Introduction

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Corticotropin-releasing factor (CRF) was first isolated from ovine hypothalamus (Vale et al., 1981) and identified as a key secretagogue for ACTH release from the anterior pituitary. In the past 10 years, considerable progress has been made in understanding the physiological and potential pathological roles of the CRF system. In addition to its endocrine role in the regulation of the hypothalamic-pituitary-adrenal axis in response to stress, CRF appears to be implicated in a variety of other central and peripheral functions including arousal, anxiety-like behaviors, learning and memory, feeding, immune, and autonomic functions (Owens and Nemeroff, 1991; De Souza and Grigoriadis, 1998; Gilligan et al., 2000; Dautzenberg and Hauger 2002).

The entire spectrum of CRF peptide effects is mediated through two known receptor subtypes, CRF₁ and CRF₂. Both receptor subtypes belong to the class B family of G protein-coupled receptors that include receptors for secretin and parathyroid hormone, among others (review Dautzenberg and Hauger, 2002). Despite their high degree of sequence homology and their common coupling through G_s proteins to cAMP signaling, CRF₁ and CRF₂ receptors differ markedly from each other in pharmacological properties and anatomic distribution (DeSouza et al., 1998; Gilligan et al., 2000; Dautzenberg and Hauger, 2002). CRF₁ receptors are widely distributed in the central nervous system. There exist three splice variants of the CRF₂ receptor (CRF₂, CRF₂, and CRF₂) with distinct anatomic localization. CRF₂ receptors are primarily located in discrete rat brain areas such as lateral septum, and CRF₂ receptors are located in rat choroid plexus, heart, lung, and skeletal muscle. CRF₁ and CRF₂ receptors are activated by several related peptides identified from various species. These include CRF, sauvagine, urotensin, and the urocortins, including recently identified urocortin II and III (Lewis et al., 2001; Reyes et al., 2001), which display differential affinity for CRF₁ and CRF₂ receptors.

Studies of the neuronal circuitry mediating fear and anxiety states (Davis, 1992) suggest that both CRF₁ and CRF₂ receptors located in differential brain areas may be involved in the regulation of various stress-induced behaviors, albeit the relative importance of CRF₂ receptors is less clear (Lewis et al., 2001; Reyes et al., 2001; Bakshi et al., 2002). Clinical findings support the hypothesis that dysfunction of the CRF system is involved in certain neuropsychiatric disorders such as anxiety and depression (Gilligan et al., 2000; Keck and Holsboer, 2001). Numerous animal studies using CRF ligands and genetically altered mice provide strong evidence for the role of CRF₁ receptors in the coordination of the behavioral response to stress and in stress-related psychiatric disorders (Gilligan et al., 2000; Bakshi et al., 2002; Dautzenberg and Hauger, 2002).

In recent years there has been much emphasis on developing orally active, nonpeptidic CRF₁ antagonists to evaluate the putative role of CRF₁ receptors in psychopathology and to test their potential as novel therapeutic agents. Schulz et al. (1996) were the first to report a pyrazolopyrimidine CRF₁ antagonist, CP-154,526, with high affinity for the CRF₁ receptor and anxiolytic activity in rats. Additional CRF₁ antagonists, such as antalarmin, NBI 27914, DMP696, R121919, and SSR125543A, also exhibit CRF₁ antagonist properties in vitro and in vivo (He et al., 2000; McCarthy et al., 1999; Habib et al., 2000; Griebel et al., 2002; Gully et al., 2002; Heinrichs et al., 2002; Maciag et al., 2002; McElroy et al., 2002). Although a variety of the iodine-125-labeled peptides such as [¹²⁵I]ovine CRF have been extensively employed in previous studies of CRF₁ receptors, the use of a small molecule CRF₁ antagonist radioligand as a tool would permit investigation of interactions between CRF and small molecule antagonists at CRF₁ receptors and allow direct mapping of the small molecule binding sites in discrete brain regions.

The aim of the present studies is to describe the in vitro pharmacological properties of tritiated (±)-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003), a small-molecule radioligand for rat and human CRF₁ receptors. The binding characteristics of [³H]SN003 were profiled in rat cortical and human CRF₁ cell membranes, and the specificity and anatomic distribution of [³H]SN003 binding sites in rat brain were illustrated by brain section phosphorimaging. The in vitro pharmacological profile of the unlabeled SN003 ligand was also studied. Parts of these studies were previously presented in abstract form (Li et al., 2001; Zhang et al., 2001). This is the first report identifying a small molecule antagonist radioligand specifically labeling CRF₁ receptors in brain tissues and slices. This nonpeptide radioligand as a tool provides an opportunity to further understand the interactions of CRF and small molecule antagonists with CRF₁ receptors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals. Male Sprague-Dawley rats weighing 250 to 350 g were obtained from Charles River Laboratories, Inc. (Wilmington, MA). They were housed two per cage in a room with controlled illumination, humidity, and temperature. Food and water were provided ad libitum. All animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Protocols were approved by the Committee on Animal Care and Use of the Bristol-Myers Squibb Company.

Materials. SN003 was obtained by directed synthesis efforts (Bakthavatchalam et al., 1997) as were other small-molecule CRF₁ antagonists, DMP696 (He et al., 2000) and DMP904 (Gilligan et al., 2000). SN003, DMP696, DMP904, and CP-154,526 were synthesized by the Department of Chemical and Physical Sciences, Bristol-Myers Squibb Company. The chemical structures of the small molecule CRF₁ receptor antagonists are shown in Fig. 1. CRF-related peptides, human/rat CRF (hrCRF), ovine CRF (oCRF), sauvagine, urocortin I (human), urocortin I (rat), -helical CRF_9-41 (-helical CRF), and [D-Phe¹²,Nle^21,38,C^a-MeLeu³⁷]-CRF_12-41 (D-PheCRF), were purchased from American Peptide Co., Inc. (Sunnyvale, CA), Bachem California (Torrance, CA), and Peninsula Laboratories (Merseyside, UK). [¹²⁵I]oCRF and [¹²⁵I]sauvagine (specific activities, 2200 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Gpp(NH)p (5'-guanylylimidodiphosphate) and other standard reagents were purchased from Sigma-Aldrich (St. Louis, MO) and Invitrogen (Carlsbad, CA).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chemical structures of SN003 and other small molecule CRF1 receptor antagonists. A, SN003 [(±)-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c] pyridin-4-amine]; B, DMP696, DMP904, and CP-154,526.

Synthesis of [³H]SN003. [³H]SN003 was labeled by the radioligand synthesis facility in the drug metabolism group of the Bristol-Myers Squibb Company (former DuPont Pharmaceuticals Company).

Cell Culture of HEK293e Cells Expressing Human CRF Receptors. Full-length human cDNAs for human CRF₁ and CRF₂ receptors were subcloned into plasmids and transfected into HEK293EBNA (HEK293e) cells (Invitrogen) using lipofectamine (Invitrogen). The details of the plasmid construct were described in previous studies (Horlick et al., 1997; Kostich et al., 1998). HEK293e cells stably expressing human CRF₁ or CRF₂ receptors were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum at 37°C in a humid environment (5% CO₂) for 10 days. The cells were then adapted to spinner culture for bulk processing. Cells were harvested, washed in phosphate-buffered saline (PBS), and counted; and the cell pellet (containing approximately 1 × 10⁸ HEK293e cells) was stored at -80°C until use.

Peptide Radioligand Binding to Homogenates. For binding assays, the total particulate fraction of rat frontal cortex and pituitary tissues or cell pellets was prepared as a crude membrane source expressing CRF₁ or CRF₂ receptors. Frozen tissues or cell pellets were thawed on ice and homogenized in tissue buffer (containing 50 mM HEPES, 10 mM MgCl₂, 2 mM EGTA, and 1 µg/ml each of aprotonin, leupeptin, and pepstatin, pH 7.0 at 23°C) using a Brinkmann Instruments (Westbury, NY) Polytron (PT-10, setting 6 for 10 s). The homogenate was centrifuged at 48,000g for 12 min, and the resulting pellet was washed by resuspension and centrifugation steps. The final pellet was suspended in tissue buffer, and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) with bovine serum albumin as standard. The membrane proteins prepared from human CRF₁ HEK293e cells (5-8 µg), rat cortex, pituitary, and human CRF₂ HEK293e cells, respectively (40-60 µg), were used in receptor binding experiments.

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[³H]SN003 Binding to Membranes. Equilibrium binding experiments in cell or tissue homogenates were performed under conditions similar to those described for the [^125I]oCRF binding with a few exceptions. GF/B filters were used in the filtration assay. The saturation experiments using [³H]SN003 as a radioligand and rat cortex and HEK293e cell homogenates as CRF₁ receptor sources were conducted in 12 concentrations of [³H]SN003 (0.60-40 nM) in triplicate at 23°C for 2 h. The nonspecific binding was defined in the presence of 5 µM DMP696. Association and dissociation assays were performed at the [³H]SN003 K_D concentration of 4.8 nM at 23°C. After 2 h of incubation, when association equilibrium was reached, dissociation reactions were initiated by addition of 5 µM DMP696 and continued for an additional 3 h.

cAMP Assays in Recombinant Human CRF₁ HEK293e Cells. Intracellular cAMP levels were measured using the Adenyl Cyclase Activation Flash Plate kit purchased from PerkinElmer Life Sciences. This radioimmunoassay-based kit enables direct detection of cAMP generated in live cells in a 96-well format. HEK293e cells expressing CRF₁ receptors were grown in the Dulbecco's modified Eagle's medium supplement with 10% fetal bovine serum, L-glutamine (2 mM), and hygromycin (400 µg/ml) at 37°C in a humid environment with 5% CO₂. On the assay day, cells were dissociated from flasks and centrifuged down at 1,200 rpm for 4 min. Cells were resuspended in 100% stimulation buffer, counted, and diluted to 0.6 × 10⁶ cell/ml. hrCRF (1 nM) in the absence and presence of SN003 in PBS containing 10% stimulation buffer (50 µl) was added to the assay plate. Drug treatment was initiated by adding HEK293e cells expressing CRF₁ receptors (50,000 cells/50 µl/well) to the Flash Plate and incubated for 15 min at 37°C in a final volume of 100 µl. Intracellular cAMP was released from cells through cell lysis resulting from adding detection buffer containing [¹²⁵I]cAMP (100 µl/well). Assay signal is based on competition of endogenous cAMP and [¹²⁵I]cAMP for cAMP antibodies coated on the Flash Plate. Radioactivity from binding of [¹²⁵I]cAMP to the plate was assessed 2 h later by a 96-well PerkinElmer Top Counter.

ACTH Release Assays in Rat Primary Pituitary Cultures. Primary pituitary culture was established as described previously (Vale et al., 1972). Pituitaries were harvested from 20 to 25 rats and washed in PBS four to six times. Pituitary cells were dissociated in collagenase buffer [1× PBS containing 25 mM HEPES, 0.2% glucose, 0.4% (w/v) bovine serum albumin, 80 µg/ml DNase II, and 0.4% (w/v) collagenase type 2] for 3 h at 37°C. Cells were spun down, decanted, and incubated with 0.25% trypsin solution [PBS containing 0.25% (w/v) trypsin, 0.4% (w/v) bovine serum albumin, 0.2% (w/v) glucose] for 5 to 10 min at 37°C. Cells were briefly centrifuged and resuspended in M199 culture medium (Invitrogen) containing 10% fetal calf serum, minimal essential medium vitamins, streptomycin/penicillin, insulin/transferrin/selenium, fibroblast growth factor, and trace elements. Cells (300,000/ml) were added to a 48-well plate (Costar) and grown in culture (37°C, 5% CO₂) for 4 days. On the assay day, medium was removed and replaced with M199 incubation medium identical to that used above except for exclusion of serum and addition of 0.1% (w/v) ovalbumin and 1 µg/ml aprotinin. The hrCRF-induced ACTH release was conducted at 37°C for 3 h. With pretreatment of various concentrations of SN003 or vehicle (control) to cells for 15 min, the antagonist assay of SN003 was performed by adding hrCRF (0.3 nM) and coincubation at 37°C for 3 h. After the incubation period, medium was removed, frozen, and stored at -20°C until assayed for ACTH measurement. ACTH levels were determined using a radioimmunoassay kit purchased from DiaSorin Inc. (Stillwater, MN).

