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.

Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding.

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Deciphering the importance of MD descriptors in designing Vitamin D Receptor agonists and antagonists using machine learning.

The Vitamin D Receptor (VDR) ligand-binding domain undergoes conformation change upon the binding of VDR agonists/antagonists. Helix 12 ((H)12) is one of the important helices at VDR ligand binding and its conformational changes are controlled by the binding of agonists and antagonists molecules. Various molecular modeling studies are available to explain the agonistic and antagonistic activity of vitamin D analogs. In this work, for the first time, we attempted to generate a machine learning model with fingerprints, 2D, 3D and MD descriptors that are specific to Vitamin D analogs and VDR. Initially, 2D and 3D descriptors and fingerprints of 1003 vitamin D analogs were calculated using CDK and RDKit. The machine learning model was generated using descriptors and fingerprints. Further, 80 Vitamin D analogs (40 VDR agonists + 40 VDR antagonists) were docked in the VDR active site. 50ns MD simulation was performed for each protein-ligand complex. Different MD descriptors such as Solvent Accessible Surface Area (SASA), radius of gyration, PC1 and PC2 were calculated and considered along with CDK and RDKit descriptors as features for machine learning calculations. A few other descriptors that are related to VDR conformational changes such as conformation of the (H)12, the angle at kink were considered for machine learning model generation. It was observed that the descriptors calculated from VDR conformational changes i) were able to distinguish between agonists and antagonists ii) provide key and comprehensive information about the unique binding characteristics of agonists and antagonists iii) provide a strong basis for the machine learning model generation. Overall, this study attempts the utilization of descriptors that are specific to a protein conformation will be helpful for the generation of an efficient machine learning model.

Structural and functional insights on vitamin D receptor and CYP24A1 deleterious single nucleotide polymorphisms: A computational and pharmacogenomics perpetual approach.

The development of chronic kidney disease (CKD) drugs remains a challenge due to the variations in the genes. The vitamin D receptor (VDR) and Cytochrome 24A1 (CYP24A1) genetic variants might affect the drug potency, efficacy and pathway. Here we have to analyse and determine the deleterious single-nucleotide polymorphisms (nsSNPs) of VDR and CYP24A1 genes and their different population's drug responses in different populations to understand the key role in CKD. Among that the large scale of nsSNP, we used certain computational tools that predicted six missense variants are observed to be significantly damaging effect and SNP variability with large differences in various populations. Molecular docking studies were carried out by clinical and our screened compounds to VDR and CYP24A1. Docking results revealed all the compounds have a good binding affinity (Score). The screened compounds (TCM_2868 and UNPD_141613) show good binding affinity when compared to known compounds. The QM/MM study revealed that the compounds have electron transfer ability and act as a donor/acceptor to mutated proteins. The structural and conformational changes of protein complexes were analysed by molecular dynamics study. Hence, this study helps to identify suitable drugs through drug discovery in CKD treatment. The abovementioned compounds have more binding affinity, efficacy, and potency of both wild and mutant of VDR and CYP24A1.

Proposal of novel inhibitors for vitamin-D receptor: Molecular docking, molecular mechanics and ab initio molecular orbital simulations.

The specific binding of active vitamin-D to the vitamin-D receptor (VDR) is closely related to the onset of immunological diseases. To inhibit the binding, various compounds have been developed as potent inhibitors against VDR. Among them, a compound NS-54c, which was developed based on the first VDR antagonist TEI-9647 (25-dehydro-1α-hydroxyvitamin D3-26,23-lactone), was revealed to posse almost 1000-fold improved antagonistic activity over the original TEI-9647. However, the reason for this significant improvement has not been elucidated. In the present study, we investigated the specific interactions between VDR and these inhibitors, using molecular simulations based on molecular docking, molecular mechanics and ab initio fragment molecular orbital calculations. Based on the results simulated, we furthermore proposed novel inhibitors and investigated their binding properties to VDR. The results elucidate that the replacement of propyl group at the 24th site of NS-54c by a phenethyl group can enhance the binding affinity of the inhibitor to VDR. This finding provides useful information for developing novel potent inhibitors against VDR.

Preparation and Use of Turn-on Fluorescent Probe for Detection and Live Cell Imaging of Vitamin D Receptor as a Target Protein.

Turn-on fluorescent probe mediated by conjugate addition and cyclization (TCC probe) is a small molecule that reacts with a protein of interest in cells. TCC probe is applicable to various types of proteins by exchanging the ligand unit for target proteins. TCC probes are a potent tool for molecular imaging and chemical proteomics. This protocol describes the synthesis of a TCC probe via unstable intermediate and how to use this probe to visualize vitamin D receptor as a target protein. For complete details on the use and execution of this protocol, please refer to Kojima et al. (2020).

