Abstract
Peroxisome proliferator-activated receptor γ (PPARγ) is a molecular target of metabolic syndrome and inflammatory disease. PPARγ is an important nuclear receptor and numerous PPARγ ligands were developed to date; thus, efficient assay methods are important. Here, we investigated the incorporation of 7-diethylamino coumarin into the PPARγ agonist rosiglitazone and used the compound in a binding assay for PPARγ. PPARγ-ligand-incorporated 7-methoxycoumarin, 1, showed weak fluorescence intensity in a previous report. We synthesized PPARγ-ligand-incorporating coumarin, 2, in this report, and it enhanced the fluorescence intensity. The PPARγ ligand 2 maintained the rosiglitazone activity. The obtained partial agonist 6 appeared to act through a novel mechanism. The fluorescence intensity of 2 and 6 increased by binding to the ligand binding domain (LBD) of PPARγ and the affinity of reported PPARγ ligands were evaluated using the probe.
Highlights
Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and are categorized into three subtypes—PPARα, β/δ, and γ [1,2,3]
We previously synthesized the rosiglitazone-based model compound 1, in which the terminal pyridine was replaced by the coumarin skeleton, using a coumarin precursor (Scheme 1)
We suggest that the compounds incorporating 7-Et2N coumarins, which are easy to synthesize, could be applied to Peroxisome proliferator-activated receptor γ (PPARγ) binding assays and did not require fluorescence anisotropy apparatus and techniques. 7-Et2N coumarin could be used for live cells imaging [51] and 2 and 6 were potent probes for cell imaging
Summary
Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and are categorized into three subtypes—PPARα, β/δ, and γ [1,2,3]. Nuclear receptor ligands, including PPARγ ligands, are often evaluated by investigating their gene transcription activity [19] This assay is useful in identifying agonists with strong efficacy, but this is likely to overlook antagonists and partial agonists because of its weak efficacy. Conventional PPAR binding assays often use competition with a radioligand [20] and such assays provide superior sensitivity, but they are costly, are possible health hazards, and require laborious experimental procedures and special facilities. Fluorescent probes overcome these drawbacks [21].
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