Data Analyses. The concentrations of compounds to inhibit 50% of radioligand binding (IC₅₀) for CRF₁ and CRF₂ receptors were calculated by fitting data through a competition equation in the iterative nonlinear regression curve-fitting program Prism (GraphPad Software, Inc.). K_i values (equilibrium dissociation constant) for inhibitors in competition experiments were calculated according to the Cheng-Prusoff equation. Saturation data were fit through hyperbolic equations to estimate apparent equilibrium constant (K_D) and the maximal number of binding sites (B_max) using Prism (GraphPad Software, Inc.). Kinetic studies to determine k_obs (observed association rate constant) and k₁ (dissociation rate constant) were generated from a slope of the line via linear regression of transformed data. The association rate constant (k₊₁) was generated based on the equation k₊₁ = (k_obs  k₁/[L], where L is the ligand concentration. The equilibrium dissociation constant derived from kinetic studies was calculated as K_D = k₁/k₊₁.

Brain Section Ligand Binding and Storage Phosphorimaging. Rats were decapitated, and the brain and pituitary were immediately collected, embedded in M-1 embedding matrix (Thermo Shandon, Pittsburgh, PA), and frozen in iso-pentane chilled with dry ice. Twenty-micrometer coronal sections were cut on a Cryostat, thaw-mounted on superfrost slides (VWR, West Chester, PA), dried, and stored at -70°C until use. Before in vitro binding, sections were brought to 23°C and preincubated for 30 min in assay solution containing 50 mM HEPES, 10 mM MgCl₂, 2 mM EGTA, 100 kallikrein-inactivating units/ml aprotinin, 0.1 M bacitracin, and 0.1% ovalbumin (pH 7.2). Sections were then incubated in the same solution containing 4 to 10 nM [³H]SN003 for 2 h at 23°C. As a comparison, one set of adjacent sections was incubated with 200 pM [¹²⁵I]sauvagine. At this concentration (200 pM), [¹²⁵I]sauvagine binds to both CRF₁ and CRF₂ receptors (Rominger et al., 1998). Nonspecific binding was defined by inclusion of 1 µM DMP696. After incubation, sections were rinsed in PBS with 0.01% Triton X-100 for 10 min and subsequently dried under a stream of cold air. Slides of the sections were then placed in cassettes against storage phosphorimaging screens (PerkinElmer Life Sciences) for 1 to 4 weeks ([³H]SN003) or for 12 h ([¹²⁵I]sauvagine), respectively. The screens were then scanned with a Cyclone phosphorimaging scanner, and captured images were analyzed with the OptiQuant requisition and analysis system (PerkinElmer Life Sciences).

	Results

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Binding Affinity of SN003 for CRF₁ and CRF₂ Receptors. The chemical structure of SN003 is shown in Fig. 1A. The binding affinity of SN003 for CRF₁ receptors was determined by competition binding experiments using membranes prepared from HEK293e cells expressing human recombinant CRF₁ receptors, and rat cortex and pituitary membranes containing native CRF₁ receptors. Like the CRF peptide antagonist -helical CRF and the small molecule CRF₁ antagonist DMP696 (Fig. 2), SN003 potently and completely inhibited [¹²⁵I]oCRF (200 pM) binding to CRF₁ receptors, with maximal inhibition identical to that of -helical CRF and DMP696. The K_i value of SN003 for rat CRF₁ receptors detected in pituitary membranes was 3.4 ± 0.5-fold of that determined in cortical membranes in paired experiments (p = 0.001), as shown in Table 1. A similar shift was seen for DMP696 in pituitary and cortical tissues with a K_i ratio (pituitary/cortex) of 2.5 ± 0.6-fold (p = 0.017). In contrast, the peptide agonist ovine CRF exhibited an equal affinity for rat CRF₁ receptors in cortical (mean K_i = 0.6 nM) and pituitary (mean K_i = 0.7 nM) tissues (p = 0.38; paired Student's t test). Although the peptide antagonist -helical CRF appeared to have a small difference in potency between rat cortex (mean K_i = 5.3 nM) and pituitary (mean K_i = 8.1 nM) tissues, this difference was not statistically significant (p = 0.22), as summarized in Table 1.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Competition of [125I]oCRF binding to rat cortical and pituitary membranes by SN003, DMP696, -helical CRF, and hrCRF. The assays were performed in duplicate in final concentration of [125I]oCRF (200 pM) and 12 concentrations of displacers. Nonspecific binding was defined by 1 µM DMP696. The data are plotted and the respective curves are expanded from the parameter estimates derived from fitting a competition equation to the data by nonlinear regression analysis using Prism software.

