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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
Received for publication July 10, 2007.
Accepted for publication November 7, 2007.
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Abstract |
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). 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)].
This article describes for the first time the pharmacological characterization of the human luteinizing hormone receptor transfected in Chinese hamster ovary (CHO) cells using a tritium-labeled form of a small molecule ligand, [3H]Org 43553 (see Table 2 for its chemical structure). Org 43553 is one of a series of thieno[2,3-d]pyrimidine derivatives that showed agonistic LH activity in a functional assay (Hanssen and Timmers, 2003). Org 43553 was chosen to be labeled with tritium, because it was one of the more potent compounds from this screen. The kinetic and equilibrium binding characteristics of the new radioligand were determined and compared with those of the radiolabeled natural hormone hCG. [3H]Org 43553 was also used in a displacement assay with four other thienopyrimidines and one quinazoline derivative. Their affinities correspond well to their potency in generating a cAMP response. Thus, this LMW radioligand provides a useful tool to further understand the interactions of the LH receptor with small molecule ligands.
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Materials and Methods |
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; Timmers and Karstens, 2002; Hanssen and Timmers, 2003; Hanssen et al., 2003). Bovine serum albumin (BSA; fraction V) was purchased from Sigma (St. Louis, MO), whereas BCA protein assay reagent was from Pierce Chemical Company (Rockford, IL). 125I-hCG (5966 Ci/mmol) was purchased from PerkinElmer Life Sciences Inc. (Waltham, MA). Chinese hamster ovary (CHO-K1) cellsstably expressing the human LH receptor and cAMP-response-element luciferase reporter gene (CRE-luc) were kindly provided by Organon BioSciences. All other chemicals and cell culture materials were obtained from standard commercial sources. Cell Culture. CHO cells with stable expression of the human LH receptor and CRE-luc (CHOhLHr_luc) were grown in culture medium consisting of Dulbecco's modified Eagle's medium and Ham's F12 medium (1:1) supplemented with 5% normal adult bovine serum, streptomycin (100 µg/ml), penicillin (100 IU/ml) at 37°C in 5% CO2. The cells were subcultured twice weekly at a ratio of 1:15. For membrane preparation, the cells were subcultured 1:10 and transferred to large 15-cm diameter plates.
Membrane Preparation. Cells were detached from the plates by scraping them into 5-ml phosphate-buffered saline (PBS), collected, and centrifuged at 700g (3000 rpm) for 5 min. Pellets derived from 30 plates were pooled and resuspended in 20 ml of ice-cold 50 mM Tris-HCl buffer containing 2 mM MgCl2, pH 7.4. An Ultra-Turrax (Heidolph Instruments, Schwabach, Germany) was used to homogenize the cell suspension. Membranes and the cytosolic fraction were separated by centrifugation at 100,000g (31,000 rpm) in an Optima LE-80K ultracentrifuge (Beckman Coulter, Fullerton, CA) at 4°C for 20 min. The pellet was resuspended in 10 ml of the Tris buffer, and the homogenization and centrifugation step was repeated. Tris buffer (12 ml) was used to resuspend the pellet and the membranes were stored in 500-µl aliquots at -80°C. Membrane protein concentrations were measured using the BCA (bicinchoninic acid) method with BSA as a standard (Smith et al., 1985).
