Pharmacological characterization of a novel nonpeptide antagonist radioligand, (±)-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([3H]SN003) for corticotropin-releasing factor receptors

Article abstract of DOI:10.1124/jpet.102.046128

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 ([³H]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 ([¹²⁵I]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 B_max of [¹²⁵I]oCRF binding by SN003 suggest that this antagonist is not simply competitive. To further explore the interaction of SN003 with the CRF₁ receptors, [³H]SN003 binding to rat cortex and human CRF₁HEK293e cell membranes was characterized and shown to be reversible and saturable, with K_D values of 4.8 and 4.6 nM, and B_max values of 0.142 and 7.42 pmol/mg protein, respectively. The association and dissociation rate constants of [³H]SN003 (k₊₁ 0.292 nM^-1 min^-1 and k_-1 0.992 × 10^-2 min^-1) were also assessed using human CRF₁EK293e cell membranes, giving an equilibrium dissociation constant of 3.4 nM. Moreover, [³H]SN003 binding displayed a single affinity state and insensitivity to 5′-guanylylimidodiphosphate, consistent with characteristics of antagonist binding. Incomplete inhibition of [³H]SN003 binding by CRF peptides also suggests that SN003 is not simply competitive with CRF at CRF₁ receptors. The distribution of [³H]SN003 binding sites was consistent with the expression pattern of CRF₁ receptors in rat brain regions. Small molecule CRF₁ antagonist radioligands like [³H]SN003 should enable a better understanding of small molecule interactions with the CRF₁ receptor.

Full text of DOI:10.1124/jpet.102.046128

0
022-3565/03/3051-57–69$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 305:57–69, 2003
Vol. 305, No. 1
46128/1052158
Printed in U.S.A.
Pharmacological Characterization of a Novel Nonpeptide
Antagonist Radioligand, (Ϯ)-N-[2-Methyl-4-methoxyphenyl]-1-
(
1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-
3
c]pyridin-4-amine ([ H]SN003) for Corticotropin-Releasing
Factor Receptors
1
GE ZHANG, NING HUANG, YU-WEN LI, XIAOPING QI, ANNE P. MARSHALL, XIAO-XIN YAN, GERALDINE HILL,
CYNTHIA ROMINGER, SHIMOGA R. PRAKASH, RAJAGOPAL BAKTHAVATCHALAM, DAVID H. ROMINGER,
PAUL J. GILLIGAN, and ROBERT ZACZEK
CNS Diseases Research (G.Z., N.H., Y.-W.L., X.Q., A.P.M., X.-X.Y., G.H., C.R., D.H.R., R.Z.), Drug Metabolism (S.R.P.), and Chemical and
Physical Sciences (R.B., P.J.G.), the Bristol-Myers Squibb Pharmaceuticals Research Institute, Wilmington, Delaware
Received October 29, 2002; accepted January 03, 2003
ABSTRACT
The in vitro pharmacological profile of a novel small molecule CRF HEK293e cell membranes was characterized and shown
1
corticotropin-releasing factor 1 (CRF ) receptor antagonist, (Ϯ)- to be reversible and saturable, with KD values of 4.8 and 4.6
1
N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl)propyl)- nM, and Bmax values of 0.142 and 7.42 pmol/mg protein, re-
6
-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine (SN003), and spectively. The association and dissociation rate constants of
3 3 Ϫ1 Ϫ1 Ϫ2
ϩ1
the characteristics of its radioligand ([ H]SN003) are described. [ H]SN003 (k
SN003 has high affinity and selectivity for CRF receptors ex- min ) were also assessed using human CRF HEK293e cell
0.292 nM
min
and kϪ1 0.992 ϫ 10
Ϫ1
1
1
pressed in rat cortex, pituitary, and recombinant HEK293EBNA membranes, giving an equilibrium dissociation constant of 3.4
3
(
(
HEK293e) cells with respective radiolabeled ovine CRF nM. Moreover, [ H]SN003 binding displayed a single affinity
[
125
I]oCRF) binding K values of 2.5, 7.9, and 6.8 nM. SN003 state and insensitivity to 5Ј-guanylylimidodiphosphate, consis-
i
was shown to be a CRF receptor antagonist inasmuch as it tent with characteristics of antagonist binding. Incomplete in-
inhibited CRF-induced cAMP accumulation in human hibition of [ H]SN003 binding by CRF peptides also suggests
1
3
CRF HEK293e cells and CRF-stimulated adrenocorticotropin that SN003 is not simply competitive with CRF at CRF recep-
1
1
3
hormone release from rat pituitary cells without agonist activi- tors. The distribution of [ H]SN003 binding sites was consistent
ties. Significant decreases in the Bmax of [ I]oCRF binding by with the expression pattern of CRF1 receptors in rat brain
SN003 suggest that this antagonist is not simply competitive. regions. Small molecule CRF1 antagonist radioligands like
To further explore the interaction of SN003 with the CRF1 [ H]SN003 should enable a better understanding of small mol-
receptors, [ H]SN003 binding to rat cortex and human ecule interactions with the CRF receptor.
125
3
3
1
Corticotropin-releasing factor (CRF) was first isolated from In the past 10 years, considerable progress has been made in
ovine hypothalamus (Vale et al., 1981) and identified as a key understanding the physiological and potential pathological
secretagogue for ACTH release from the anterior pituitary. 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
Article, publication date, and citation information can be found at
of other central and peripheral functions including arousal,
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.046128.
anxiety-like behaviors, learning and memory, feeding, im-
ABBREVIATIONS: CRF, corticotropin-releasing factor; ACTH, adrenocorticotropin hormone; CP-154,526, butyl-[2,5-dimethyl-7-(2,4,6-trimethyl-
phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]ethylamine; SN003, (Ϯ)-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-
triazolo[4,5-c] pyridin-4-amine; NBI 27914, 5-chloro-N-(cyclopropylmethyl)-2-methyl-N-propyl-NЈ-(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-meth-
yl-N-(2-propynyl)-1,3-thiazol-2-amine hydrochloride; hrCRF, human/rat corticotropin-releasing factor; oCRF, ovine corticotropin-releasing factor;
1
2
21,38
a
37
␣
-helical CRF, ␣-helical corticotropin-releasing factor9 – 41
;
D-PheCRF, [D-Phe ,Nle
,C -MeLeu ]-CRF12– 41; Gpp(NH)p, 5Ј-
guanylylimidodiphosphate; HEK, human embryonic kidney; PBS, phosphate-buffered saline; DMP696, 4-(1,3-dimethoxyprop-2-ylamino)-2,7-
dimethyl-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.
57

