In vitro activity: ML224, also known as NCGC00242364 and ANTAG3, is a selective and inverse agonist of Thyroid Stimulating Hormone Receptor(TSHR). It inhibits TSH-stimulated cAMP production with an IC50 of 2.3 μM. ANTAG3 was selective for TSHR inhibition and the half-maximal inhibitory doses were 2.1 μM for TSHR and greater than 30 μM for LH and FSH receptors. In mice treated with TRH, ANTAG3 lowered serum free T4 by 44% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 75% and 83%, respectively. In mice given M22, ANTAG3 lowered serum free T4 by 38% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 73% and 40%, respectively. In conclusion, we developed a selective TSHR antagonist that is effective in vivo in mice. This is the first report of a small-molecule TSHR antagonist active in vivo and may lead to a drug to treat Graves' disease.

Kinase Assay: ML224, also known as NCGC00242364 and ANTAG3, is a selective and inverse agonist of Thyroid Stimulating Hormone Receptor(TSH)R). It inhibits TSH-stimulated cAMP production with an IC50 of 2.3 μM. ANTAG3 was selective for TSHR inhibition and the half-maximal inhibitory doses were 2.1 μM for TSHR and greater than 30 μM for LH and FSH receptors.

Cell Assay: