Effect of 25-hydroxyl group orientation on biological activity and binding to the 1alpha,25-dihydroxy vitamin D3 receptor.

The hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D), generates many biological actions by interactions with its nuclear receptor (VDR). The presence of a carbon-25 hydroxyl group is necessary for optimizing binding to the VDR. To examine the effect of spatial orientation of the 25-hydroxyl, two pairs of 22,23-allene sidechain analogs were studied. The 22R orientation in analogs HR (52+/-2%) and LA (154+/-19%) resulted in higher affinity binding than the 22S orientation of analogs HQ (21+/-3%) and LB (3.5+/-1.3%; 1,25D=100%). Limited trypsin proteolysis showed that 22R analogs induced VDR conformational changes better able to protect VDR from digestion than 22S analogs. 22R analogs were also able to induce gene transcription at 10-100-fold lower concentrations than 1,25D; 22S analogs were less effective. Analog LA was at least 10-fold more potent than 1,25D at inducing differentiation, while the other analogs were less potent. None of the analogs were as potent as 1,25D in promoting in vivo intestinal calcium absorption or bone calcium mobilization. LA was the most potent of the analogs but required 20-30-fold higher doses than 1,25D. The 25-hydroxyl orientation combined with the 16,17-ene functionality of analog LA enhances its ability to interact with VDR and induce biological actions.

Ligand structure-function relationships in the vitamin D endocrine system from the perspective of drug development (including cancer treatment).

It has become readily apparent to many scientists and pharmaceutical companies that the vitamin D endocrine system offers a wide array of drug development opportunities. There are already successes, as noted by 1alpha,25(OH)2D3 (Roche, and Abbott) for renal osteodystrophy and osteoporosis and 1alpha(OH)D3 (Leo, Chugai, Teijin) for renal osteodystrophy and (in Japan) osteoporosis, 1alpha,24(OH)2-24-cyclopropyl-D3 (Dovonex) and 1alpha,24(OH)2D3 (Teijin) for psoriasis, and 19-nor-1alpha,25(OH)2D2 (Abbott) for renal osteodystrophy, as well as drugs under active development. Yet there are still many important and challenging drug development frontiers, particularly in the area of cancer treatment and immune system disorders where exploration is only in the initial early stages. In addition, the application of vitamin D-related drugs in neurology and brain pathology should not be overlooked. It is to be hoped that the cellular and molecular basis for the vexing problem of analog-induced hypercalcemia will be elucidated. Given that there are believed to be over 2000 analogs of 1alpha,25(OH)2D3 already available for consideration, it is to be expected that over the next decade a significant number of new vitamin D structure-function drug development projects will be brought to conclusion.

Update on biological actions of 1alpha,25(OH)2-vitamin D3 (rapid effects) and 24R,25(OH)2-vitamin D3.

All biologic responses to vitamin D are now known to arise as a consequence of the metabolism of this seco-steroid into its two principal biologically active metabolites 1alpha,25(OH)(2)-vitamin D(3) (1ALPHA;,25(OH)(2)D(3)) and 24R,25(OH)(2)-vitamin D(3) (24R,25(OH)(2)D(3)). 1alpha,25(OH)(2)D(3) is the dominant metabolite and produces a wide array of biological responses via interacting both with the classical vitamin D nuclear receptor (VDR(nuc)) that regulates gene transcription in over 30 target organs and with a putative cell membrane receptor (VDR(mem1,25)) that mediates rapid (within seconds to minutes) biological responses. Ligand occupancy of VDR(mem1,25) is linked to signal transduction systems that can mediate the opening of Ca(2+) and chloride voltage gated channels as well as activation of MAP-kinase. MAP-kinase activation in some cells containing VDR(mem1,25)+VDR(nuc) then results in "cross-talk" from VDR(mem1,25) to VDR(nuc) which modulates transactivation of 1alpha,25(OH)(2)D(3) responsive gene promoters. The 24R,25(OH)(2)D(3) metabolite has been shown to be an essential hormone for the process of bone fracture healing. The activity of the enzyme responsible for the production of 24R,25(OH)(2)D(3), the renal 25(OH)D-24-hydroxylase, becomes elevated within 4-11 days after imposition of a tibial fracture, thereby increasing the blood concentrations of 24R,25(OH)(2)D(3) by threefold. The 24R,25(OH)(2)D(3) likely initiates its biological responses via binding to the ligand binding domain of a second cell membrane receptor, the VDR(mem24,25), which is stereospecific for 24R,25(OH)(2)D(3) in comparison with 24S,25(OH)(2)D(3) and 1alpha,25(OH)(2)D(3). This report summarizes the status of several current research frontiers in this arena of the vitamin D endocrine system.

1alpha,25-dihydroxyvitamin D3 inhibits uncoupling protein 2 expression in human adipocytes.

We recently demonstrated that suppressing 1alpha,25-(OH)2-D3 by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, and inhibits lipogenesis. High calcium diets also increase core temperature and white adipose tissue uncoupling protein 2 (UCP2) expression in aP2-agouti transgenic mice. Accordingly, we have evaluated the role of 1alpha,25-(OH)2-D3 in regulating human adipocyte UCP2 expression. Treatment of human adipocytes for 48 h with 1 nM 1alpha,25-(OH)2-D3 inhibited UCP2 mRNA and protein levels by 50% (P<0.002) and completely blocked isoproterenol- or fatty acid-stimulated two- to threefold increases in UCP2 expression. However, a specific agonist for the membrane vitamin D receptor (mVDR), 1alpha,25-dihydroxylumisterol3, was unable to inhibit basal, isoproterenol-stimulated, or fatty acid-stimulated UCP2 expression, whereas a specific mVDR antagonist,1beta,25-dihydroxyvitamin D3, was unable to prevent the 1alpha,25-(OH)2-D3 inhibition of UCP2 expression. In contrast, nuclear vitamin D receptor (nVDR) knockout via antisense oligodeoxynucleotide (ODN) prevented the inhibitory effect of 1alpha,25-(OH)2-D3 on adipocyte UCP2 expression and protein levels. These data indicate that 1a,25-(OH)2-D3 exerts an inhibitory effect on adipocyte UCP2 expression via the nVDR. Thus, suppression of 1alpha,25-(OH)2-D3 and consequent up-regulation of UCP2 may contribute to our previous observation of increased thermogenesis in mice fed with high calcium diets.

Molecular tools for study of genomic and rapid signal transduction responses initiated by 1 alpha,25(OH)(2)-vitamin D(3).

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) [1 alpha,25(OH)(2)D(3)] mediates through its widely distributed nuclear receptor (VDR(nuc)) regulation of gene transcription (genomic responses) and through a putative membrane receptor (VDR(mem)) a variety of rapid responses. Rapid responses studied in our laboratories include opening of voltage-gated calcium and chloride channels in ROS 17/2.8 osteoblast cells, activation of MAP-kinase in human leukemia NB4 cells and chick intestinal cells, release of insulin by rat pancreatic beta-cells, and in chick duodena transcaltachia (the rapid hormonal stimulation of intestinal Ca(2+) transport). 1 alpha,25(OH)(2)D(3) is conformationally flexible (side chain, seco B-ring and A-ring) and accordingly is able to generate a large array of different shapes to serve as ligands for available receptors (VDR(nuc) and VDR(mem)) in the vitamin D endocrine system. Our laboratories have utilized a number of conformationally restricted analogs of 1 alpha,25(OH)(2)D(3) (from a library of several hundred analogs) to evaluate the preferred shape of the ligands for rapid and genomic responses. The determination of the X-ray structure of the 1 alpha,25(OH)(2)D(3)-occupied VDR(nuc) revealed that the preferred ligand shape was a twisted 6-s-trans bowl shape [Molecular Cell 5 (2000) 173-179]. Optimal agonists for genomic responses include 1 alpha,25(OH)(2)D(3) and other side chain conformationally flexible analogs such as 20-epi-1 alpha,25(OH)(2)D(3) [approximately equal to 200-500-fold more potent than 1 alpha,25(OH)(2)D(3)] and 21-(3'-hydroxy-3-methylbutyl)-1 alpha,25(OH)(2)D(3) [an analog with two side chains] all which can achieve the preferred VDR(nuc) shape. In contrast, rapid responses require a 6-s-cis shape of the agonist ligand such as can be achieved by the natural hormone 1 alpha,25(OH)(2)D(3) or by analogs permanently locked in the 6-s-cis shape such as 1 alpha,25(OH)(2)lumisterol(3) or 1 alpha,25(OH)(2)-7-dehydrocholesterol. Additionally, we have discovered analogs that are specific in their antagonist properties for either rapid or genomic responses. Thus, 1 beta,25(OH)(2)D(3) is an antagonist of only rapid responses [via the VDR(mem)], while 23S-25-dehydro-1 alpha,25(OH)D(3)-26,23-lactone is an antagonist of only nuclear responses [via the VDR(nuc)]. In conclusion, we have presented evidence that 1 alpha,25(OH)(2)D(3) mediated rapid response and genomic response signal transduction pathways utilize differing shapes of ligand, both as agonists and antagonists.

An 8pi electron electrocyclization leading to a 9,19-methano-bridged analogue of 1 alpha,25-dihydroxyvitamin D3.

[reaction: see text] Lindlar semihydrogenation of a vitamin D type trienyne leads spontaneously to 9 alpha,19-methano-1 alpha,25-dihydroxyvitamin D3. The intermediate tetraene resulting from the reduction undergoes a rapid, stereoselective 8pi electron electrocyclization affording a novel steroid containing a linearly fused ABC (six-eight-six) 1,3,5-cyclooctatriene carbon framework.

Synthesis and NMR studies of (13)C-labeled vitamin D metabolites.

Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar solutions as compared to that in the solid state is most easily rationalized on the basis that the 5(10)-torsion angle in 58 is decreased in micellar solutions as compared to that in the solid state.