5
8
Zhang et al.
mune, and autonomic functions (Owens and Nemeroff, 1991; pharmacological properties of tritiated (Ϯ)-N-[2-methyl-4-
De Souza and Grigoriadis, 1998; Gilligan et al., 2000; Daut- methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1H-
3
zenberg and Hauger 2002).
1,2,3-triazolo[4,5-c]pyridin-4-amine ([ H]SN003), a small-
The entire spectrum of CRF peptide effects is mediated molecule radioligand for rat and human CRF receptors. The
1
3
through two known receptor subtypes, CRF1 and CRF2. binding characteristics of [ H]SN003 were profiled in rat
Both receptor subtypes belong to the class B family of G cortical and human CRF cell membranes, and the specificity
1
3
protein-coupled receptors that include receptors for secre- and anatomic distribution of [ H]SN003 binding sites in rat
tin and parathyroid hormone, among others (review Daut- brain were illustrated by brain section phosphorimaging. The
zenberg and Hauger, 2002). Despite their high degree of in vitro pharmacological profile of the unlabeled SN003 li-
sequence homology and their common coupling through Gs gand was also studied. Parts of these studies were previously
proteins to cAMP signaling, CRF1 and CRF2 receptors presented in abstract form (Li et al., 2001; Zhang et al.,
differ markedly from each other in pharmacological prop- 2001). This is the first report identifying a small molecule
erties and anatomic distribution (DeSouza et al., 1998; antagonist radioligand specifically labeling CRF receptors
1
Gilligan et al., 2000; Dautzenberg and Hauger, 2002). in brain tissues and slices. This nonpeptide radioligand as a
CRF receptors are widely distributed in the central ner- tool provides an opportunity to further understand the inter-
1
vous system. There exist three splice variants of the CRF2 actions of CRF and small molecule antagonists with CRF1
receptor (CRF2␣, CRF2␤, and CRF2␥) with distinct ana- receptors.
tomic 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,
Materials and Methods
Animals. Male Sprague-Dawley rats weighing 250 to 350 g
lung, and skeletal muscle. CRF and CRF receptors are
1
2
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 accor-
dance with the National Institutes of Health Guide for the Care
activated by several related peptides identified from vari-
ous 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 CRF and CRF receptors. and Use of Laboratory Animals. Protocols were approved by the
1
2
Studies of the neuronal circuitry mediating fear and Committee on Animal Care and Use of the Bristol-Myers Squibb
anxiety states (Davis, 1992) suggest that both CRF and Company.
1
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 (Gilli-
gan et al., 2000). SN003, DMP696, DMP904, and CP-154,526 were
synthesized by the Department of Chemical and Physical Sci-
ences, Bristol-Myers Squibb Company. The chemical structures of
CRF receptors located in differential brain areas may be
2
(
involved in the regulation of various stress-induced behav-
iors, 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 neu-
ropsychiatric disorders such as anxiety and depression
the small molecule CRF receptor antagonists are shown in Fig. 1.
1
CRF-related peptides, human/rat CRF (hrCRF), ovine CRF
(
Gilligan et al., 2000; Keck and Holsboer, 2001). Numerous (oCRF), sauvagine, urocortin I (human), urocortin I (rat), ␣-helical
animal studies using CRF ligands and genetically altered
mice provide strong evidence for the role of CRF receptors
1
in the coordination of the behavioral response to stress and
in stress-related psychiatric disorders (Gilligan et al.,
2
000; Bakshi et al., 2002; Dautzenberg and Hauger, 2002).
In recent years there has been much emphasis on devel-
oping orally active, nonpeptidic CRF antagonists to eval-
1
uate the putative role of CRF receptors in psychopathol-
1
ogy and to test their potential as novel therapeutic agents.
Schulz et al. (1996) were the first to report a pyrazolopy-
rimidine CRF antagonist, CP-154,526, with high affinity
1
for the CRF receptor and anxiolytic activity in rats. Ad-
1
ditional CRF1 antagonists, such as antalarmin, NBI
2
7914, DMP696, R121919, and SSR125543A, also exhibit
CRF antagonist properties in vitro and in vivo (He et al.,
1
2
000; 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
1
25
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 inter-
actions between CRF and small molecule antagonists at
CRF1 receptors and allow direct mapping of the small
molecule binding sites in discrete brain regions.
Fig. 1. Chemical structures of SN003 and other small molecule CRF1
receptor antagonists. A, SN003 [(Ϯ)-N-[2-methyl-4-methoxyphenyl]-1-(1-
(
methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c] pyridin-4-
The aim of the present studies is to describe the in vitro amine]; B, DMP696, DMP904, and CP-154,526.

A Nonpeptide CRF1 Antagonist Radioligand
59
1
2
21,38
a
37
CRF9–41 (␣-helical CRF), and [D-Phe ,Nle
D-PheCRF), were purchased from American Peptide Co., Inc. (Sunny- CRF2␣ receptors, respectively. Binding assays were terminated by
vale, CA), Bachem California (Torrance, CA), and Peninsula Labora- rapid filtration over GF/C glass-fibers [presoaked in 0.3% (v/v)
,C -MeLeu ]-CRF12–41 of 1 ␮M DMP696 for CRF1 receptors and 1 ␮M ␣-helical CRF for
(
1
25
125
tories (Merseyside, UK). [ I]oCRF and [ I]sauvagine (specific polyethyleneimine for more than 2 h] using a 96-well PerkinElmer
activities, 2200 Ci/mmol) were purchased from PerkinElmer Life Unifilter harvester followed by three washes with 0.3 ml of cold
Sciences (Boston, MA). Gpp(NH)p (5Ј-guanylylimidodiphosphate) wash buffer (PBS, pH 7.0, containing 0.01% Triton X-100). After
and other standard reagents were purchased from Sigma-Aldrich the filter was dried, scintillation cocktail was added and the plate
(
St. Louis, MO) and Invitrogen (Carlsbad, CA).
was counted in a 96-well PerkinElmer Top Counter. The CRF1
Synthesis of [ H]SN003. [ H]SN003 was labeled by the radioli- competition binding to membranes from rat frontal cortex and
gand synthesis facility in the drug metabolism group of the Bristol- pituitary was performed similarly except for the radioligand con-
3
3
1
25
Myers Squibb Company (former DuPont Pharmaceuticals Compa- centration of [ I]oCRF (200 pM) used in the binding.
ny). To determine the nature of inhibition (competitive and noncom-
Compound 1 (ST613) was prepared from Compound 2a (SN003) by petitive) of SN003, homologous isotopic displacement of [ I]oCRF
demethylation with sodium thiomethoxide in dimethylformamide (Fig. (150 pM) binding by ovine CRF was conducted in membranes pre-
125
1
A). To a solution of compound 1 in dimethylformamide, potassium pared from HEK293e cells expressing human CRF receptors in the
1
carbonate was added and the mixture was stirred. Tritium-labeled absence and presence of SN003. The KD and Bmax values from ho-
methyl iodide (250 mCi, 80 Ci/mmol) in toluene was transferred to this mologous competition curves were calculated using the nonlinear
reaction mixture, followed by rinsing, extraction, and separation by regression analysis in Prism (1999; GraphPad Software, Inc., San
chromatography on a reversed-phase column (Vydac Protein-peptide Diego, CA). A Scatchard plot of the homologous binding with
1
25
C18 Semiprep column). Fractions containing the product were lyophi-
[
I]oCRF was generated for visualization of any KD or Bmax
3
lized to provide radiochemically pure compound 2 ([ H]SN003, purity changes. Specific bound (femtomoles per milligram of protein) and
Ͼ99%). [ H]SN003 was dissolved and stored as an ethanolic solution Bound/Free ligand data were transformed. These transformations
3
(1mCi/ml; specific activity 74 Ci/mmol).
were performed according to similar procedures described in Prism
Cell Culture of HEK293e Cells Expressing Human CRF Re- (1999; GraphPad Software, Inc.).
3
ceptors. Full-length human cDNAs for human CRF1 and CRF2␣ re-
[ H]SN003 Binding to Membranes. Equilibrium binding exper-
ceptors were subcloned into plasmids and transfected into iments in cell or tissue homogenates were performed under condi-
125I
HEK293EBNA (HEK293e) cells (Invitrogen) using lipofectamine (In- tions similar to those described for the [
]oCRF binding with a few
vitrogen). The details of the plasmid construct were described in previ- exceptions. GF/B filters were used in the filtration assay. The satu-
3
ous studies (Horlick et al., 1997; Kostich et al., 1998). HEK293e cells ration experiments using [ H]SN003 as a radioligand and rat cortex
stably expressing human CRF1 or CRF2␣ receptors were grown in and HEK293e cell homogenates as CRF receptor sources were con-
1
3
Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine ducted in 12 concentrations of [ H]SN003 (0.60–40 nM) in triplicate
serum at 37°C in a humid environment (5% CO ) for 10 days. The cells at 23°C for 2 h. The nonspecific binding was defined in the presence
2
were then adapted to spinner culture for bulk processing. Cells were of 5 ␮M DMP696. Association and dissociation assays were per-
3
harvested, washed in phosphate-buffered saline (PBS), and counted; formed at the [ H]SN003 KD concentration of 4.8 nM at 23°C. After
8
and the cell pellet (containing approximately 1 ϫ 10 HEK293e cells) 2 h of incubation, when association equilibrium was reached, disso-
was stored at –80°C until use.
ciation reactions were initiated by addition of 5 ␮M DMP696 and
Peptide Radioligand Binding to Homogenates. For bind- continued for an additional 3 h.
ing assays, the total particulate fraction of rat frontal cortex and
cAMP Assays in Recombinant Human CRF1 HEK293e
pituitary tissues or cell pellets was prepared as a crude membrane Cells. Intracellular cAMP levels were measured using the Adenyl
source expressing CRF or CRF receptors. Frozen tissues or cell Cyclase Activation Flash Plate kit purchased from PerkinElmer
1
2␣
pellets were thawed on ice and homogenized in tissue buffer
Life Sciences. This radioimmunoassay-based kit enables direct
(
containing 50 mM HEPES, 10 mM MgCl , 2 mM EGTA, and 1 detection of cAMP generated in live cells in a 96-well format.
2
␮
g/ml each of aprotonin, leupeptin, and pepstatin, pH 7.0 at 23°C) HEK293e cells expressing CRF1 receptors were grown in the
using a Brinkmann Instruments (Westbury, NY) Polytron (PT-10, Dulbecco’s modified Eagle’s medium supplement with 10% fetal
setting 6 for 10 s). The homogenate was centrifuged at 48,000g for bovine serum, L-glutamine (2 mM), and hygromycin (400 ␮g/ml) at
1
2 min, and the resulting pellet was washed by resuspension and 37°C in a humid environment with 5% CO . On the assay day,
2
centrifugation steps. The final pellet was suspended in tissue cells were dissociated from flasks and centrifuged down at 1,200
buffer, and protein concentrations were determined using the rpm for 4 min. Cells were resuspended in 100% stimulation buffer,
6
bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) with bovine counted, and diluted to 0.6 ϫ 10 cell/ml. hrCRF (1 nM) in the
serum albumin as standard. The membrane proteins prepared absence and presence of SN003 in PBS containing 10% stimula-
from human CRF1 HEK293e cells (5–8 ␮g), rat cortex, pituitary, tion buffer (50 ␮l) was added to the assay plate. Drug treatment
and human CRF2␣ HEK293e cells, respectively (40–60 ␮g), were was initiated by adding HEK293e cells expressing CRF receptors
1
used in receptor binding experiments.
(50,000 cells/50 ␮l/well) to the Flash Plate and incubated for 15
Equilibrium competition binding experiments were performed min at 37°C in a final volume of 100 ␮l. Intracellular cAMP was
using a modification of the methods described previously to deter- released from cells through cell lysis resulting from adding detec-
1
25
mine binding potencies (IC50 values) of compounds at CRF1 and tion buffer containing [ I]cAMP (100 ␮l/well). Assay signal is
1
25
CRF2␣ receptors (Rominger et al., 1998). All small molecule li- based on competition of endogenous cAMP and [ I]cAMP for
gands were initially prepared in 100% dimethyl sulfoxide at a cAMP antibodies coated on the Flash Plate. Radioactivity from
Ϫ2
125
concentration of 10
M and diluted in assay buffer that was binding of [ I]cAMP to the plate was assessed 2 h later by a
identical to the tissue buffer except for inclusion of 0.15 mM 96-well PerkinElmer Top Counter.
bacitracin and 0.1% (w/v) ovalbumin. Competition assays were
ACTH Release Assays in Rat Primary Pituitary Cultures.
conducted in disposable polypropylene 96-well plates (Costar, Primary pituitary culture was established as described previously
Cambridge, MA) and initiated by the addition of 150 ␮l of mem- (Vale et al., 1972). Pituitaries were harvested from 20 to 25 rats
brane homogenate to 150 ␮l of assay buffer containing the radio- and washed in PBS four to six times. Pituitary cells were disso-
1
25
ligand (100 ␮l) in a final concentration of 150 pM ([ I]oCRF for ciated in collagenase buffer [1ϫ PBS containing 25 mM HEPES,
1
25
the CRF1 or [ I]sauvagine for CRF2␣ receptors) and competing 0.2% glucose, 0.4% (w/v) bovine serum albumin, 80 ␮g/ml DNase
ligands (50 ␮l). The reaction mixtures were incubated to equilib- II, and 0.4% (w/v) collagenase type 2] for 3 h at 37°C. Cells were
rium for 2 h at 23°C. Specific binding was defined in the presence spun down, decanted, and incubated with 0.25% trypsin solution

