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Updated: Apr 1, 2026
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Identification of Bexarotene as a PPARγ Antagonist with HDX
David P Marciano1, Dana S Kuruvilla1, Bruce D Pascal1
1Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
Abstract:
The retinoid x receptors (RXRs) are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL). The RXRs form heterodimers with several nuclear receptors (NRs), including peroxisome proliferator-activated receptor gamma (PPARγ), to regulate target gene expression through cooperative recruitment of transcriptional machinery. Here we have applied hydrogen/deuterium exchange (HDX) mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγ heterodimer. Interestingly, addition of Bexarotene to PPARγ in the absence of RXRα induced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγ antagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.
Insights
Bexarotene, used for cutaneous T cell lymphoma, directly binds to PPARγ, acting as an antagonist. This study reveals complex drug interactions using HDX mass spectrometry.
Area of Science:
- Pharmacology
- Structural Biology
- Molecular Biology
Background:
- Retinoid X Receptors (RXRs) are targeted by Bexarotene, an anti-cancer drug for CTCL.
- RXRs form heterodimers with nuclear receptors (NRs) like PPARγ to control gene expression.
Purpose of the Study:
- To investigate Bexarotene's effects on the RXRα:PPARγ heterodimer's conformational flexibility.
- To characterize Bexarotene's direct interactions with nuclear receptors using HDX mass spectrometry.
Main Methods:
- Hydrogen/deuterium exchange (HDX) mass spectrometry was employed.
- Competitive binding assays confirmed direct receptor interactions.
- Cell-based reporter assays assessed transactivation modulation.
Main Results:
- Bexarotene directly binds to PPARγ, independent of RXRα.
- HDX MS revealed conformational changes upon Bexarotene binding.
- Bexarotene antagonized rosiglitazone-induced PPARγ transactivation.
Conclusions:
- Bexarotene exhibits complex polypharmacology, interacting directly with PPARγ.
- HDX mass spectrometry is a valuable tool for characterizing small molecule interactions with nuclear receptor complexes.
- Understanding these interactions is crucial for developing targeted therapies.
