Functional and Structural Insights into Human PPAR alpha/delta/gamma Subtype Selectivity of Bezafibrate, Fenofibric Acid, and Pemafibrate

Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPAR alpha (fibrates) and PPAR gamma (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPAR delta 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 PPAR alpha/delta/gamma 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 PPAR delta/gamma when used as anti-NAFLD drugs. Therefore, this study examined their PPAR alpha/delta/gamma 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 PPAR delta-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPAR delta/gamma-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPAR alpha-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPAR delta/gamma-bezafibrate, PPAR gamma-fenofibric acid, and PPAR delta/gamma-pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment.