GQ-16, a novel peroxisome proliferator-activated receptor (PPAR ) ligand, promotes insulin sensitization without weight gain

ABSTRACTBackground: PPAR agonists improve insulin sensitivity but also evoke weight gain. Results: GQ-16 is a PPAR partial agonist that blocks receptor phosphorylation by Cdk5 and improves insulin sensitivity in diabetic mice in the absence of weight gain. Conclusion: The unique binding mode of GQ-16 appears to be responsible for the compound’s advantageous pharmacological profile. Significance: Similar compounds could have promise as anti-diabetic therapeutics

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

Contribution of Long-Range Interactions to the Secondary Structure of an Unfolded Globin

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an α-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable α-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins

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

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

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