Current Topics in Medicinal Chemistry - Volume 6, Issue 12, 2006

The recognition that Vitamin D exerts its activity through a nuclear mechanism led to the discovery of the binding protein, the vitamin-D receptor or VDR. As a consequence of this agonist binding in the ligand-binding domain (LBD), the VDR changes its topology and, after translocation from the cytoplasm to the nucleus, permits the VDR to function as a ligandactivated transcription factor by virtue of its association with the response elements (VDREs) in some 50 vitamin-D regulated genes. This binding phenomenon to DNA at the VDRE sites is a complex process and occurs by heterodimerization with the retinoid X-receptor (RXR) and recruitment of a complex machinery of co-modulators and co-activators. The hormonally active form of vitamin D, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), is produced by a sequence of steps commencing with a photochemically induced conrotatory, electrocyclic ring opening of 7-dehydrocholesterol in the skin leading to previtamin D3 which is subject to a thermal, antarafacial [1,7]-hydrogen shift, followed by 25-hydroxylation in the liver to generate 25(OH)D3, and 1α-hydroxylation, preferentially in the kidney. The VDR is present in multitudinous tissues, including many cancer cells, some with the capability to 1α-hydroxylate 25(OH)D3 to create the active 1,25(OH)2D3 and hence enabling locally initiated cellular proliferation and differentiation processes. While today's picture of the genomic responses, mediated by 1,25(OH)2D3, is somewhat coherent, the imputed non-genomic 'rapid responses' are more enigmatic. The medicinal chemist who seeks to intervene therapeutically in vitamin-D controlled gene transcription activity has reduced this intricate scenario to a working hypothesis based on a few facts and lemmas: Similar to other hormonal nuclear receptors, the VDR can modify gene expression by hormone-initiated signal transduction; 1,25(OH)2D3 induces co-localization of the VDR and RXR, the receptor activation unit, to the VDRE gene promoter rendering 1,25(OH)2D3 an essential component for gene expression. Cloning of the VDR and the availability of the VDR LBD crystal structure has not only illuminated the role of the receptor and its ligand in transcription regulation but also, to some extent, the physico-chemical requirements of the ligand. Add to this the dynamic behavior of the LBD with the resulting variations of transcriptional activity, the known endocrinal steering mechanism for the required cellular hormone levels through complex CYP-mediated metabolic pathway, and the chemical, biological and even clinical properties of vitamin D analogs previously prepared, and one arrives at an intriguing and exciting prospect to view vitamin D and its analogs as multifaceted tools implicated in a myriad of established and conjectured biological activities with a set of predefined preferences of molecular features. And therein are anchored the opportunities and challenges for the medicinal chemist. 1,25(OH)2D3 can suppress several growth factor and cytokine genes thus inhibiting the release of IL2, GM-CSF, interferon-γ, and PTH, for example. It regulates mineral homeostasis, cellular proliferation and differentiation, and autoimmune activities. The disease targets, envisioned for vitamin D analogs, appropriately, include osteoporosis by balancing bone-mineral mobilization, secondary hyperparathyroidism to reduce PTHgene transcription and blocking chief cell hyperplasia, autoimmune diseases such as psoriasis and asthma, organ-transplant rejection, benign prostate hyperplasia, involuntary bladder control, blood pressure control by suppressing renin biosynthesis, type 1 diabetes and insulin secretion by affecting pancreatic β-cell function, anti-inflammatory events via cyclooxygenase-2 inhibition, and cancer via the established antiproliferative and prodifferentiating effects on a variety of cell lines, such as breast, prostate and colon. The chemist must strive for selective molecular modifications of vitamin D to balance the potential function as a nuclear receptor agonist, antagonist or reverse agonist, and at the same time maintain tissue specificity and sufficient metabolic stability with a constant look out for hypercalcemia and hyperphosphatemia. While the discovery of agonist or even "superagonist" activity is the more popular goal in molecular design, the prospect of finding ligands that selectively stabilize an antagonistic conformation of the VDR LBD within the VDR-RXR-DVRE construct, to actually prevent induction of transactivation, is also of potential therapeutic value. The adaptability of the VDR to accept ligands that are deprived of many structural elements common to 1,25(OH)2D3 and demand drastically changed space requirements, however, underlines the difficulty to employ modern drug design technologies........

