title: The "TSH Receptor Glo Assay" - A High-Throughput Detection System for Thyroid Stimulation.
abstract: BACKGROUND
  To identify novel small molecules against the TSH receptor, we developed a sensitive transcription-based luciferase high-throughput screening (HTS) system named the TSHR-Glo Assay (TSHR-Glo).
  
  
  METHODS
  This assay uses double-transfected Chinese hamster ovary cells stably expressing the human TSHR and a cAMP-response element (CRE) construct fused to an improved luciferase reporter gene.
  
  
  RESULTS
  The assay was highly responsive toward TSH in a dose-dependent manner with a TSH sensitivity of 10(-10)M (10 ± 1.12 μU/ml) and thyroid-stimulating antibodies, a hallmark of Graves' disease, could also be detected. The assay was validated against the standard indicator of HTS performance - the Z-factor (Z') - producing a score of 0.895. Using the TSHR-Glo assay, we screened 48,224 compounds from a diverse chemical library in duplicate plates at a fixed dose of 17 μM. Twenty molecules with the greatest activity out of 62 molecules that were identified by this technique were subsequently screened against the parent luciferase stable cell line in order to eliminate false positive stimulators.
  
  
  CONCLUSIONS
  Using this approach, we were able to identify specific agonists against the TSH receptor leading to the characterization of several TSH agonist molecules. Hence, the TSHR-Glo assay was a one-step cell-based HTS assay, which was successful in the discovery of novel small molecular agonists and for the detection of stimulating antibodies to the TSH receptor.
authors: Thyroid Research Unit, Department of Medicine, Icahn School of Medicine at Mount Sinai and the James J. Peters VA Medical Center , New York, NY , USA.
authors: Rauf
authors: R
authors: Latif
authors: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
authors: Zerlina
authors: Z
authors: Lau
authors: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
authors: Pamela
authors: P
authors: Cheung
authors: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
authors: Dan P
authors: DP
authors: Felsenfeld
authors: Thyroid Research Unit, Department of Medicine, Icahn School of Medicine at Mount Sinai and the James J. Peters VA Medical Center , New York, NY , USA.
authors: Terry F
authors: TF
authors: Davies
keywords: TSH receptor; high-throughput; screening; small molecule; thyroid-stimulating hormone