Identification de ligands pour les récepteurs nucléaires humains PXR (NR112) et CAR (NR113)

Les récepteurs nucléaires humains PXR (Pregnane X Receptor, NR1I2) et CAR (Constitutive Androstane Receptor, NR1I3) modulent l'expression de plusieurs familles de gènes impliquées dans des voies de détoxification de l'organisme. Ainsi, des agonistes affins et spécifiques de ces deux récepteurs pourraient être utilisés pour protéger l organisme des effets néfastes dus à l accumulation d agents toxiques endogènes et exogènes. Par ailleurs, des antagonistes de PXR et CAR permettraient de traiter certains cancers ou d éviter des interactions médicamenteuses. Ce projet a donc consiste à mettre en place une stratégie allant de l identification de ces ligands par une méthode de conception rationnelle basée sur la structure 3D de PXR et CAR, à leur caractérisation biologique, voire à leur optimisation moléculaire pour améliorer leur activité. Ce travail a ainsi permis l identification d un agoniste et de 9 antagonistes pour CAR. Pour PXR, un agoniste doté d une forte activité a été identifié.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

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

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

Discovery of a highly active ligand of human pregnane x receptor: a case study from pharmacophore modeling and virtual screening to "in vivo" biological activity.

International audienceThe human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and II drug-metabolizing enzymes as well as that of drug transporters. In addition, this receptor plays a critical role in cholesterol homeostasis and in protecting tissues from potentially toxic endobiotics. hPXR is activated by a broad spectrum of low-affinity compounds including xenobiotics and endobiotics such as bile acids and their precursors. Crystallographic studies revealed a ligand binding domain (LBD) with a large and conformable binding pocket that is likely to contribute to the ability of hPXR to respond to compounds of varying size and shape. Here, we describe an in silico method that allowed the identification of nine novel hPXR agonists. We further characterize the compound 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide (C2BA-4), a methanesulfonamide that activates PXR specifically and more potently than does the reference compound 4-[2,2-bis(diethoxyphosphoryl)ethenyl]-2,6-ditert-butyl-phenol (SR12813) in our stable cell line expressing a Gal4-PXR and a GAL4 driven luciferase reporter gene. Furthermore treatment of primary human hepatocytes with C2BA-4 results in a marked induction of the mRNA expression of hPXR target genes, such as cytochromes P450 3A4 and 2B6. Finally, C2BA-4 is also able to induce hPXR-mediated in vivo luciferase expression in HGPXR stable bioluminescent cells implanted in mice. The study suggests new directions for the rational design of selective hPXR agonists and antagonists

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 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