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 Analysis of VDR Complex with ZK168281 Antagonist.

Vitamin D receptor (VDR) antagonists prevent the VDR activation function helix 12 from folding into its active conformation, thus affecting coactivator recruitment and antagonizing the transcriptional regulation induced by 1α,25-dihydroxyvitamin D3. Here, we report the crystal structure of the zebrafish VDR ligand-binding domain in complex with the ZK168281 antagonist, revealing that the ligand prevents optimal folding of the C-terminal region of VDR. This interference was confirmed by hydrogen-deuterium exchange mass spectrometry (HDX-MS) in solution.

Modulating effect of vitamin D3 on the mutagenicity and carcinogenicity of doxorubicin in Drosophila melanogaster and in silico studies.

Vitamin D3 (VD3) deficiency increases DNA damage, while supplementation may exert a pro-oxidant activity, prevent viral infections and formation of tumors. The aim of this study was to investigate the mutagenicity and carcinogenicity of VD3 alone or in combination with doxorubicin (DXR) using the Somatic Mutation and Recombination Test and the Epithelial Tumor Test, both in Drosophila melanogaster. For better understanding of the molecular interactions of VD3 and receptors, in silico analysis were performed with molecular docking associated with molecular dynamics. Findings revealed that VD3 alone did not increase the frequency of mutant spots, but reduced the frequency of mutant spots when co-administered with DXR. In addition, VD3 did not alter the recombinogenic effect of DXR in both ST and HB crosses. VD3 alone did not increase the total frequency of tumor, but significantly reduced the total frequency of tumor when co-administered with DXR. Molecular modeling and molecular dynamics between calcitriol and Ecdysone Receptor (EcR) showed a stable interaction, indicating the possibility of signal transduction between VD3 and EcR. In conclusion, under these experimental conditions, VD3 has modulatory effects on the mutagenicity and carcinogenicity induced by DXR in somatic cells of D. melanogaster and exhibited satisfactory interactions with the EcR.

Vitamin D receptor exhibits different pharmacodynamic features in tumoral and normal microenvironments: A molecular modeling study.

The vitamin D receptor (VDR) constitutes a promising therapeutic target for the treatment of cancer. Unfortunately, its natural agonist calcitriol does not have clinical utility due to its potential to induce hypercalcemic effects at the concentrations required to display antitumoral activity. For this reason, the search for new calcitriol analogues with adequate therapeutic profiles has been actively pursued by the scientific community. We have previously reported the obtaining and the biological activity evaluation of new calcitriol analogues by modification of its sidechain, which exhibited relevant antiproliferative and selectivity profiles against tumoral and normal cells. In this work we conducted molecular modeling studies (i.e. molecular docking, molecular dynamics, constant pH molecular dynamics (CpHMD) and free energy of binding analysis) to elucidate at an atomistic level the molecular basis related to the potential of the new calcitriol analogues to achieve selectivity between tumoral and normal cells. Two histidine residues (His305 and His397) were found to exhibit a particular tautomeric configuration that produces the observed bioactivity. Also, different acid-based properties were observed for His305 and His307 with His305 showing an increased acidity (pKa 5.2) compared to His397 (pKa 6.8) and to the typical histidine residue. This behavior favored the pharmacodynamic interaction of the calcitriol analogues exhibiting selectivity for tumoral cells when VDR was modeled at the more acidic tumoral environment (pH ≅ 6) compared to the case when VDR was modeled at pH 7.4 (normal cell environment). On the other hand, non-selective compounds, including calcitriol, exhibited a similar interaction pattern with VDR when the receptor was modeled at both pH conditions. The results presented constitute the first evidence on the properties of the VDR receptor in different physicochemical environments and thus represent a significant contribution to the in silico screening and design of new calcitriol analogues.

Cyclization Reaction-Based Turn-on Probe for Covalent Labeling of Target Proteins.

Fluorescent molecules have contributed to basic biological research but there are currently only a limited number of probes available for the detection of non-enzymatic proteins. Here, we report turn-on fluorescent probes mediated by conjugate addition and cyclization (TCC probes). These probes react with multiple amino acids and exhibit a 36-fold greater emission intensity after reaction. We analyzed the reactions between TCC probes and nuclear receptors by electrospray ionization mass spectrometry, X-ray crystallography, spectrofluorometry, and fluorescence microscopy. In vitro analysis showed that probes consisting of a protein ligand and TCC could label vitamin D receptor and peroxisome proliferator-activated receptor γ. Moreover, we demonstrated that not only a ligand unit but also a peptide unit can label the target protein in a complex mixture.

