Cellular and Biophysical Pipeline for the Screening of Peroxisome Proliferator-Activated Receptor Beta/Delta Agonists: Avoiding False Positives

Figure 5

Validation assays of PPARß/δ ligand screening. (a) Thermal shift assay of hit candidates. Ten micromolar PPARß/δ with commercial agonists (GW0742, GW501516, L-165,041, and Bezafibrate) at 30 μM or extracts at 0.14 mg/mL. The vehicle is DMSO. Experiments were performed in triplicate. Data are the mean ± SD. (b) Table with variation and standard deviation for the extracts/compounds from the thermal shift. The experiment was performed in triplicate. ND: not defined. It is possible to verify that two possible hit candidates stabilized the receptor structure by more than 2.5°C, indicating direct binding to the receptor. (c)-(d) Dissociation curves for PPARß/δ ligands and hit candidates. Normalized fluorescence intensity at the emission maximum (480 nm) versus the ligand/fraction concentration, adjusted by the Hill1 approach with OriginPro 8.0. Dissociation curves for the commercial agonists varied from 10⁻⁷ to 10⁻⁵ μM. In the dissociation curve, the concentration varied from 0.003 to 1 mg/mL. Data are the mean ± SD ( independent replicates).

(a)

(b)

(c)

(d)