Superagonism at the human somatostatin receptor subtype 4

Article abstract of DOI:10.1124/jpet.104.075531

We have discovered a novel compound, J-2156 [(1′S, 2S)-4-amino-N-(1′-carbamoyl-2′-phenylethyl)-2-(4″-methyl- 1″-naphthalenesulfonylamino)butanamide], that belongs to a new class of somatostatin receptor ligands. J-2156 binds with nanomolar affinity to the human somatostatin receptor subtype 4 and is over 400-fold subtype-selective against the other somatostatin receptors. When evaluated in a [³⁵S]guanosine- 5′-O-(3-thio) triphosphate binding assay, J-2156 elicited a response 2 to 3 times as large as that of somatostatin-28 and somatostatin-14. That somatostatin-14 is clearly not a maximally efficacious agonist could be verified by demonstrating that it displays the typical behavior of a partial agonist when tested against J-2156. Increasing concentrations of somatostatin-14 cause a concentration-dependent rightward shift of the dose-response curves for J-2156, without affecting its maximal response. This lack of reduction of the maximal response and the fact that the superior efficacy of J-2156 is detected in membranes argue against desensitization and internalization as possible explanations for the superior efficacy of J-2156. More likely is that somatostatin-14 and J-2156 stabilize distinct receptor conformations that differ in their ability to interact with G-proteins. In a cyclic AMP assay, J-2156, somatostatin-28, and somatostatin-14 all act as full agonists. However, this outcome is most likely due to the presence of a receptor reserve in the cyclic AMP assay since there is a large gain of apparent potency in the cyclic AMP assay and the gain is larger for J-2156 than for somatostatin. We conclude that the endogenous ligands somatostatin-1 4 and somatostatin-28 do not define maximal agonism on the human somatostatin receptor subtype 4 and that J-2156 represents a so-called superagonist.

Full text of DOI:10.1124/jpet.104.075531

0022-3565/05/3121-332–338$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics
JPET 312:332–338, 2005
Vol. 312, No. 1
75531/1182902
Printed in U.S.A.
Superagonism at the Human Somatostatin Receptor Subtype 4
Mia Engstro¨ m, Jussi Tomperi, Kamel El-Darwish, Mikaela Åhman, Juha-Matti Savola, and
Siegfried Wurster
Juvantia Pharma Ltd., Turku, Finland
Received August 2, 2004; accepted August 27, 2004
ABSTRACT
We have discovered a novel compound, J-2156 [(1ЈS, 2S)-4- lack of reduction of the maximal response and the fact that the
amino-N-(1Ј-carbamoyl-2Ј-phenylethyl)-2-(4Љ-methyl-1Љ-naph- superior efficacy of J-2156 is detected in membranes argue
thalenesulfonylamino)butanamide], that belongs to a new class against desensitization and internalization as possible explana-
of somatostatin receptor ligands. J-2156 binds with nanomolar tions for the superior efficacy of J-2156. More likely is that
affinity to the human somatostatin receptor subtype 4 and is somatostatin-14 and J-2156 stabilize distinct receptor confor-
over 400-fold subtype-selective against the other somatostatin mations that differ in their ability to interact with G-proteins. In
receptors. When evaluated in a [35S]guanosine-5Ј-O-(3-thio) a cyclic AMP assay, J-2156, somatostatin-28, and somatosta-
triphosphate binding assay, J-2156 elicited a response 2 to 3 tin-14 all act as full agonists. However, this outcome is most
times as large as that of somatostatin-28 and somatostatin-14. likely due to the presence of a receptor reserve in the cyclic
That somatostatin-14 is clearly not a maximally efficacious AMP assay since there is a large gain of apparent potency in
agonist could be verified by demonstrating that it displays the the cyclic AMP assay and the gain is larger for J-2156 than for
typical behavior of a partial agonist when tested against somatostatin. We conclude that the endogenous ligands so-
J-2156. Increasing concentrations of somatostatin-14 cause a matostatin-14 and somatostatin-28 do not define maximal ago-
concentration-dependent rightward shift of the dose-response nism on the human somatostatin receptor subtype 4 and that
curves for J-2156, without affecting its maximal response. This J-2156 represents a so-called superagonist.
