A phosphorimager-based filter binding thyroid hormone receptor competition assay for chemical screening

IntroductionA phosphorimager-based filter binding thyroid hormone receptor (THR) competition assay has been developed for use in verifying hits from compound library screens.MethodsThis method employs in vitro translated ligand binding domains (LBDs) of THRα and THRβ, separation through nitrocellulose via a 96-well vacuum manifold, and analysis of receptor-bound radioactivity by phosphorimaging.ResultsA standard curve of [I125]T3 showed a linear response over the dynamic range of a competition assay, and a comparison of Sephadex G-25 column separation and gamma counting with en masse filtration and phosphorimaging revealed similar IC50 and Ki values when using unlabeled T3 as competitor. In addition, this method produced IC50 and Ki values for the known T3 competitors [3,5-Dimethyl-4-(4′-hydoxy-3′-isopropylbenzyl) phenoxy] acetic acid (GC-1) and 3,5-diiodothyropropionic acid (DITPA) similar to those reported elsewhere.DiscussionThese data suggest that filtration and phosphorimaging adequately and properly reproduces binding values associated with THR competition. Further, this method gave a 3-fold reduction in time and a 40-fold reduction in radioactive waste over the column-based method. These reductions allow for a substantial increase in assay throughput. Taken together, these data suggest that en masse filtration and phosphorimaging is an efficient and tractable method for verifying large groups of putative T3 competitors in vitro.