The Vitamin D Analog, KH1060, Is Rapidly Degraded Both in Vivo and in Vitro via Several Pathways: Principal Metabolites Generated Retain Significant Biological Activity*

Dilworth, F. J.; Williams, Graham R.; Kissmeyer, Anne-Marie; Nielsen, Jeanet L.; Binderup, Ernst; Calverley, Martin J.; Makin, H. L. J.; Jones, Glenville

doi:10.1210/endo.138.12.5594

Cited by 42 publications

(20 citation statements)

References 38 publications

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“…Limited digest proteolysis shows that the 20-epi analog-receptor complexes are more resistant to digestion and suggests that they are more stable. Additionally, an important role might be played by the rate of assimilation and how the synthetic agonists are metabolized vs. the natural ligand in different cell types (25,31). Altogether, these data suggest that the life time of the active conformation is then the main factor responsible for the formation of a more potent complex with coactivators like DRIP and the subsequent higher transcription activity.…”

Section: Discussionmentioning

confidence: 98%

“…Similarly, the naturally occurring hydroxylation of C24␣ in KH1060 and C23 in 1␣,25(OH) 2 D 3 would form steric clashes with His-305, affecting the hydrogen bond network with 25-OH. The resulting conformational changes can thus explain the reported lack of transcriptional activity (25). Based on the crystal structures, this activity would depend on the capacity of the pocket to accommodate these additional groups.…”

Section: Conformation Of the Bound Ligandsmentioning

confidence: 99%

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Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

Tocchini-Valentini

Rochel

Wurtz

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

221

198

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The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1␣,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A-to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17␤-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain. A different class of proteins has been characterized that stimulate the transcriptional activity of liganded NRs in an activation function 2 (AF2)-dependent way. These coactivators are thought to bridge the NRs to the transcriptional apparatus. In particular, VDR is regulated by coactivators belonging to the steroid receptor activator (SRC)͞p160 family of proteins, which contain several LXXLL motifs (6). This motif has been shown to form an amphipatic ␣-helical structure that can interact with the AF2 region of NRs (7). Another class of coactivators [vitamin D receptor-interacting protein (DRIP), thyroid hormone receptor-associated protein (TRAP), activator-recruited cofactor (ARC); refs. 8-11] has been isolated as multiprotein complexes and strongly potentiated transcription mediated by VDR͞RXR in a ligand-dependent manner on DNA templates assembled into chromatin (8). One of their components, DRIP205, interacts directly with the ligand-binding domain (LBD) in the presence of ligand and anchors the rest of the subunits to the receptor.Among the several synthetic analogs of vitamin D, the 20-epi compounds, which exhibit an inverted stereochemistry at position 20 in the flexible aliphatic chain, have attracted much attention. They are potent growth inhibitors and inducers of cell differentiation, while showing an affinity similar to vitamin D for VDR (11). KH1060 ( Fig. 1), a member of this 20-epi family, exhibits similar properties, with decreased calcemic side effects. These compounds induce VDR-dependent transcription at concentrations at least 100-fold lower than the natural ligand and present antiproliferative activity several orders of magnitude higher than the natural ligand (11-13). The differences in biological activity of 1␣,25(OH) 2 D 3 and the 20-epi molecules in general, and KH1060 in particular, are known to be VDR-LBD dependent, but are not yet understood. Th...

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Section: Discussionmentioning

confidence: 98%

Section: Conformation Of the Bound Ligandsmentioning

confidence: 99%

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

Tocchini-Valentini

Rochel

Wurtz

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

221

198

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“…In addition, we have demonstrated that for the 1,25-(OH) 2 D 3 analog KH1060 a cellular/ nuclear context (i.e. the absence or presence of nuclear cofactors) is crucial for observing the ligand-induced VDR-VDRE binding that reflects its increased biological potency (15)(16)(17)(18). Thereby, this study implicates the significance of these nuclear cofactors for determining the extent of transcriptional activity.…”

Section: Discussionmentioning

confidence: 58%

“…In addition, the present paper shows that the increased biological potency of the vitamin D analog KH1060 (15)(16)(17)(18) is not reflected by an increased binding of in vitro synthesized VDR and RXR␣ to various VDREs. KH1060-induced binding of in vitro synthesized VDR/RXR␣ to the mouse osteopontin VDRE was slightly increased, whereas binding to human osteocalcin and rat osteocalcin VDREs was comparable with or even less than 1,25-(OH) 2 RO 24-2637 and RO 23-7553 also demonstrated a lack of parallelism between the potency of these analogs to induce the binding of recombinant human VDR and RXR␣ to the human osteocalcin VDRE and the ability of these analogs to activate a human osteocalcin VDRE-driven reporter gene.…”