Somatostatin, also known as somatotropin release-inhibit- somatostatin in humans, which consists of 28 amino acids
ing factor (SRIF), was first identified as a peptide that inhib-
its growth hormone release from the anterior pituitary (Kru-
lich et al., 1968; Brazeau et al., 1973). Later, SRIF has been
shown to also have other physiological functions, including
the inhibition of both endocrine secretion, e.g., thyroid-stim-
ulating hormone, prolactin, insulin, and glucagon, and exo-
crine secretion, e.g., gastric acid, intestinal fluid, and pan-
creatic enzymes (for review, see Patel, 1999). In addition,
SRIF functions as a modulator of neuronal activity and may
act as a neurotransmitter (Patel, 1999). The wide range of
physiological actions of SRIF are mediated by a family of
G-protein-coupled receptors (GPCRs), which comprises five
distinct receptor subtypes (Reisine and Bell, 1995) termed
sst1 through sst5. In addition to the initially identified 14-
amino acid peptide (SRIF-14), there is also a second form of
(SRIF-28) (Pradayrol et al., 1980). Both endogenous SRIF
forms bind to the five receptor subtypes with approximately
equal affinity, even though SRIF-28 has been reported to
show some preference for the sst5 (Reisine and Bell, 1995).
Somatostatin receptors are widely expressed in many tis-
sues. Among the broad spectrum of SRIF effects, several
responses have been identified as being mediated through
distinct receptor subtypes. The inhibition of gastric acid se-
cretion, for example, is considered to occur through the sst2,
whereas the inhibition of insulin release appears to be me-
diated by the sst2 and sst5 (Weckbecker et al., 2003).
Although SRIF has attracted considerable attention due to
its wide range of biological functions, it is one of the least
exploited hormones in medical therapeutics. One reason is
the rather short biological half-lives of the endogenous forms
of SRIF. More stable analogs, such as the octapeptides oct-
reotide and lanreotide, have been synthesized to overcome
this problem and are in clinical use for the treatment of
acromegaly and neuroendocrine tumors (Lamberts, 1988;
Weckbecker et al., 1993; Chaudhry and Kvols, 1996). How-
This work was supported by the National Technology Agency of Finland
(Tekes).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.075531.
ABBREVIATIONS: SRIF, somatotropin release-inhibiting factor; GPCR, G-protein-coupled receptor; sst1–5, somatostatin receptor subtypes 1
through 5; h sst1–5, human sst1–5; J-2156, (1ЈS, 2S)-4-amino-N-(1Ј-carbamoyl-2Ј-phenylethyl)-2-(4Љ-methyl-1Љ-naphthalenesulfonylamino)buta-
namide; DMF, N,N-dimethylformamide; DCM, dichloromethane; Fmoc, 9-fluorenylmethoxycarbonyl; Boc, tert-butyloxycarbonyl; CHO, Chinese
hamster ovary; 125I-LTT-SRIF-28, (125I-Tyr)-[Leu8,DTrp22]-somatostatin-28; [35S]GTP␥S, [35S]guanosine-5Ј-O-(3-thio)triphosphate.
332

Superagonism at the Human Somatostatin Receptor Subtype 4
333
ever, these shortened peptides do not interact equally well ros, Tournai, Belgium), and 1-hydroxybenzotriazole (0.57 mmol, Ac-
ros) was added. After 16 h, the solvent was evaporated, and DCM
was added. The organic phase was washed three times with water,
once with brine, and subsequently evaporated. The Fmoc protection
was removed by treating (1ЈS,2S)-4-(N-Boc-amino)-NЈ-(1Ј-car-
bamoyl-2Ј-phenylethyl)-2-(NЈЈ-Fmoc-amino)butanamide with 4.5 ml
of 20% (v/v) piperidine in DMF for 45 min. After solvent evaporation,
the residue was dissolved in 9 ml of dry tetrahydrofuran/DMF (1/1)
and 4-methyl-1-naphthalenesulfonyl chloride (1.5 Eq; Maybridge
Chemicals, Trevillet, UK) plus triethylamine (0.85 mmol; Fluka)
were added. After 16 h, the solvent was evaporated, and the residue
was purified via silica column chromatography (mobile phase from
5% MeOH in DCM up to 20% MeOH in DCM). As a final step, the Boc
protection was removed by treating the product with 2.5 ml of 25%
(v/v) trifluoroacetic acid in DCM for 1 h. Then the solvent was
evaporated, and the residue was purified via reverse-phase high-
performance liquid chromatography to give 52.5 mg of (1ЈS, 2S)-4-
amino-N-(1Ј-carbamoyl-2Ј-phenylethyl)-2-(NЈ-(4Љ-methyl-1Љ-naph-
thalenesulfonyl)amino)butanamide. The structure was verified by
1H NMR and liquid chromatography-tandem mass spectroscopy
analysis.
