Functional and Structural Insights into Human PPARα/δ/γ Subtype Selectivity of Bezafibrate, Fenofibric Acid, and Pemafibrate

Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPARα (fibrates) and PPARγ (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPARδ agonists are expected to be the next-generation metabolic disease drug. In addition, some dual/pan PPAR agonists are currently being investigated via clinical trials as one of the first curative drugs against nonalcoholic fatty liver disease (NAFLD). Because PPARα/δ/γ share considerable amino acid identity and three-dimensional structures, especially in ligand-binding domains (LBDs), clinically approved fibrates, such as bezafibrate, fenofibric acid, and pemafibrate, could also act on PPARδ/γ when used as anti-NAFLD drugs. Therefore, this study examined their PPARα/δ/γ selectivity using three independent assays—a dual luciferase-based GAL4 transactivation assay for COS-7 cells, time-resolved fluorescence resonance energy transfer-based coactivator recruitment assay, and circular dichroism spectroscopy-based thermostability assay. Although the efficacy and efficiency highly varied between agonists, assay types, and PPAR subtypes, the three fibrates, except fenofibric acid that did not affect PPARδ-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPARδ/γ-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPARα-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPARδ/γ–bezafibrate, PPARγ–fenofibric acid, and PPARδ/γ–pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment.