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On the Mechanism of Interaction of Potent Surmountable and Insurmountable Antagonists with the Prostaglandin D2 Receptor CRTH2

7TM Pharma A/S, Hørsholm, Denmark (J.M.M., E.K., T.U.); Department of Pharmacology, Monash University, Victoria, Australia (A.C.); Department of Experimental and Clinical Pharmacology, Medical University Graz, Graz, Austria (J.F.R., A.H.); Laboratorio de Medicina Computacional, Unidad de Bioestadistica, Facultad de Medicina, Universidad Autonoma de Barcelona, Barcelona, Spain (M.C., L.P.); and Department of Chemistry, University of Southern Denmark, Odense, Denmark (T.U.)

Received for publication August 8, 2005.

Accepted for publication January 17, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion Appendix References

 
Chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2) has attracted interest as a potential therapeutic target in inflammatory diseases. Ramatroban, a thromboxane A₂ receptor antagonist with clinical efficacy in allergic rhinitis, was recently found to also display potent CRTH2 antagonistic activity. Here, we present the pharmacological profile of three ramatroban analogs that differ chemically from ramatroban by either a single additional methyl group (TM30642), or an acetic acid instead of a propionic acid side chain (TM30643), or both modifications (TM30089). All three compounds bound to human CRTH2 stably expressed in human embryonic kidney 293 cells with nanomolar affinity. [³H]Prostaglandin D₂ (PGD2) saturation analysis reveals that ramatroban and TM30642 decrease PGD2 affinity, whereas TM30643 and TM30089 exclusively depress ligand binding capacity (B_max). Each of the three compounds acted as potent CRTH2 antagonists, yet the nature of their antagonism differed markedly. In functional assays measuring inhibition of PGD2-mediated 1) guanosine 5'-O-(3-thio)triphosphate binding, 2) -arrestin translocation, and 3) shape change of human eosinophils endogenously expressing CRTH2, ramatroban, and TM30642 produced surmountable antagonism and parallel rightward shifts of the PGD2 concentration-response curves. For TM30643 and TM30089, this shift was accompanied by a progressive reduction of maximal response. Binding analyses indicated that the functional insurmountability of TM30643 and TM30089 was probably related to long-lasting CRTH2 inhibition mediated via the orthosteric site of the receptor. A mechanistic understanding of insurmountability of CRTH2 antagonists could be fundamental for development of this novel class of anti-inflammatory drugs.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Structures of ramatroban and its analogs TM30642, TM30643, and TM30089.

Antagonists of G protein-coupled receptors can be distinguished as either surmountable or insurmountable (Lew et al., 2000; Vauquelin et al., 2002). Surmountable antagonists produce parallel rightward shifts of agonist concentration-response curves without altering the maximal agonist response. Insurmountable antagonists partially or completely decrease the maximal agonist response and may or may not induce concomitant rightward shifts of agonist dose-response curves. Insurmountable antagonism has been observed for a variety of GPCR systems, including those for angiotensin II, histamine, acetylcholine, serotonin, substance P, bradykinin, cysteinyl-leukotrienes, ADP, glutamate, and anaphylatoxin C5a (Schambye et al., 1994; Aramori et al., 1997; Lew et al., 2000; Carroll et al., 2001; Gillard et al., 2002; Vauquelin et al., 2002; Marteau et al., 2003; Rashid et al., 2003; March et al., 2004; Takezako et al., 2004). It is a priori unclear whether one type of antagonism is desired over the other to obtain clinical efficacy; nevertheless, insurmountable behavior of antagonists may be a means to obtain long-lasting receptor blockade in vivo.

We have previously reported the synthesis and selectivity profile of three novel ramatroban analogs (Fig. 1), which represented the first highly selective and potent CRTH2 antagonists (Ulven and Kostenis, 2005). In the current study, we present a detailed pharmacological analysis of their antagonistic profile, in comparison with ramatroban as a reference antagonist, using mammalian cells overexpressing CRTH2 or human eosinophils that naturally express CRTH2. Despite their close structural resemblance and similar binding affinities to CRTH2, the compounds display significant differences in the nature of their antagonism. Elucidation of the molecular mechanism underlying the divergent modes of CRTH2 blockade (surmountable versus insurmountable) of the compounds is presented. This is the first report disclosing both surmountable and insurmountable selective and potent antagonists for CRTH2 as valuable tools for further exploring the role of CRTH2 in vitro and in vivo.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion Appendix References

 
Materials White 96-well Optiplates and DeepBlueC were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Tissue culture media and reagents were purchased from the Invitrogen (Breda, The Netherlands). PGD2 was from Cayman Chemical (Ann Arbor, MI) and [³H]PGD2 was from PerkinElmer Life and Analytical Sciences. TM27868 was obtained from ChemDiv (San Diego, CA). Eotaxin was from Preprotech EC (London, UK). CellFix and FACS-Flow were from BD Immunocytometry Systems (Vienna, Austria). Fixative solution was prepared by diluting Cellfix 1:10 in distilled water and 1:4 in FACSFlow. Ramatroban was obtained from Bayer AG (Wuppertal, Germany). Synthesis of the CRTH2 antagonists TM30642, TM30643, and TM30089 was described previously (Ulven and Kostenis, 2005). All other laboratory reagents were from Sigma-Aldrich (St. Louis, MO), unless explicitly specified.