6
0
Zhang et al.
[
PBS containing 0.25% (w/v) trypsin, 0.4% (w/v) bovine serum
Results
albumin, 0.2% (w/v) glucose] for 5 to 10 min at 37°C. Cells were
briefly centrifuged and resuspended in M199 culture medium
Binding Affinity of SN003 for CRF1 and CRF2 Re-
ceptors. The chemical structure of SN003 is shown in Fig.
(
Invitrogen) containing 10% fetal calf serum, minimal essential
medium vitamins, streptomycin/penicillin, insulin/transferrin/se- 1A. The binding affinity of SN003 for CRF
lenium, fibroblast growth factor, and trace elements. Cells determined by competition binding experiments using
300,000/ml) were added to a 48-well plate (Costar) and grown in membranes prepared from HEK293e cells expressing hu-
receptors was
1
(
culture (37°C, 5% CO ) for 4 days. On the assay day, medium was man recombinant CRF receptors, and rat cortex and pi-
2
1
removed and replaced with M199 incubation medium identical to tuitary membranes containing native CRF receptors. Like
1
that used above except for exclusion of serum and addition of 0.1%
the CRF peptide antagonist ␣-helical CRF and the small
(
w/v) ovalbumin and 1 ␮g/ml aprotinin. The hrCRF-induced ACTH
molecule CRF1 antagonist DMP696 (Fig. 2), SN003 po-
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
125
tently and completely inhibited [ I]oCRF (200 pM) bind-
ing to CRF receptors, with maximal inhibition identical to
that of ␣-helical CRF and DMP696. The K value of SN003
for rat CRF1 receptors detected in pituitary membranes
was 3.4 Ϯ 0.5-fold of that determined in cortical mem-
branes in paired experiments (p ϭ 0.001), as shown in
Table 1. A similar shift was seen for DMP696 in pituitary
1
i
–
20°C until assayed for ACTH measurement. ACTH levels were
determined using a radioimmunoassay kit purchased from Dia-
Sorin Inc. (Stillwater, MN).
and cortical tissues with a K ratio (pituitary/cortex) of
Data Analyses. The concentrations of compounds to inhibit
0% of radioligand binding (IC50) for CRF1 and CRF2␣ receptors
i
2
.5 Ϯ 0.6-fold (p ϭ 0.017). In contrast, the peptide agonist
recep-
the iterative nonlinear regression curve-fitting program Prism tors in cortical (mean K ϭ 0.6 nM) and pituitary (mean K
5
were calculated by fitting data through a competition equation in ovine CRF exhibited an equal affinity for rat CRF
1
i
i
(
GraphPad Software, Inc.). Ki values (equilibrium dissociation ϭ 0.7 nM) tissues (p ϭ 0.38; paired Student’s t test).
constant) for inhibitors in competition experiments were calcu- Although the peptide antagonist ␣-helical CRF appeared
lated according to the Cheng-Prusoff equation. Saturation data
to have a small difference in potency between rat cortex
were fit through hyperbolic equations to estimate apparent equi-
librium constant (K ) and the maximal number of binding sites
D
(
Bmax) using Prism (GraphPad Software, Inc.). Kinetic studies to
determine kobs (observed association rate constant) and kϪ1 (dis-
sociation rate constant) were generated from a slope of the line via
linear regression of transformed data. The association rate con-
stant (kϩ1) was generated based on the equation kϩ1 ϭ (kobs
Ϫ
kϪ1/[L], where L is the ligand concentration. The equilibrium
dissociation constant derived from kinetic studies was calculated
as KD ϭ kϪ1/kϩ1
.
For cell-based functional studies, the potency for agonists (EC50
)
and for antagonists (IC50) was estimated by fitting raw data through
the sigmoidal dose-response equation using the iterative nonlinear
regression curve-fitting programs in Prism (GraphPad Software,
Inc.).
Brain Section Ligand Binding and Storage Phosphorim-
aging. 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 MgCl , 2
2
mM EGTA, 100 kallikrein-inactivating units/ml aprotinin, 0.1 M
bacitracin, and 0.1% ovalbumin (pH 7.2). Sections were then in-
3
cubated in the same solution containing 4 to 10 nM [ H]SN003 for
2
h at 23°C. As a comparison, one set of adjacent sections was
1
25
incubated with 200 pM [ I]sauvagine. At this concentration (200
1
25
pM), [ I]sauvagine binds to both CRF1 and CRF2 receptors
Rominger et al., 1998). Nonspecific binding was defined by inclu-
(
sion of 1 ␮M 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
125
Fig. 2. Competition of [ I]oCRF binding to rat cortical and pituitary
in cassettes against storage phosphorimaging screens
membranes by SN003, DMP696, ␣-helical CRF, and hrCRF. The assays
3
125
(
1
PerkinElmer Life Sciences) for 1 to 4 weeks ([ H]SN003) or for
were performed in duplicate in final concentration of [ I]oCRF (200 pM)
1
25
and 12 concentrations of displacers. Nonspecific binding was defined by 1
2 h ([ I]sauvagine), respectively. The screens were then
␮
M DMP696. The data are plotted and the respective curves are ex-
scanned with a Cyclone phosphorimaging scanner, and captured
images were analyzed with the OptiQuant requisition and analy-
sis system (PerkinElmer Life Sciences).
panded from the parameter estimates derived from fitting a competition
equation to the data by nonlinear regression analysis using Prism soft-
ware.