Cell Culture and Transfections. Chinese hamster ovary (CHO)
cells (K1 strain) were cultured in Ham’s F-12 medium (Nutrient
Mixture Ham’s F-12; Invitrogen, Carlsbad, CA) containing 10% fetal
calf serum. The cDNA encoding the h sst1 or h sst4 (received from Dr.
Einari Aavik, University of Helsinki, Finland) was subcloned from a
construct in pBluescript (Stratagene, La Jolla, CA) into a bicistronic
mammalian expression construct pIRESNeo2 (BD Biosciences Clon-
tech, Palo Alto, CA) with an amino-terminal hemagglutinin tag (BD
Biosciences Clontech) and EF1 promoter (Invitrogen, Groningen,
Netherlands) in h sst4 cloning or CMV promoter (BD Biosciences
Clontech) in h sst1 cloning. The cloned construct was then trans-
fected into CHO cells using FuGENE 6 Transfection Reagent (Roche
Diagnostics, Indianapolis, IN). Stable transfectants were selected in
Ham’s F-12 containing the neomycin analog G418 (Calbiochem, San
Diego, CA) at 500 ␮g/ml. After selection, transfected CHO-h sst4 cells
were sorted and the highest expressing cells collected by fluores-
cence-activated cell sorting (Flow Cytometry Core Facility, Turku,
Finland). Hereafter, both the sorted CHO-h sst4 and the unsorted
CHO-h sst1 cells were cloned by dilution-plating. The highest ex-
pressing clone was isolated in SRIF binding assays using (125I-Tyr)-
[Leu8,DTrp22]-SRIF-28 (125I-LTT-SRIF-28) (Anawa, Zurich, Switzer-
with all the five subtypes but show distinct preferences for
the subtypes sst2 and sst5, and to a somewhat lesser degree,
sst3. Since this pattern of subtype preference has been a
general feature of octapeptide or hexapeptide SRIF analogs,
it has given rise to an operational subdivision of the five
somatostatin receptors into two subfamilies, one consisting of
sst2, sst3, and sst5 and the other of sst1 and sst4 (Hoyer et al.,
1995).
Until recently, the lack of sst4-selective ligands has made it
difficult to connect sst4 more closely to particular (patho-
)physiological functions. However, the expression of both sst1
and sst4 has been reported to be associated with smooth
muscle cell proliferation and vascular intimal thickening
(Aavik et al., 2002). Pharmacological agents acting on sst1
and/or sst4 may therefore be advantageous for the treatment
of diseases involving pathological vascular proliferation, e.g.,
in connection with pathological angiogenesis and restenosis
(Curtis et al., 2000; Ha¨yry and Patel, 2003). Compounds
acting on sst4 have also been suggested to be useful as anti-
glaucoma agents (Mori et al., 1997). Furthermore, based on
the high sst4 expression levels in cerebral cortex, hippocam-
pus, amygdala, and hypothalamus, sst4-selective ligands
may have therapeutic potential in the treatment of various
central nervous system disorders (Bito et al., 1994; Har-
rington et al., 1995; Perez and Hoyer, 1995; Liu et al., 1999).
Considerable progress has been made in recent years in
the identification of nonpeptide ligands for somatostatin re-
ceptor. Most prominently, a research group at Merck has
been able to identify subtype-selective, nonpeptide agonists
for each of the five somatostatin receptor subtypes by using a
combinatorial chemistry approach (Rohrer et al., 1998).
With the promises of subtype-selective somatostatin recep-
tor ligands in mind, we initiated a chemistry program to
identify new, nonpeptide chemical entities selective for hu-
man sst4 (h sst4). In the current communication, we report
that a member of a novel class of somatostatin receptor
ligands, i.e., sulfonamido-peptidomimetics, displays the
unique property of superagonism at the h sst4.
land; specific activity, 2000 Ci/mmol) as
a tracer. CHO cells
expressing the h sst2, h sst3, and h sst5 subtypes were a kind gift
¨
from Prof. Kjell Oberg (Uppsala University Hospital, Uppsala, Swe-
Materials and Methods
den). The recombinant CHO cells expressing the somatostatin recep-
tor subtypes were grown in Ham’s F-12 medium supplemented with
5% fetal bovine calf serum and 200 to 250 ␮g/ml G418.