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View larger version (20K): [in this window] [in a new window]   Fig. 3.   Selectivity profile of SN003 for human CRF1 and CRF2 receptors. Competition binding experiments were conducted using [125I]oCRF (150 pM) and [125I]sauvagine (150 pM) as radioligands to label human CRF1 (A) and CRF2 (B) receptors stably expressed in HEK293e cells, respectively. Nonspecific binding was defined by 1 µM DMP696 at CRF1 receptors or 1 µM -helical CRF at CRF2 receptors. Data presented are mean specific binding values of duplicate determinations from one of three to six experiments yielding similar results. IC50 and Ki values of compounds were derived from fitting a competition equation to the data by means of iterative nonlinear least squares regression analysis.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of SN003 on [125I]ovine CRF binding parameters in HEK293 cells expressing human CRF1 receptors. The homologous competition experiment was performed in recombinant CRF1 HEK293e cell homogenates, and saturation of [125I]ovineCRF (150 pM) labeled CRF1 binding sites was affected by increasing concentrations of unlabeled CRF in the absence (control) and presence of SN003. Nonspecific binding was defined by 1 µM DMP696. The data were transformed to representative Scatchard plots to visualize the KD (1/slope) and the Bmax (the x-intercept) changes in the top panel, that experiment was performed three times with essentially identical results. Data shown in the middle and bottom panels are mean ± S.E.M. values calculated from three independent experiments performed in triplicate.

Inhibition of CRF-Mediated cAMP Accumulation and ACTH Release by SN003. To evaluate functional activities of SN003 in vitro, SN003 was assessed for inhibition of CRF-mediated responses in rat pituitary and human CRF₁ HEK293e cells. In cell-based functional assays, the antagonist properties of SN003 in CRF-stimulated ACTH secretion in cultured rat pituitary cells were examined. In parallel, evaluation of the agonist concentration-response was included in each experiment (Fig. 5A). Basal secretion of ACTH was markedly enhanced in response to CRF in a concentration-dependent manner. CRF-induced maximal stimulation of ACTH release (3400-4093 pg/100-µl sample), which is 5- to 9-fold of the basal level (400-520 pg/100-µl sample), was achieved at 10 nM CRF during a 3-h incubation (n = 4). The mean EC₅₀ ± S.E.M. of CRF for stimulation of pituitary ACTH secretion was calculated as 0.30 ± 0.05 nM (n = 4). As shown in Fig. 5B, SN003 dose dependently suppressed CRF (0.3 nM)-induced ACTH release with an IC₅₀ value of 241.5 ± 48.4 nM (n = 4) and completely abolished CRF-stimulated ACTH secretion at higher concentrations, suggesting full antagonist properties at pituitary CRF₁ receptors. To test potential agonist activity, SN003 (1 and 10 µM), exposed alone to pituitary cells for 3 h, did not alter basal secretion of ACTH from cells (data not shown), indicating a lack of partial or inverse agonist activities.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   SN003 inhibition of CRF-stimulated ACTH release from primary rat pituitary cultures. The agonist and antagonist assays were parallel for each experiment. Data in the graph are the means ± S.E.M. of four independent experiments performed in triplicate. A, the agonist concentration-response relationship of hrCRF-induced ACTH release. The raw data are normalized to the percentage of maximum stimulation provided by the highest concentration of hrCRF, and the basal level of ACTH secretion with vehicle treatment is defined as 0% stimulation. B, the antagonism of CRF (0.3 nM)-stimulated ACTH secretion by SN003. Rat pituitary cells were exposed to CRF (0.3 nM) and/or various concentrations of SN003 for 3 h at 37°C. ACTH levels were normalized to percentage of control (0.3 nM hrCRF alone). The mean ± S.E.M. values were obtained with a statistical analysis of four individual EC50 or IC50 determinations.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Antagonism of CRF-induced cAMP accumulation in HEK293e cells expressing human CRF1 receptors by SN003. CRF-elicited intracellular cAMP elevation was determined in the absence and presence of coadministered concentrations of SN003 after 15 min incubation at 37°C. Data in the graph are the mean percentage of stimulated cAMP accumulation, which was normalized as 100% of control maximal response obtained with 30 nM CRF, and basal cAMP level was defined as 0% stimulation in the absence of CRF. Statistical difference in the presence of SN003 from control CRF (1 nM) responses was assessed by Student's t test; *, p < 0.05 was considered as significant. **, p <0.01; ***, p <0.001.

