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【关键词】 [3H]Org 43553
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 luteinizing hormone (LH) receptor is a member of the glycoprotein hormone receptor family within class A of G protein-coupled receptors (Vassart et al., 2004). A unique feature of the LH receptor is that it recognizes two endogenous ligands with high molecular weight, namely human chorionic gonadotropin (hCG) and LH. Both hormones bind with high affinity and selectivity to the large N terminus of the receptor (Smits et al., 2003). Together with other gonadotropins, LH and hCG play a pivotal role in reproduction in which LH is responsible for ovulation induction in women and control testosterone production in men, whereas hCG maintains the early stages of pregnancy (Ascoli et al., 2002). Gonadotropins are currently used clinically in infertility treatment. Here, either urinary or recombinant gonadotropins are used, which need to be administered by parenteral (subcutaneous or intramuscular) injection (Loumaye et al., 1996). The advantage of low molecular weight (LMW) agonists is that they have the potential to become orally available drugs (van Straten et al., 2002). This will alleviate the necessity of parenteral administration, which may result in enhanced patient compliance and convenience compared with current methods.
In the past few years, medicinal chemists have therefore been challenged to find LMW ligands for receptors that have high molecular weight endogenous ligands (e.g., polypeptides and protein hormones). Although LMW ligands have already been described for the gonadotropin hormone receptors (van Straten et al., 2002, 2005), radioligands have not thus far. Small molecule radioligands have been reported for other receptors with high molecular weight endogenous ligands [for example, an antagonist for the corticotropin-releasing factor1 receptor (Zhang et al., 2003), an agonist for the insulin receptor (Zhang et al., 1999), and an agonist for the glucagon-like peptide1 receptor (Knudsen et al., 2007)].
作者单位：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 Che