To identify novel vitamin D receptor (VDR) ligands that induce a novel architecture within the ligand-binding pocket (LBP), we have investigated eight 22-butyl-1alpha,24-dihydroxyvitamin D(3) derivatives (3-10), all having a butyl group as the branched alkyl side chain. We found that the 22S-butyl-20-epi-25,26,27-trinorvitamin D derivative 5 was a potent VDR agonist, whereas the corresponding compound 4 with the natural configuration at C(20) was a potent VDR antagonist. Analogues with the full vitamin D(3) side chain were less potent agonist, and whether they were agonists or antagonists depended on the 24-configuration. X-ray crystal structures demonstrated that the VDR-LBD accommodating the potent agonist 5 has an architecture wherein the lower side and the helix 11 side of the LBP is simply expanded relative to the canonical active-VDR situation; in contrast, the potent antagonist 4 induces an extra cavity to accommodate the branched moiety. This is the first report of a VDR antagonist that generates a new cavity to alter the canonical pocket structure of the ligand occupied VDR.

To investigate the allosteric effects of ligands in the function of nuclear receptors, we performed exhaustive alanine scanning mutational analysis (ASMA) of the residues lining the ligand-binding pocket (LBP) of the human vitamin D receptor. The effects of ligands were examined in this system (termed two-dimensional (2D) ASMA) using 10 structurally and biologically characteristic ligands that included agonists, partial agonists, and a full antagonist. The results clearly revealed the role and importance of all the amino acid residues lining the LBP and the relationships between ligand binding and transcriptional potency. 2D ASMA indicated ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the ligand. Taking the results as a whole, we suggest a ligand-mediated allosteric network through which information from ligands is transmitted to the interfaces with protein cofactors and which was shown to be linked to part of the network found by statistical coupling analysis.

We previously reported that 22S-butyl-25,26,27-trinor-1alpha,24-dihydroxyvitamin D(3) 2 was a potent VDR antagonist. The X-ray crystal structure of the ligand binding domain of VDR complexed with 2 indicated that this ligand induces an extra cavity within the ligand-binding pocket. The structure also showed that the ligand forms only poor hydrophobic interactions with helix 12 of the protein. Here, to study the effects of the induction of the extra cavity and of insufficient interactions with helix 12 on antagonism, we designed and synthesized a series of vitamin D(3) analogues with or without a 22-alkyl substituent and evaluated their biological potency. We found that the 22-butyl analogues 3c and 5c act as full antagonists, the 22-ethyl analogues 3b, 4b, 5b, and 22-butyl analogue 4c act as partial agonists, and the others (3a, 4a, 5a, 6a, 6b, and 6c) act as full agonists for VDR. It is intriguing that 6c is a potent agonist for VDR, whereas its 26,27-dinor analogue 5c is a potent antagonist. Analogue 6c recruited coactivator SRC-1 well, but 5c did not. These results indicate that a combination of induction of the extra cavity and insufficient hydrophobic interactions with helix 12 is important for VDR antagonism in this class of ligands.

The vitamin D receptor (VDR) mediates the biological actions of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], the active form of vitamin D, which regulates calcium homeostasis, immunity, cellular differentiation, and other physiological processes. We investigated the effects of three 1,25(OH) 2 D 3 derivatives on VDR function. AD47 has an adamantane ring and LAC67a and LAC67b have lactone ring substituents at the side chain position. These vitamin D derivatives bind to VDR but do not stabilize an active cofactor conformation. In a VDR transfection assay, AD47 and LAC67b act as partial agonists and all three compounds inhibit VDR activation by 1,25(OH) 2 D 3 . The derivatives enhanced the heterodimerization of VDR with the retinoid X receptor, an effect unrelated to agonist/antagonist activity. AD47 and LAC67b weakly induced recruitment of the SRC-1 cofactor to VDR, and all three derivatives inhibited the recruitment of p160 family cofactors to VDR induced by 1,25(OH) 2 D 3 . It is noteworthy that AD47 induced DRIP205 recruitment as effectively as 1,25(OH) 2 D 3 , whereas LAC67a and LAC67b were not effective. We examined the expression of endogenous VDR target genes and the nuclear protein levels of VDR and cofactors in several cell lines, including cells derived from intestine, bone, and monocytes, and found that the vitamin D 3 derivatives act as cell type-selective VDR modulators. The data indicate that side chain modification is useful in the development of VDR antagonists and tissue-selective modulators. Further elucidation of the molecular mechanisms of action of selective VDR modulators will be essential for their clinical application.The vitamin D receptor (VDR; NR1I1) is a member of the nuclear receptor superfamily that regulates physiological processes, including cell growth and differentiation, embryonic development, and metabolic homeostasis (Makishima, 2005). Nuclear receptor transcriptional activity is modulated by ligands such as steroids, retinoids, and other lipid-soluble compounds. Upon ligand binding, nuclear receptors undergo a conformational change in the cofactor binding site and activation function 2 (AF2) helices, which results in dynamic exchange of cofactor complexes, allowing nuclear receptors to modulate the transcription of specific target genes (Rosenfeld et al., 2006). In the absence of ligand, corepressors bind to the AF2 surface composed of portions of helix (H) 3, loop 3-4, H4/5, and H11. Ligand binding reduces the receptor affinity for corepressors and recruits coactivators to the altered AF2 surface formed by repositioning of H12. Recent studies suggest that DNA sequence-specific effects of transcription factor activity are associated with site-specific interaction with cofactor complexes (Rosenfeld et al., 2006). Chemical modification of ligands may induce AF2 conformations and cofactor interactions distinct from those of natural ligands and can result in cell type-selective modulation of target gene expression.The active form of vitamin D 3 , 1,25-dihydroxyvitamin D...

The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly­(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/β-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/β-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.

The X-ray crystal structures of the rat VDR ligand-binding domain complexed with 19-norvitamin D compounds that contain an adamantyl substituent at the side-chain terminus, 2a (ADTT), 2b (ADNY), and 2c (ADMI4) and a coactivator peptide derived from DRIP205 are reported. These compounds show a series of partial agonistic (10-75% efficacy)/antagonistic activities. All of these complexed receptors are crystallized in the canonical active conformation, regardless of their activity profiles. The bulky adamantyl side chain does not crowd helix 12 but protrudes into the gap formed by helix 11, loop 11-12, helix 3, and loop 6-7, thereby widening the ligand binding pocket. We suggest that these structural changes destabilize the active protein conformation and reduce its contribution to equilibrium among the active and inactive conformations. The coactivator peptide traps the minor active conformation, and the equilibrium shifts to the active conformation. As a result, these ligands show partial agonistic activities.

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