Generation/Origin of the cDNA Constructs The coding sequence of human CRTH2 (GenBank accession no. NM_004778 [GenBank] ) was amplified by PCR from a human hippocampus cDNA library and inserted into the pcDNA3.1(+) expression vector (Invitrogen) via 5' HindIII and 3' EcoRI. To generate a CRTH2-Renilla reniformis luciferase (CRTH2-Rluc) fusion protein, the CRTH2 coding sequence without a STOP codon and Rluc were amplified, fused in frame by PCR, and subcloned into the pcDNA3.1(+)Zeo expression vector. For ELISA experiments, the 78-base pair M1 FLAG-epitope tag was introduced by PCR at the extreme N terminus, and the resulting construct was inserted via 5' NheI and 3' EcoRI into pcDNA3.1(+). Human -arrestin2 (-arr2) N-terminally tagged with GFP² (GFP²/-arr2) and R. reniformis luciferase were purchased from Packard BioSignal Inc. The -arr2 mutant incapable of interacting with the endocytic machinery (-arr2, R393E, R395E) was a generous gift from R. Jørgensen (7TM Pharma A/S, Hørsholm, Denmark) and has been described previously (Vrecl et al., 2004). The sequence identity of the constructs was verified by restriction endonuclease digests and sequencing in both directions on an ABI Prism 310 DNA sequencer (Applied Biosystems, Foster City, CA).

Cell Culture and Transfection COS-7 cells were grown in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal bovine serum and 10 µg/ml gentamicin and kept at 37°C in a 10% CO₂ atmosphere. HEK293 cells were maintained in minimal essential medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM GlutaMAX-I, 1% nonessential amino acids, 1% sodium pyruvate, and 10 µg/ml gentamicin. For functional inositol phosphate assays, COS-7 cells were transiently cotransfected with CRTH2 and a promiscuous G protein facilitating inositol phosphate production by the G_i-selective CRTH2 receptor (Kostenis et al., 2005) using a calcium phosphate-DNA coprecipitation method with the addition of chloroquine (Mathiesen et al., 2005). To perform the functional bioluminescence resonance energy transfer (BRET) assays, a HEK293 cell clone stably expressing arr2-R393E, R395E-GFP², and CRTH2-Rluc was generated. This cell line will hereafter be referred to as CRTH2-HEK293 stable cells.

Binding Experiments Whole Cell Binding. CRTH2-HEK293 cells were seeded into 96-well plates at a density of 30,000 cells/well. Competition binding experiments on whole cells were then performed approximately 18 to 24 h later using 1.2 nM [³H]PGD2 (172 Ci/mmol; NEN) in a binding buffer consisting of HBSS (Invitrogen) and 10 mM HEPES, pH 7.4. Competing ligands were diluted in DMSO, which was kept constant at 1% (v/v) of the final incubation volume. Total and nonspecific binding were determined in the absence and presence of 10 µM PGD2. Binding reactions were routinely conducted for 3 h at 4°C and terminated by two washes (100 µl each) with ice-cold binding buffer. Radioactivity was determined by liquid scintillation counting in a TopCount liquid scintillation counter (PerkinElmer Life and Analytical Sciences) after overnight incubation in MicroScint 20. For saturation binding experiments, CRTH2-HEK293 cells were incubated with 1.5 to 48 nM [³H]PGD2 for 3 h in the absence and presence of equivalent concentrations (with respect to receptor occupancy) of CRTH2 antagonists, and nonspecific binding determined in the presence of 10 µM PGD2. The exact concentration of [³H]PGD2 used was determined from experiment to experiment. Determinations were made in duplicates.

Dissociation Kinetics. CRTH2-HEK293 whole cells (250,000 cells/ml) were incubated at 4°C with 3 nM [³H]PGD2 in binding buffer (HBSS + 10 mM HEPES, pH 7.4) for 60 min to obtain equilibrium. Dissociation was initiated by adding 10 µM PGD2 alone or in combination with 20 µM CRTH2 antagonists ramatroban, TM30089, or TM27868, respectively. After various time intervals, 200-µl aliquot samples were taken, and the reaction was terminated by sample filtration on a Millipore vacuum manifold using Whatman GF/F filters (presoaked in 0.5% BSA for at least 1 h). The filters were washed rapidly three times with 3 ml of ice-cold binding buffer, and radioactivity was determined in a beta counter (PerkinElmer Life and Analytical Sciences). In a separate set of dissociation kinetics, dissociation was initiated with an excess of PGD2 (10 µM), ramatroban (20 µM), or TM30089 (20 µM) alone.

Association Kinetics. The rate of [³H]PGD2 binding to CRTH2 receptors in whole CRTH2-HEK293 cells at 4°C was measured after preincubation with vehicle (DMSO) or equivalent concentrations of CRTH2 antagonists (K_i x 10) for 30 min. CRTH2-HEK293 whole cells were preincubated with antagonists or vehicle for 30 min, washed in 10 ml of binding buffer to remove nonbound antagonist, and resuspended, followed by addition of 4 nM [³H]PGD2 to initiate association using 500,000 cells/ml. After various time intervals, 200-µl aliquot samples (100,000 cells) were taken and processed as described under Dissociation Kinetics. Binding equilibrium was reached after 60 min at 4°C.

BRET² Assay Functional BRET² (hereafter referred to as BRET) assays were performed on HEK293 cells stably expressing human CRTH2-Rluc and GFP²--arr2, R393E, R395E essentially as described previously (Vrecl et al., 2004). Before the assay, cells were detached and resuspended in Dulbecco's PBS with 1000 mg/l L-glucose at a density of 2 x 10⁶ cells/ml. DeepBlueC was diluted to 50 µM in Dulbecco's PBS with 1000 mg/l L-glucose (light sensitive). Cell suspension (100 µl) was transferred to wells in a 96-well microplate (white OptiPlate) and placed in the Mithras LB 940 instrument (Berthold Technologies, Bad Wildbad, Germany). Agonist (12 µl/well) was then injected by injector 1, and 10 µl/well DeepBlueC was injected simultaneously by injector 2. Five seconds after the injections, the light output from the well was measured sequentially at 400 and 515 nm, and the BRET signal [milliBRET (mBRET) ratio] was calculated by the ratio of the fluorescence emitted by GFP²--arr2 (515 nm) over the light emitted by the receptor-Rluc (400 nm). Antagonists were preincubated with the cells for 15 min before the addition of agonist and DeepBlueC. Compounds were dissolved in DMSO, and the final DMSO concentration was kept constant at 1% in the assay. For BRET experiments in the presence of pertussis toxin, cells were incubated overnight in the presence of the toxin at a final concentration of 100 ng/ml. Use of the BRET² assay and the GFP²--arr2, R393E, R395E mutant for BRET² requires a license from 7TM Pharma A/S.

