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 ([H]SN003) for Corticotropin-Releasing Factor1 Receptors

The in vitro pharmacological profile of a novel small molecule corticotropin-releasing factor 1 (CRF1) receptor antagonist, ( )N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl)propyl)6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine (SN003), and the characteristics of its radioligand ([H]SN003) are described. SN003 has high affinity and selectivity for CRF1 receptors expressed in rat cortex, pituitary, and recombinant HEK293EBNA (HEK293e) cells with respective radiolabeled ovine CRF ([I]oCRF) binding Ki values of 2.5, 7.9, and 6.8 nM. SN003 was shown to be a CRF1 receptor antagonist inasmuch as it inhibited CRF-induced cAMP accumulation in human CRF1HEK293e cells and CRF-stimulated adrenocorticotropin hormone release from rat pituitary cells without agonist activities. Significant decreases in the Bmax of [ I]oCRF binding by SN003 suggest that this antagonist is not simply competitive. To further explore the interaction of SN003 with the CRF1 receptors, [H]SN003 binding to rat cortex and human CRF1HEK293e cell membranes was characterized and shown to be reversible and saturable, with KD values of 4.8 and 4.6 nM, and Bmax values of 0.142 and 7.42 pmol/mg protein, respectively. The association and dissociation rate constants of [H]SN003 (k 1 0.292 nM 1 min 1 and k 1 0.992 10 2 min ) were also assessed using human CRF1HEK293e cell membranes, giving an equilibrium dissociation constant of 3.4 nM. Moreover, [H]SN003 binding displayed a single affinity state and insensitivity to 5 -guanylylimidodiphosphate, consistent with characteristics of antagonist binding. Incomplete inhibition of [H]SN003 binding by CRF peptides also suggests that SN003 is not simply competitive with CRF at CRF1 receptors. The distribution of [H]SN003 binding sites was consistent with the expression pattern of CRF1 receptors in rat brain regions. Small molecule CRF1 antagonist radioligands like [H]SN003 should enable a better understanding of small molecule interactions with the CRF1 receptor. 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, imArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.046128. ABBREVIATIONS: CRF, corticotropin-releasing factor; ACTH, adrenocorticotropin hormone; CP-154,526, butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]ethylamine; SN003, ( )-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3triazolo[4,5-c] pyridin-4-amine; NBI 27914, 5-chloro-N-(cyclopropylmethyl)-2-methyl-N-propyl-N -(2,4,6-trichlorophenyl)-4,6-pyrimidinediamine hydrochloride; SSR125543A, 4-(2-chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thiazol-2-amine hydrochloride; hrCRF, human/rat corticotropin-releasing factor; oCRF, ovine corticotropin-releasing factor; -helical CRF, -helical corticotropin-releasing factor9 – 41; D-PheCRF, [D-Phe ,Nle,C-MeLeu]-CRF12– 41; Gpp(NH)p, 5 guanylylimidodiphosphate; HEK, human embryonic kidney; PBS, phosphate-buffered saline; DMP696, 4-(1,3-dimethoxyprop-2-ylamino)-2,7dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine; DMP904, 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo[1,5-a]-pyrimidine; R-121919, 3-[6-dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimidin-7-amine. 0022-3565/03/3051-57–69$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 46128/1052158 JPET 305:57–69, 2003 Printed in U.S.A. 57 at A PE T Jornals on A uust 6, 2017 jpet.asjournals.org D ow nladed from mune, 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 Gs 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 [I]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-4methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1H1,2,3-triazolo[4,5-c]pyridin-4-amine ([H]SN003), a smallmolecule radioligand for rat and human CRF1 receptors. The binding characteristics of [H]SN003 were profiled in rat cortical and human CRF1 cell membranes, and the specificity and anatomic distribution of [H]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 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 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-4amine]; B, DMP696, DMP904, and CP-154,526. 58 Zhang et al. at A PE T Jornals on A uust 6, 2017 jpet.asjournals.org D ow nladed from CRF9 – 41 ( -helical CRF), and [D-Phe ,Nle,C-MeLeu]-CRF12–41 (D-PheCRF), were purchased from American Peptide Co., Inc. (Sunnyvale, CA), Bachem California (Torrance, CA), and Peninsula Laboratories (Merseyside, UK). [I]oCRF and [I]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). Synthesis of [H]SN003. [H]SN003 was labeled by the radioligand synthesis facility in the drug metabolism group of the BristolMyers Squibb Company (former DuPont Pharmaceuticals Compa-