Section: Discussionmentioning

confidence: 62%

“…12-14 and Table I). To gain insight into the biological relevance of nucleotide variation in naturally occurring VDREs for the action of 1,25-(OH) 2 D 3 and its potent analog KH1060 (15)(16)(17)(18), we examined the binding and activity of the osteocalcin and osteopontin VDREs. The main finding of our study is that nucleotide differences between osteopontin and osteocalcin VDREs affect DNA binding and transactivation in response to 1,25-(OH) 2 D 3 and its potent analog, KH1060.…”

mentioning

confidence: 99%

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A Central Dinucleotide within Vitamin D Response Elements Modulates DNA Binding and Transactivation by the Vitamin D Receptor in Cellular Response to Natural and Synthetic Ligands

Bemd

Jhamai

Staal

et al. 2002

Journal of Biological Chemistry

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includes regulating the expression of genes involved in calcium and bone metabolism (1). In addition, the hormone is an important mediator of cell growth and differentiation (2, 3). In a large number of 1,25-(OH) 2 D 3 -dependent target genes, distinct vitamin D response elements (VDREs) have been identified that are composed of two hexameric half-sites separated by three base pairs (DR3-type VDRE). The vitamin D receptor (VDR)/retinoid X receptor (RXR) complex binds with defined polarity to these VDREs with the RXR occupying the distal half-site and the VDR the proximal half-site (4 -7).The spacing of the half-sites plays an important role in specifying the type of receptor pair that interacts with hormone response elements (7). There is considerable divergence in the nucleotide sequence of the distal and proximal half-site, which is attributable at least in part to redundancy in the consensus recognition motifs of the VDR and the RXR (8). The sequences of steroid hormone half-elements also influence the relative affinities for different steroid hormone receptors and thus may support receptor-specific gene activation by discriminating between different receptors (7). In addition, specific nucleotides in the VDRE appear to influence the conformation of bound VDR/RXR heterodimers, reflected by a VDRE-specific alteration in epitope accessibility (6). Furthermore, minor changes in the nucleotide sequence of half-elements can change a negative VDRE that suppresses transcription into a positive VDRE, which transactivates in a 1,25-(OH) 2 D 3 -dependent manner (9). The impact of minor changes in nucleotide sequences on receptor DNA binding is not restricted to VDR/ VDRE interactions, as evidenced by half-element point mutations that have been shown to alter the affinity (10) or identity of the cognate receptor (11).Two major 1,25-(OH) 2 D 3 -regulated genes are the two noncollagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. These genes have sequence variations in their VDREs (Refs. 12-14 and Table I). To gain insight into the biological relevance of nucleotide variation in naturally occurring VDREs for the action of 1,25-(OH) 2 D 3 and its potent analog KH1060 (15-18), we examined the binding and activity of the osteocalcin and osteopontin VDREs. The main finding of our study is that nucleotide differences between osteopontin and osteocalcin VDREs affect DNA binding and transactivation in response to 1,25-(OH) 2 D 3 and its potent analog, KH1060. Our findings show that the primary sequence of VDREs may influence the biological activities of natural and synthetic ligands that bind to the VDR. Finally, the strong biological potency of the analog KH1060 is only reflected by DNA binding of VDR/RXR heterodimers in the environment of nuclear proteins and not by the binding of in vitro synthesized VDR and RXR␣, underscoring the importance of a cellular context for ligand-induced DNA binding.

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Mechanism of action of superactive vitamin D analogs through regulated receptor degradation

et al. 2000

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The Vitamin D Analog, KH1060, Is Rapidly Degraded Both in Vivo and in Vitro via Several Pathways: Principal Metabolites Generated Retain Significant Biological Activity*

Cited by 42 publications

References 38 publications

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

A Central Dinucleotide within Vitamin D Response Elements Modulates DNA Binding and Transactivation by the Vitamin D Receptor in Cellular Response to Natural and Synthetic Ligands

Mechanism of action of superactive vitamin D analogs through regulated receptor degradation

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