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[³H]Org 43553, the First Low-Molecular-Weight Agonistic and Allosteric Radioligand for the Human Luteinizing Hormone Receptor

Division of Medicinal Chemistry (L.H.H., A.P.IJ.), Leiden/Amsterdam Center for Drug Research and Department of Bio-Organic Synthesis (K.M.B.), Leiden Institute of Chemistry, University of Leiden, Leiden, The Netherlands; and Molecular Pharmacology (J.O.), Medicinal Chemistry (C.M.T.) and Process Chemistry (P.H.G.W.), Organon BioSciences, Oss, The Netherlands

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 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., 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.

Address correspondence to: A. P. IJzerman, Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: ijzerman{at}lacdr.leidenuniv.nl