Ferdinand Molnár

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand.

25-Hydroxyvitamin D(3) 1alpha-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), a vitamin D receptor ligand. 25-Hydroxyvitamin D(3) has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1alpha-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D(3) has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D(3) internalisation, in Cyp27b1(-/-) cells explains their higher sensitivity to 25-hydroxyvitamin D(3). 25-Hydroxyvitamin D(3) action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D(3). Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D(3) in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D(3) with 1alpha,25-dihydroxyvitamin D(3) in Cyp27b1(-/-) cells further demonstrates the agonistic action of 25-hydroxyvitamin D(3) and suggests that a synergism between 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) might be physiologically important. In conclusion, 25-hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.

Current Status of Vitamin D Signaling and Its Therapeutic Applications

Vitamin D and in particular its biologically most active metabolite, 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃), are central endocrine molecules that influence many aspects of human physiology, which are not only the well-known calcium and phosphorus up-take and transport controlling bone formation, but also the control of immune functions and of cellular growth and differentiation. Basically all actions of 1α,25(OH)₂D₃ are mediated by the transcription factor vitamin D receptor (VDR). The crystal structure of the VDR and detailed knowledge on its molecular interactions with the ligand provide significant insight into the mechanisms of vitamin D signaling. This applies also on the action of the huge number of synthetic 1α,25(OH)₂D₃ analogues, which have been developed with the goal of a therapeutic application in hyper-proliferative diseases, such as psoriasis, benign prostate hyperplasia and different types of cancer, in immune functions, such as autoimmune diseases and microbial infections, or in bone disorders, such as osteoporosis. Moreover, detailed investigations on many VDR target genes and in particular the recently available genome-wide view on vitamin D signaling allows a more complete view on the potential of the nuclear hormone. In this review we discuss the latest insight into vitamin D signaling in context with the most prominent 1α,25(OH)₂D₃ analogues.

Vitamin D Receptor Agonists Specifically Modulate the Volume of the Ligand-binding Pocket *□

Existing crystal structure data has indicated that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3) and its analogues bind the ligand-binding pocket (LBP) of the human vitamin D receptor in a very similar fashion. Because docking of a ligand into the LBP is a more flexible process than crystallography can monitor, we analyzed 1α,25(OH)2D3, its 20-epi derivative MC1288, the two side-chain analogues Gemini and Ro43-83582 (a hexafluoro-derivative) by molecular dynamics simulations in a complex with the vitamin D receptor ligand-binding domain and a co-activator peptide. Superimposition of the structures showed that the side chain of MC1288, the first side chain of the conformation II of Gemini, the second side chain of Ro43-83582 in conformation I and the first side chain of Ro43-83582 in conformation II take the same agonistic position as the side chain of 1α,25(OH)2D3. Compared with the LBP of the natural hormone MC1288 reduced the volume by 17%, and Gemini expanded it by 19%. The shrinking of the LBP of MC1288 and its expansion to accommodate the second side chain of Gemini or Ro43-83582 is the combined result of minor movements of more than 30 residues and major movements of a few critical amino acids. The agonist-selective recognition of anchoring OH groups by the conformational flexible residues Ala-303, Leu-309, and His-397 was confirmed by in vitro assays. In summary, variations in the volume of agonists lead to adaptations in the volume of the LBP and alternative contacts of anchoring OH-groups.

Detailed Molecular Understanding of Agonistic and Antagonistic Vitamin D Receptor Ligands

The vitamin D receptor (VDR) is an endocrine member of the nuclear receptor superfamily and binds the biologically most active vitamin D metabolite, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). The VDR ligand-binding domain is a molecular switch, since its ligand-triggered interactions with corepressor and coactivator proteins are the central molecular events of nuclear 1alpha,25(OH)2D3 signaling. 1alpha,25(OH)2D3 analogues have been developed with the goal to improve the biological profile of the natural hormone for a therapeutic application either in hyperproliferative diseases, such as psoriasis and different types of cancer, or in bone disorders, such as osteoporosis. Most of the analogues described to date are agonists, with a few having been identified as antagonists. Only the two side chain analogue Gemini and some of its derivatives act under restricted conditions as inverse agonists. In this review we discuss the molecular mechanisms of these different type of analogues based on crystal structure data, molecular dynamics simulations and biochemical assays.

Vitamin D receptor ligands: the impact of crystal structures.

Introduction: In the past years, the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), has received large appreciation due to the broad physiological impact of the hormone and its nuclear receptor, the transcription factor vitamin D receptor (VDR). Recently, the understanding of VDR actions has progressed greatly, due to VDR crystal structures with various ligands. Areas covered: This review will present and discuss new synthetic agonistic and antagonistic 1α,25(OH)2D3 analogs in the context of the recent insights provided by VDR crystal structures. Expert opinion: During the last 5 years, a large number of new 1α,25(OH)2D3 analogs, many of which have an interesting functional profile, have been patented. Moreover, for a surprisingly high number of 1α,25(OH)2D3 analogs, the crystal structure data of their complex with the VDR is available. This structural information provides important insight into the functional potential of the VDR ligands and explains their agonistic and antagonistic action. However, so far, only for a few VDR ligands, a rational design, based on crystal structure information, has been applied. The design of future analogs may also take the specificity of co-factor interaction into account, in order to create selective VDR modulators.

