INTRODUCTION: A phosphorimager-based filter binding thyroid hormone receptor (THR) competition assay has been developed for use in verifying hits from compound library screens. METHODS: This method employs in vitro translated ligand binding domains (LBDs) of THRalpha and THRbeta, separation through nitrocellulose via a 96-well vacuum manifold, and analysis of receptor-bound radioactivity by phosphorimaging. RESULTS: A standard curve of [I(125)]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 IC(50) and K(i) values when using unlabeled T3 as competitor. In addition, this method produced IC(50) and K(i) 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. DISCUSSION: These 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.
INTRODUCTION: A phosphorimager-based filter binding thyroid hormone receptor (THR) competition assay has been developed for use in verifying hits from compound library screens. METHODS: This method employs in vitro translated ligand binding domains (LBDs) of THRalpha and THRbeta, separation through nitrocellulose via a 96-well vacuum manifold, and analysis of receptor-bound radioactivity by phosphorimaging. RESULTS: A standard curve of [I(125)]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 IC(50) and K(i) values when using unlabeled T3 as competitor. In addition, this method produced IC(50) and K(i) 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. DISCUSSION: These 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.
Authors: Charles F Verge; Daniel Konrad; Michal Cohen; Caterina Di Cosmo; Alexandra M Dumitrescu; Teresa Marcinkowski; Shihab Hameed; Jill Hamilton; Roy E Weiss; Samuel Refetoff Journal: J Clin Endocrinol Metab Date: 2012-09-19 Impact factor: 5.958