[³⁵S]GTPS Binding Assays Scintillation proximity assay [³⁵S]GTPS binding was performed on membranes from CHO-K1 cells stably expressing CRTH2 essentially as described in Mathiesen et al. (2005). Four micrograms of membrane protein was incubated in GTPS binding buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1% BSA, and 10 µg/ml saponin) with 50 nCi of [³⁵S]GTPS, 1 µM GDP, and 0.4 mg of wheat germ agglutinin-coupled scintillation proximity assay beads (RPNQ0001; GE Healthcare, Little Chalfont, Buckinghamshire, UK) with or without increasing concentrations of PGD2 in the absence or presence of the various CRTH2 antagonists. Parallel assays containing 100 µM nonradioactive GTPS defined nonspecific binding. Samples were incubated for 30 min at ambient temperature on a plate shaker, centrifuged for 5 min, and radioactivity was counted in a TopCount liquid scintillation counter.

Inositol Phosphate Accumulation Assays Twenty-four hours after transfection cells were seeded in 24-well tissue culture plates and loaded with 5 µCi of [2-³H]myo-inositol (TRK911; Amersham Biosciences). The next day, cells were washed twice in HBSS buffer (including CaCl₂ and MgCl₂; Invitrogen) and stimulated with the respective agonists in HBSS buffer supplemented with 5 mM LiCl for 45 min at 37°C. Antagonists are routinely preincubated for 15 min before the 45-min agonist incubation period. The reactions were terminated by aspiration and addition of 10 mM ice-cold formic acid and incubated for 30 min on ice. The lysate was applied to AG 1-X8 anion exchange resin (Bio-Rad, Hercules, CA) and washed twice with buffer containing 60 mM sodium formate and 5 mM borax. The [³H]inositol phosphate fraction was then eluted by adding 1 M ammonium formate and 100 mM formic acid solution and counted after addition of HiSafe3 scintillation fluid (PerkinElmer Life and Analytical Sciences).

Enzyme-Linked Immunosorbent Assay Determination of cell surface expression levels of CRTH2 was performed using an N-terminally FLAG-tagged CRTH2 receptor in an ELISA assay as described previously (Mathiesen et al., 2005). Twenty-four hours after transfection cells were seeded in poly-D-lysine-coated 48-well tissue culture plates at a density of 100,000 cells/well. Approximately 48 h after transfection, cells were washed once in minimal essential medium + 0.1% BSA and exposed to the indicated compounds diluted in the same buffer for 30 or 180 min at both 37 and 4°C. Cells were then fixed with 4% paraformaldehyde, and CRTH2 surface expression levels were determined with the 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) substrate. All experiments were performed in triplicate determinations.

Human Eosinophil Shape Change Assay Blood was sampled from healthy volunteers according to a protocol approved by the Ethics Committee of the University of Graz and processed as described previously (Bohm et al., 2004). Preparations of polymorphonuclear leukocytes (containing eosinophils and neutrophils) were prepared by dextran sedimentation of citrated whole blood and Histopaque gradients. The resulting cells were washed and resuspended in assay buffer (comprising PBS with Ca²⁺/Mg²⁺ supplemented with 0.1% BSA, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 5 x 10⁶ cells/ml. Cells were incubated with the antagonists or vehicle (PBS or DMSO) for 10 min at 37°C and then stimulated with various concentrations of the agonists (PGD2 or eotaxin) for 4 min at 37°C. To stop the reaction, samples were transferred to ice and fixed with 250 µl of fixative solution. Samples were immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences), and eosinophils were identified according to their autofluorescence in the FL-1 and FL-2 channels. Shape change responses were quantified as percentage of the maximal response to PGD2 or eotaxin in the absence of an antagonist.

Calculations and Data Analysis Analysis was performed using Prism 4.03 (GraphPad Software Inc., San Diego, CA). Data sets of saturation binding isotherms were analyzed via nonlinear regression according to a hyperbolic, one-site binding model, and individual estimates for total receptor number (B_max) and radioligand dissociation constant (K_A) were calculated. The following equation was used:

(1)

where [A] denotes the concentration of radioligand, and B_max and K_A denote the true PGD₂ binding capacity and affinity, respectively.

Specific binding data from the [³H]PGD2 competition binding assays using the test antagonists were normalized and fitted to the following empirical one-site model for competitive interaction:

(2)

where Y denotes percentage of specific binding, Top denotes maximal asymptotic binding, Bottom denotes the minimal asymptotic binding, [A] denotes the concentration of radioligand, [B] denotes the concentration of inhibitor, and K_A and K_B denote their respective equilibrium dissociation constants.

