Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor.

No therapeutic drugs are currently available for nonalcoholic steatohepatitis (NASH) that progresses from nonalcoholic fatty liver via oxidative stress-involved pathways. Three cognate peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) are considered as attractive targets. Although lanifibranor (PPARα/δ/γ pan agonist) and saroglitazar (PPARα/γ dual agonist) are currently under investigation in clinical trials for NASH, the development of seladelpar (PPARδ-selective agonist), elafibranor (PPARα/δ dual agonist), and many other dual/pan agonists has been discontinued due to serious side effects or little/no efficacies. This study aimed to obtain functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of three current and past anti-NASH investigational drugs: lanifibranor, seladelpar, and elafibranor. Ligand activities were evaluated by three assays to detect different facets of the PPAR activation: transactivation assay, coactivator recruitment assay, and thermal stability assay. Seven high-resolution cocrystal structures (namely, those of the PPARα/δ/γ-ligand-binding domain (LBD)-lanifibranor, PPARα/δ/γ-LBD-seladelpar, and PPARα-LBD-elafibranor) were obtained through X-ray diffraction analyses, six of which represent the first deposit in the Protein Data Bank. Lanifibranor and seladelpar were found to bind to different regions of the PPARα/δ/γ-ligand-binding pockets and activated all PPAR subtypes with different potencies and efficacies in the three assays. In contrast, elafibranor induced transactivation and coactivator recruitment (not thermal stability) of all PPAR subtypes, but the PPARδ/γ-LBD-elafibranor cocrystals were not obtained. These results illustrate the highly variable PPARα/δ/γ activation profiles and binding modes of these PPAR ligands that define their pharmacological actions.

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.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.