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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 TLX and PNR transcriptional activities by ccrp1, ccrp2 and ccrp3. A.Transfections of TLX LBD repress the UAS promoter leading to a decrease in luciferase activities compared to the control (cells transfected with empty GAL4 vector). Compounds ccrp1, ccrp2 and ccrp3 respectively enhance repressive transcriptional activity of TLX only in cells transfected with TLX LBD. HeLa cells transiently transfected with TLX LBD or empty GAL4 vector and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at different concentrations (indicated). Following 16 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines. B. HeLa cells transiently transfected with PNR LBD or empty GAL4 vector and the luciferase reporter gene were treated with DMSO (0.1%, solvent control) or ccrp1, ccrp2 and ccrp3 at 10 µM. Following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.
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pone-0099440-g006: Modulation of TLX and PNR transcriptional activities by ccrp1, ccrp2 and ccrp3. A.Transfections of TLX LBD repress the UAS promoter leading to a decrease in luciferase activities compared to the control (cells transfected with empty GAL4 vector). Compounds ccrp1, ccrp2 and ccrp3 respectively enhance repressive transcriptional activity of TLX only in cells transfected with TLX LBD. HeLa cells transiently transfected with TLX LBD or empty GAL4 vector and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at different concentrations (indicated). Following 16 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines. B. HeLa cells transiently transfected with PNR LBD or empty GAL4 vector and the luciferase reporter gene were treated with DMSO (0.1%, solvent control) or ccrp1, ccrp2 and ccrp3 at 10 µM. Following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.

Mentions: To verify that selected ligands modulate TLX activity, we assessed transcriptional activity of the TLX LBD in the absence and the presence of varying concentrations of ccrp1, ccrp2, and ccrp3. As predicted, binding of TLX LBD repressed a GAL4 UAS-dependent promoter (in the pGL4.35 reporter gene vector) (Fig. 6 panel A). This agrees with suggestions that TLX is a constitutive transcriptional repressor [6], [45]. In presence of the compounds of interest, we observed further repression of the GAL4-dependent promoter in the presence of GAL4-TLX LBD compared to DMSO-treated cells. Defined EC50 values for each compound were 9.2±1.0 µM for ccrp1, 1.0±0.3 µM for ccrp2, and 250±100 nM for ccrp3 (Fig. 6 panel A and Fig. S2). No analogous effects were seen in cells that were transfected with GAL4 DBD vector (Fig. S3). Thus, we conclude that hit TLX compounds potentiate TLX transcriptional repressive activity.


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 TLX and PNR transcriptional activities by ccrp1, ccrp2 and ccrp3. A.Transfections of TLX LBD repress the UAS promoter leading to a decrease in luciferase activities compared to the control (cells transfected with empty GAL4 vector). Compounds ccrp1, ccrp2 and ccrp3 respectively enhance repressive transcriptional activity of TLX only in cells transfected with TLX LBD. HeLa cells transiently transfected with TLX LBD or empty GAL4 vector and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at different concentrations (indicated). Following 16 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines. B. HeLa cells transiently transfected with PNR LBD or empty GAL4 vector and the luciferase reporter gene were treated with DMSO (0.1%, solvent control) or ccrp1, ccrp2 and ccrp3 at 10 µM. Following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 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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Show All Figures
getmorefigures.php?uid=PMC4060991&req=5

pone-0099440-g006: Modulation of TLX and PNR transcriptional activities by ccrp1, ccrp2 and ccrp3. A.Transfections of TLX LBD repress the UAS promoter leading to a decrease in luciferase activities compared to the control (cells transfected with empty GAL4 vector). Compounds ccrp1, ccrp2 and ccrp3 respectively enhance repressive transcriptional activity of TLX only in cells transfected with TLX LBD. HeLa cells transiently transfected with TLX LBD or empty GAL4 vector and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at different concentrations (indicated). Following 16 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines. B. HeLa cells transiently transfected with PNR LBD or empty GAL4 vector and the luciferase reporter gene were treated with DMSO (0.1%, solvent control) or ccrp1, ccrp2 and ccrp3 at 10 µM. Following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.
Mentions: To verify that selected ligands modulate TLX activity, we assessed transcriptional activity of the TLX LBD in the absence and the presence of varying concentrations of ccrp1, ccrp2, and ccrp3. As predicted, binding of TLX LBD repressed a GAL4 UAS-dependent promoter (in the pGL4.35 reporter gene vector) (Fig. 6 panel A). This agrees with suggestions that TLX is a constitutive transcriptional repressor [6], [45]. In presence of the compounds of interest, we observed further repression of the GAL4-dependent promoter in the presence of GAL4-TLX LBD compared to DMSO-treated cells. Defined EC50 values for each compound were 9.2±1.0 µM for ccrp1, 1.0±0.3 µM for ccrp2, and 250±100 nM for ccrp3 (Fig. 6 panel A and Fig. S2). No analogous effects were seen in cells that were transfected with GAL4 DBD vector (Fig. S3). Thus, we conclude that hit TLX compounds potentiate TLX transcriptional repressive activity.

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