Characterization of [³H]SN003 Binding to Membranes Expressing CRF₁ Receptors. To examine the binding characteristics of [³H]SN003 to CRF₁ receptors, a time course of [³H]SN003 (~4.8 nM) equilibrium binding was performed in rat frontal cortex homogenates at 23°C. Under these conditions, [³H]SN003 binding was time-dependent (Fig. 7), and its association reaction (>99%) was completed by 120 min. Nonspecific binding was increased slightly within 5 min and remained constant, representing approximately 50% of total binding. The mean ± S.E.M. value for k_obs (1.62 ± 0.3 × 10² min¹) and the corresponding t_1/2 of 43 min was determined based on linear regression analysis of data from three independent experiments. Subsequent saturation experiments were conducted using a 120-min incubation time.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Equilibrium association binding of [3H]SN003 to rat frontal cortical membrane. Time course of [3H]SN003 binding to rat cortical homogenates was performed using the radioligand concentration of 4.8 nM at 23°C. Nonspecific binding was defined at each time point by 5 µM DMP696. Inset, linear transformation of specific association data, where Beq represents specific binding at the steady state and Bt represents the specific binding at time t. The graph shows the representative data of three independent measurements performed in triplicate.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Saturation isotherm of [3H]SN003 binding to membranes from rat frontal cortex (A) and HEK293e cells expressing human CRF1 receptors (B). Data presented are from one of three independent experiments performed in triplicate. KD and Bmax values were determined from nonlinear regression analysis of the data using the one-site model that fits the data significantly better than the two-site model. Inset, Scatchard plot of transformed saturation data. Nonspecific binding was defined as that occurring in the presence of 5 µM DMP696.

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View larger version (25K): [in this window] [in a new window]   Fig. 9.   Kinetic studies of [3H]SN003 binding to membrane prepared from HEK293e cells recombinantly expressing human CRF1 receptors. A, equilibrium association of [3H]SN003 binding to human CRF1 receptors. Inset: linear conversion of association data, where Beq represents specific binding at the steady state, and Bt represents the specific binding at time t. B, dissociation of [3H]SN003 binding to human CRF1 receptors where (inset) Bt represents specific binding at time t and B0 represents binding at equilibrium. Kinetic data were fit to mono- and biexponential curves by weighted nonlinear curve-fitting; in all cases the biexponential model did not give a significant improvement over the monoexponential model (p > 0.05, F-test). Nonspecific binding was defined at each time point by 5 µM DMP696. Data shown are the mean from three independent experiments performed in triplicate.

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Effects of the GTP analog on [3H]SN003 and [125I]ovine CRF binding to human CRF1 receptors. [3H]SN003 (5 nM) and [125I]oCRF (150 pM) binding to human CRF1 receptors expressed in HEK293e cell membranes was examined in the absence (control) and presence of 50 µM Gpp(NH)p, respectively. Nonspecific binding was defined by 5 µM DMP696. Data presented as the bar are the mean ± S.E.M. values from four experiments performed in triplicate. ***, significant difference from control (p < 0.001) determined by Student's t test.

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Competition of [3H]SN003 binding to human CRF1 receptors expressed in HEK293e cell membranes by small molecule CRF1 antagonists and CRF peptides. Nonspecific binding was defined by 1 µM DMP696. Shown is a representative measurement of six to nine independent experiments performed in duplicate.

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Anatomical Distribution of [³H]SN003 Binding Sites. The distribution of specific [³H]SN003 binding sites in slide-mounted rat brain sections was assessed and compared with that of [¹²⁵I]sauvagine binding sites. Figure 12A exhibits storage phosphorimages of [³H]SN003 binding in the absence and presence of 1 µM DMP696. Specific [³H]SN003 binding was measured by digitally subtracting nonspecific binding (Fig. 12A, right panel), defined by 1 µM DMP696, from total binding (Fig. 12A, left panel). In the cortex and other brain regions, in which there were higher levels of binding, specific binding accounted for 50 to 60% of the total binding. [³H]SN003 binding sites appeared as granular particles with space resolution close to that labeled with [¹²⁵I]sauvagine but not in lateral septal nucleus and choroid plexus.

