RT Journal Article SR Electronic T1 [3H]Org 43553, the First Low-Molecular-Weight Agonistic and Allosteric Radioligand for the Human Luteinizing Hormone Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 518 OP 524 DO 10.1124/mol.107.039875 VO 73 IS 2 A1 Laura H. Heitman A1 Julia Oosterom A1 Kimberly M. Bonger A1 Cornelis M. Timmers A1 Peter H. G. Wiegerinck A1 Adriaan P. IJzerman YR 2008 UL http://molpharm.aspetjournals.org/content/73/2/518.abstract AB 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, [3H]5-amino-2-methylsulfanyl-4-[3-(2-morpholin-4-yl-acetylamino)-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 [3H]Org 43553 to CHO-K1 cell membranes expressing the human LH receptor and a cAMP response element-luciferase reporter gene was saturable with a KD value of 2.4 ± 0.4 nM and a Bmax 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 Ki 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., 125I-hCG) is not suitable as it is not displaced by a LMW compound. Therefore, [3H]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. The American Society for Pharmacology and Experimental Therapeutics