The receptor densities (Bmax) and radioligand Kd values for the
CHO-h sst4 clones were determined in saturation binding experi-
ments using 125I-LTT-SRIF-28 as radioligand (Siehler et al., 1999). A
CHO-h sst4 cell clone having a receptor density of 5.2 pmol/mg
membrane protein and a Kd of 0.48 nM for 125I-LTT-SRIF-28 (aver-
age of two experiments performed with triplicate concentrations)
was chosen for further pharmacological studies.
Ligands. J-2156 (Fig. 1) was synthesized as follows. H-Phe-NH2
hydrochloride (0.57 mmol; Advanced Chemtech, Louisville, KY) was
dissolved in 2 ml of dry N,N-dimethylformamide (DMF)/dichlo-
romethane (DCM) (1/1) and triethylamine (0.68 mmol; Fluka, Buchs,
Switzerland) was added. After 30 min, a DMF/DCM (1/1.4 ml) solu-
tion containing 4-(tert-butyloxycarbonyl)amino-2-(9-fluorenylme-
thoxycarbonyl)aminobutanoic acid (Fmoc-Dbu(Boc)-OH) (0.57 mmol;
Advanced Chemtech), N,N-diisopropylcarbodiimide (0.57 mmol; Ac-
Receptor Ligand-Binding Assays. Recombinant CHO cells ex-
pressing 1 of the 5 human somatostatin receptor subtypes were
harvested in phosphate-buffered saline and frozen at Ϫ70°C. Mem-
branes were prepared as previously described (Engstro¨m et al.,
2003). In competition binding assays membranes (3–20 ␮g of total
protein per sample, depending on the expression level of individual
subtypes) were incubated in assay buffer (10 mM Hepes, 1 mM
EDTA, 5 mM MgCl2, 5 mg/ml bovine serum albumin, and 30 ␮g/ml
bacitracin, pH 7.6) with 20 to 40 pM 125I-LTT-SRIF-28 and six
concentrations of the test ligand. Each concentration was tested in
duplicate. Nonspecific binding was defined with 1 ␮M SRIF-14 and
corresponded to 5 to 25% of total binding. After 60 min at room
temperature, incubations were terminated by rapid vacuum filtra-
Fig. 1. Structure of J-2156.

334
Engstro¨ m et al.
tion through GF/B glass fiber filter mats (presoaked at 4°C in 200 ml (Arunlakshana and Schild, 1959). When the 95% confidence interval
of 10 mM Hepes, 1 mM EDTA, and 5 mM MgCl2, pH 7.6) and three for the regression line of the Schild plot accommodated a straight
5-ml washes with ice-cold wash buffer (20 mM Tris, 1 mM EDTA, line with the slope of Ϫ1, pA2 values were calculated with the slope
and 5 mM MgCl2, pH 7.4). The filters were then dried and impreg- constrained to unity. Data are given as the average of at least three
nated with scintillate, and their radioactivity was measured by scin- experiments and statistical significance was determined using Stu-
tillation counting.
dent’s two-tailed paired t test.
The affinity profiling on a number of GPCRs other than sst1–5 was
conducted by Cerep (Le Bois L’Eveˆque, Celle L’Evescault, France)
using documented standard procedures.