A Nonpeptide CRF1 Antagonist Radioligand
61
Table 1
1
25
Potency of compounds inhibiting ͓ I͔oCRF binding to rat CRF1
receptors
Parallel competition assays were conducted to generate IC50 values of compounds
inhibiting 50% of ͓1 I͔oCRF (200 pM) binding to rat pituitary and cortical mem-
branes. Data shown are the mean Ki (Ϯ S.E.M.) from six to nine experiments
performed in duplicate. The number of independent paired experiments performed is
25
shown in parentheses. The statistical significance of K values of compounds between
i
cortex and pituitary was assessed by a paired Student’s t test, and two-tailed p
values (ء, Ͻ0.05) were considered as significant.
Pituitary
Mean K
Cortex
Mean K
Pituitary/Cortex
Mean K Ratio
Significance
(p Values) (n)
Compounds
i
i
i
nM
SN003
DMP696
7.9 Ϯ 0.8
2.6 Ϯ 0.5
2.5 Ϯ 0.4
1.0 Ϯ 0.1
5.3 Ϯ 1.9
0.7 Ϯ 0.2
3.4 Ϯ 0.5
2.5 Ϯ 0.6
3.3 Ϯ 2.8
0.9 Ϯ 0.3
**0.001(6)
*0.017(6)
0.222(9)
␣
-helical CRF 8.1 Ϯ 2.3
hrCRF 0.6 Ϯ 0.1
0.388(6)
(
mean K ϭ 5.3 nM) and pituitary (mean K ϭ 8.1 nM)
i i
tissues, this difference was not statistically significant
p ϭ 0.22), as summarized in Table 1.
Figure 3A shows that SN003 specifically inhibited [ I]oCRF
(
125
binding to human CRF receptors expressed in HEK293e cells
1
with high affinity (K ϭ 6.8 Ϯ 0.8 nM) like DMP696 (K ϭ 2.8 Ϯ
i
i
0
.5 nM) and ␣-helical CRF9–41 (K ϭ 7.9 Ϯ 1.5 nM). The spec-
i
ificity of SN003 for the CRF receptor was also assessed by
1
125
examining its ability to compete with [ I]sauvagine binding to
the human CRF2␣ receptors. As depicted in Fig. 3B, ␣-helical
125
CRF was a potent inhibitor of [ I]sauvagine binding to human
CRF2␣ receptors (K ϭ 11.2 Ϯ 1.8 nM), whereas SN003 was
i
inactive at the CRF2␣ receptor up to 10 ␮M concentrations (n ϭ
3). The data indicate that SN003 is highly selective in its affin-
ity for CRF receptors, being approximately 1000 times more
1
125
potent against [ I]oCRF binding to human CRF (K ϭ 6.8
1
i
125
nM) than against [ I]sauvagine binding to human CRF2␣ (Ki
Ͼ10 ␮M).
Homologous competition experiments were conducted to
determine SN003 effects on the K and B
values of oCRF
D
max
binding in the human CRF HEK293e cell membranes. The
1
Scatchard plot in Fig. 4 shows that SN003 dramatically de- Fig. 3. Selectivity profile of SN003 for human CRF and CRF receptors.
1
2␣
1
25
creased the Bmax (the x-intercept) to 85.3 Ϯ 1.9%, 67.4 Ϯ Competition binding experiments were conducted using [ I]oCRF (150
1
25
pM) and [ I]sauvagine (150 pM) as radioligands to label human CRF1
A) and CRF2␣ (B) receptors stably expressed in HEK293e cells, respec-
tively. Nonspecific binding was defined by 1 ␮M DMP696 at CRF recep-
1
0.7%, and 46.4 Ϯ 3.0% of the control value without change
(
of K (1/slope) values of ovine CRF at concentrations of 3, 10,
and 30 nM, respectively, suggesting that SN003 is not simply tors or 1 ␮M ␣-helical CRF at CRF2␣ receptors. Data presented are mean
competitive.
D
1
specific binding values of duplicate determinations from one of three to
six experiments yielding similar results. IC50 and K values of compounds
i
Inhibition of CRF-Mediated cAMP Accumulation
were derived from fitting a competition equation to the data by means of
and ACTH Release by SN003. To evaluate functional iterative nonlinear least squares regression analysis.
activities of SN003 in vitro, SN003 was assessed for inhi-
bition of CRF-mediated responses in rat pituitary and
human CRF HEK293e cells. In cell-based functional as-
1
says, the antagonist properties of SN003 in CRF-stimu- duced ACTH release with an IC50 value of 241.5 Ϯ 48.4 nM
lated ACTH secretion in cultured rat pituitary cells were (n ϭ 4) and completely abolished CRF-stimulated ACTH
examined. In parallel, evaluation of the agonist concentra- secretion at higher concentrations, suggesting full antag-
tion-response was included in each experiment (Fig. 5A). onist properties at pituitary CRF1 receptors. To test po-
Basal secretion of ACTH was markedly enhanced in re- tential agonist activity, SN003 (1 and 10 ␮M), exposed
sponse to CRF in a concentration-dependent manner. CRF- alone to pituitary cells for 3 h, did not alter basal secretion
induced maximal stimulation of ACTH release (3400–4093 of ACTH from cells (data not shown), indicating a lack of
pg/100-␮l sample), which is 5- to 9-fold of the basal level partial or inverse agonist activities.
(
400–520 pg/100-␮l sample), was achieved at 10 nM CRF
The antagonist properties of SN003 for human CRF1
during a 3-h incubation (n ϭ 4). The mean EC50 Ϯ S.E.M. receptors were examined in HEK293e cells expressing re-
of CRF for stimulation of pituitary ACTH secretion was combinant human CRF1 receptors (Fig. 6). CRF (1 nM)
calculated as 0.30 Ϯ 0.05 nM (n ϭ 4). As shown in Fig. 5B, elicited 60.2 Ϯ 3.9% stimulation of cAMP production as
SN003 dose dependently suppressed CRF (0.3 nM)-in- normalized to the maximal stimulation (100%) obtained by

