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Screening for ligands of human retinoid X receptor-alpha using ultrafiltration mass spectrometry
Dongting Liu1, Jian Guo, Yan Luo
1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, 833 South Wood Street, Chicago, Illinois 60612, USA.
Abstract:
Retinoid X receptors (RXRs) function as ligand-activated transcription factors and are obligatory components of a large number of nuclear receptor heterodimers. RXRs help regulate diverse physiological responses including the cancer prevention responses of cell proliferation, inflammation, cell differentiation, and apoptosis. Since RXRs represent important targets for cancer chemoprevention, an ultrafiltration mass spectrometry-based assay was developed to facilitate the discovery of potential chemoprevention agents that bind to human RXRalpha. Natural and synthetic ligands for RXRalpha including 9-cis-retinoic acid, docosahexaenoic acid, and LG100268 could be detected and identified in DMSO (dimethyl sulfoxide) or even complex matrixes such as extracts of marine bacteria. Specific binding of ligands to RXRalpha was demonstrated through competitive binding using ultrafiltration LC-MS/MS (liquid chromatography-tandem mass spectrometry), and ligands could be ranked in order of affinity for RXRalpha. Therefore, ultrafiltration LC-MS/MS is suitable for the screening of complex mixtures such as natural product extracts for the discovery of new ligands to RXRalpha.
Insights
A new ultrafiltration mass spectrometry assay identifies Retinoid X Receptor (RXR) ligands. This method aids in discovering cancer chemoprevention agents from complex natural product extracts.
Area of Science:
- Molecular Biology
- Pharmacology
- Analytical Chemistry
Background:
- Retinoid X Receptors (RXRs) are crucial transcription factors regulating cell proliferation, inflammation, differentiation, and apoptosis.
- RXRs are key targets for cancer chemoprevention strategies.
- Identifying novel RXR ligands is essential for developing new therapeutic agents.
Purpose of the Study:
- To develop and validate an ultrafiltration mass spectrometry-based assay for discovering ligands that bind to human RXRalpha.
- To demonstrate the assay's capability in detecting and identifying RXRalpha ligands in various sample types.
Main Methods:
- Development of an ultrafiltration mass spectrometry (UF-MS) assay.
- Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection and identification.
- Employing competitive binding assays to rank ligand affinity for RXRalpha.
Main Results:
- The UF-MS assay successfully detected and identified known RXRalpha ligands, including 9-cis-retinoic acid and docosahexaenoic acid.
- Ligands were identified in both simple (DMSO) and complex matrices (marine bacteria extracts).
- Competitive binding demonstrated specific ligand interaction and allowed for affinity ranking.
Conclusions:
- Ultrafiltration LC-MS/MS is a suitable method for screening complex mixtures, such as natural product extracts, for novel RXRalpha ligands.
- This assay facilitates the discovery of potential cancer chemoprevention agents targeting RXRs.
- The developed method provides a robust platform for identifying and characterizing RXR-ligand interactions.
