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

Figure 7

Screening pipeline for PPARß/δ agonists. Our proposed screening pipeline for PPARß/δ agonists is composed of 3 complementary assays. The primary transactivation reporter-gene assay screening and the confirmatory transactivation reporter-gene assay utilize a cellular transactivation reporter-gene assay that has been optimized to a 3-day long experiment with 40,000 cells and only 25 μL of the luciferases substrates per well, reducing the time and cost of the screening assay. The two following validation assays are the thermal shift (TSA) to check if the compounds/extracts previously selected stabilize the PPARß/δ tertiary structure and the ANS fluorescence quenching to determine the compound affinity to the hydrophobic pocket of PPARß/δ. We submitted a 560-natural extract library to the proposed pipeline and found 31 possible hit candidates in the primary transactivation screening. Ten hit candidates were selected in the confirmatory cellular transactivation. The TSA selected 2 extracts, and one of them showed a 0.02 mg/mL affinity constant in the ANS quenching assay.