6
2
Zhang et al.
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 Ϯ S.E.M. of four
independent experiments performed in triplicate. A, the agonist concen-
tration-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 concentra-
tions of SN003 for 3 h at 37°C. ACTH levels were normalized to percent-
age of control (0.3 nM hrCRF alone). The mean Ϯ S.E.M. values were
obtained with a statistical analysis of four individual EC50 or IC50 deter-
minations.
3
0 nM CRF. As displayed in Fig. 6, SN003 significantly
1
25
Fig. 4. Effects of SN003 on [ I]ovine CRF binding parameters in
decreased the control CRF response from 60.2 Ϯ 3.9 to
HEK293 cells expressing human CRF1 receptors. The homologous com-
petition experiment was performed in recombinant CRF HEK293e cell 26.4 Ϯ 4.9% and 12.9 Ϯ 2.8% in a dose-dependent manner
1
1
25
homogenates, and saturation of [ I]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
at concentrations of 100 nM and 300 nM, respectively. As
observed in the case of ACTH release, SN003 showed no
defined by 1 ␮M DMP696. The data were transformed to representative effect on basal cAMP production in the absence of CRF
Scatchard plots to visualize the KD (1/slope) and the Bmax (the x-intercept) stimulation. The Schild analysis of SN003 antagonism of
changes in the top panel, that experiment was performed three times
CRF-induced cAMP responses was also performed in this
with essentially identical results. Data shown in the middle and bottom
recombinant cell line that highly expresses human CRF1
panels are mean Ϯ S.E.M. values calculated from three independent
experiments performed in triplicate.
receptors, showing rightward shifts of CRF concentration-

A Nonpeptide CRF1 Antagonist Radioligand
63
Fig. 6. Antagonism of CRF-induced cAMP accumulation in HEK293e
cells expressing human CRF1 receptors by SN003. CRF-elicited intra-
cellular cAMP elevation was determined in the absence and presence
of coadministered concentrations of SN003 after 15 min incubation at
3
Fig. 7. Equilibrium association binding of [ H]SN003 to rat frontal
3
cortical membrane. Time course of [ H]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
␮M DMP696. Inset, linear transformation of specific association data,
3
7°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; ء, p Ͻ 0.05 was considered as significant. ءء, p Ͻ0.01;
ءءء, p Ͻ0.001.
where Beq represents specific binding at the steady state and B
t
represents the specific binding at time t. The graph shows the repre-
sentative data of three independent measurements performed in trip-
licate.
4
.8 Ϯ 0.2 nM for CRF receptors expressed in rat frontal
1
response curves with no significant decrease in CRF effi- cortex (Fig. 8A), and a Bmax of 7.42 Ϯ 0.70 pmol/mg protein
cacy (Emax) by SN003 (antagonist potency constant Kb and a KD of 4.6 Ϯ 0.5 nM for recombinant human CRF1
8 Ϯ 3 nM). This may be due to the large number of spare receptors expressed in HEK293e cells (Fig. 8B), respec-
ϭ
5
receptors expressed in this system. Even after eliminating tively. In comparison, expression of CRF receptors in the
1
a large percentage of receptors by SN003 via a noncompet- recombinant CRF HEK293 cell line is remarkably higher,
1
itive mode of inhibition, a small portion of receptors re- by 50-fold, than that in the rat frontal cortex, but with
mains adequate for CRF to achieve the maximal responses. identical affinity of this radioligand for rat and human
Therefore, Schild analysis was performed in human reti- CRF1 receptors. The transformation of saturation data
noblastoma Y-79 cells (Hauger et al., 1997), which natively generated a monophasic Scatchard plot (Fig. 8, insets),
3
express a low level of human CRF receptors. SN003 re- suggesting a single affinity binding of [ H]SN003 that was
1
duced CRF potency (1EC50) in a concentration-dependent consistent with the characteristic of antagonist binding.
3
manner, and high concentrations of SN003 (1 and 3 ␮M) These data indicate that the affinity of [ H]SN003 for
significantly decreased Emax to 89 Ϯ 2% and 77 Ϯ 3%, CRF1 receptors is independent of the coupling state of
3
respectively, of the control level in the Y-79 cell line (n ϭ receptors. Specific binding of [ H]SN003 to rat frontal
3
; p Ͻ 0.01 by Student’s t test).
Characterization of [ H]SN003 Binding to Mem- and 90%, respectively, of total binding at the radioligand
cortex and CRF cell homogenates was approximately 50%
1
3
branes Expressing CRF Receptors. To examine the concentration equal to its K value.
1
D
3
3
binding characteristics of [ H]SN003 to CRF receptors, a
time course of [ H]SN003 (ϳ4.8 nM) equilibrium binding was performed at a 4.8 nM concentration of the ligand in hu-
[ H]SN003 association and dissociation studies were
1
3
performed in rat frontal cortex homogenates at 23°C. Under man CRF HEK293e cell homogenates using 5 to 8 ␮g of
1
3
3
these conditions, [ H]SN003 binding was time-dependent protein at 23°C. [ H]SN003 binding kinetics were
Fig. 7), and its association reaction (Ͼ99%) was completed monophasic (Fig. 9, insets), with mean t1/2 values of 27.2 Ϯ
(
by 120 min. Nonspecific binding was increased slightly 9.2 min for association and 60.7 Ϯ 8.8 min for dissociation
within 5 min and remained constant, representing approxi- obtained from three experiments. As kinetic constants de-
mately 50% of total binding. The mean Ϯ S.E.M. value for rived from linear regression (Fig. 9, insets), the slope
Ϫ2
Ϫ1
kobs (1.62 Ϯ 0.3 ϫ 10 min ) and the corresponding t1/2 of yielded an observed association rate constant (kobs) of
Ϫ2
Ϫ1
4
3 min was determined based on linear regression analysis of 2.42 Ϯ 0.6 ϫ 10 min and a dissociation rate constant
Ϫ2 Ϫ1
data from three independent experiments. Subsequent satu- (kϪ1) of 0.992 Ϯ 0.05 ϫ 10
ration experiments were conducted using a 120-min incuba- association rate constant (kϩ1) of 0.292 nM
min , respectively. The
Ϫ1 Ϫ1
min
was
tion time. calculated for SN003 using the equation described under
As presented in the saturation analysis (Fig. 8), Materials and Methods. The equilibrium dissociation con-
3
[
H]SN003 bound saturably, specifically with high affinity stant (K ) of 3.4 nM was calculated from kinetic studies,
D
to an apparently homogeneous population of recognition which is comparable with the KD of 4.8 nM derived from
sites, with a Bmax of 142 Ϯ 14 fmol/mg protein and KD of saturation analysis.

6
4
Zhang et al.
3
Fig. 9. Kinetic studies of [ H]SN003 binding to membrane prepared from
HEK293e cells recombinantly expressing human CRF receptors. A, equi-
1
3
librium association of [ H]SN003 binding to human CRF1 receptors.
Inset: linear conversion of association data, where Beq represents specific
binding at the steady state, and B represents the specific binding at time
t
3
t. B, dissociation of [ H]SN003 binding to human CRF receptors where
1
(
inset) B represents specific binding at time t and B represents binding
t
0
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 Ͼ
0.05, F-test). Nonspecific binding was defined at each time point by 5 ␮M
DMP696. Data shown are the mean from three independent experiments
performed in triplicate.
3
Fig. 8. Saturation isotherm of [ H]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 ex-
periments performed in triplicate. KD and Bmax values were deter-
mined 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. Nonspe-
cific binding was defined as that occurring in the presence of 5 ␮M
DMP696.
3
In initial [ H]SN003 competition experiments performed
in human CRF1 HEK293e cell homogenates, hrCRF was
To further distinguish antagonist and agonist behaviors used as a competitor and DMP696 was included as the
in binding experiments, the effect of a nonhydrolyzable reference compound. It was of particular interest that
3
GTP analog, Gpp(NH)p (50 ␮M), on specific binding of hrCRF was able to inhibit [ H]SN003 binding, but the
3
125
[
H]SN003 (5 nM) and [ I]oCRF (150 pM) was examined. maximal reduction of binding was less compared with that
As depicted in Fig. 10, addition of Gpp(NH)p in reaction achieved by DMP696. To further characterize the recipro-
mixtures resulted in a marked decrease (55 Ϯ 1%) of spe- cal interactions between CRF-related peptides and the
1
25
cific [ I]oCRF binding as compared with the control (p ϭ small molecule antagonist at CRF receptors, the displace-
0
1
1
25
3
.0001 by Student’s t test; n ϭ 5). In contrast to [ I]oCRF ment of [ H]SN003 binding by CRF peptides including two
3
binding, the [ H]SN003 binding was not affected by inclu- peptide antagonists and another small molecule antago-
sion of 50 ␮M Gpp(NH)p (p ϭ 0.85 by Student’s t test; n ϭ nist, DMP904, was assessed. Several key observations
1
25
4
). The reduction of [ I]oCRF binding is consistent with were made from these studies. First, maximal inhibitions
the elimination of high-affinity sites of agonist binding via provided by DMP696 and DMP904 did not vary substan-
3
uncoupling of G proteins to CRF receptors in the presence tially between inhibitions of the small molecule [ H]SN003
1
3
125
of Gpp(NH)p. In contrast, [ H]SN003 binding was insensi- and peptide [ I]oCRF binding, although K values of
tive to guanine nucleotides, demonstrating characteristics small molecules derived from [ H]SN003 binding were
i
3
1
25
of antagonist binding.
slightly lower than that determined from [ I]oCRF bind-