Data sets of [³H]PGD2 homologous competition binding experiments (total binding), performed in the absence or presence of increasing concentrations of each test antagonist, were initially globally fitted to the following model for simple homologous competition:

(3)

where B_max denotes the apparent maximal density of binding sites, K_A denotes the apparent equilibrium dissociation constant of PGD₂, [A_Hot] denotes the concentration of radioligand, [A_Cold] denotes the concentration of unlabeled PDG₂ (the independent variable), and NS denotes the fraction of nonspecific binding (Motulsky and Christopoulos, 2004). Note that the estimates of B_max and K_A are only estimates of true PGD₂ binding capacity and affinity, respectively, for the control curve in the absence of added antagonist. Subsequent to this fit, an F-test was used to determine whether the data could be better fitted by sharing the B_max and estimating a separate K_A for each curve (consistent with the expectations of competitive antagonism) or by sharing the K_A across the curves and estimating a separate B_max for each curve (indicative of noncompetitive antagonism; see Fig. 4 under Results for example). Data sets that were better described by assuming no change in B_max with increasing antagonist concentrations were then globally fitted to the following homologous binding model, which explicitly describes a surmountable competitive interaction between radioligand, homologous displacer and a second antagonist:

(4)

View larger version (37K): [in this window] [in a new window]   Fig. 4. Homologous competition binding between [3H]PGD2 (1.2 nM) and unlabeled PGD2 in the absence or presence of the indicated concentrations of CRTH2 antagonist. The data for ramatroban and TM30642 represent the best global fit according to a competitive binding model where the effect of each antagonist is to reduce the apparent binding affinity of PGD2 with no effect on the Bmax (as determined by F-test). The data for TM30643 and TM30089 represent the best global fit according to a noncompetitive binding model, where the effect of each antagonist is to reduce the Bmax of PGD2 with no effect on its binding affinity (as determined by F-test). Also shown is the relationship between the best-fit values for apparent Bmax and logKA for each data set. Data shown are representative of a single experiment, performed in duplicate, which was repeated five times.

(5)

Functional concentration-response curves for PGD2 obtained in the absence or presence of CRTH2 antagonists were fitted via nonlinear regression analysis to the following four-parameter logistic equation:

(6)

where Top denotes maximal asymptotic binding, Bottom denotes the minimal asymptotic binding, [A] denotes the concentration of radioligand, and and n_H is the Hill coefficient. EC₅₀ values were obtained as a measure of agonist potency and represent the effective concentrations of half-maximal responses.

pA₂ values were estimated from dose ratios (DR) calculated from the EC₅₀ values of the individual dose response curves obtained in the absence (EC_{50,agonist alone}) and presence (EC_50,+[B]) of CRTH2 antagonists by fitting to the following equation using linear regression:

(7)

where [B] denotes the antagonist concentration used when estimating the EC₅₀ for the agonist, and K_B is the dissociation constant of the antagonist. pA₂ was estimated as the interception of the regression line with the x-axis.

Data from association and dissociation kinetic experiments were analyzed to calculate the dissociation rate constants (K_-1) and the observed association rate constants (K_app). To determine the dissociation rate constants of PGD2, data were fitted by nonlinear regression to the following equation:

(8)

where K_-1 is the dissociation rate constant, and Y_max denotes the amount of specific binding at 0 min.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Affinities of ramatroban and the three analogs TM30642, TM30643, and TM30089 for the cloned human CRTH2 receptor stably expressed in HEK293 cells (CRTH2-HEK293 cells). CRTH2-HEK293 whole cells were incubated at 4°C for 3 h with increasing concentrations of the competitor compounds. Nonspecific binding was defined in the presence of 10 µM PGD2. Ki values were calculated by transforming the IC50 values according to the Cheng-Prusoff equation as described under Materials and Methods. Symbols represent the mean ± S.E. of three independent experiments conducted in duplicate.

(9)

where the curve ascends to Y_max1+ Y_max2 via a biphasic exponential association; K_app1 and K_app2 denote the individual apparent association rate constants.

In practice, all estimates of ligand potency or affinity were obtained as logarithms.

	Results

Top Abstract Materials and Methods Results Discussion Appendix References

 
The Tetrahydrocarbazoles TM30642, TM30643, and TM30089 Are Almost Equipotent Inhibitors of [³H]PGD2 Binding at Human CRTH2 Receptors At the outset, ramatroban and the three analogs were tested for their ability to compete for [³H]PGD2 specific binding to human CRTH2 receptors stably expressed in HEK293 cells (CRTH2-HEK293 cells). All three compounds displaced [³H]PGD2-specific binding concentration dependently (Fig. 2) with estimated antagonist dissociation constants as shown in Table 1. The affinity of the reference antagonist ramatroban as determined in this set of assays is congruent with previously reported data (Sugimoto et al., 2003; Ulven and Kostenis, 2005; Fig. 2). To characterize the effects of the compounds on [³H]PGD2 binding in more detail, [³H]PGD2 saturation isotherms were generated in the absence and presence of equivalent concentrations (with respect to receptor occupancy) of inhibitor compounds (approx. 3 x K_i) (Fig. 3A). Compounds interacting with a receptor in a competitive and reversible manner alter radioligand affinity without affecting ligand binding capacity (B_max). Conversely, compounds interacting in a noncompetitive, slowly reversible or irreversible manner reduce binding capacity but have minimal, or no, effect on the affinity of the radiotracer. In the absence of competitor compounds, [³H]PGD2 binding to CRTH2-HEK293 whole cells was saturable, and data were fitted adequately to a one-site binding model [eq. 1; equilibrium dissociation constant (K_D) and B_max values amounted to K_D = 11.1 ± 2.7 nM, B_max = 3.1 ± 0.6 pmol/mg]. To better visualize the apparent effects of the compounds on [³H]PGD2 affinity and receptor number, Scatchard plots were derived from the saturation isotherms (Fig. 3B). In the presence of ramatroban and its N-methylated analog TM30642 the affinity of [³H]PGD2 seemed to decrease as indicated by flatter slopes of the Scatchard regression lines, whereas B_max values remained unaffected as shown by similar interceptions of regression lines with the x-axis (Fig. 3B). In contrast, incubation of CRTH2-HEK293 cells with TM30643 and TM30089, in which the side chain linking the tetrahydrocarbazole scaffold with the carboxylic acid moiety is shortened by one methylene unit, seemed to decrease the number of PGD2 binding sites but did not alter PGD2 affinity.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of ramatroban, TM30642, TM30643, and TM30089 on [3H]PGD2 saturation binding in CRTH2-HEK293 cells. A, representative saturation analysis of [3H]PGD2 binding to CRTH2 receptors in whole cells in the absence or presence of equivalent concentrations (with respect to receptor occupancy) of the compounds. Concentrations of compounds chosen inhibited approximately 75% of [3H]PGD2 specific binding at equilibrium (approximately 30 x Ki; see Fig. 2). Data were fitted best to a one-binding site model, and KD and Bmax values were determined. B, Scatchard transformation of the saturation isotherms shown in A. TM30643 and TM30089 significantly lowered Bmax (p < 0.05) compared with vehicle- and ramatroban/TM30642-treated cells. [3H]PGD2 KD values were significantly higher (p < 0.05) in the presence of ramatroban and TM30642 compared with vehicle-treated cells.