Preparation of [3H]Org 43553. The tritiation of Org 43553 was carried out by RC Tritec AG (Teufen, Switzerland). In short, 10 mg of Org 43553 was dissolved in 250 µl of THF containing 0.1% (v/v) water under nitrogen atmosphere. This solution was stirred for approximately 20 min, while cooling the flask to -78°C. Then 120 µl of 1.3 M sec-butyl-lithium in hexane/cyclohexane (final concentration, 10 eq) was added drop-wise. The color of the solution changed from yellow to dark red/brown, indicative for the molecule to be deprotonated. The solution was stirred for another2hat -78°C. Next, the reaction was quenched with 20 Ci (i.e., an excess) of tritiated water at -78°C. The color of the solution changed back to yellow, showing that the deprotonated molecule was tritiated. The reaction mixture was stirred for another 1.5 h at -78°C, after which the reaction mixture was allowed to warm up to room temperature in approximately 30 min. Next, a large excess of diethyl ether (50 ml) was added. The organic layer was washed twice with water and once with brine, dried, and concentrated under reduced pressure. The residue was dissolved in ethanol to give a solution of 250 mCi of crude [3H]Org 43553 in 25 ml of ethanol. This solution was then purified by high-performance liquid chromatography on a Symmetry C18 column eluting with acetonitrile/water [40:60 (v/v) containing 0.1% trifluoroacetic acid] at Organon BioSciences. After purification, 140 mCi of [3H]Org 43553 with a radiochemical purity 
95%, and a specific activity of 16.6 Ci/mmol was obtained.
Radioligand Displacement and Saturation Assays. [3H]Org 43553 membrane aliquots containing 20 µg of protein were incubated in a total volume of 100 µl of assay buffer (25 mM Tris-HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.1% BSA) at 30°C for 1 h. For saturation experiments, total binding was determined at increasing concentrations (0.2-20 nM) of [3H]Org 43553, whereas nonspecific binding was determined at three concentrations of radioligand in the presence of 10 µM Org 43553 and analyzed by linear regression. Displacement experiments were performed using 10 concentrations of competing ligand in the presence of 20 nM [3H]Org 43553. Nonspecific binding was determined in the presence of 10 µM Org 43553 and represented approximately 50% of the total binding. [3H]Org 43553 did not bind specifically to membranes prepared from CHO cells lacking the LH receptor. Total binding was determined in the presence of buffer and was set at 100% in all experiments, whereas nonspecific binding was set at 0%. Incubations were terminated by dilution with 1 ml of ice-cold Tris-HCl buffer. Bound from free radioligand was immediately separated by rapid filtration through Whatman (Clifton, NJ) GF/B filters using a Millipore (Billerica, MA) manifold. Filters were subsequently washed three times with ice-cold wash buffer (25 mM Tris HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.05% BSA). Filter-bound radioactivity was determined by scintillation spectrometry (Tri-Carb 2900 TR; PerkinElmer Life and Analytical Sciences) after addition of 3.5 ml of PerkinElmer Emulsifier Safe.
125I-hCG membrane aliquots containing 15 µg of protein were incubated in a total volume of 100 µl of assay buffer (25 mM Tris-HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.1% BSA) at 30°C for 2.5 h. For 125I-hCG, displacement experiments were performed using 10 concentrations of recLH in the presence of 80,000 cpm (
0.1 nM) radioligand. Nonspecific binding was determined in the presence of 50 U/ml (70 nM) recLH and represented approximately 50% of the total binding. 125I-hCG did not bind specifically to membranes prepared from CHO cells lacking the LH receptor. Total binding was determined in the presence of buffer and was set at 100% in all experiments, whereas nonspecific binding was set at 0%. Incubations were terminated by dilution with 1 ml of ice-cold Tris-HCl buffer. Bound from free radioligand was immediately separated by rapid filtration through Whatman GF/C filters presoaked with 0.25% polyethylenimine (PEI) for 1 h using a Millipore manifold. Filters were subsequently washed three times with ice-cold wash buffer (25 mM Tris HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.05% BSA). Filter-bound radioactivity was determined in a 
-counter (Wizard 1470; PerkinElmer Life and Analytical Sciences).
Radioligand Association and Dissociation Assays. Association experiments were performed by incubating membrane aliquots containing 20 µg of protein in a total volume of 100 µl of assay buffer (25 mM Tris HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.1% BSA) at 30°C for 3 h with 80,000 cpm of 125I-hCG or for 90 min with 20 nM [3H]Org 43553. The amount of radioligand bound to the receptor was measured at various time intervals during incubation. Dissociation experiments were performed by preincubating membrane aliquots containing 20 µg of protein in a total volume of 100 µl of assay buffer (25 mM Tris HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.1% BSA) at 30°C for 2.5 h with 80,000 cpm 125I-hCG or for 1 h with 20 nM [3H]Org 43553, respectively. After preincubation, dissociation was initiated by addition of 50 U/ml recLH for 125I-hCG. For [3H]Org 43553, dissociation was initiated by addition of 10 µM Org 43553 (control), 10 ml of assay buffer (100-fold dilution), or 50 U/ml recLH or combinations thereof, as explained under Results. The amount of radioligand still bound to the receptor was measured at various time intervals for a total of 240 min (125I-hCG) or 180 min ([3H]Org 43553). Incubations were terminated, and samples were obtained and analyzed as described above.
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Data Analysis. All binding data were analyzed using the nonlinear regression curve-fitting program Prism ver. 4.02 (GraphPad Software Inc., San Diego, CA). EC50 values were directly obtained from the dose-response curves and inhibitory binding constants (Ki values) were derived from the IC50 values according to Ki = IC50/(1 + [C]/Kd) where [C] is the concentration of the radioligand and Kd its dissociation constant (Cheng and Prusoff, 1973). The Kd value of [3H]Org 43553 at CHOhLHr_luc membranes was obtained by computer analysis of saturation curves. Dissociation constants, koff, were obtained by computer analysis of the exponential decay of either 125I-hCG or [3H]Org 43553 bound to the receptor. Association rates were calculated according to the equation kon = (kobs - koff)/[L], where kobs was obtained by computer analysis of the exponential association of either 125I-hCG or [3H]Org 43553 bound to the receptor, and [L] is the amount of radioligand used for the association experiments. All values obtained are means of at least three independent experiments performed in duplicate.
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Results |
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). We started our optimization efforts with [3H]Org 43553 and 15 µg of protein in a simple buffer of low ionic strength (25 mM Tris HCl, pH 7.4) to which 2 mM MgCl2 was added, as is often done with agonist radioligands. Figure 1 summarizes the results of these experiments, in which we studied further buffer components, filters and filter pretreatment, and membrane concentration. First, a relatively high concentration (20 nM) of [3H]Org 43553 was needed for an appreciable window of specific binding, which was greatly improved by the addition of BSA or CHAPS. It was decided to continue with 0.1% BSA in the assay buffer. Second, the initial choice to use uncoated GF/B glass fiber filters to separate free from membrane-bound radioligand resulted in the highest specific binding; coating with PEI was not favorable. Next, the amount of protein used was increased to 20 µg, as this yielded a desired window of more than 1500 dpm. Initial kinetic association experiments taught us that the optimal incubation time was 60 min at 30°C. Radioligand Saturation Experiments. Saturation binding assays were performed with [3H]Org 43553. The results of a representative saturation experiment are shown in Fig. 2. Binding of [3H]Org 43553 to membranes of CHO cells expressing the human LH receptor was saturable and best described by a one-site model. The KD value and Bmax value obtained from the saturation experiments were 2.4 ± 0.4 nM and 1.6 ± 0.2 pmol/mg of protein, respectively. The KD value for [3H]Org 43553 obtained with these experiments was used to derive Ki rather than IC50 values for analogs of Org43553 (see Radioligand Displacement Assays, below). In the presence of 70 nM recLH, the KD value was unaffected, whereas the Bmax value was decreased by 24 ± 2% (Fig. 2), indicative of a noncompetitive interaction.
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Radioligand Displacement Assays. After this characterization of the radioligand, the affinities of nonlabeled compounds 1 to 6 for the human luteinizing hormone receptor were determined (Table 2 and Fig. 5). Displacement experiments were carried out with [3H]Org 43553 on CHOhLHr-_luc membranes, because the small molecule compounds were not able to displace 125I-hCG from the human luteinizing hormone receptor (Table 2). Compound 1 (Org 41841) was potent, displacing [3H]Org 43553 with a Ki value of 17 ± 5 nM. Replacement of the S-Me group of 1 with a phenyl group had a negative effect on receptor affinity (2, Org 41247). Enlargement of the meta-oriented substituent on the 4-phenylgroup had a positive impact, resulting in compounds 3 to 5 (Org 42619, Org 43311, and Org 43553, respectively) with a 4- to 10-fold higher affinity. Replacement of the thienyl moiety in the scaffold of 1 (Org 41841) by a phenyl ring (6, Org 43983) resulted in an approximately 5-fold lower affinity. This value was comparable with the affinity of compound 2 (Org 41247). Org 43553 was chosen to be labeled with tritium, because it had a proven nanomolar potency in the functional assay, and it was assumed also to exhibit a nanomolar binding affinity. In comparison, the endogenous ligand recLH was able to displace 125I-hCG with a 3-fold higher affinity of 0.61 ± 0.1 nM, whereas it only modestly displaced [3H]Org 43553 at a high concentration (Table 2). Labeling Org 43553 also had a practical reason, because the relatively acidic proton next to the morpholino-group was easily exchanged with tritium without a need for the synthesis of precursor molecules.
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Agonistic Activity and Selectivity at the LH Receptor. In addition to radioligand displacement experiments, cAMP-induced luciferase assays were performed with compounds 1 to 6. It follows from Fig. 6 that all compounds were able to almost fully activate the receptor in comparison to recLH (87-95%). Their potencies were determined from the relatively steep dose-response curves and ranged from 1.3 nM (4) to 119 ± 18 nM (2) (Table 2). In comparison, the endogenous ligand, recLH, had an EC50 value of 78 ± 2 pM in this cellular assay. There was a high correlation between data obtained from binding and functional assays (r = 0.99; P < 0.0001) (Fig. 7). To obtain a full selectivity profile, Org 43553 was examined on 59 different drug targets, of which 39 were GPCRs. At the other glycoprotein hormone GPCRs, Org 43553 was at least 10-fold selective for the LH receptor (data not shown). LH receptor selectivity was more than 3000-fold for all other targets.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), kinase inhibitor (Dai et al., 2005), and antimicrobial agent (Chambhare et al., 2003)]. The substitution pattern can be diverse, however, resulting in selectivity toward different targets. Org 43553 is selective for the LH receptor by at least 10-fold compared with other glycoprotein hormone receptors and more than 3000-fold selective for a whole panel of drug targets, including serotonin receptors.
The results of this study show that [3H]Org 43553 is a highly potent and selective agonistic radioligand that represents a novel tool for the screening of low molecular weight ligands for the LH receptor. The radioligand's receptor binding was saturable with a high affinity (KD = 2.4 ± 0.4 nM). Kinetic experiments showed that both association and dissociation were much faster for [3H]Org 43553 than for 125I-hCG (Table 1). In addition, 125I-hCG did not fully dissociate from the receptor. This has been described before for 125I-hCG, where dissociation of specific binding was only 40% after 8 h at 37°C (Henderson et al., 1984). This pseudo-irreversibility of dissociation most likely precluded the determination of the radioligand's KD and Bmax values from equilibrium saturation studies in the present experimental set-up. Others have reported a KD value of 0.1 nM for 125I-hCG when binding to intact COS-7 cells transiently expressing the rat LH receptor (Bhowmick et al., 1996; Angelova et al., 2003). This value is in good agreement with the KD value of 0.064 nM obtained from the kinetic experiments presented here. The dissociation of another glycoprotein hormone, thyroid-stimulating hormone, however, was shown to be complete and much faster, already at 22°C (Powell-Jones et al., 1981). In the kinetic comparison in the present study the binding of [3H]Org 43553, unlike that of 125I-hCG, was fully reversible. This feature rendered [3H]Org 43553 a more suitable radioligand for further displacement studies.
To further explore the binding characteristics of this novel radioligand, assays were performed where the dissociation was initiated by the "infinite dilution" method (Christopoulos et al., 1997). A 100-fold ("infinite") dilution gave a comparable dissociation rate as the rate obtained by the addition of excess unlabeled Org 43553 (Fig. 3), which suggests that maximal radioligand dissociation was achieved and that Org 43553 binds to a single noninteracting site. Infinite dilution in the presence of excess unlabeled Org 43553 or recLH did not alter the dissociation rate of the radioligand, further proof of Org 43553's binding to a single, noninteracting site. To get more insight into the fact that recLH showed some displacement of Org 43553, the effect of recLH on the dissociation of [3H]Org 43553 was monitored. As shown in Fig. 3, recLH induces some dissociation of the radioligand, which correlates with the effect seen on the saturation (Fig. 2) and displacement equilibrium binding of [3H]Org 43553 (Table 2).
Characterization of the new radioligand was continued by radioligand displacement assays with nonlabeled Org 43553 and a number of derivatives. Recently, a high throughput screen identified compounds with a thieno[2,3-d]pyrimidine core as potent and selective receptor agonists for the LH receptor (van Straten et al., 2002). It was shown that the amide group at position 6 of the heterocyclic core was crucial for low nanomolar activity. From this series, Org 41841 (1) was the most potent. In addition, a more bulky meta-substituent on the phenyl group at position 4 resulted in more potent compounds (Hanssen and Timmers, 2003; Hanssen et al., 2003). The brief structure-activity relationship study presented here shows that the affinity of Org 41841 can be improved 5-fold through meta substitution [e.g., resulting in Org 43553 (Table 2)]. When compound 1 was compared with compound 3, it became clear that the introduction of an additional H-bond donor by the amide-group in combination with steric bulk from the benzoyl group was favorable for receptor affinity. Introduction of the ethanolamine- (4) and morpholino group (5) was allowed, and helped to increase water solubility. The compounds were not able to displace 125I-hCG in a binding assay (Table 2), which is in accordance with similar experiments on the FSH receptor (van Straten et al., 2005). It has been shown in docking and mutational studies that Org 41841 has its putative binding site in the seven-transmembrane part of the receptor (Jäschke et al., 2006; Moore et al., 2006), unlike the presumed binding of hCG and LH to the N terminus of the receptor. This is in line with many other class A GPCRs [e.g., the adenosine A2A (Kim et al., 1995), D3 dopamine (Alberts et al., 1998), and GnRH receptors (Söderhäll et al., 2005), which bind their cognate ligands in the seven-transmembrane domain as retinal in the rhodopsin receptor (Palczewski et al., 2000)]. Therefore, we assume that all small-molecule ligands reported here also bind in this domain of the LH receptor, explaining why the endogenous ligand, 125I-hCG, is not displaced. Conversely, recLH has some effect on [3H]Org 43553 displacement. It is feasible that the recLH-occupied rather than "empty" receptor is seen differently by LMW compounds such as Org43553.
In the cAMP-mediated luciferase assay, the compounds studied exhibited a rank order of potency similar to that observed in binding experiments (Fig. 5). Although the endogenous ligand, recLH, was more potent, the small molecule ligands are potent in binding (Ki values ranging from 3.3 to 100 nM) and in functional assays (EC50 values ranging from 1.3 to 119 nM).
In conclusion, equilibrium saturation and displacement and kinetic association and dissociation assays have been performed to elucidate the binding characteristics of the first small molecule radioligand, [3H]Org 43553, at the human LH receptor. A high correlation exists between affinity and activity of low molecular weight ligands. Moreover, the development of this binding assay will aid in the identification and elucidation of the SAR of newly synthesized small molecule LH receptor ligands.