A Nonpeptide CRF1 Antagonist Radioligand
65
Fig. 11, performed in the recombinant CRF HEK293 cell
1
3
line, with maximal inhibition of [ H]SN003 binding by
DMP696 (100%), hrCRF (66%), and ␣-helical CRF or D-
PheCRF (0%), respectively.
To further extend these observations, we determined
3
whether partial inhibition of [ H]SN003 binding is a general
characteristic of CRF-related peptide agonists at human
CRF receptors expressed in HEK293e cells. Twelve-point
1
Ϫ12
Ϫ6
(
10
-10
M) inhibition curves were generated using rat
and human urocortin I, hrCRF, ovine CRF, and sauvagine in
3
the [ H]SN003 binding assay as described above. The peptide
agonists all exhibited incomplete inhibition of specific
3
[
H]SN003 binding to human CRF receptors with maximal
1
inhibition ranging from 58 to 84% as compared with small
molecule CRF1 antagonists (DMP696, DMP904, and
CP-154,526), which led to complete displacement of specific
3
[
H]SN003 binding. Similar to the earlier studies, ␣-helical
CRF and D-PheCRF were inactive up to 1 ␮M in this exper-
3
125
Fig. 10. Effects of the GTP analog on [ H]SN003 and [ I]ovine CRF
3
125
iment. For comparison, binding data for CRF peptides and
binding to human CRF receptors. [ H]SN003 (5 nM) and [ I]oCRF (150
1
1
25
pM) binding to human CRF receptors expressed in HEK293e cell mem- nonpeptide CRF antagonists against [ I]oCRF peptide and
1
1
branes was examined in the absence (control) and presence of 50 ␮M
Gpp(NH)p, respectively. Nonspecific binding was defined by 5 ␮M
DMP696. Data presented as the bar are the mean Ϯ S.E.M. values from
3
[
H]SN003 binding, respectively, were summarized in Table
2. Unlike small molecules, an increase in endogenous peptide
four experiments performed in triplicate. ءءء, significant difference from concentrations did not eventually provide a complete dis-
control (p Ͻ 0.001) determined by Student’s t test.
3
placement of specific [ H]SN003 binding, suggesting that
CRF does not interact with the small molecule CRF antag-
1
onist, SN003, at the CRF receptor in a simple competitive
1
manner.
ing. As shown in Fig. 1, these small molecules share a
somewhat structural similarity in contrast to the structure
difference between small molecules and peptides. Second,
3
Anatomical Distribution of [ H]SN003 Binding
Sites. The distribution of specific [ H]SN003 binding sites
in slide-mounted rat brain sections was assessed and com-
pared with that of [ I]sauvagine binding sites. Figure
3
3
hrCRF led to partial inhibition of [ H]SN003 binding with
1
25
a mean maximum suppression of 67 Ϯ 3% when compared
with the level of 100% inhibition by DMP696 in the exper-
iments (p Ͻ 0.001 by Student’s t test). In addition, hrCRF
3
1
2A exhibits storage phosphorimages of [ H]SN003 bind-
ing in the absence and presence of 1 ␮M DMP696. Specific
3
[
H]SN003 binding was measured by digitally subtracting
nonspecific binding (Fig. 12A, right panel), defined by 1
M DMP696, from total binding (Fig. 12A, left panel). In
was 10-fold less potent (p Ͻ 0.01 by Student’s t test) in
3
competition with [ H]SN003 binding (mean K ϭ 14.1 nM)
i
1
25
␮
as compared with inhibition of [ I]oCRF binding (mean
K ϭ 1.4 nM). The same case was observed with oCRF the cortex and other brain regions, in which there were
i
(
Table 2). Third, the peptide antagonists ␣-helical CRF and higher levels of binding, specific binding accounted for 50
3
D-PheCRF, which lack the N-terminal sequence of CRF, to 60% of the total binding. [ H]SN003 binding sites ap-
were inactive up to 1 ␮M concentrations, as shown in Fig. peared as granular particles with space resolution close to
1
25
1
1 and Table 2. Identical results and inhibition patterns that labeled with [ I]sauvagine but not in lateral septal
were also seen with rat CRF receptors natively expressed nucleus and choroid plexus.
1
in the frontal cortex tissue, such as the one presented in
Figure 12B shows the distribution pattern of specific
Table 2
1
25
3
Summary data for peptides and nonpeptide ligands inhibiting ͓ I͔oCRF and ͓ H͔SN003 binding to human CRF receptors
1
values were calculated from IC50 values of compounds inhibiting 50% of ͓1 I͔oCRF (150 pM) and ͓ H͔SN003 (4.7 nM) binding to human CRF
25
3
receptors recombinantly
K
i
1
3
expressed in HEK293e cell membranes, respectively. K values for hrCRF and oCRF, derived from inhibition of ͓ H͔SN003 binding, were from automatic curve fitting, and
i
the bottom of the peptide curves was not constrained to the reference compound DMP696 value. The maximum inhibition provided by compounds was normalized to the value
of DMP696 (100%). Data shown are the mean (Ϯ S.E.M.) from three to nine experiments performed in duplicate. The number of independent experiments performed is shown
in parentheses.
͓3
Mean K
H͔SN003
͓ H͔SN003
3
͓125I͔oCRF
͓125I͔oCRF
Mean Maximum Inhibition
Compounds
i
Mean Maximum Inhibition
Mean K
i
nM
% control
nM
% control
DMP696 (Control)
SN003
1.8 Ϯ 0.2
4.9 Ϯ 0.5
0.4 Ϯ 0.1
0.4 Ϯ 0.1
14.1 Ϯ 1.9
23.1 Ϯ 6
Ͼ1000
100 Ϯ 1(8)
100 Ϯ 1(8)
100 Ϯ 2(6)
100 Ϯ 2(3)
67 Ϯ 3(9)
58 Ϯ 8(3)
3 Ϯ 1(7)
2.8 Ϯ 0.5
6.8 Ϯ 0.8
1.0 Ϯ 0.1
1.2 Ϯ 0.5
1.4 Ϯ 0.2
3.3 Ϯ 0.3
7.9 Ϯ 1.5
22.4 Ϯ 3.8
100 Ϯ 2(6)
101 Ϯ 1(5)
100 Ϯ 1(4)
99 Ϯ 2(3)
99 Ϯ 3(3)
100 Ϯ 1(4)
99 Ϯ 7(6)
96 Ϯ 8(3)
DMP904
CP-154,526
hrCRF
OCRF
␣
-Helical CRF
D-PheCRF
Ͼ1000
2 Ϯ 2(7)