Although saturation binding experiments and Scatchard analysis indicated a reduced [³H]PGD2 binding capacity in the presence of TM30643 and TM30089, a rigorous test of this assumption would have required a larger range of antagonist concentrations, and, as a consequence, appreciably larger (and practically unobtainable) tracer concentrations. In an attempt to circumvent this limitation but retain an ability to reliably quantify antagonist effects on radioligand binding affinity versus binding capacity, we developed a novel analytical method that is described in detail under Materials and Methods. To this end, a series of [³H]PGD2 homologous competition binding experiments were performed in the absence and presence of increasing concentrations of the respective CRTH2 antagonists. As shown in Fig. 4, a global analysis of the data according to eq. 3 under Materials and Methods, followed by F-test, revealed that the interaction between PGD2 and either ramatroban or TM30642 was best fitted by a model that assumed a change in the apparent affinity of PGD2 with no change in PGD2 B_max; a single best-estimate of the latter parameter was able to describe all the curves in the data set for each antagonist. This behavior is consistent with simple surmountable antagonism. In contrast, application of the same test to the data measuring the interaction between PGD2 and either TM0643 or TM30089 revealed that the entire family of curves could best be fitted by assuming no change in PGD2 affinity (described by a single estimate for logK_A) but a progressive reduction in B_max with increasing antagonist concentration —behavior that is consistent with insurmountable antagonism. As a consequence, the ramatroban and TM30642 data sets were refitted to eq. 4 under Materials and Methods, which is based on a competitive model of interaction, to explicitly estimate the dissociation constant for either antagonist when interacting with PGD2; the resulting values are shown in Table 1. In contrast, the TM0643 or TM30089 data sets were globally refitted to eq. 5 under Materials and Methods, which is based on the standard model for noncompetitive antagonism. The estimated affinity values for these latter two antagonists are also shown in Table 1. It is noteworthy that a comparison of the antagonist affinity estimates obtained by this approach with those obtained by the more traditional inhibition binding method (Fig. 2), which assumes a strictly competitive, surmountable antagonism between ligands, yielded very similar values. An explanation as to why this may be so is outlined under Discussion and the Appendix. Together, the inhibition and saturation binding experiments revealed that the tetrahydrocarbazole CRTH2 antagonists display similar receptor binding affinities but belong to two different pharmacological classes (i.e., surmountable and insurmountable antagonists).

Effects of the Compounds on PGD2-Mediated Responses in Different Functional Assays in Mammalian Cells, Overexpressing CRTH2 To determine the functional consequences of the divergent effects on [³H]PGD2 binding, concentration-dependent inhibition of PGD2 responses by the CRTH2 antagonists was evaluated in a set of different functional assays. We were particularly curious about the nature of antagonism of TM30643 and TM30089, because both compounds clearly reduced the available number of PGD2 sites on the cell surface, which may correlate to insurmountable inhibition of agonist responses in functional assays. First, the compounds were tested for their ability to antagonize PGD2-mediated stimulation of [³⁵S]GTPS binding in membrane preparations from stably transfected CRTH2 cells (Fig. 5). Ramatroban and TM30642 caused parallel rightward shifts of the PGD2 concentration-response curves without altering the maximal PGD2 response, consistent with competitive and reversible antagonism. Estimated pA₂ values and slopes of the Schild regressions amounted to pA₂ = 7.44 ± 0.14, n_H = 0.91 ± 0.05 (n = 5) for ramatroban, and pA₂ = 7.72 ± 0.13, n_H = 0.89 ± 0.075 (n = 3) for TM30642, respectively. In contrast, TM30089 simultaneously shifted the PGD2 dose-response curve to the right and decreased the maximal PGD2 response, indicating insurmountable antagonism. TM30643 induced clear rightward shifts of PGD2 concentration-effect curves in concentrations up to 1 µM; at a concentration of 10 µM, however, E_max of PGD2 seemed to be depressed. A pA₂ value for TM30643 was computed excluding the PGD2 dose-response curve in the presence of the highest applied concentration of TM30643 and amounted to pA₂ = 8.37 ± 0.08, n_H = 1.06 ± 0.02 (n = 3).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effects of ramatroban, TM30642, TM30643, and TM30089 on PGD2-mediated stimulation of [35S]GTPS binding in Chinese hamster ovary cell membranes stably expressing human CRTH2. PGD2 dose-response curves were performed in the presence of the indicated concentrations of the compounds (left) and dose ratios were transformed into Schild plots (right). The plots of the dose ratios over antagonist concentration were subjected to linear regression analysis, and pA2 values were determined as the x-intercepts at log (DR - 1) = 0. Data are means ± S.D. of one of three representative experiments each performed in duplicate.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effects of ramatroban, TM30642, TM30643, and TM30089 on PGD2-mediated stimulation of -arrestin recruitment by CRTH2 in BRET assays. HEK293 cells stably transfected with a modified -arrestin-GFP2 and CRTH2-Rluc (for details, see Materials and Methods) were preincubated for 15 min at 37°C with fixed concentrations of the compounds before arrestin translocation was initiated by addition of increasing concentrations of PGD2. mBRET ratios were calculated as described under Materials and Methods. Shown are means ± S.D. of one of three independent experiments, each performed in duplicate.

