Fig. 7: MTX-531 acts as an agonist of PPARγ.
a, Left, in a competitive binding assay, MTX-531 displaced the PPARγ agonist GW1929 with an EC50 of 2.5 µM (R² = 0.9712). Right, MTX-531 was further shown to be a weak activator of PPARγ in a cell-based transcription assay carried out in PPARγ-UAS-bla HEK 293H cells with an EC50 of 3.4 µM (R² = 0.9898). Data from both studies are representative of two individual experiments carried out at concentrations tested in duplicate. b, MTX-531 did not exhibit activity when titrated across a ten-point concentration range to test for competitive binding to PPARα against GW7647 (left) and PPARδ against GW0742 (right). Data are representative of two studies carried out at concentrations tested in duplicate. c, Left, PPARγ target gene expression levels were analyzed in quadruplicate by RT–qPCR analysis of total RNA extracts from 3T3-L1 cells treated with differentiation medium containing rosiglitazone or MTX-531 for 8 or 24 h. Data are shown as the mean and upper–lower limits of RQ. A two-tailed, unpaired t-test or Mann–Whitney test was used to determine the statistical differences between treatment groups. *P = 0.0286, **P = 0.0213, ***P = 0.0001 and ****P < 0.0001. Right, an immunoblot analysis of PPARγ expression was carried out in 3T3-L1 adipocytes at 7 days after induction of differentiation. PCR and immunoblotting data are representative of two independent experiments. d, Crystal structure of PPARγ cocomplexed with MTX-531 solved at 1.9 Å (PDB 8SC9). Left, the secondary structure; right, a view of MTX-531 bound to PPARγ from the ligand-binding pocket. e, Electron density maps of the MTX-531-binding site of PPARγ. Top, the initial Fo − Fc difference electron density map of the model (contoured at 3.0 σ) before modeling of the compound with BUSTER. Shown is the region of the compound-binding site in chain B and the final refined coordinates. Bottom, the final 2Fo − Fc electron density map (contoured at 1.0 σ) resulting from refinement of the final model. Shown is the region of the compound-binding site. f, Overlay of MTX-531 X-ray binding models with commercial PPARγ agonists rosiglitazone (PDB 4EMA) and pioglitazone (PDB 2XKW).