Ligand-based pharmacophore modeling and docking studies on vitamin D receptor inhibitors.

In recent years, pharmacophore modeling and molecular docking approaches have been extensively used to characterize the structural requirements and explore the conformational space of a ligand in the binding pocket of the selected target protein. Herein, we report a pharmacophore modeling and molecular docking of 45 compounds comprising of the indole scaffold as vitamin D receptor (VDR) inhibitors. Based on the selected best hypothesis (DRRRR.61), an atom-based three-dimensional quantitative structure-activity relationships model was developed to rationalize the structural requirement of biological activity modulating components. The developed model predicted the binding affinity for the training set and test set with R2(training) = 0.8869 and R2(test) = 0.8139, respectively. Furthermore, molecular docking and dynamics simulation were performed to understand the underpinning of binding interaction and stability of selected VDR inhibitors in the binding pocket. In conclusion, the results presented here, in the form of functional and structural data, agreed well with the proposed pharmacophores and provide further insights into the development of novel VDR inhibitors with better activity.

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence.

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR-dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P. cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α-hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR-specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL-60 with EC50 values of 21.0 ± 0.52, 6.7 ± 0.37, and 9.4 ± 0.65 µM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR-dependent pathway may play an important role in their anti-acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P. cocos are revealed as naturally occurring RXR selective agonists with the potential for anti-cancer activity. These results suggest a novel approach to the treatment or prevention of APL.

Identification of a novel melatonin-binding nuclear receptor: Vitamin D receptor.

Previous studies confirmed that melatonin regulates Runx2 expression but the mechanism is unclear. There is a direct interaction between Runx2 and the vitamin D receptor (VDR). Herein, we observed a direct interaction between melatonin and the VDR but not Runx2 using isothermal titration calorimetry. Furthermore, this direct binding was detected only in the C-terminal ligand binding domain (LBD) of the VDR but not in the N-terminal DNA-binding domain (DBD) or the hinge region. Spectrophotometry indicated that melatonin and vitamin D3 (VD3) had similar uptake rates, but melatonin's uptake was significantly inhibited by VD3 until the concentration of melatonin was obviously higher than that of VD3 in a preosteoblastic cell line MC3T3-E1. GST pull-down and yeast two-hybrid assay showed that the interactive smallest fragments were on the 319-379 position of Runx2 and the N-terminus 110-amino acid DBD of the VDR. Electrophoretic mobility shift assay (EMSA) demonstrated that Runx2 facilitated the affinity between the VDR and its specific DNA substrate, which was further documented by a fluorescent EMSA assay where Cy3 labeled Runx2 co-localized with the VDR-DNA complex. Another fluorescent EMSA assay confirmed that the binding of the VDR to Runx2 was significantly enhanced with an increasing concentrations of the VDR, especially in the presence of melatonin; it was further documented using a co-immunoprecipitation assay that this direct interaction was markedly enhanced by melatonin treatment in the MC3T3-E1 cells. Thus, the VDR is a novel melatonin-binding nuclear receptor, and melatonin indirectly regulates Runx2 when it directly binds to the LBD and the DBD of the VDR, respectively.

Potentially repurposable drugs for schizophrenia identified from its interactome.

We previously presented the protein-protein interaction network of schizophrenia associated genes, and from it, the drug-protein interactome which showed the drugs that target any of the proteins in the interactome. Here, we studied these drugs further to identify whether any of them may potentially be repurposable for schizophrenia. In schizophrenia, gene expression has been described as a measurable aspect of the disease reflecting the action of risk genes. We studied each of the drugs from the interactome using the BaseSpace Correlation Engine, and shortlisted those that had a negative correlation with differential gene expression of schizophrenia. This analysis resulted in 12 drugs whose differential gene expression (drug versus normal) had an anti-correlation with differential expression for schizophrenia (disorder versus normal). Some of these drugs were already being tested for their clinical activity in schizophrenia and other neuropsychiatric disorders. Several proteins in the protein interactome of the targets of several of these drugs were associated with various neuropsychiatric disorders. The network of genes with opposite drug-induced versus schizophrenia-associated expression profiles were significantly enriched in pathways relevant to schizophrenia etiology and GWAS genes associated with traits or diseases that had a pathophysiological overlap with schizophrenia. Drugs that targeted the same genes as the shortlisted drugs, have also demonstrated clinical activity in schizophrenia and other related disorders. This integrated computational analysis will help translate insights from the schizophrenia drug-protein interactome to clinical research - an important step, especially in the field of psychiatric drug development which faces a high failure rate.

Vitamin D and Its Synthetic Analogs.