View larger version (95K): [in this window] [in a new window]   Fig. 12.   Storage phosphate images showing [3H]SN003 binding sites in the rat brain. A, [3H]SN003 binding to coronal sections of the rat brain in the absence (left panel) and presence (right panel) of 1 µM DMP696. B, distribution pattern of [3H]SN003 binding sites in the brain slice section. Olf, olfactory bulb; FC, frontal cortex; CP, caudate-putamen; Pi, pituitary; PC, parietal cortex; Hi, hippocampus, Th, thalamus; PAG, periaqueductal gray; Ceb, cerebellum. Scale bar = 2 mm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study describes the in vitro pharmacological characteristics of a novel nonpeptide radioligand, [³H]SN003, for the rat and human CRF₁ receptors. SN003 is a high-affinity and selective ligand for rat and human CRF₁ receptors having more than 1000-fold selectivity over human CRF₂ receptors. SN003 in vitro functions as a CRF₁ receptor antagonist against CRF-mediated responses. Equilibrium binding studies revealed that specific binding of tritium-labeled SN003 to CRF₁ receptors was saturable, reversible, and of high affinity. Antagonist characteristics of [³H]SN003 were also suggested by a single class of [³H]SN003 binding sites and insensitivity to guanine nucleotides. The distribution pattern of [³H]SN003 binding sites was consistent with CRF₁ receptor mapping in the brain, further supporting the specific labeling of CRF₁ receptors by [³H]SN003.

Both SN003 and DMP696 had 2- to 3-fold higher affinities (lower K_i values) for rat CRF₁ receptors in the frontal cortex than in the pituitary and in recombinant CRF₁ receptors expressed in HEK293e cells. The lower potency of small molecules against CRF binding in the pituitary and the recombinant cell line may be in part attributable to the higher density of CRF₁ receptors in the membranes from these receptor sources. On the other hand, the disparity in inhibition of CRF binding observed for small molecules in these two rat tissues may reflect a differential nature of CRF₁ receptors expressed in various cell types. The differential effects of the small molecule antagonists on CRF binding in rat pituitary and cortical tissues could be the result of differing post-translational modification or accessory proteins of CRF₁ receptors from various cell types (Grigoriadis and De Souza, 1988). Differential glycosylation of CRF₁ receptor proteins in anterior pituitary and brain tissues during post-translational modification (Grigoriadis and De Souza, 1989) has been demonstrated. Additional experiments are required to determine whether differential post-translational modifications of CRF₁ receptors have functional consequences.

In functional assays of CRF-mediated ACTH release and cAMP accumulation, SN003 completely antagonized CRF effects without partial or inverse agonist properties. We noted that the functional potency of SN003 as determined in the ACTH assay was approximately 10-fold lower than its binding affinity at rat pituitary CRF₁ receptors. This weaker potency in functional assays is also seen with other GPCR antagonists and, more specifically, CRF₁ antagonists (Bymaster and Falcone, 2000; Heinrichs et al., 2002). The discrepancy between binding and functional potencies of SN003 is not well understood. It could result, in part, from different assay conditions in the functional and binding assays, in particular, the use of intact cells versus membrane preparations and physiological buffer versus hypotonic buffer (Bymaster and Falcone, 2000). Besides assay conditions, differential properties of small molecule CRF antagonists such as their binding affinity and coupling, as well as competitive versus noncompetitive nature, may also have an impact on the potency of the difference between binding and functional activity of CRF ligands in the hypothalamic-pituitary-adrenal axis.

The present work suggests that SN003 is not simply competitive with CRF at the CRF₁ receptor. The maximal number of binding sites for CRF is significantly reduced by SN003 in a concentration-dependent manner, indicative of a noncompetitive interaction. Incomplete reciprocal binding inhibition of peptide agonists and the small molecule antagonist also suggests that the interaction is not simply competitive at CRF₁ receptors; at least SN003 may provide mixed competitive and/or noncompetitive inhibition. Partial inhibition could result from allosteric modulation and noncompetitive inhibition by small molecules or accessibility of peptides to the small molecule binding domains. The underlying mechanisms need to be explored in a future investigation.