We have recently shown that CRTH2-mediated -arrestin recruitment has a major G protein-independent component and is uncoupled from G_i/o activation in HEK293 cells (Mathiesen et al., 2005). To test whether the set of CRTH2 antagonists retains its pharmacological profile when -arrestin recruitment is exclusively mediated in a G protein-independent manner, CRTH2-expressing cells were pretreated with PTX, a selective inhibitor of G_i/o proteins. PTX treatment decreased PGD2-mediated arrestin recruitment in CRTH2-HEK293 cells by approximately 20%, confirming the substantial G_i-independent component. However, the nature of antagonism of the compounds was essentially unchanged in PTX-treated cells (Supplemental Fig. 3), suggesting that the nature of antagonism is independent of the cellular signaling pathway used by CRTH2.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Inhibition by ramatroban and the analogs TM30642, TM30643, and TM30089 of flow cytometric shape change responses of eosinophils exposed to PGD2. Samples of polymorphonuclear leukocytes were pretreated with the antagonists or their vehicle for 30 min and then stimulated with PGD2. Eosinophils were identified according to their autofluorescence, and shape change responses were quantified as percentage of the maximal response to PGD2 in the absence of an antagonist. Although ramatroban and TM30642 shifted the concentration-response curve to PGD2 rightward in a parallel manner, TM30643 and TM30089 induced both affinity shifts and depression of maximal efficacy of PGD2 dose-response curves. Data are shown as mean ± S.E., n = 3.

Molecular Mechanism of Insurmountable Antagonism Insurmountable Antagonists Do Not Decrease CRTH2 Receptor Cell Surface Expression. To investigate a potential link between insurmountable antagonism and the ability of the compounds to internalize receptors, we analyzed CRTH2 cell surface expression in the absence and presence of different concentrations of antagonists (20, 200, and 2000 nM) in HEK293 cells stably expressing CRTH2. ELISA assays showed that none of the antagonists significantly decreased CRTH2 cell surface numbers as opposed to PGD2 (data not shown). This finding together with the effects of the antagonists on [³H]PGD2 B_max in saturation and homologous competition binding analyses is congruent with the notion that insurmountability is not merely related to the disappearance of CRTH2 cell surface receptors but that antagonists rather make CRTH2 inaccessible to its agonist PGD2.

Is Insurmountable Antagonism Mediated via an Allosteric Binding Site? To determine whether the insurmountable antagonism of PGD2 by TM30089 and TM30643 observed in the functional assays could be because of the compounds interacting with an allosteric site different from the orthosteric PGD2 binding domain, CRTH2-HEK293 cells were incubated with 3 nM [³H]PGD2 until equilibrium was reached. Subsequently, dissociation of bound radioligand was monitored over time by adding an excess of unlabeled PGD2 (1000 x K_D) to prevent radioligand reassociation, in the absence or presence of a high concentration of insurmountable antagonist. If the dissociation rate of [³H]PGD2 is altered by the simultaneous presence of an antagonist, it must be because of antagonist interacting with an allosteric site distinct from the PGD2 binding domain. Figure 8A shows that none of the tested antagonists was capable of affecting the dissociation rate of [³H]PGD2, in contrast to the positive control compound TM27868, which has recently been shown to significantly delay [³H]PGD2 dissociation from CRTH2 receptors (Mathiesen et al., 2005). In another set of kinetic experiments, we tested whether [³H]PGD2 dissociation was differentially affected when monitored only in the presence of excess PGD2, surmountable or insurmountable antagonists. To this end, CRTH2-HEK293 cells were incubated with [³H]PGD2 until equilibrium was reached and dissociation was monitored over time by adding either a large excess of PGD2 (10 µM) or surmountable (20 µM ramatroban) or insurmountable (20 µM TM30089) ligand. Under these experimental conditions, acceleration or retardation of [³H]PGD2 dissociation could be indicative of a cooperative mechanism of binding, whereas dissociation coinciding with that induced by PGD2 would indicate action via the orthosteric site. Dissociation of [³H]PGD2, however, was essentially unchanged when reassociation was precluded by excess PGD2, ramatroban, or TM30089, respectively. Together, the different sets of kinetic experiments clearly support the notion that insurmountability of the antagonists does not arise from cooperative or allosteric interactions with CRTH2, occupation of which may lead to a conformational change refractory to agonist activation.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Time course of [3H]PGD2 dissociation from human CRTH2 receptors stably expressed in HEK293 cells. A, CRTH2-HEK293 whole cells were incubated with 4 nM [3H]PGD2 for 60 min at 4°C until binding equilibrium was attained. Dissociation of [3H]PGD2 was initiated by adding a large excess of PGD2 (10 µM) in the absence or presence of the indicated compounds. Nonspecific binding was determined in the presence of 10 µM PGD2. Binding levels after the initial equilibration phase were set 100% for all dissociation data sets. Shown are representative experiments performed as single point determinations. Two additional experiments gave similar results. B, dissociation was initiated by adding either the surmountable antagonist ramatroban or the insurmountable antagonist TM30089. Shown is a representative experiment.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Effects of ramatroban, TM30642, TM30643, and TM30089 on [3H]PGD2 association kinetics in CRTH2-HEK293 cells. [3H]PGD2 association kinetics were performed at 4°C in the presence of a single concentration of CRTH2 antagonist (chosen to correspond to Ki x 10) or their vehicle (control). Cells were pre-exposed to the antagonists for 30 min at 4°C before [3H]PGD2 association was initiated by ligand addition. [3H]PGD2 association curves were analyzed by nonlinear regression analysis using a biexponential model as outlined in detail under Materials and Methods. Curves are representative of a single experiment. Four additional experiments gave similar results.