For many individuals, in particular during winter, supplementation with the secosteroid vitamin D3 is essential for the prevention of bone disorders, muscle weakness, autoimmune diseases, and possibly also different types of cancer. Vitamin D3 acts via its metabolite 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as potent agonist of the transcription factor vitamin D receptor (VDR). Thus, vitamin D directly affects chromatin structure and gene regulation at thousands of genomic loci, i.e., the epigenome and transcriptome of its target tissues. Modifications of 1,25(OH)2D3 at its side-chain, A-ring, triene system, or C-ring, alone and in combination, as well as nonsteroidal mimics provided numerous potent VDR agonists and some antagonists. The nearly 150 crystal structures of VDR's ligand-binding domain with various vitamin D compounds allow a detailed molecular understanding of their action. This review discusses the most important vitamin D analogs presented during the past 10 years and molecular insight derived from new structural information on the VDR protein.

Alternative binding sites at the vitamin D receptor and their ligands.

In recent decades, the majority of ligands developed for the vitamin D receptor (VDR) bind at its deeply buried genomic ligand binding pocket. Theses ligands can be categorized into agonists and partial agonists/antagonists. A limited number of ligands, most of them peptides, bind the VDR‒coactivator binding site that is formed in the presence of an agonist and inhibit coactivator recruitment, and therefore transcription. Another solvent exposed VDR‒ligand binding pocket was identified for lithocholic acid, improving the overall stability of the VDR complex. Additional proposed interactions with VDR are discussed herein that include the alternative VDR‒ligand binding pocket that may mediate both non-genomic cellular responses and binding function 3 that was identified for the androgen receptor. Many VDR ligands increase blood calcium levels at therapeutic concentrations in vivo, thus the identification of alternative VDR‒ligand binding pockets might be crucial to develop non-calcemic and potent ligands for VDR to treat cancer and inflammatory disease.

Nuclear hormone receptors: Ancient 9aaTAD and evolutionally gained NCoA activation pathways.

In higher metazoans, the nuclear hormone receptors activate transcription trough their specific adaptors, nuclear hormone receptor adaptors NCoA, which are absent in lower metazoans. The Nine amino acid TransActivation Domain, 9aaTAD, was reported for a large number of the transcription activators that recruit general mediators of transcription. In this study, we demonstrated that the 9aaTAD from NHR-49 receptor of nematode C.elegans activates transcription as a small peptide. We showed that the ancient 9aaTAD domains are conserved in the nuclear hormone receptors including human HNF4, RARa, VDR and PPARg. Also their small 9aaTAD peptides effectively activated transcription in absence of the NCoA adaptors. We also showed that adjacent H11 domains in ancient and modern hormone receptors have an inhibitory effect on their 9aaTAD function.

Ligand chirality can affect histidine protonation of vitamin-D receptor: ab initio molecular orbital calculations in water.

Vitamin D is recognized to play important roles in the onset of immunological diseases as well as the regulation of the amount of Ca in the blood. Since these physiological actions caused by active vitamin D are triggered by the specific interaction between the vitamin D receptor (VDR) and active vitamin D, many types of compounds have been developed as potent ligands against VDR. It was found that the binding affinity between VDR and its ligand depends significantly on the chirality of the ligand. However, the reason for the dependence has, thus far, not been elucidated. In the present study, we investigated the specific interactions between VDR and some ligands with different chirality, using ab initio fragment molecular orbital (FMO) calculations. The FMO results reveal that two histidine residues of VDR contribute significantly to the binding between VDR and ligand and that their protonation states can affect the specific interactions between VDR and ligand. We therefore considered other possible protonation states of these histidine residues and determined their most stable states, using the ab initio FMO calculations. The results illustrate the possibility that the difference in the chirality of a ligand can induce the change in protonation states of the histidine residues of VDR existing near the ligand. This finding provides an important warning that the protonation states of histidine residues existing near the ligand should be considered more precisely in the molecular simulations for investigating the specific interactions between protein and ligand.

3D-QSAR studies of D3R antagonists and 5-HT1AR agonists.

Combination of dopamine D3 antagonism and serotonin 5-HT1A agonism leads to an effective way to atypical antipsychotics. In this work, two predictive 3D-QSAR models were bulit for D3R antagonists and 5-HT1AR agonists, respectively. Based on the steric and electrostatic information of contour maps, four compounds with improved predicted activities were newly designed. In addition, molecular docking and ADMET properties suggested that designed molecules had strong interactions with receptors and low hepatotoxicity. This work sheds light on the design of bifunctional novel antipsychotic drugs for D3R antagonists and 5HT1AR agonists.