The Human Orphan Nuclear Receptor Tailless (TLX, NR2E1) Is Druggable

<div><p>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.</p></div

Results of direct binding assays for ccrp1, ccrp2 and ccrp3 on the TLX LBD using the Octet RED 384 instrument.

<p>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.</p

Modulation of TLX and PNR transcriptional activities by ccrp1, ccrp2 and ccrp3. A.

<p>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.</p

Results of direct binding assays using the DSF method.

<p>A. This panel represents the melting curve of purified TLX LBD in the presence of 5% DMSO. B. This panel allows a rapid visualization on how compounds binding to TLX LBD can shift the reference Tm. We plotted the first derivatives of the melting curves for unliganded TLX LBD and TLX LBD in presence of ccrp1, ccrp2 and ccrp3. While these curves were utilized to accurately calculate T_m, a rough approximation of the Tm would be the minimum of the first derivative curves. In presence of the three compounds this minimum shifted downward. C. This panel displays the chemical structures of ccrp1, ccrp2, and ccrp3.</p

Sequence alignment of TLX LBD with COUP-TFII, RXRα and PNR LBDs.

<p>The nomenclature of the helices and β-sheet is indicated. Predicted residues belonging to the LBP are indicated by pink stars. Residues involved in the binding of TLX corepressors are highlighted in yellow. An exposed cysteine C338 is highlighted in green.</p

Biochemical characterization of purified TLX LBD. A.

<p>Analytical ultracentrifugation sedimentation analysis on purified TLX LBD. Analytical ultracentrifugation sedimentation equilibrium profiles were recorded at 4°C after 70 hour incubation at three rotor speeds: 11,000 r.p.m. (cyan), 18,000 r.p.m. (green) and 22,000 r.p.m. (blue), for a 10 µM TLX sample, dissolved in 20 mM Tris-HCl, 150 mM NaCl, 5 mM DTT, 10% (v/v) glycerol, 1% (v/v) DMSO and 2 mM CHAPS at pH 8.0. The upper panel shows the sedimentation equilibrium profiles with the lines of best fit shown in red. The best fit was obtained for a monomer-dimer equilibrium model with a Kd of 10 µM. 95% confidence interval limits for this dissociation constant were determined to be 4 µM</p

Models of TLX LBD and TLX LBP.

<p>A. This panel represents TLX LBD in a putative agonist conformation. Helix α11 is in blue/green, helix α12 in orange/yellow. The LBP appears in pink circles behind helix α12. B. This panel illustrates the key amino-acids inside the LBP, hydrophobic residues are in yellow, hydrophilic residues in red.</p