Kinase experiment:

Transfected cells are cultured for 48 h before incubation for 1 h in serum-free Dulbecco's modified Eagle's medium containing 1 mM 3-isobutyl-1-methylxanthine and bovine thyroid-stimulating hormone (TSH) (0 to 1.8 μM) or human LH (0.1 to 1000 ng) or Org41841 (0 to100 μM) in a humidified 5% CO2 incubator. Following aspiration of the medium, cells are lysed using lysis buffer 1 of the cAMP Biotrak Enzymeimmunoassay System. The cAMP content of the cell lysate is determined and data are analyzed[1].

Org41841 is a partial agonist of both luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and thyroid-stimulating hormone receptor (TSHR) with EC50s of 0.2 and 7.7 μM, respectively.

Functional assays demonstrate that Org41841 is a partial agonist of both luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and thyroid-stimulating hormone receptor (TSHR) with EC50s of 0.2 and 7.7 μM, respectively. Treatment of L570F with Org41841 reveals an improved EC50 of 800 nM. M9 responds to Org41841 with an improved EC50 of 2700 nM and a greatly improved efficacy for signaling to 99% of the maximal value observed for thyroid-stimulating hormone (TSH) stimulation of thyroid-stimulating hormone receptor (TSHR)[1].

[1]. Holger Jaschke, et al. A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor(LHCGR). J Biol Chem. 2006 Apr 14;281(15):9841-4.