	Discussion

Top Abstract Materials and Methods Results Discussion Appendix References

 
Approximately 15 years ago, the orally available, small molecule ramatroban was developed as a thromboxane A₂ receptor antagonist for clinical use in various cardiovascular, cerebrovascular, and pulmonary diseases, but it has only recently been discovered to also antagonize CRTH2 (Sugimoto et al., 2003). We previously disclosed three ramatroban analogs that differ in structure only slightly from ramatroban (Fig. 1), yet exhibit very weak or no affinity for or functional activity on the thromboxane A₂ receptor, whereas their antagonistic activity on CRTH2 is preserved or potentiated (Ulven and Kostenis, 2005). In this study, we present a detailed pharmacological profile of these compounds and propose receptor binding modes to rationalize the observed pharmacological differences. All compounds inhibited PGD2 binding to CRTH2, but their nature of antagonism differed markedly (surmountable versus insurmountable). We were interested in exploring the molecular mechanisms underlying the experimental observations and also the structural features conferring insurmountability, because this is important from both a basic scientific and drug development point of view.

Proposed mechanisms for insurmountable antagonism include 1) slow dissociation of antagonist-receptor complexes; 2) interaction with allosteric binding sites inducing a conformational change in the receptor that compromises its interaction with the agonist; 3) antagonist-mediated conformational changes rendering receptors refractory to agonist stimulation; 4) antagonist-mediated desensitization or internalization; 5) slow antagonist removal from tissue compartments, cells, or matrix surrounding the receptor; 6) slowly interconverting receptor conformations; and 7) irreversible covalent ligand binding (for review, see Lew et al., 2000, Vauquelin et al., 2002).

Saturation and homologous inhibition binding experiments in the presence of the CRTH2 antagonists performed in this study indicated that TM30643 and TM30089 lead to a decrease of CRTH2 receptors capable of binding ligand; this pattern of behavior is also manifested in the functional effects of these antagonists (Figs. 3, 4, 5, 6 and 7). It is now well established that receptor desensitization and internalization can occur in the absence of receptor activation (Whistler et al., 2002), and several examples of GPCRs undergoing antagonist-mediated internalization have been described previously (Perry et al., 2005). We therefore investigated whether insurmountable inhibition of PGD2 responses by the ramatroban analogs TM30089 and TM30643 was because of such a property. Although binding analyses indicated that the two insurmountable antagonists significantly depressed B_max, ELISA assays (data not shown) revealed that the total number of CRTH2 receptors on the cell surface was not reduced in their presence. Insurmountable antagonism of the compounds is therefore independent of receptor internalization.

To ascertain whether the insurmountability of the compounds reflected a noncompetitive mechanism that was possibly mediated through an allosteric site, [³H]PGD2 dissociation kinetic studies were performed. Occupation by the compounds of an allosteric site may lead to a conformational change of CRTH2 that may perturb its interaction with PGD2 itself and hence its ability to elicit a cellular response. The dissociation kinetics presented in Fig. 8, however, imply that it is unlikely that the insurmountable antagonists interact with an allosteric site, because neither compound altered the rate of [³H]PGD2 dissociation from CRTH2 receptors (Fig. 8A). Furthermore, when dissociation of [³H]PGD2 was monitored in the presence of excess PGD2, ramatroban, or TM30089 alone, the [³H]PGD2 dissociation curves coincided for all three ligands, again suggesting interaction with a common binding site (Fig. 8B). Allosterically acting compounds, on the other hand, could have caused an acceleration or retardation of radioligand dissociation under these conditions because of cooperative binding.

Another possible explanation for insurmountability of orthosteric antagonists is the longevity of antagonist-receptor complexes caused by slow dissociation of antagonists from the receptors (Lew et al., 2000; Vauquelin et al., 2002). As outlined in the Appendix, binding behavior that is consistent with that observed in our current study can arise when the kinetics of one orthosteric ligand in the presence of another are so slow that insufficient readjustment of receptor occupancy occurs over the time course of the experiment. This phenomenon has previously been described as the "hemi-equilibrium" condition (Paton and Rang, 1966; Kenakin, 1997). Because a true state of equilibrium may not be practically reached during the course of such an experiment, affinity estimates for the antagonists have to be obtained under nonequilibrium conditions and hence are likely to deviate from the true affinities of the antagonists. However, as outlined in the Appendix, use of very low radioligand concentrations relative to its dissociation constant is a means to circumvent this problem. In fact, the lower the radioligand concentration used for competition binding assays, the closer will the apparent estimate of orthosteric antagonist affinity approach its true affinity, irrespective of whether antagonism is surmountable or insurmountable (shown by curve simulations in the Appendix). It is clear, however, that the drawback of using low radioligand concentrations is the introduction of uncertainty to the data because of a potentially low signal-to-noise ratio. In our study (Fig. 2; Table 1), the signal-to-noise ratio at low radioligand concentrations did not represent an experimental obstacle because binding data were very clean even under these conditions.

View larger version (20K): [in this window] [in a new window]   Fig. 10. Simulations of the interaction between an orthosteric ligand, A, in the absence (leftmost curve) or presence of a slowly dissociating orthosteric ligand, B, according to eq. 10. For all simulations, the following parameters were used: KA = 10-8 M, KB = 10-9 M, and koff = 10-4 s-1. Concentrations of B ranged from 10-9 M to 10-7 M in half-log units. The incubation times were 0.5 s (A), 3000 s (B), and 100,000 s (equilibrium) (C). Ordinates, fractional occupancy of ligand A (A); abscissa, concentration of ligand A.