New in Vitro Tools to Study Human Constitutive Androstane Receptor (CAR) Biology: Discovery and Comparison of Human CAR Inverse Agonists

The human constitutive androstane receptor (CAR, NR1I3) is one of the key regulators of xenobiotic and endobiotic metabolism. The unique properties of human CAR, such as the high constitutive activity and the complexity of signaling, as well as the lack of functional and predictive cell-based assays to study the properties of the receptor, have hindered the discovery of selective human CAR ligands. Here we report a novel human CAR inverse agonist, 1-[(2-methylbenzofuran-3-yl)methyl]-3-(thiophen-2-ylmethyl) urea (S07662), which suppresses human CAR activity, recruits the corepressor NCoR in cell-based assays, and attenuates the phenytoin- and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO)-induced expression of CYP2B6 mRNA in human primary hepatocytes. The properties of S07662 are also compared with those of known human CAR inverse agonists by using an array of different in vitro and in silico assays. The identified compound S07662 can be used as a chemical tool to study the biological functions of human CAR and also as a starting point for the development of new drugs for various conditions involving the receptor.

Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view 1

Vitamin D3 is one of the few natural compounds that has, via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein-protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.

An update on the constitutive androstane receptor (CAR)

Abstract The constitutive androstane receptor (CAR; NR1I3) has emerged as one of the main drug- and xenobiotic-sensitive transcriptional regulators. It has a major effect on the expression of several oxidative and conjugative enzymes and transporters, and hence, CAR can contribute to drug/drug interactions. Novel functions for CAR are also emerging: it is able to modulate the metabolic fate of glucose, lipids, and bile acids, and it is also involved in cell-cell communication, regulation of the cell cycle, and chemical carcinogenesis. Here, we will review the recent information available on CAR and its target gene expression, its interactions with partner proteins and mechanisms of action, interindividual and species variation, and current advances in CAR ligand selectivity and methods used in interrogation of its ligands.

Molecular mechanism of allosteric communication in the human PPARα-RXRα heterodimer

The peroxisome proliferator‐activated receptor α (PPARα) is a nuclear receptor (NR) that forms a heterodimeric transcription factor complex with the retinoid X receptor α (RXRα). The phenomenon that the heterodimer can be activated by both PPARα and RXRα ligands, while both ligands have a synergistic effect on its activity suggests that there is an allosteric communication within the heterodimer. In this study, the molecular mechanism of this allosteric signaling was studied by molecular dynamics (MD) simulations and some of the residues involved in this communication were tested experimentally. Multiple MD simulations were done for the PPARα‐RXRα heterodimer ligand‐binding domains (LBDs) without ligands, with agonistic ligand bound to RXRα or PPARα, and ligand bound to both receptors. Fluctuation calculations and structural clustering analysis of the heterodimer MD simulations showed that ligand binding to RXRα decreases fluctuations of large parts of PPARα, most notably helices 3 and 4 at the coactivator binding site, which presumably stabilizes the coactivator binding to heterodimer complex. The dynamics of helix 8–9 loop and helix 10/11 located at the heterodimeric interface were affected by RXRα ligand binding, suggesting that these parts of the dimer are involved in allosteric communication. Experimental data complemented this view by showing that a large set of residues at the heterodimerization surface has a role in the communication. These results provided evidence that RXRα ligand binding‐induced stabilization of PPARα coactivator binding site has a role in the permissive and synergistic activation of the PPARα‐RXRα heterodimer. Proteins 2010.

Vitamin D receptor 2016: novel ligands and structural insights

ABSTRACT Introduction: Vitamin D3 activates via its hormonal form 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the transcription factor vitamin D receptor (VDR). VDR is expressed in most human tissues and has more than 1,000 target genes. Thus, 1α,25(OH)2D3 and its synthetic analogs have a broad physiological impact. The crystal structures of the VDR ligand-binding domain (LBD), and its various ligands, allows further the understanding of the receptor’s molecular actions. Areas covered: We discuss the most important novel VDR ligands and the further insight derived from new structural information on VDR. Expert opinion: There is an increasing appreciation of the impact of vitamin D and its receptor VDR not only in bone biology, but also for metabolic diseases, immunological disorders, and cancer. Detailed structural analysis of the interaction of additional novel ligands with VDR highlight helices 6 and 7 of the LBD as being most critical for stabilizing the receptor for an efficient interaction with co-activator proteins, i.e. for efficient agonistic action. This permits the design of even more effective VDR agonists. In addition, chemists took more liberty in replacing major parts of the 1α,25(OH)2D3 molecule, such as the A- and CD-rings or the side chain, with significantly different structures, such as carboranes, and still obtained functional VDR agonists.