The vitamin D receptor, a member of the nuclear receptor subgroup NR1I, is regulated by 1α,25(OH)2D3 to control calcium metabolism, cell proliferation and differentiation and immunomodulation. The therapeutic applications of vitamin D metabolites are wide. To develop efficient therapy, the elucidation of the structure-function relationships of VDR and its ligands are essential. In this review we will focus on the current structural understanding of the interactions of ligands in the ligand binding pocket of the VDR. These structures revealed the mutual adaptability of the ligands and the protein. In silico modeling has further revealed a possible new pocket in the VDR LBD responsible of the nongenomic action mediated by VDR. With the availability of all these structural information on VDR LBD, new ligands that are more selective, such as non-steroidal ligands, could be designed by taking into account the flexibility of some VDR regions. Tissue selectivity may also be achieved by developing ligands that specifically activate the non-genomic pathway.

The vitamin D receptor (VDR) is an endocrine member of the nuclear receptor superfamily and binds the biologically most active vitamin D metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). The VDR ligandbinding domain is a molecular switch, since its ligand-triggered interactions with corepressor and coactivator proteins are the central molecular events of nuclear 1α,25(OH)2D3 signaling. 1α,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.

We performed total alanine scanning mutational analysis (ASMA) of the residues lining the ligand binding pocket (LBP) of the human vitamin D receptor (hVDR) to investigate allosteric effects of ligands in the function of nuclear receptors (NRs). This was accomplished for the first time in the NR superfamily. The effects of ligand structure were also examined in this system (termed 2D-ASMA) using 8 representative VDR ligands. The results clearly revealed the role and importance of all amino acid residues lining the LBP and the relationships between ligand binding and transcriptional potency. 2D-ASMA indicated ligand-specific ligand-protein interactions, which are essential in determining the transactivation potency of the ligand. Taking the results as a whole, we suggest a ligand-mediated allosteric network, which allows transmission of information from ligands to the interfaces of the VDR in association with protein cofactors and was shown to be linked to a part of the network identified by statistical coupling analysis (SCA).

Among the many important physiological functions of the activated vitamin D receptor (VDR) is the signaling of monocytic differentiation, first demonstrated by conversion of malignant myeloid leukemia cells to nonproliferating cells with mature monocyte/macrophage appearance. However, the understanding of how 1, 25-dihydroxyvitamin D3 (1,25D) signals monocytic differentiation is still developing. Recent advances summarized here include the role of the principal "mitogen-activated protein kinase" (MAPK) pathways, their potential downstream target the CCAAT/enhancer binding protein β (C/EBP β), cell cycle related proteins, and cyclin-dependent kinase 5 (Cdk5) in 1,25D-induced differentiation. The precise steps by which activated VDR signals differentiation are incompletely understood in any of the cell types known to respond to 1,25D. We have focused our studies on HL60 cells, a widely available cell line derived from a patient with promyeloblastic leukemia, with the goal of achieving as clear a picture as possible with the currently available tools. In this model, outlined in Fig. 1, a plausible sequence of events is presented, with the caveats that these are not the only pathways activated by liganded VDR, and that several other pathways, also operative, remain to be convincingly demonstrated. The details of the scheme will be discussed in the sections below.