These data indicate that the recognition sites for small molecule antagonists and peptide agonists are not mutually exclusive on CRF₁ receptors. In general, the peptide agonist binding domain of class B receptors of the GPCR family, such as CRF₁ receptors, is predominantly formed from the large extracellular N-terminal domain and portions of the extracellular loops of the receptors. Small molecule CRF₁ antagonists appear to bind to transmembrane sites in a pocket formed by helices III to VII (Liaw et al., 1997). It is possible that, depending on the affinity state of the receptor, limited overlap of binding valencies may result in a low-affinity interaction of the N-terminal sequence of CRF with the small molecule binding site. The fact that CRF peptide antagonists without N-terminal amino acids fail to inhibit [³H]SN003 binding suggests that it is the N-terminus of CRF that interacts with the small molecule recognition sites of the receptor, possibly sharing binding valences in the transmembrane region. The findings of previous studies demonstrating that the N-terminus of CRF is important for receptor activation (Vale et al., 1981; De Souza, 1987; Beyermann et al., 1996) may be relevant to the present findings in which the N-terminus of CRF was also crucial for CRF inhibition of small molecule binding.

Further studies employing radiolabeled CRF peptides and small molecule antagonists will greatly aid understanding of molecular aspects of CRF₁ receptor signaling and small molecule antagonism. Additionally, this information may have implications for other G protein-coupled receptors with endogenous peptide agonists. This does appear to be the case for the neurokinin-1 and -2 receptors, since partial inhibition and low potencies of endogenous peptides, substance P and neurokinin A, in displacement of small molecule antagonist radioligand binding (Rosenkilde et al., 1994) have been observed with those receptors. Considered together, the data suggest that the interactions between small molecules are more competitive than that between the small molecule and peptide, and binding domains for peptides and small molecule antagonists are not mutually exclusive.

Two nonpeptide CRF₁ antagonists, CP-154,526 and an antalarmin analog, have recently been radiolabeled (Tian et al., 2001; Keller et al., 2002). The tritium-labeled CP-154,526 was used for assessing pharmacokinetics and blood-brain barrier penetration, and the antalarmin analog was developed as a positron emission tomography ligand. However, in these studies there are no descriptions of radioligand binding profiles in either tissues or cells expressing CRF₁ receptors. Tritiated SN003 is the first nonpeptidic CRF₁ antagonist radioligand shown to be capable of detecting specific binding in rat brain tissues. The binding pattern of [³H]SN003 in the brain was consistent with the distribution of CRF₁ receptors revealed previously by binding autoradiography using the radioligand [¹²⁵I]oCRF (De Souza et al., 1985; Aguilera et al., 1987) or by in situ hybridization of CRF₁ mRNA (Potter et al., 1994). The sensitivity of [³H]SN003 binding was moderate considering that it was 50 to 60% specific over total binding in both the homogenized membrane and slide-mounted brain section binding assays. The moderate sensitivity was, perhaps, caused by several factors, including the "sticky" lipophilic property of SN003 and the low energy status of the labeled isotope tritium. In addition, storage phosphorimaging used in the autoradiography study may have compromised the sensitivity in terms of space resolution. However, this technique proved to be very effective for acquiring images marked with tritium-labeled ligands such as [³H]SN003 in as little as 7 days, compared with longer exposure times required for autoradiographic films. These studies may provide a foundation for studying in vivo binding of CRF₁ antagonists and developing high-energy isotope-labeled, small molecule ligands with applications in clinical positron emission tomography/single photon emission computed tomography studies of CRF₁ antagonists.

In summary, we describe the in vitro characterization of a high-affinity, selective nonpeptidic antagonist radioligand, [³H]SN003, for CRF₁ receptors. Since this radioligand possesses an excellent signal/noise ratio in a recombinant human CRF₁ cell line and distinct features compared with peptide ligands, it provides a useful tool to understand small molecule antagonism of CRF at CRF₁ receptors. This information may prove helpful in drug design and development of small molecule CRF₁ antagonists for treatment of affective disorders and stress-related diseases.