6
6
Zhang et al.
tors highly expressed in these areas, and their functional role
and relevance, are not well understood. In the neocortex, the
binding density was clearly higher in lamina IV than in other
laminae. Among subcortical regions, moderate to high den-
sities were seen in the amygdala, especially its basolateral
portion, the nucleus accumbens, and the hippocampus.
3
Fairly uniform, moderate densities of [ H]SN003 binding
sites were present in the caudate-putamen and thalamus.
3
[
H]SN003 binding density was low in the hypothalamus but
high in the anterior portion of the pituitary. In the brain-
stem, low to moderate levels of binding sites were seen in the
superior and inferior colliculi, the periaqueductal gray, and
3
Fig. 11. Competition of [ H]SN003 binding to human CRF receptors
1
expressed in HEK293e cell membranes by small molecule CRF antago-
1
nists and CRF peptides. Nonspecific binding was defined by 1 ␮M the dorsal raphe nucleus.
DMP696. Shown is a representative measurement of six to nine indepen-
We compared the binding pattern and density of
H]SN003 with those of [ I]sauvagine in the brain. In
dent experiments performed in duplicate.
3
125
[
the lateral septal nucleus and the choroid plexus there
1
25
3
were dense levels of [ I]sauvagine, but not [ H]SN003,
binding sites, which were not displaceable by DMP696,
1
25
3
indicating that unlike [ I]sauvagine, [ H]SN003 did not
bind to CRF2 receptors, which are predominant in these
regions (Primus et al., 1997; Rominger et al., 1998). We
3
further compared the relative density of [ H]SN003 with
1
25
[
I]sauvagine, as summarized in Table 3. The percentage
of the relative density in each brain region was calculated
by normalizing the specific binding value with reference to
that in the neocortex, where binding density was defined
as 100%. In most brain regions, the percentage of the
3
relative binding density of [ H]SN003 was similar to that
1
25
revealed by [ I]sauvagine. The higher percentage of
1
25
[
I]sauvagine binding in the hypothalamus reflects a
higher density of CRF receptors existing in the hypotha-
2
lamic ventromedial nucleus receptors. Another apparent
difference was that in the caudate-putamen, hippocampus,
and thalamus, there was moderately higher binding den-
3
125
sity for [ H]SN003 compared with [ I]sauvagine. Specific
1
25
CRF receptor sites labeled by [ I]oCRF in these brain
regions have been reported previously by De Souza et al.
(
[
1985). Interestingly, the density and pattern of specific
1
25
I]oCRF binding in the regions (see Figs. 8 and 9 in De
Souza et al., 1985) were essentially the same as those
3
labeled by [ H]SN003 in this study.
Table 3
3
125
Percentage of binding density of ͓ H͔SN003 and ͓ I͔sauvagine in the
rat brain regions
Percentage of binding density in each brain region was calculated by normalizing the
specific density value with reference to that in the neocortex (control), which was
defined as 100%. Data presented are the mean Ϯ S.E.M. values from three indepen-
dent experiments using three rat brains. Sets of the adjacent sections were incubated
with different radioligands in the absence and presence of 1 ␮M DMP696.
3
Fig. 12. Storage phosphate images showing [ H]SN003 binding sites in
3
the rat brain. A, [ H]SN003 binding to coronal sections of the rat brain in
the absence (left panel) and presence (right panel) of 1 ␮M DMP696. B,
3
͓3H͔SN003
͓125I͔Sauvagine
distribution pattern of [ H]SN003 binding sites in the brain slice section.
Brain Region
Olf, olfactory bulb; FC, frontal cortex; CP, caudate-putamen; Pi, pitu-
itary; PC, parietal cortex; Hi, hippocampus, Th, thalamus; PAG, periaq-
ueductal gray; Ceb, cerebellum. Scale bar ϭ 2 mm.
%
control
Neocortex (control)
Olfactory bulb
Hippocampus
Amygdala
100 Ϯ 4
105 Ϯ 9
25 Ϯ 2
57 Ϯ 8
32 Ϯ 6
29 Ϯ 1
9 Ϯ 2
101 Ϯ 12
93 Ϯ 7
115 Ϯ 8
100 Ϯ 5
118 Ϯ 7
10 Ϯ 3
59 Ϯ 3
14 Ϯ 3
16 Ϯ 1
22 Ϯ 4
138 Ϯ 8
68 Ϯ 6
86 Ϯ 6
3
[
[
H]SN003 binding sites in the rat brain. Abundant
H]SN003 binding was found throughout the cortical regions
3
Striatum
Thalamus
Hypothalamus
Pituitary
Superior colliculus
Cerebellar cortex
and was especially intense in the prefrontal, somatosensory,
cingulate, and entorhinal cortex. High levels of binding were
also apparent in the olfactory bulb and cerebellar cortex,
which is consistent with previous findings that CRF recep-
1

A Nonpeptide CRF1 Antagonist Radioligand
67
competitive with CRF at the CRF receptor. The maximal
Discussion
1
number of binding sites for CRF is significantly reduced by
SN003 in a concentration-dependent manner, indicative of
a noncompetitive interaction. Incomplete reciprocal bind-
ing inhibition of peptide agonists and the small molecule
antagonist also suggests that the interaction is not simply
The present study describes the in vitro pharmacological
characteristics of
[
a
novel nonpeptide radioligand,
3
H]SN003, for the rat and human CRF receptors. SN003
1
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 re-
sponses. Equilibrium binding studies revealed that spe-
cific binding of tritium-labeled SN003 to CRF1 receptors
was saturable, reversible, and of high affinity. Antagonist
characteristics of [ H]SN003 were also suggested by a
single class of [ H]SN003 binding sites and insensitivity to
guanine nucleotides. The distribution pattern of
H]SN003 binding sites was consistent with CRF recep-
competitive at CRF receptors; at least SN003 may provide
1
mixed competitive and/or noncompetitive inhibition. Par-
tial inhibition could result from allosteric modulation and
noncompetitive inhibition by small molecules or accessibil-
ity of peptides to the small molecule binding domains. The
underlying mechanisms need to be explored in a future
investigation.
3
3
These data indicate that the recognition sites for small
molecule antagonists and peptide agonists are not mutually
3
[
1
exclusive on CRF receptors. In general, the peptide agonist
1
tor mapping in the brain, further supporting the specific
binding domain of class B receptors of the GPCR family, such
3
labeling of CRF receptors by [ H]SN003.
1
as CRF receptors, is predominantly formed from the large
1
Both SN003 and DMP696 had 2- to 3-fold higher affini-
extracellular N-terminal domain and portions of the extra-
ties (lower K values) for rat CRF receptors in the frontal
i
1
cellular loops of the receptors. Small molecule CRF antago-
1
cortex than in the pituitary and in recombinant CRF1
nists 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
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 CRF receptors in the membranes
1
from these receptor sources. On the other hand, the dis-
parity in inhibition of CRF binding observed for small
molecules in these two rat tissues may reflect a differential
3
without N-terminal amino acids fail to inhibit [ H]SN003
binding suggests that it is the N-terminus of CRF that inter-
acts with the small molecule recognition sites of the receptor,
possibly sharing binding valences in the transmembrane re-
gion. 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.
nature of CRF receptors expressed in various cell types.
1
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 glyco-
sylation 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
Further studies employing radiolabeled CRF peptides
and small molecule antagonists will greatly aid under-
differential post-translational modifications of CRF1 re- standing of molecular aspects of CRF receptor signaling
1
ceptors have functional consequences.
and small molecule antagonism. Additionally, this infor-
In functional assays of CRF-mediated ACTH release and mation may have implications for other G protein-coupled
cAMP accumulation, SN003 completely antagonized CRF ef- receptors with endogenous peptide agonists. This does ap-
fects without partial or inverse agonist properties. We noted pear to be the case for the neurokinin-1 and -2 receptors,
that the functional potency of SN003 as determined in the since partial inhibition and low potencies of endogenous
ACTH assay was approximately 10-fold lower than its bind- peptides, substance P and neurokinin A, in displacement of
ing affinity at rat pituitary CRF1 receptors. This weaker small molecule antagonist radioligand binding (Rosenkilde
potency in functional assays is also seen with other GPCR et al., 1994) have been observed with those receptors.
antagonists and, more specifically, CRF1 antagonists (By- Considered together, the data suggest that the interac-
master and Falcone, 2000; Heinrichs et al., 2002). The dis- tions between small molecules are more competitive than
crepancy between binding and functional potencies of SN003 that between the small molecule and peptide, and binding
is not well understood. It could result, in part, from different domains for peptides and small molecule antagonists are
assay conditions in the functional and binding assays, in not mutually exclusive.
particular, the use of intact cells versus membrane prepara-
Two nonpeptide CRF1 antagonists, CP-154,526 and an
tions and physiological buffer versus hypotonic buffer (By- antalarmin analog, have recently been radiolabeled (Tian
master and Falcone, 2000). Besides assay conditions, differ- et al., 2001; Keller et al., 2002). The tritium-labeled CP-
ential properties of small molecule CRF antagonists such as 154,526 was used for assessing pharmacokinetics and
their binding affinity and coupling, as well as competitive blood-brain barrier penetration, and the antalarmin ana-
versus noncompetitive nature, may also have an impact on log was developed as a positron emission tomography li-
the potency of the difference between binding and functional gand. However, in these studies there are no descriptions
activity of CRF ligands in the hypothalamic-pituitary-adre- of radioligand binding profiles in either tissues or cells ex-
nal axis.
pressing CRF receptors. Tritiated SN003 is the first nonpep-
1
The present work suggests that SN003 is not simply tidic CRF antagonist radioligand shown to be capable of de-
1