The structure-activity relationships of 1α,25-dihydroxyvitamin D3 on simultaneous modification at both C2α and CD-ring side chain, including 20-epimerization, double side chain (gemini), and vitamin D receptor (VDR) antagonists TEI-9647 and TEI-9648 lactone rings, and also on simultaneous modifications at both C2 and C10 positions, i.e., C2 modified active 19-norvitamin D3, have been studied in our laboratory to find new seeds of B-seco-steroidal medicine for treating bone diseases, psoriasis, secondary hyperparathyroidism, and certain kinds of cancers. We developed an efficient and systematic route to the 2α-substituted 1α,25-dihydroxyvitamin D3 analogs, i.e., VDR-agonists (20-epi-2-4, double side chain 13a-c, 19-nor 15a-c) and antagonists (36a-c, 37a-c). The A-ring precursors (11a-o) for these analogs were synthesized from D-glucose as a chiral template. In the 19-nor series, we used radical coupling reaction for preparing the A-ring parts from (-)-quinic acid, and the resulting 2-substituted A-ring moiety was coupled with 25-hydroxy Grundmann's ketone utilizing Julia olefination to connect between the C5 and C6 positions. We also synthesized the highly potent VDR-antagonists by introducing the 2α-functional group to the TEI-9647 and TEI-9648 skeletons.

The formal C-20 methylation of 1,25-dihydroxy vitamin D3 (calcitriol) and bridging of two methyl groups produces spiro[cyclopropane-1, 20'-calcitriol], colloquially referred to as C-20 cyclopropylcalcitriol, which is much more active in MLR for suppression of interferon-γ release than calcitriol, and hypercalcemia in mice is elicited at a ten-fold lower dose when compared to calcitriol. Introduction of the Δ16,17-double bond, modification of the side chain by 23- unsaturation and replacement of the methyl groups at C-26 and C-27 with trifluoromethyl moieties create a highly active series of vitamin D analogs. As previously observed in the calcitriol series, the presence of the C-16 double bond in the cyclopropyl analogs also arrests metabolic side-chain oxidation in the at the C-24 oxo level in UMR 106 cells. The enhanced biological activity is ascribed, at least in part, to the improved resistance toward metabolic degradation.

Vitamin D receptor (VDR) agonists can inhibit cell growth, promote apoptosis, and induce differentiation of many cell types, in addition to inhibiting metastasis and angiogenesis, all desirable properties for a drug to control cancer. However, from an immunological perspective, the immunomodulatory properties of VDR agonists are apparently just opposite to the main aims of cancer immunotherapy: boosting the immune response and breaking tumor-related tolerance. While it may be possible to identify VDR agonists with enhanced anti-proliferative/pro-differentiative and reduced immunomodulatory activities as anti-cancer agents, a complementary approach could rely on identifying clinical indications where their systemic immunomodulatory properties could be minimized. Superficial bladder cancer, where treatments are usually administered by vesical instillation, may represent such an indication. We have observed a strong synergism in vitro between calcitriol and doxorubicin or epirubicin in the inhibition of bladder cancer cell proliferation. Thus, calcitriol and doxorubicin or epirubicin in combination may have clinical value in the management of superficial bladder cancer.

1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an important hormone that regulates metabolism of calcium and phosphorus in small intestine, kidney, and bone, and its physiological action is expressed as ligand-dependent transcription activity mediated by vitamin D receptor (VDR). The VDR is found in various organs and cells including small intestine, kidney, and bone. In addition to the regulation of calcium metabolism, 1,25(OH)2D3 is involved in various biological reactions such as differentiation induction, antiproliferative effect, immunomodulatory effect, and regulation of cytokine and parathyroid hormone secretion. Thus, 1,25(OH)2D3 is expected to become a therapeutic drug for various related diseases. At present, a number of vitamin D derivatives are clinically applied to psoriasis, secondary hyperparathyroidism and osteoporosis but hypercalcemia and hypercalciuria are major concerns. Therefore, the current focus is directed toward new vitamin D derivatives with weak calcemic effects and a wide therapeutic window. In this summary, recent developments of new vitamin D derivatives for application in clinical treatment are described.