Results
Compound J-2156 belongs to a new class of somatostatin
G-Protein Activation. The ability of ligands to stimulate the
receptor-mediated binding of
[
35S]guanosine-5Ј-O-(3-thio)triphos- ligands, i.e., sulfonamido-peptidomimetics (Fig. 1). We found
phate ([35S]GTP␥S) (PerkinElmer Life and Analytical Sciences, Bos-
ton, MA; specific activity, 1250 Ci/mmol) to G-proteins in membranes
of CHO cells expressing the h sst4 was measured. Membranes (about
10 ␮g of protein per sample), prepared as previously described (Eng-
stro¨m et al., 2003), were incubated in 20 mM Hepes, pH 7.4, 10 mM
MgCl2, 10 ␮M GDP, 100 mM NaCl, and 10 ␮g/ml saponin (incubation
buffer) with different concentrations of test compound. Each concen-
tration was tested in duplicate. After a 45-min incubation period at
30°C (15-min preincubation without label followed by a 30 min-
stimulation period past the addition of the radiolabel), the reaction
that this compound possesses an affinity of 1.2 nM for the h
sst4 (Table 1). This provides J-2156 with equal or even a
slightly higher h sst4 affinity compared with the endogenous
ligands SRIF-14 and SRIF-28 or cortistatin-17, which has
also been suggested to act via somatostatin receptors (Fuku-
sumi et al., 1997). Since the affinity of J-2156 on the four
other human somatostatin receptor subtypes is 500 nM or
lower, the compound is endowed with a minimum selectivity
of about 400-fold for the h sst4. The profiling of J-2156 at a
was terminated by rapid vacuum filtration through GF/B glass fiber concentration of 1 ␮M against a number of other peptide
filter mats. Filters were then washed four times with 5 ml of ice-cold
wash buffer (20 mM Tris, 1 mM EDTA, and 5 mM MgCl2, pH 7.4),
dried, and counted for radioactivity in a scintillation counter. To
ascertain that the stimulatory effect of J-2156 on [35S]GTP␥S bind-
ing was mediated exclusively via the h sst4, control experiments with
membranes of CHO cells transfected with the ␣2A-adrenoceptor (Po-
hjanoksa et al., 1997) were conducted under identical conditions as
the experiments with membranes of CHO-h sst4 cells.
GPCRs shows very little interaction (Table 2), suggesting the
affinity value of the compound on these receptors is well
above 1 ␮M.
When tested for its ability to stimulate the binding of
[
35S]GTP␥S to membranes of CHO cells stably expressing the
h sst4, J-2156 surprisingly produced a much higher response
than the endogenous ligands SRIF-14 and SRIF-28 (Fig. 2A;
Table 3). This enhanced response toward J-2156 is clearly
mediated through pertussis toxin-sensitive G-proteins since
The effects of SRIF-14 on the J-2156-induced stimulation of
[
35S]GTP␥S binding in membranes of CHO cells expressing the h sst4
were determined by establishing dose-response curves for J-2156 in the stimulatory effect of J-2156, as well as that of SRIF-14, is
the presence of fixed concentrations of SRIF-14 (100 nM, 300 nM,
and 1 ␮M). The assays were run as described above for the determi-
nation of agonist activities, except that the total incubation time was
75 min (15-min preincubation without label followed by a 60-min
stimulation after the addition of the radiolabel).
Adenylyl Cyclase Activity. CHO cells expressing h sst4 (2.5 ϫ
104 cells per well in 96-well plates) were cultured for 20 h at 37°C in
Ham’s F-12 medium containing 5% fetal calf serum and thereafter in
serum-free medium for 1 h. Cells were further incubated for 10 min
with 0.03 mM indomethacine and 0.03 mM rolipram in the cell
incubator before the addition of various concentrations of agonists
and/or 10 ␮M forskolin. After 30 min, incubations were terminated
by removing the medium and adding 20 ␮l of lysis buffer
(PerkinElmer Wallac, Turku, Finland) per well. Cyclic AMP was
measured using a DELFIA cyclic AMP kit as described by the man-
ufacturer (PerkinElmer Wallac).
completely ablated in membranes from h sst4-expressing
CHO cells that have been pretreated with pertussis toxin, an
agent known to uncouple Gi/o proteins from GPCRs (Fig. 2A).
Neither J-2156 nor SRIF-14 at micromolar concentrations
produce any significant increase in [35S]-GTP␥S binding with
membranes from CHO cells that lack the h sst4 but which
instead stably express the human ␣2A adrenergic receptors
(Fig. 2B). In contrast, the ␣2-adrenoceptor agonist epineph-
rine produces the expected dose-dependent increase in
[
35S]GTP␥S binding in these membranes.
Increasing amounts of SRIF-14 cause a concentration-de-
pendent right-shift of the dose-response curve for J-2156
without affecting its maximal response (Fig. 3A). For the six
analyses conducted, the linear regression analysis yielded a
slope of 1.3 Ϯ 0.2 (r2 ϭ 0.8119). The confidence limits of the
Schild regression comfortably accommodate a slope of unity
and a linear regression computed with the slope constrained
to such a value yielded a pA2 value of 8.00 (Fig. 3B).
