Functional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662
Abstract
In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma. The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta. Corepressor binding was decreased. In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.
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GW9662 covalently and irreversibly binds to the PPARγ ligand binding pocket, preventing activating ligand binding and interrupting PPARγ signal transduction.
“GW9662 has been shown to covalently and irreversibly bind to the PPARg ligand binding pocket (Leesnitzer et al., 2002;Brust et al., 2018).The binding of activating ligands is thereby prevented, and the signal transduction via PPARg is interrupted.”
GW9662 is known as a selective PPARγ antagonist and is widely accepted to demonstrate participation of PPARγ in signaling cascades.
“GW9662, known as a selective peroxisome proliferator (PPAR)g antagonist, is widely accepted to demonstrate participation of PPARg in signaling cascades (Leesnitzer et al., 2002;Lea et al., 2004;Nielsen et al., 2006).”
Previous studies show that GW9662 is not entirely specific for PPARgamma.
“Previous studies using GW9662 revealed that this compound is not entirely specific for PPARg (Leesnitzer et al., 2002).”
The authors of the initial study were not able to confirm PPARδ antagonism in an assay based on the full-length receptor and a luciferase reporter.
“Interestingly, the authors of the initial study were not able to confirm the PPARd antagonism in an assay based on the full-length receptor and a luciferase reporter.”
GW9662 has been shown to bind to all PPAR subtypes.
“The proposed ability of GW9662 to bind to all PPAR subtypes (vide supra) has indeed been shown.”
GW9662 was identified as a potent antagonist of PPARγ.
“Although GW9662 was identified as a potent antagonist of PPARg, it also covalently modifies PPARa and PPARd at higher concentrations (Leesnitzer et al., 2002).”
GW9662 covalently modifies PPARα and PPARδ at higher concentrations.
“Although GW9662 was identified as a potent antagonist of PPARg, it also covalently modifies PPARa and PPARd at higher concentrations (Leesnitzer et al., 2002).”
GW9662 was initially characterized as an antagonist of PPARδ using an assay based on a fusion protein containing the ligand binding domain of PPARδ and the galactose-responsive transcription factor 4.
“Contradictory to this, GW9662 has initially been characterized as an antagonist of PPARd using an assay based on a fusion protein containing the ligand binding domain of PPARd and the galactose-responsive transcription factor 4 (Leesnitzer et al., 2002)”
GW9662 is able to activate gene expression via PPARδ in native cellular systems like THP-1 macrophages.
“Here, even an activation of PPARd was observed (Leesnitzer et al., 2002), indicating that GW9662 is indeed able to activate gene expression via PPARd in”
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- Authors
- Leesnitzer, L. M., Parks, D. J., Bledsoe, R. K., Cobb, J. E., Collins, J. L., Consler, T. G., Davis, R. G., Hull-Ryde, E. A., Lenhard, J. M., Patel, L., Plunket, K. D., Shenk, J. L., Stimmel, J. B., Therapontos, C., Willson, T. M., & Blanchard, S. G.
- Affiliations
- GlaxoSmithKline (United Kingdom) (GB)
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2002-04-30
- Volume
- 41
- Issue
- 21
- Pages
- 6640-6650
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2089687829
- Semantic Scholar
- eb83d0a083a66374a0cc902dc0121e7f0a88c63d
- Cited by
- 566
- References
- 22
- Citations by year
- 2026: 9 · 2025: 18 · 2024: 24 · 2023: 16 · 2022: 16 · 2021: 21 · 2020: 15 · 2019: 27 · 2018: 23 · 2017: 18 · 2016: 22 · 2015: 31 · 2014: 23 · 2013: 29 · 2012: 26
- FWCI
- 8.53
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Peroxisome Proliferator-Activated Receptors
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
- Health Sciences › Medicine › Surgery › Cholesterol and Lipid Metabolism
- Keywords
- GW9662, PPARγ, cysteine modification, covalent modification, PPARα, PPARδ, coactivator binding, corepressor binding, PPAR antagonists, adipogenesis, ligand selectivity
- MeSH terms
- Anilides, Binding Sites, Cell Differentiation, Cysteine, Dose-Response Relationship, Drug, Escherichia coli, Humans, Ligands, Nuclear Proteins, Repressor Proteins, Transcription Factors, Trans-Activators, Adipocytes, Receptors, Cytoplasmic and Nuclear, Receptors, Retinoic Acid, Dimerization, Retinoid X Receptors, CREB-Binding Protein, Nuclear Receptor Co-Repressor 1
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