AUTHOR=Latif Rauf , Lau Zerlina , Cheung Pamela , Felsenfeld Dan P. , Davies Terry F. TITLE=The “TSH Receptor Glo Assay” – A High-Throughput Detection System for Thyroid Stimulation JOURNAL=Frontiers in Endocrinology VOLUME=7 YEAR=2016 URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2016.00003 DOI=10.3389/fendo.2016.00003 ISSN=1664-2392 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 10−10M (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.

Conclusion

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.