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Acknowledgements |
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Footnotes |
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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
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Angelova K, Narayan P, and Puett D (2003) The luteinizing hormone receptor: influence of buffer composition on ligand binding and signaling of wild type and mutant receptors. Mol Cell Endocrinol 204: 1-9.[CrossRef][Medline]
Ascoli M, Fanelli F, and Segaloff DL (2002) The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr Rev 23: 141-174.
Bhowmick N, Huang J, Puett D, Isaacs NW, and Lapthorn AJ (1996) Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. Mol Endocrinol 10: 1147-1159.
Chambhare RV, Khadse BG, Bobde AS, and Bahekar RH (2003) Synthesis and preliminary evaluation of some N-[5-(2-furanyl)-2-methyl-4-oxo-4H-thieno[2,3-d]pyrimidin-3-yl]-carboxamide and 3-substituted-5-(2-furanyl)-2-methyl-3H-thieno[2,3-d]pyrimidin-4-ones as antimicrobial agents. Eur J Med Chem 38: 89-100.[CrossRef][Medline]
Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099-3108.[CrossRef][Medline]
Christopoulos A, Lanzafame A, Ziegler A, and Mitchelson F (1997) Kinetic studies of co-operativity at atrial muscarinic M2 receptors with an "infinite dilution" procedure. Biochem Pharmacol 53: 795-800.[CrossRef][Medline]
Dai Y, Guo Y, Frey RR, Ji Z, Curtin ML, Ahmed AA, Albert DH, Arnold L, Arries SS, Barlozzari T, et al. (2005) Thienopyrimidine ureas as novel and potent multitargeted receptor tyrosine kinase inhibitors. J Med Chem 48: 6066-6083.[CrossRef][Medline]
Gerritsma GG, Van Straten NCR and Adang AEP (2000), inventors. Preparation of bicyclic heteroaromatic compounds as LH agonists. World Patent no. WO0061586. 2000 Oct 19.
Hanssen RGJM and Timmers CM (2003), inventors; AKZO Nobel NV, assignee. Thieno[2,3-d]pyrimidines with combined LH and FSH agonistic activity. World Patent WO03020726. 2003 Mar 13.
Hanssen RGJM, Timmers CM and Kelder J (2003), inventors; AKZO Nobel NV, assignee. Glycine-substituted thieno[2,3-d]pyrimidines with combined LH and FSH agonistic activity. World Patent WO03020727. 2003 Mar 13.
Heitman LH, Mulder-Krieger T, Spanjersberg RF, von Frijtag Drabbe Kunzel JK, Dalpiaz A, and AP IJ (2006) Allosteric modulation, thermodynamics and binding to wild-type and mutant (T277A) adenosine A1 receptors of LUF5831, a novel nonadenosine-like agonist. Br J Pharmacol 147: 533-541.[CrossRef][Medline]
Henderson KM, Kieboom LE, McNatty KP, Lun S, and Heath DA (1984) [125I]hCG binding to bovine thecal tissue from healthy and atretic antral follicles. Mol Cell Endocrinol 34: 91-98.[CrossRef][Medline]
Jäschke H, Neumann S, Moore S, Thomas CJ, Colson A-O, Costanzi S, Kleinau G, Jiang J-K, Paschke R, Raaka BM, et al. (2006) A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH-CGR). J Biol Chem 281: 9841-9844.
Kim J, Wess J, van Rhee AM, Schoneberg T, and Jacobson KA (1995) Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2a adenosine receptor. J Biol Chem 270: 13987-13997.
Knudsen LB, Kiel D, Teng M, Behrens C, Bhumralkar D, Kodra JT, Holst JJ, Jeppesen CB, Johnson MD, de Jong JC, et al. (2007) Small-molecule agonists for the glucagon-like peptide 1 receptor. Proc Natl Acad Sci U S A 104: 937-942.
Loumaye E, Martineau I, Piazzi A, O'Dea L, Ince S, Howles C, Decosterd G, Van Loon K, and Galazka A (1996) Clinical assessment of human gonadotrophins produced by recombinant DNA technology. Hum Reprod 11(Suppl 1): 95-107; discussion 117-109.