Data Analysis. Results from individual experiments were deter-
mined via nonlinear least-squares curve fitting procedures. Affinity
constants (Ki values), as determined in competition binding assays,
were calculated from the IC50 values according to the Cheng-
Prusoff’s equation (Cheng and Prusoff, 1973). Apparent pA2 values
To exclude the possibility that the higher efficacy of J-2156
and their 95% confidence limits were estimated by Schild analysis at the h sst4 might be due to metabolic instability of SRIF-14,
TABLE 1
Binding affinity (Ki) of ligands for human recombinant SRIF receptor subtypes (h sst1–5) stably expressed in CHO cells
The data are given as the mean Ϯ S.E.M. of at least three experiments. If the competition curve was incomplete, the Ki is given as greater than the highest concentration
tested.
Ki
h sst1
h sst2
h sst3
h sst4
h sst5
nM
SRIF-14
1.4 Ϯ 0.2
0.57 Ϯ 0.09
0.77 Ϯ 0.32
500 Ϯ 150
0.62 Ϯ 0.15
0.34 Ϯ 0.14
1.9 Ϯ 0.4
Ͼ5000
1.6 Ϯ 0.2
0.28 Ϯ 0.11
1.9 Ϯ 0.4
4.1 Ϯ 0.6
1.9 Ϯ 0.3
3.5 Ϯ 0.3
1.2 Ϯ 0.4
4.1 Ϯ 0.4
0.30 Ϯ 0.12
3.3 Ϯ 0.3
540 Ϯ 80
SRIF-28
Cortistatin-17
J-2156
1400 Ϯ 100

Superagonism at the Human Somatostatin Receptor Subtype 4
335
TABLE 2
The effect of 1 ␮M J-2156 on specific radioligand binding at various
receptor targets
The data are given as a percentage of inhibition of specific binding (control set as
0%).
Receptor
Inhibition
%
ANP
0
0
Bombesina
Bradykinin-1
Bradykinin-2
Glucagon
4
14
8
MCH
0
Melanocortin-1
Melanocortin-3
Melanocortin-4
Melanocortin-5
Motilin
0
0
3
0
0
Neuropeptide Y
Opioid-␦2
Opioid-␬
Opioid-␮
8
0
0
5
Urotensin II
VIP-1
12
5
VIP-2
5
Vasopressin-1a
Vasopressin-1b
Vasopressin-2
0
0
1
ANP, atrial natriuretic peptide; MCH, melanin-concentrating hormone; VIP,
vasoactive intestinal peptide.
a Nonselective, tissue source rat brain.
we measured the stimulation kinetics of saturating concen-
trations of both compounds at various time points up to 60
min. As can be seen from Fig. 4, the extent of binding elicited
by both compounds, as well as the basal binding of
Fig. 2. Stimulation of [35S]GTP␥S binding in membranes of CHO cells
expressing h sst4 (A) or human ␣2A-adrenergic receptor (CHO-C10; B). A,
CHO-h sst4 cells either nonpretreated or pretreated with pertussis toxin.
Trace amounts of [35S]GTP␥S were added to membrane CHO-h sst4 cells
(about 10 ␮g/sample) that had been preincubated with the indicated
[
35S]GTP␥S, is linear over the whole time period with the
stimulation of J-2156 possessing a clearly steeper slope. concentration of SRIF-14 (Ⅺ, f), SRIF-28 (E), or J-2156 (‚, Œ) in the
presence of 10 mM MgCl2, 10 ␮M GDP, 100 mM NaCl, and 10 ␮g/ml
Thus, the higher efficacy J-2156 is observable already at the
earliest time points measured, and the response toward
saponin. The membranes were prepared from nonpretreated cells (open
symbols) or from cells that had been pretreated with 100 ng/ml pertussis
toxin for 16 h (closed symbols). B, trace amounts of [35S]GTP␥S added to
membranes of transfected cells (about 10 ␮g/sample) that had been pre-
incubated with the indicated concentration of SRIF-14 (Ⅺ), J-2156 (‚), or
epinephrine (ƒ) in the presence of 10 mM MgCl2, 10 ␮M GDP, 100 mM
NaCl, and 10 ␮g/ml saponin. Basal [35S]GTP␥S binding to membranes of
the nonpretreated and pretreated CHO-h sst4 cells were 103 Ϯ 9 and 29 Ϯ
3 fmol/mg protein, respectively, whereas the corresponding value in mem-
SRIF-14 does not show signs of compound depletion. Further-
more, a stability analysis by high-performance liquid chro-
matography revealed that maximally 25% of SRIF-14 is be-
ing degraded during the 1-h incubation (data not shown).
