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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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Results of direct binding assays for ccrp1, ccrp2 and ccrp3 on the TLX LBD using the Octet RED 384 instrument.A, B, C. Those panels represent the plotted steady-state response levels and the fitted binding isotherms. The purified TLX protein was immobilized onto the surfaces of Super-Streptavidin biosensors. Solutions of compounds ccrp1 (panel A), ccrp2 (panel B), ccrp3 (panel C) at 0.4–100 µM concentrations were tested against immobilized TLX LBD and reference surfaces composed of blocked biotinylated Streptavidin.
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pone-0099440-g005: Results of direct binding assays for ccrp1, ccrp2 and ccrp3 on the TLX LBD using the Octet RED 384 instrument.A, B, C. Those panels represent the plotted steady-state response levels and the fitted binding isotherms. The purified TLX protein was immobilized onto the surfaces of Super-Streptavidin biosensors. Solutions of compounds ccrp1 (panel A), ccrp2 (panel B), ccrp3 (panel C) at 0.4–100 µM concentrations were tested against immobilized TLX LBD and reference surfaces composed of blocked biotinylated Streptavidin.

Mentions: To verify DSF hits observed in primary screens and to determine if the interactions were truly stoichiometric, we performed an orthogonal direct binding assay. This approach relied upon the Octet Red 384 (FortéBio), an optical biosensor, to determine the binding kinetics for small molecules in a 384-well plate format. For this analysis, purified avi-tagged TLX LBD protein was immobilized onto the surface of Super-Streptavidin sensors. For reference surfaces, blocked biotinylated Streptavidin was immobilized onto another set of Super-Streptavidin sensors. Biosensors coated with TLX LBD and blocked biotinylated Streptavidin, were dipped separately into solutions of each of the putative 190 hits at a final concentration of 100 µM. This method confirmed binding of 24/190 compounds. Compounds defined as not binding to TLX LBD in this assay were removed from further considerations. Binding of 24 compounds was then evaluated at concentrations ranging from 100 µM to 400 nM. This revealed that three compounds (ccrp1, ccrp2 and ccrp3) produced a steady-state response in a dose-dependent manner (Fig. 5). Since purified avi-tagged TLX protein has a molecular weight of 27,000 Da and a loading signal of 10 nm; the maximum binding response for compounds with molecular weights around 300 Da would be 0.08–0.1 nm in these experiments. A maximum binding response above 0.1 would signify that the compounds bind with a super-stoichiometry. As expected, we reached 0.06–0.1 nm for the maximum response plateau of ccrp2 and ccrp3. At the highest concentrations tested, ccrp1 only attained a maximum binding response of 0.04 nm, likely due to a less effective loading of the protein onto the sensors. The equilibrium dissociation constant (Kd) was determined using steady state analysis of binding affinities, assuming 1∶1 ligand-protein stoichiometry. Curve-fitting analysis of the corrected response isotherm estimated Kd values of 6.6±0.07 µM for ccrp1, 650±100 nM for ccrp2, and 27.5±3.5 µM for ccrp3.


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)

Results of direct binding assays for ccrp1, ccrp2 and ccrp3 on the TLX LBD using the Octet RED 384 instrument.A, B, C. Those panels represent the plotted steady-state response levels and the fitted binding isotherms. The purified TLX protein was immobilized onto the surfaces of Super-Streptavidin biosensors. Solutions of compounds ccrp1 (panel A), ccrp2 (panel B), ccrp3 (panel C) at 0.4–100 µM concentrations were tested against immobilized TLX LBD and reference surfaces composed of blocked biotinylated Streptavidin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099440-g005: Results of direct binding assays for ccrp1, ccrp2 and ccrp3 on the TLX LBD using the Octet RED 384 instrument.A, B, C. Those panels represent the plotted steady-state response levels and the fitted binding isotherms. The purified TLX protein was immobilized onto the surfaces of Super-Streptavidin biosensors. Solutions of compounds ccrp1 (panel A), ccrp2 (panel B), ccrp3 (panel C) at 0.4–100 µM concentrations were tested against immobilized TLX LBD and reference surfaces composed of blocked biotinylated Streptavidin.
Mentions: To verify DSF hits observed in primary screens and to determine if the interactions were truly stoichiometric, we performed an orthogonal direct binding assay. This approach relied upon the Octet Red 384 (FortéBio), an optical biosensor, to determine the binding kinetics for small molecules in a 384-well plate format. For this analysis, purified avi-tagged TLX LBD protein was immobilized onto the surface of Super-Streptavidin sensors. For reference surfaces, blocked biotinylated Streptavidin was immobilized onto another set of Super-Streptavidin sensors. Biosensors coated with TLX LBD and blocked biotinylated Streptavidin, were dipped separately into solutions of each of the putative 190 hits at a final concentration of 100 µM. This method confirmed binding of 24/190 compounds. Compounds defined as not binding to TLX LBD in this assay were removed from further considerations. Binding of 24 compounds was then evaluated at concentrations ranging from 100 µM to 400 nM. This revealed that three compounds (ccrp1, ccrp2 and ccrp3) produced a steady-state response in a dose-dependent manner (Fig. 5). Since purified avi-tagged TLX protein has a molecular weight of 27,000 Da and a loading signal of 10 nm; the maximum binding response for compounds with molecular weights around 300 Da would be 0.08–0.1 nm in these experiments. A maximum binding response above 0.1 would signify that the compounds bind with a super-stoichiometry. As expected, we reached 0.06–0.1 nm for the maximum response plateau of ccrp2 and ccrp3. At the highest concentrations tested, ccrp1 only attained a maximum binding response of 0.04 nm, likely due to a less effective loading of the protein onto the sensors. The equilibrium dissociation constant (Kd) was determined using steady state analysis of binding affinities, assuming 1∶1 ligand-protein stoichiometry. Curve-fitting analysis of the corrected response isotherm estimated Kd values of 6.6±0.07 µM for ccrp1, 650±100 nM for ccrp2, and 27.5±3.5 µM for ccrp3.

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