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Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay.

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.

SUBMITTER: Yoshikawa C

PROVIDER: S-EPMC8070791 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay.

Yoshikawa Chisato C Ishida Hiroaki H Ohashi Nami N Itoh Toshimasa T

International journal of molecular sciences 20210414 8

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, <b>1</b>, showed weak fluorescence intens ...[more]

PMID: 33919837

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Project description:Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY(®)-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY(®)-PbTx-2 conjugate was found to displace [(3)H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [(3)H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Dataset Information

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay.

Publications

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay.

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