To further characterize the functional activity of J-2156 at
the h sst4, we evaluated its effect on forskolin-stimulated
cyclic AMP production in the same CHO-h sst4 cell line, from
which membranes were prepared for use in the [35S]GTP␥S
binding studies. The addition of 10 ␮M forskolin increases
cellular cyclic AMP accumulation by about 10-fold in the
CHO-h sst4 cells. SRIF-14, SRIF-28, and J-2156 act as
apparent full agonists by completely inhibiting the forskolin-
stimulated adenylyl cyclase activity (Fig. 5; Table 3). How-
ever, in the cyclic AMP assay, the compounds are consider-
ably more potent than in the [35S]GTP␥S binding assay
(Table 3). In control CHO cells, expressing the human ␣2A
adrenergic receptors, there is no effect of J-2156 on forskolin-
stimulated adenylyl cyclase activity (data not shown).
branes of CHO-C10 cells was 185
Ϯ 7 fmol/mg protein. Maximal
[
35S]GTP␥S binding to membranes of CHO-h sst4 cells was 396 Ϯ 13
fmol/mg protein (stimulated by J-2156; A); maximal binding to mem-
branes of CHO-C10 cells was 661 Ϯ 21 (stimulated by epinephrine; B).
Stimulation is given as a percentage over basal. The combined data of at
least three different experiments with each concentration run in dupli-
cate are shown.
is comparable with that of SRIF-14 and SRIF-28, its func-
tional properties are uniquely different by demonstrating
superagonism at the h sst4.
We used two functional assays to measure agonist-induced
responses at the h sst4: first, the binding of [35S]GTP␥S in
membranes containing the h sst4 and, second, the agonist-
mediated inhibition of cyclic AMP accumulation in forskolin-
stimulated cells expressing the receptor. The [35S]GTP␥S
binding assay measures the most proximal event after recep-
Discussion
In this report, we describe a novel compound, J-2156, that tor activation, whereas the inhibition of cyclic AMP accumu-
belongs to a new class of somatostatin receptor ligands, i.e., lation represents a more distal step at the level of second
sulfonamido-peptidomimetics. J-2156 possesses nanomolar messenger production. In the [35S]GTP␥S binding assay,
affinity for the h sst4 and is at least 400-fold selective for that J-2156 produces a significantly greater response compared
receptor over any of the other four human somatostatin re- with SRIF. This stimulation of [35S]GTP␥S binding by J-2156
ceptor subtypes. Although the affinity of J-2156 on the h sst4 is fully dependent on pertussis toxin-sensitive G-proteins

336
Engstro¨ m et al.
TABLE 3
Agonist effects of ligands at the human sst4
Agonist activity (EC50 and efficacy) was assessed as the ability of the ligand to stimulate ͓35S͔GTP␥S binding in membranes of CHO cells expressing human sst4 (CHO-h sst4)
or as the ability of the compound to inhibit forskolin-stimulated cyclic AMP accumulation in CHO-h sst4 cells (see Figs. 2A and 5). The data are given as the mean Ϯ S.E.M.
of at least three experiments.
͓
35S͔GTP␥S Binding Assay
Cyclic AMP Assay
Efficacy (Inhibition
of Cyclic AMP
Accumulation)
EC50
Efficacy (Stimulation over Basal)
EC50
nM
%
nM
%
SRIF-14
SRIF-28
J-2156
4.9 Ϯ 0.4
81 Ϯ 24
17 Ϯ 2
80 Ϯ 10
130 Ϯ 10*
290 Ϯ 20**
0.42 Ϯ 0.12
0.73 Ϯ 0.18
0.07 Ϯ 0.03
98 Ϯ 3
93 Ϯ 5
102 Ϯ 2
* Efficacy significantly different from SRIF-14 (p Ͻ 0.05, Student’s paired t test).
** Efficacy significantly different from SRIF-14 (p Ͻ 0.01, Student’s paired t test).
Fig. 4. [35S]GTP␥S binding in membranes of CHO cells expressing the h
sst4 after different stimulation times. Trace amounts of [35S]GTP␥S,
membranes of transfected cells (about 10 ␮g/sample), and buffer (basal;
E) or saturating concentrations of SRIF-14 (3 ␮M; Ⅺ) or J-2156 (10 ␮M;
‚) were incubated for the indicated times in the presence of 10 mM
MgCl2, 10 ␮M GDP, 100 mM NaCl, and 10 ␮g/ml saponin. One represen-
tative example of three different experiments performed in triplicate is
shown.