6
8
Zhang et al.
receptor modulators: progress and opportunities for new therapeutic agents.
J Med Chem 43:1641–1660.
tecting specific binding in rat brain tissues. The binding
pattern of [ H]SN003 in the brain was consistent with the
3
Griebel G, Simiand J, Steinberg R, Jung M, Gully D, Roger P, Geslin M, Scatton B,
Maffrand JP, and Soubri e´ P (2002) 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 (SSR125543A), a potent and selective corticotro-
phin-releasing factor1 receptor antagonist. II. Characterization in rodent models
of stress-related disorders. J Pharmacol Exp Ther 301:333–345.
Grigoriadis DE and De Souza EB (1988) The brain corticotropin-releasing factor
(CRF) receptor is of lower apparent molecular weight than the CRF receptor in
anterior pituitary: evidence from chemical cross-linking studies. J Biol Chem
distribution of CRF receptors revealed previously by bind-
1
1
25
ing autoradiography using the radioligand [ I]oCRF (De
Souza et al., 1985; Aguilera et al., 1987) or by in situ
hybridization of CRF1 mRNA (Potter et al., 1994). The
3
sensitivity of [ H]SN003 binding was moderate consider-
ing that it was 50 to 60% specific over total binding in both
the homogenized membrane and slide-mounted brain sec-
tion binding assays. The moderate sensitivity was, per-
haps, caused by several factors, including the ”sticky“ li-
pophilic property of SN003 and the low energy status of the
labeled isotope tritium. In addition, storage phosphorim-
aging used in the autoradiography study may have com-
promised the sensitivity in terms of space resolution. How-
ever, this technique proved to be very effective for
acquiring images marked with tritium-labeled ligands
263:10927–10931.
Grigoriadis DE and De Souza EB (1989) Heterogeneity between brain and pituitary
corticotropin-releasing factor receptors is due to differential glycosylation. Endo-
crinology 125:1877–1888.
Gully D, Geslin M, Serva L, Fontaine E, Roger P, Lair C, Darre V, Marcy C, Rouby
PE, Simiand J, et al. (2002) 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 (SSR125543A): a potent and selective corticotrophin-
releasing factor1 receptor antagonist. I. Biochemical and pharmacological
characterization. J Pharmacol Exp Ther 301: 322–332.
Habib KE, Weld KP, Rice KC, Pushkas J, Champoux M, Listwak S, Webster EL,
Atkinson AJ, Schulkin J, Contoreggi C, et al. (2000) Oral administration of a
corticotropin-releasing hormone receptor antagonist significantly attenuates be-
havioral, neuroendocrine and autonomic responses to stress in primates. Proc Natl
Acad Sci USA 97:6079–6084.
3
such as [ H]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 appli-
cations in clinical positron emission tomography/single
photon emission computed tomography studies of CRF1
antagonists.
Hauger RL, Dautzenberg FM, Flaccus A, Liepold T, and Spiess J (1997) Regulation
of corticotropin-releasing factor receptor function in human Y-79 retinoblastoma
cells: rapid and reversible homologous desensitization but prolonged recovery.
J Neurochem 68:2308–2316.
He L, Gilligan PJ, Zaczek R, Fitzgerald LW, McElroy J, Shen HS, Saye JA, Kalin
NH, Shelton S, Christ D, et al. (2000) 4-(1,3-Dimethylthoxyprop-2-ylamino)-2,7-
dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bio-
available CRF1 receptor antagonist. J Med Chem 43:449–456.
Heinrichs SC, De Souza EB, Schulteis G, Lapsansky JL, and Grigoriadis DE (2002)
Brain penetrance, receptor occupancy and antistress in vivo efficacy of a small
molecule corticotropin releasing factor type I receptor selective antagonist. Neu-
ropsychopharmacology 27:194–202.
Horlick RA, Sperle K, Breth LA, Reid CC, Shen ES, Robbins AK, Cooke GM, and
Largent BL (1997) Rapid generation of stable cell lines expressing corticotropin-
releasing hormone receptor for drug discovery. Protein Expr Purif 9:301–308.
Keck ME and Holsboer F (2001) Hyperactivity of CRH neuronal circuits as a target
for therapeutic interventions in affective disorders. Peptides 22:835–844.
Keller C, Bruelisauer A, Lemaire M, and Enz A (2002) Brain pharmacokinetics of a
non-peptidic corticotropin-releasing factor antagonist. Drug MetabDispos 30:173–
176.
In summary, we describe the in vitro characterization of a
high-affinity, selective nonpeptidic antagonist radioligand,
3
[
H]SN003, for CRF receptors. Since this radioligand pos-
1
sesses an excellent signal/noise ratio in a recombinant hu-
man CRF1 cell line and distinct features compared with
peptide ligands, it provides a useful tool to understand small
molecule antagonism of CRF at CRF receptors. This infor-
1
Kostich WA, Chen A, Sperle K, and Largent BL (1998) Molecular identification and
analysis of a novel human corticotropin-releasing factor (CRF) receptor: the
CRF2␥ receptor. Mol Endocrinol 12:1077–1085.
mation may prove helpful in drug design and development of
small molecule CRF antagonists for treatment of affective
1
disorders and stress-related diseases.
Lewis K, Li C, Perrin MH, Blount A, Kunitake KS, Donaldson C, Vaughan J, Reyes
TM, Gulyas J, Fischer W, et al. (2001) Identification of urocortin III, an additional
member of the corticotropin-releasing factor (CRF) with high affinity for the CRF2
receptors. Proc Natl Acad Sci USA 98:7570–7575.
Acknowledgments
Li YW, Yan XX, Hill G, Gilligan PJ, Bakthavatchalam R, Prakash SR, and Robert
3
We would like to thank Dr. Rebecca Taub for review and helpful
suggestions on the manuscript.
Zaczek (2001) Autoradiographic of [ H]-SN003, a small molecule corticotropin
releasing factor receptor type-1 antagonist, in the rat brain. Soc Neurosci Abstr
27:414.1.
Liaw CW, Grigoriadis DE, Lorang MT, De Souza EB, and Maki RA (1997) Localiza-
tion of agonist- and antagonist-binding domains of human corticotropin-releasing
factor receptors. Mol Endocrinol 11:2048–2053.
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Address correspondence to: Dr. Ge Zhang, The Bristol-Myers Squibb Com-
pany, Building 21/Room 2344A, 311 Pennington-Rocky Hill Rd., Hopewell, NJ
08534. E-mail: ge.zhang@bms.com