Development of a Nonradioactive Steroid Receptor Ligand Binding Assay Using Fluorescence Polarization for Studying Hsp90‐Mediated Chaperone Protein Activity

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While glucocorticoid receptor (GR) ligand binding has historically been measured using radiolabeled agonists, such as [ 3 H]dexamethasone, new methods have sought to use fluorescently conjugated ligands, such as dexamethasone‐fluorescein (Dex‐Fl), as more sustainable alternatives. The molecular chaperone protein hsp90 has been shown to be essential for facilitating GR ligand binding, and structurally unrelated hsp90 inhibitors are currently in various phases of clinical investigation. The purpose of our study was to develop a fluorescence polarization (FP)‐based assay for measuring GR binding activity suitable for use in undergraduate research environments, and then apply this method to evaluating the extent to which specific agonists and antagonists of the GR and hsp90 chaperone machinery affect ligand binding. We constructed a versatile FP assay with comparable results to the previously used radiation‐based method. Interestingly, GR agonists dexamethasone and cortisol produced equivalent Dex‐Fl competition to the GR antagonist RU486, and did so in a concentration‐dependent manner. Data from hsp90 and hsp70 inhibitors showed mixed results, with hsp90 antagonists radicicol, NVP‐AUY922, and BIIB012 having limited effects, while the hsp70 antagonist novobiocin showed a potentiation of FP‐detectable steroid binding. Using the described FP method to evaluate steroid binding can be applied to broader studies examining clinically relevant GR and hsp90‐based drug therapies.

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