It is well known that the degree of insurmountable antagonism is related to the level of receptor expression in a given cell. In our study, the extent of depression of maximal PGD2 responses was inversely correlated to receptor expression such that insurmountability was most evident in eosinophil shape change assays with "physiological levels" of receptor expression but least evident in GTPS assays with very high levels of CRTH2 expression. In addition, we noted a significant discrepancy between the concentrations of insurmountable antagonist required to depress B_max in radioligand binding assays and the ability to depress E_max in functional assays. This discrepancy was particularly evident for TM30643 and may reflect differences in both receptor reserve but also temperature in binding (4°C) and functional assays (37°C), because binding processes are significantly slower at lower temperatures, leading to apparent overestimation of potency for slowly dissociating ligands.

In conclusion, our study explored in detail the pharmacological profile of three structurally closely related ramatroban analogs that display high potency and selectivity for CRTH2. It also provided insight into the structural features required to elicit insurmountable antagonism and the underlying molecular mechanism showing that slow dissociation from the receptor is sufficient to explain the pharmacological behavior. To date, TM30643 and TM30089 are the only insurmountable CRTH2 antagonists reported in the literature. Yet, it is premature to conclude that slowly dissociating antagonists with the potential to produce long-lasting receptor blockade will be therapeutically advantageous in clinical settings. Although it is clear that many additional pharmacokinetic factors govern duration of compound action in vivo, insurmountability may contribute to this duration and may allow compounds to act much longer as would be predicted from their plasma half-lives.

	Appendix

Top Abstract Materials and Methods Results Discussion Appendix References

 
The standard analyses applied to the agonist-antagonist interaction assume that the measured response, be it binding or function, reflects an equilibrium interaction between the ligands. If this assumption is not met by the experimental conditions, complex behaviors can be observed in the data that may lead the investigator to misinterpret the mechanism underlying the interaction. To explore some of the consequences of lack of appropriate equilibration between two orthosteric ligands, the following equation was used to simulate the re-adjustment in receptor occupancy between an orthosteric ligand, A, exposed to a receptor that has been pre-equilibrated with a second orthosteric ligand, B, as a function of time, t (Paton and Rang, 1966; Kenakin, 1997).

(A1)

View larger version (14K): [in this window] [in a new window]   Fig. 11. Simulations of the interaction between a fixed concentration of orthosteric ligand, A, in the absence or presence of increasing concentrations of a slowly dissociating orthosteric ligand, B, according to eq. 10. Parameter values were as described for Fig. 10. Incubation times are indicated in the figure. The concentrations of ligand A were 0.1 x KA (A), 1]times] KA (B), or 10 x KA (C). Ordinates, fractional occupancy of ligand A (A); abscissa, concentration of ligand B.

A more common method that is used experimentally to monitor the fractional occupancy of one ligand in the presence of another is the inhibition binding assay. Figure 11 shows a series of simulations based on this type of paradigm whereby the concentration of ligand A is held constant, and the effects on its fractional occupancy are monitored as the concentration of ligand B is increased. At a concentration of A that is 10-fold less (Fig. 11A) or equal to (Fig. 11B), its K_A value, it can be seen that no, or minimal, discrepancy occurs in the estimate of the affinity of ligand B. In contrast, at a concentration that is 10 times above the K_A, a dramatic effect of incubation time is evident on the location and shape of the inhibition binding curve. These latter simulations are particularly useful, because they indicate that the most accurate estimates of affinity for a slowly reversible orthosteric ligand are obtained by using as low a concentration as possible of the orthosteric probe ligand.

	Acknowledgements

 
We are grateful to Helle Z. Andresen for excellent technical assistance, to Kate Hansen for excellent cloning support, and to Anders Heding (7TM Pharma A/S) for development of the improved BRET² assay using the GFP²--arr2, R393E, R395E mutant.

	Footnotes

 
This work was supported by the European Community's Sixth Framework Programme, Grant LSHB-CT-2003-503337.

ABBREVIATIONS: PDG2, prostaglandin D₂; DP, prostaglandin D receptor; CRTH2, chemoattractant receptor-homologous molecule expressed on T helper 2 cells; Th2, T helper 2; TM27868, 1-(4-ethoxyphenyl)-5-methoxy-2-methylindole-3-carboxylic acid; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; -arr2, -arrestin2; arr2-GFP², green fluorescent protein--arrestin2-(R393E, R395E) fusion protein; CRTH2-Rluc, Renilla reniformis luciferase-CRTH2 fusion protein; HEK, human embryonic kidney; BRET, bioluminescence resonance energy transfer; HBSS, Hanks' balanced salt solution; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GTPS, guanosine 5'-O-(3-thio)triphosphate; PTX, pertussis toxin; EXP3174, 2-butyl-4-chloro-1-((2'-(1H-tetrazol-5-yl)(1,1'-biphenyl)-4-yl)methyl)-1H-imidazole-5-carboxylic acid; SC-54629, 1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-((6-(2-(1H-tetrazol-5-yl)phenyl)-3-pyridinyl)methyl)-2H-imidazol-2-one; SC-54628, 1-(2-methylphenyl)-4-butyl-1,3-dihydro-3-((6-(2-(1H-tetrazol-5-yl)phenyl)-3-pyridinyl)methyl)-2H-imidazol-2-one; TM30642, 3-{3-[(4-fluoro-benzenesulfonyl)-methyl-amino]-1,2,3,4-tetrahydro-carbazol-9-yl}-propionic acid; TM30643, [3-(4-fluoro-benzenesulfonylamino)-1,2,3,4-tetrahydro-carbazol-9-yl]-acetic acid; TM30089, {3-[(4-fluoro-benzenesulfonyl)-methyl-amino]-1,2,3,4-tetrahydro-carbazol-9-yl}-acetic acid.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Evi Kostenis, 7TM Pharma A/S, Fremtidsvej 3, 2970 Hørsholm, Denmark. E-mail: ek{at}7tm.com

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