Modica M, Romeo G, Materia L, Russo F, Cagnotto A, Mennini T, Gaspar R, Falkay G, and Fulop F (2004) Synthesis and binding properties of novel selective 5-HT3 receptor ligands. Bioorg Med Chem 12: 3891-3901.[CrossRef][Medline]
Moore S, Jaeschke H, Kleinau G, Neumann S, Costanzi S, Jiang JK, Childress J, Raaka BM, Colson A, Paschke R, et al. (2006) Evaluation of small-molecule modulators of the luteinizing hormone/choriogonadotropin and thyroid stimulating hormone receptors: structure-activity relationships and selective binding patterns. J Med Chem 49: 3888-3896.[CrossRef][Medline]
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, et al. (2000) Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289: 739-745.
Powell-Jones CH, Saltiel AR, Thomas CG, Jr. and Nayfeh SN (1981) Dissociation kinetics of the thyrotropin-receptor complex. Characterization of a slowly dissociable component. Mol Cell Endocrinol 24: 219-231.[CrossRef][Medline]
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85.[CrossRef][Medline]
Smits G, Campillo M, Govaerts C, Janssens V, Richter C, Vassart G, Pardo L, and Costagliola S (2003) Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J 22: 2692-2703.[CrossRef][Medline]
Söderhäll JA, Polymeropoulos EE, Paulini K, Gunther E, and Kuhne R (2005) Antagonist and agonist binding models of the human gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun 333: 568-582.[CrossRef][Medline]
Timmers CM and Karstens WFJ (2002), inventors; AK20 Nobel NV, assignee. Preparation of thienopyrimidinecarboxamides, quinazolinecarboxamides, and related compounds as luteinizing hormone agonists. WO02024703. 2002 Mar 28.
van Straten NC, Schoonus-Gerritsma GG, van Someren RG, Draaijer J, Adang AE, Timmers CM, Hanssen RG, and van Boeckel CA (2002) The first orally active low molecular weight agonists for the LH receptor: thienopyr(im)idines with therapeutic potential for ovulation induction. Chembiochem 3: 1023-1026.[CrossRef][Medline]
van Straten NC, van Berkel TH, Demont DR, Karstens WJ, Merkx R, Oosterom J, Schulz J, van Someren RG, Timmers CM, and van Zandvoort PM (2005) Identification of substituted 6-amino-4-phenyltetrahydroquinoline derivatives: potent antagonists for the follicle-stimulating hormone receptor. J Med Chem 48: 1697-1700.[CrossRef][Medline]
Vassart G, Pardo L, and Costagliola S (2004) A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 29: 119-126.[CrossRef][Medline]
Zhang B, Salituro G, Szalkowski D, Li Z, Zhang Y, Royo I, Vilella D, iacute, ez MT, Pelaez F, et al. (1999) Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science 284: 974-977.
Zhang G, Huang N, Li Y-W, Qi X, Marshall AP, Yan X-X, Hill G, Rominger C, Prakash SR, Bakthavatchalam R, et al. (2003) Pharmacological characterization of a novel nonpeptide antagonist radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([3H]SN003) for corticotropin-releasing factor 1 receptors. J Pharmacol Exp Ther 305: 57-69.
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 S. Neumann, W. Huang, S. Titus, G. Krause, G. Kleinau, A. T. Alberobello, W. Zheng, N. T. Southall, J. Inglese, C. P. Austin, et al. Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice PNAS, July 28, 2009; 106(30): 12471 - 12476. [Abstract] [Full Text] [PDF]
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) was determined by subtracting the nonspecific binding (
) from the total binding (
) curve. The control KD value was 2.4 ± 0.4 nM and the Bmax value was 1.6 ± 0.2 pmol/mg protein (n = 5). A similar experiment was performed in the presence of 70 nM recLH, of which only the specific binding is shown (
). Representative graphs from one experiment performed in duplicate.
