Screening for ligands of human retinoid X receptor-alpha using ultrafiltration mass spectrometry

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.

Figure 1

Structures of the RXRα ligands and other test compounds used during this investigation.

Figure 2

Experimental design of ultrafiltration LC-MS-MS screening of solutions for ligands to human RXRα.

Figure 3

Ultrafiltration LC-MS-MS screening of LG100268, 9-cis-RA, DHA, 13-cis-RA, and all trans-RA for binding to RXRα. The concentration of RXRα and each compound was 1 μM, and the incubations were carried out for 2 h. TTNPB was added to the samples as an internal standard immediately before analysis using LC-MS-MS with negative ion electrospray. The control incubations (dashed lines) containing denatured RXRα were used to test for non-specific binding and adsorption of sample to the ultrafiltration apparatus. Enhancement of HPLC peak areas in the experimental incubations (solid lines) indicates specific binding of ligands to RXRα. Note that the known ligands, LG100268, 9-cis-RA and DHA, but not 13 -cis-RA and all trans-RA, which are not known to bind to RXRα, was shown to bind.

Figure 4

Comparison of specific and non-specific binding of LG100268 and 9-cis-RA to recombinant human RXRα during ultrafiltration LC-MS-MS screening using the one-filter and two-filter procedures. In the MRM chromatograms, the solid lines represent incubations with active RXRα, and the dashed lines represent the use of denatured RXRα as a control for non-specific binding. The concentrations of ligand and protein were each 1 μM, and each incubation was carried out for 2 h. A) When the RXRα-ligand complexes were washed with ammonium acetate to remove unbound compounds and then dissociated using methanol/water (90:10; v/v) using the same ultrafiltration filter, non-specific binding to the membrane produced a strong signal for LG100268 and 9-cis-RA. B) Transferring the RXRα-ligand complexes after the washing step to a clean ultrafiltration filter for dissociation resulted in much less non-specific binding.