Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

Abstract

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.

New Concepts, Experimental Approaches, and Dereplication Strategies for the Discovery of Novel Phytoestrogens from Natural Sources

“…Both determination of biological activity and chemical characterization were performed in parallel during a single chromatographic run. A more complex screening approach combining RP-HPLC online to an ERα affinity detection system using fluorescence polarization has been recently described by Reinen et al [138]. This method is a sensitive screening platform for measuring the ERα binding affinities of individual components in mixtures.…”

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

“…Previously identified with NMR by Ambrus et al[48]. 15␤) 1 15. 1.20 (m, 1 H, 14␣) 1.20-1.27 (m, 1 H, 11␤) 1.34-1.42 (m, 1 H, 8␤) 1.43-1.49 (m, 1 H, 1␣) 1.50-1.55 (m, 1 H, 12␤) 1.59-1.67 (m, 1 H, 12␣) 1.74-1.79 (m, 1 H, 7␤) 1.79-1.84 (m, 1 H, 11␣) 2.12-2.18 (m, 1 H, 15␣) 2.15-2.18 (m, 1 H, 10␤) 2.17-2.21 (m, 1 H, 1␤) 2.20-2.24 (m, 2 H, 2␣, 2␤) 2.23-2.32 (m, 1 H, 6␤) 2.36-2.45 (m, 1 H, 6␣) 3.94-4.00 (m, 1 H, 16␣) 4.64 (s, 1 H, OH) 5.26-5.27 (d, 1 H, 16␤OH) 5.72 (s, 1 H, 4).…”

“…These receptor affinity detection (RAD) systems integrate liquid chromatography (LC) with affinity assessment and therefore allow the direct correlation between individual ligands and their affinity towards target proteins. Biochemical detection systems reported previously are based on fluorescence readout [12][13][14], fluorescent polarization [15] or mass spectrometry based reaction detection [16][17][18][19][20]. LC coupled to mass spectrometry (MS) is by far the most used tool in drug metabolism studies.…”

Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment

“…A modified protocol described by Reinen et al [ 34 ] was used for microsomal incubations. Pig liver microsomes (∼20 mg/mL protein) were diluted 1:10 in incubation buffer (50 mM KH 2 PO 4 buffer pH 7.4, 5 mM MgCl 2 , 5 mM glucose-6-phosphate, and 5 activity units/mL glucose-6-phosphate dehydrogenase) at 4 °C.…”