found convenient for the target compounds 2, 3a, 3b, and 3c of this report. These modifications enhance vitamin D nuclear receptor (VDR) interactions and consequently VDR-associated biological properties compared to parental PG-136 compound while maintaining normal calcium levels.

Design, Synthesis, Biological Activity, and Structural Analysis of Novel Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3.

The toxic calcemic effects of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3, 1,25-dihydroxycholecalciferol) in the treatment of hyperproliferative diseases demand the development of highly active and noncalcemic vitamin D analogues. We report the development of two highly active and noncalcemic analogues of 1,25D3 that lack the C-ring and possess an m-phenylene ring that replaces the natural D-ring. The new analogues (3a, 3b) are characterized by an additional six-carbon hydroxylated side chain attached either to the aromatic nucleus or to the triene system. Both compounds were synthesized by the Pd-catalyzed tandem cyclization/cross coupling approach starting from alkyne 6 and diphenol 8. Key steps include a stereoselective Cu-assisted addition of a Grignard reagent to an aromatic alkyne and a Takai olefination of an aromatic aldehyde. The new compounds are noncalcemic and show transcriptional and antiproliferative activities similar to 1,25D3. Structural analysis revealed that they induce a large conformational rearrangement of the vitamin D receptor around helix 6.

Advances in Vitamin D Receptor Function and Evolution Based on the 3D Structure of the Lamprey Ligand-Binding Domain.

1α,25-dihydroxyvitamin D3 (1,25D3) regulates many physiological processes in vertebrates by binding to the vitamin D receptor (VDR). Phylogenetic analysis indicates that jawless fishes are the most basal vertebrates exhibiting a VDR gene. To elucidate the mechanism driving VDR activation during evolution, we determined the crystal structure of the VDR ligand-binding domain (LBD) complex from the basal vertebratePetromyzon marinus, sea lamprey (lVDR). Comparison of three-dimensional crystal structures of the lVDR-1,25D3 complex with higher vertebrate VDR-1,25D3 structures suggests that 1,25D3 binds to lVDR similarly to human VDR, but with unique features for lVDR around linker regions between H11 and H12 and between H9 and H10. These structural differences may contribute to the marked species differences in transcriptional responses. Furthermore, residue co-evolution analysis of VDR across vertebrates identifies amino acid positions in H9 and the large insertion domain VDR LBD specific as correlated.

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.

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.

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.

DNA recognition by retinoic acid nuclear receptors.

Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) to regulate gene expression. The heterodimer recognizes the genome via a large and diverse repertoire of DNA response elements. Assessing the binding mode of RAR and RXR with various DNA response elements is important for understanding how they select their binding site and how DNA sequence and topology allosterically regulate RAR function. A number of complementary assays are often employed for analysis of the binding mode. To biochemically and structurally characterize RAR and RXR-DNA complexes, we describe how to express and purify RAR and RXR-DNA binding domains (DBDs) and multidomain constructs. We also describe the use of electrospray ionization mass spectrometry (ESI MS) and isothermal titration calorimetry (ITC) that give information about stoichiometry and binding affinity, as well as our approaches for co-crystallization of RAR and RXR DBDs with DNA.

Recurrent activating mutations of PPARγ associated with luminal bladder tumors.

The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein-including N-terminal, DNA-binding and ligand-binding domains-and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.

Modulation of RXR-DNA complex assembly by DNA context.

Retinoid X Receptors (RXRs) act as dimer partners for several nuclear receptors including itself, binding to genomic DNA response elements and regulating gene transcription with cell and gene specificity. As homodimers, RXRs bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1) and little variability of this consensus site is observed for natural DR1s. However, these variations are responsible of the modulation of RXR receptors function through differential binding affinity and conformational changes. To further our understanding of the molecular mechanisms underlying RXR-DNA interactions, we examined how RXR DBDs bind to different DR1s using thermodynamics, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. We show that the half-site sequences modulate the binding cooperativity that results from the protein-protein contacts between the two DBDs. Chemical shifts perturbation NMR experiments revealed that sequence variations in half-sites induce changes that propagate from the protein-DNA interface to the dimerization interface throughout the DBD fold.

Investigation of 20S-hydroxyvitamin D3 analogs and their 1α-OH derivatives as potent vitamin D receptor agonists with anti-inflammatory activities.

20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.

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.

Monitoring of the retinoic acid receptor-retinoid X receptor dimerization upon DNA binding by native mass spectrometry.

Identifying protein-DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor-retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR-RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR-RXR heterodimerization.

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.

Common architecture of nuclear receptor heterodimers on DNA direct repeat elements with different spacings.

Nuclear hormone receptors (NHRs) control numerous physiological processes through the regulation of gene expression. The present study provides a structural basis for understanding the role of DNA in the spatial organization of NHR heterodimers in complexes with coactivators such as Med1 and SRC-1. We have used SAXS, SANS and FRET to determine the solution structures of three heterodimer NHR complexes (RXR-RAR, PPAR-RXR and RXR-VDR) coupled with the NHR interacting domains of coactivators bound to their cognate direct repeat elements. The structures show an extended asymmetric shape and point to the important role played by the hinge domains in establishing and maintaining the integrity of the structures. The results reveal two additional features: the conserved position of the ligand-binding domains at the 5' ends of the target DNAs and the binding of only one coactivator molecule per heterodimer, to RXR's partner.

The "Phantom Effect" of the Rexinoid LG100754: structural and functional insights.

Retinoic acid receptors (RARs) and Retinoid X nuclear receptors (RXRs) are ligand-dependent transcriptional modulators that execute their biological action through the generation of functional heterodimers. RXR acts as an obligate dimer partner in many signalling pathways, gene regulation by rexinoids depending on the liganded state of the specific heterodimeric partner. To address the question of the effect of rexinoid antagonists on RAR/RXR function, we solved the crystal structure of the heterodimer formed by the ligand binding domain (LBD) of the RARα bound to its natural agonist ligand (all-trans retinoic acid, atRA) and RXRα bound to a rexinoid antagonist (LG100754). We observed that RARα exhibits the canonical agonist conformation and RXRα an antagonist one with the C-terminal H12 flipping out to the solvent. Examination of the protein-LG100754 interactions reveals that its propoxy group sterically prevents the H12 associating with the LBD, without affecting the dimerization or the active conformation of RAR. Although LG100754 has been reported to act as a 'phantom ligand' activating RAR in a cellular context, our structural data and biochemical assays demonstrate that LG100754 mediates its effect as a full RXR antagonist. Finally we show that the 'phantom ligand effect' of the LG100754 is due to a direct binding of the ligand to RAR that stabilizes coactivator interactions thus accounting for the observed transcriptional activation of RAR/RXR.

Detection of nucleic acid-nuclear hormone receptor complexes with mass spectrometry.

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR x RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR x RXR x DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR.