[3H]Org 43553, the first low-molecular-weight agonistic and allosteric Radioligand for the human luteinizing hormone receptor

The luteinizing hormone (LH) receptor plays a pivotal role in reproduction. The high-molecular-weight (HMW) human chorionic gonadotropin (hCG) and LH are the endogenous ligands of this receptor and bind to its large N terminus. The present study characterizes the binding of a new low-molecular-weight (LMW) radioligand, [H-3]5-amino-2-methylsulfanyl-4-[3-(2-morpholin-4-ylacetylamino)phenyl]- thieno[2,3-d] pyrimidine-6-carboxylic acid tert-butylamide (Org 43553), at the LH receptor. Equilibrium saturation and displacement assays were developed and optimized. Specific binding of [H-3]Org 43553 to CHO-K1 cell membranes expressing the human LH receptor and a cAMP response element-luciferase reporter gene was saturable with a K-D value of 2.4 +/- 0.4 nM and a B-max value of 1.6 +/- 0.2 pmol/mg protein. Affinities of five LMW analogs of Org 43553 were determined. All displaced the radioligand competitively, with K-i values ranging from 3.3 to 100 nM. Finally, the potency of these compounds in a cAMP-induced luciferase assay was also determined. There was a high correlation between affinity and potency (r = 0.99; P < 0.0001) of these compounds. In the search for LMW ligands, which bind allosterically to the seven-transmembrane domain of the LH receptor, a HMW radioligand (e.g., I-125-hCG) is not suitable as it is not displaced by a LMW compound. Therefore, [H-3] Org 43553, a new radioligand with good binding properties, allows screening for new LMW ligands that mimic the action of the endogenous hormone at the LH receptor.