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The human orphan nuclear receptor tailless (TLX, NR2E1) is druggable.

Benod C, Villagomez R, Filgueira CS, Hwang PK, Leonard PG, Poncet-Montange G, Rajagopalan S, Fletterick RJ, Gustafsson JÅ, Webb P - PLoS ONE (2014)

Bottom Line: As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity.We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands.While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.

View Article: PubMed Central - PubMed

Affiliation: Department of Genomic Medicine, Houston Methodist Research Institute (HMRI), Houston, Texas, United States of America.

ABSTRACT
Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.

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Modulation of RXRα, COUP-TFII and ERβ transcriptional activities by ccrp1, ccrp2 and ccrp3.A, B. HeLa cells transiently transfected with RXRα LBD (panel A) or COUP-TFII LBD (panel B) and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at 10 µM in absence or presence of 9-cis retinoic acid (RA) at different concentrations (100 nM for RXRα and 5 µM for COUP-TFII). C. HeLa cells transiently transfected with ERβ LBD and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or ccrp1; ccrp2 and ccrp3 at 10 µM in absence or presence of E2 at 100 nM. For all panels, following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown relative to control (0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.
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pone-0099440-g008: Modulation of RXRα, COUP-TFII and ERβ transcriptional activities by ccrp1, ccrp2 and ccrp3.A, B. HeLa cells transiently transfected with RXRα LBD (panel A) or COUP-TFII LBD (panel B) and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at 10 µM in absence or presence of 9-cis retinoic acid (RA) at different concentrations (100 nM for RXRα and 5 µM for COUP-TFII). C. HeLa cells transiently transfected with ERβ LBD and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or ccrp1; ccrp2 and ccrp3 at 10 µM in absence or presence of E2 at 100 nM. For all panels, following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown relative to control (0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.

Mentions: We also tested ccrp1, ccrp2, and ccrp3 for their ability to modulate the transcriptional activity of several human NR LBDs using a luciferase reporter assay. Because of the high sequence identity between TLX and PNR, we investigated if the three TLX ligands we identified could modulate PNR transcriptional activity. None of the compounds affected PNR transcriptional activity (Fig. 6 Panel B). Because our homology model of TLX LBP was mainly designed using RXRα as a template and COUP-TFII displays high sequence identity with TLX, we decided to investigate the ability of ccrp1, ccrp2, and ccrp3 to modulate the transcriptional activity of both receptors. No effect on luciferase activity was detected when the compounds were tested alone or in competition with the 9-cis retinoic acid, a potent agonist for RXRα and COUP-TFII (Fig. 8 Panels A and B). We also tested in transactivation assays our compounds of interest on a steroid hormone receptor, ERβ. No effect on luciferase activity was detected when the compounds were tested alone or in competition with E2, a potent agonist (Fig. 8 Panel C). This experiment also confirmed our DSF findings: no binding was observed between ccrp1, ccrp2, or ccrp3 and the purified recombinant ERβ LBD protein. Based on these data, we conclude that the identified ligands bind preferentially to TLX.


The human orphan nuclear receptor tailless (TLX, NR2E1) is druggable.

Benod C, Villagomez R, Filgueira CS, Hwang PK, Leonard PG, Poncet-Montange G, Rajagopalan S, Fletterick RJ, Gustafsson JÅ, Webb P - PLoS ONE (2014)

Modulation of RXRα, COUP-TFII and ERβ transcriptional activities by ccrp1, ccrp2 and ccrp3.A, B. HeLa cells transiently transfected with RXRα LBD (panel A) or COUP-TFII LBD (panel B) and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at 10 µM in absence or presence of 9-cis retinoic acid (RA) at different concentrations (100 nM for RXRα and 5 µM for COUP-TFII). C. HeLa cells transiently transfected with ERβ LBD and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or ccrp1; ccrp2 and ccrp3 at 10 µM in absence or presence of E2 at 100 nM. For all panels, following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown relative to control (0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4060991&req=5

pone-0099440-g008: Modulation of RXRα, COUP-TFII and ERβ transcriptional activities by ccrp1, ccrp2 and ccrp3.A, B. HeLa cells transiently transfected with RXRα LBD (panel A) or COUP-TFII LBD (panel B) and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at 10 µM in absence or presence of 9-cis retinoic acid (RA) at different concentrations (100 nM for RXRα and 5 µM for COUP-TFII). C. HeLa cells transiently transfected with ERβ LBD and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or ccrp1; ccrp2 and ccrp3 at 10 µM in absence or presence of E2 at 100 nM. For all panels, following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown relative to control (0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.
Mentions: We also tested ccrp1, ccrp2, and ccrp3 for their ability to modulate the transcriptional activity of several human NR LBDs using a luciferase reporter assay. Because of the high sequence identity between TLX and PNR, we investigated if the three TLX ligands we identified could modulate PNR transcriptional activity. None of the compounds affected PNR transcriptional activity (Fig. 6 Panel B). Because our homology model of TLX LBP was mainly designed using RXRα as a template and COUP-TFII displays high sequence identity with TLX, we decided to investigate the ability of ccrp1, ccrp2, and ccrp3 to modulate the transcriptional activity of both receptors. No effect on luciferase activity was detected when the compounds were tested alone or in competition with the 9-cis retinoic acid, a potent agonist for RXRα and COUP-TFII (Fig. 8 Panels A and B). We also tested in transactivation assays our compounds of interest on a steroid hormone receptor, ERβ. No effect on luciferase activity was detected when the compounds were tested alone or in competition with E2, a potent agonist (Fig. 8 Panel C). This experiment also confirmed our DSF findings: no binding was observed between ccrp1, ccrp2, or ccrp3 and the purified recombinant ERβ LBD protein. Based on these data, we conclude that the identified ligands bind preferentially to TLX.

Bottom Line: As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity.We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands.While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.

View Article: PubMed Central - PubMed

Affiliation: Department of Genomic Medicine, Houston Methodist Research Institute (HMRI), Houston, Texas, United States of America.

ABSTRACT
Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.

Show MeSH
Related in: MedlinePlus