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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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Specificity of ccp1, ccrp2 and ccrp3 towards unrelated nuclear receptors.A. This panel represents the melting curve of purified ERβ LBD in the absence of ligand. B. Melting temperature shifts for ERβ LBD in absence of compounds or treated with estradiol (E2), ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by estradiol. C. This panel represents the melting curve of purified LXRβ LBD in the absence of ligand. D. Melting temperature shifts for LXRβ LBD in absence of compounds or treated with T0901317, ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by T0901317.
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pone-0099440-g007: Specificity of ccp1, ccrp2 and ccrp3 towards unrelated nuclear receptors.A. This panel represents the melting curve of purified ERβ LBD in the absence of ligand. B. Melting temperature shifts for ERβ LBD in absence of compounds or treated with estradiol (E2), ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by estradiol. C. This panel represents the melting curve of purified LXRβ LBD in the absence of ligand. D. Melting temperature shifts for LXRβ LBD in absence of compounds or treated with T0901317, ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by T0901317.

Mentions: We examined whether ccrp1, ccrp2, and ccrp3 could bind two unrelated nuclear receptors ERβ and LXRβ (Fig. 7). For accurate comparisons, we employed analogous DSF methods to that employed for TLX in absence or presence of compounds of interest (500 µM) or DMSO (5%, solvent control). Human ERβ LBD produced a denaturation curve with a melting point (Tm) of ∼51°C (Fig. 7 Panel A) and LXRβ LBD produced a denaturation curve with a melting point (Tm) of ∼39°C (Fig. 7 Panel C). Unliganded ERβ and LXRβ seem to behave like molten globular proteins with high initial fluorescence values at low temperatures, indicative of exposed hydrophobic residues that are accessible to Sypro Orange dye. Additionally, as the temperature increases, thermal energy decreases the affinity of the dye for exposed hydrophobic residues of the protein and the fluorescence is decreasing, resulting in a downward slope in the initial part of the melting curve.


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)

Specificity of ccp1, ccrp2 and ccrp3 towards unrelated nuclear receptors.A. This panel represents the melting curve of purified ERβ LBD in the absence of ligand. B. Melting temperature shifts for ERβ LBD in absence of compounds or treated with estradiol (E2), ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by estradiol. C. This panel represents the melting curve of purified LXRβ LBD in the absence of ligand. D. Melting temperature shifts for LXRβ LBD in absence of compounds or treated with T0901317, ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by T0901317.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4060991&req=5

pone-0099440-g007: Specificity of ccp1, ccrp2 and ccrp3 towards unrelated nuclear receptors.A. This panel represents the melting curve of purified ERβ LBD in the absence of ligand. B. Melting temperature shifts for ERβ LBD in absence of compounds or treated with estradiol (E2), ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by estradiol. C. This panel represents the melting curve of purified LXRβ LBD in the absence of ligand. D. Melting temperature shifts for LXRβ LBD in absence of compounds or treated with T0901317, ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by T0901317.
Mentions: We examined whether ccrp1, ccrp2, and ccrp3 could bind two unrelated nuclear receptors ERβ and LXRβ (Fig. 7). For accurate comparisons, we employed analogous DSF methods to that employed for TLX in absence or presence of compounds of interest (500 µM) or DMSO (5%, solvent control). Human ERβ LBD produced a denaturation curve with a melting point (Tm) of ∼51°C (Fig. 7 Panel A) and LXRβ LBD produced a denaturation curve with a melting point (Tm) of ∼39°C (Fig. 7 Panel C). Unliganded ERβ and LXRβ seem to behave like molten globular proteins with high initial fluorescence values at low temperatures, indicative of exposed hydrophobic residues that are accessible to Sypro Orange dye. Additionally, as the temperature increases, thermal energy decreases the affinity of the dye for exposed hydrophobic residues of the protein and the fluorescence is decreasing, resulting in a downward slope in the initial part of the melting curve.

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