Discovery of peroxisome proliferator–activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line

Abstract

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both and Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Structural Basis for PPARα Activation by 1H-pyrazolo-[3,4-b]pyridine Derivatives

“…The activation of PPARs by compounds was measured by using a chimeric receptor consisting of the GAL4-DNA-binding domain and PPAR-LBD with a luciferase (LUC) reporter system, p4×UAS-tk-luc, which is a LUC reporter construct containing four copies of the GAL4 binding site followed by the thymidine kinase promoter. Assays were performed as described previously 17 . Briefly, HepG2 cells were transfected with p4×UAS-tk-luc together with the pBIND-hPPARα-LBD, pBIND-hPPARβ/δ-LBD, or pBIND-hPPARγ1-LBD expression vector (an expression plasmid for GAL4-hPPAR chimera protein) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.…”

“…We recently developed a novel screening system for PPARα agonists and discovered a new structural class these agonists, 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives (hereafter referred to as 1H-pyrazolo-[3,4-b]pyridine derivatives), which strongly activate PPARα 17 . The basic skeleton of these compounds, 1H-pyrazolo- [3,4-b]pyridine, is found in several bioactive compounds, e.g.…”

“…Furthermore, they could be expected to exhibit more potent hypolipidemic effects in vivo with reduced toxicities. Thus, 1H-pyrazolo- [3,4-b]pyridine derivatives could be promising lead compounds for dyslipidemia owing to their low associated risk 17 . Most recently, it was shown that the structure-activity-relationships (SAR) of 1H-pyrazolo-[3,4-b]pyridine derivatives were somewhat different from those of fibrates 20 .…”

Chemical Screening of Nuclear Receptor Modulators

“…Tachibana et al performed chemical screening for the activators of PPARα (NR1C1) using a two-step cell-based assay that regulates PPARα (NR1C1) expression via Tet [ 102 ]. They selected compounds that act directly on PPARα (NR1C1), rather than on the heterodimer with RXR (NR2B1-3) or various cofactors in the assays.…”

Development of a Ligand Screening Tool Using Full-Length Human Peroxisome Proliferator-Activated Receptor-Expressing Cell Lines to Ameliorate Metabolic Syndrome

“…5) This emphasizes the need to construct a ligand screening system to accurately assess the effects of ligands using full-length PPAR. In previous work, we established tightly tetracycline (Tet)-regulatable human hepatoblastoma cell lines that can be induced to express full-length human PPARs (HepG2-tet-off-hPPARs) in HY-Toff cells 6) expressing RXRα proteins (data not shown). 1) The PPAR/RXRα heterodimers bind to the PPRE and activate target gene expression in these cells.…”