Fig. 3. Inhibition of J-2156-induced stimulation of [35S]GTP␥S binding in
membranes of CHO cells expressing the h sst4. Trace amounts of
[
35S]GTP␥S were added to membranes of transfected cells (about 10
␮g/sample) that had been preincubated without (Ⅺ) or with increasing
concentrations of SRIF-14, 100 nM (E), 300 nM (ƒ), and 1 ␮M (‚), in the
presence of 10 mM MgCl2, 10 ␮M GDP, 100 mM NaCl, and 10 ␮g/ml
saponin (A). B, Schild analysis of antagonism by SRIF-14 of J-2156-
induced stimulation of [35S]GTP␥S binding. Basal [35S]GTP␥S binding to
membranes of CHO-h sst4 cells that had been preincubated without or
with increasing concentrations of SRIF-14 were 57 Ϯ 4 (w/o SRIF), 123 Ϯ
5 (100 nM SRIF-14), 132 Ϯ 8 (300 nM SRIF-14), and 139 Ϯ 9 (1 ␮M
SRIF-14) fmol/mg protein. Maximal [35S]GTP␥S binding after preincuba-
tion (without or with SRIF-14) and stimulation with J-2156 were 188 Ϯ
13 (without SRIF-14), 207 Ϯ 9 (100 nM SRIF-14), 206 Ϯ 20 (300 nM
SRIF-14), and 203 Ϯ 11 (1 ␮M SRIF-14) fmol/mg protein. To calculate the
dose ratios, EC50 values were taken as the equiactive agonist concentra-
tions. In A, the combined data of three different experiments with each
concentration run in duplicate are shown, and stimulation is given as a
percentage of the maximal effect produced by J-2156 in the absence of
SRIF-14 (control, set as 100%). In B, a Schild plot was generated from the
data of six experiments of the type shown in A. The 95% confidence
interval for the regression line in the Schild analysis easily accommo-
dated a slope of unity; therefore, the slope was constrained to that value.
Under these circumstances, the apparent pA2 was 8.00.
Fig. 5. Inhibition of forskolin-stimulated cyclic AMP accumulation. Cyclic
AMP accumulation in forskolin-stimulated CHO cells stably transfected
with h sst4 was measured in the presence of increasing concentrations of
SRIF-14 (f), SRIF-28 (E), or J-2156 (‚). The signal in the assay was 95 Ϯ
15 pmol/well, the readouts for blank (measured in the absence of cells)
and total forskolin-stimulated cyclic AMP accumulation (measured in
CHO-h sst4 cells in absence of ligand) being 7.3 Ϯ 1.4 and 102 Ϯ 16
pmol/well, respectively. The readout for maximal inhibition by SRIF-14
was 11 Ϯ 5 pmol/well, by SRIF-28 10 Ϯ 3 pmol/well, and by J-2156 6.0 Ϯ
2.4 pmol/well. The data are normalized against the forskolin-induced
cyclic AMP concentration in the absence of agonist (control, set as 100%).
The combined data of three different experiments with each concentra-
tion run in triplicate are shown.
and on the presence of the h sst4 in the CHO cell membranes.
The latter observation excludes a possible contribution from
other GPCRs in the background of the CHO cells toward the
response caused by J-2156. The amplitude of the J-2156

Superagonism at the Human Somatostatin Receptor Subtype 4
337
dose-response curve implies that the endogenous ligands confidence interval for the regression line easily accommo-
SRIF-14 and SRIF-28 are not able to cause the maximal dates a straight line with a slope of unity. It is, therefore, not
possible degree of receptor activation on the h sst4. Consis- likely that J-2156 acts as an allosteric ligand on the h sst4.
tent with this hypothesis, SRIF-14 was found to behave like Such an allosteric interaction has been proposed for a com-
a typical partial agonist when tested against J-2156. That is pound designated ASWL, which has been described as a
to say, increasing concentrations of SRIF-14 cause a right- superagonist of the chemokine receptor CXCR4 (Sachpatzi-
ward shift of J-2156 dose-response curves, without affecting dis et al., 2003). Sachpatzidis et al. (2003) have suggested
its maximal response. Therefore, J-2156 can be classified as that the superagonism of ASWL might stem from its inability
a superagonist at h sst4.
to induce receptor internalization, which may lead to contin-
In the cyclic AMP assay, for which the same CHO-h sst4 uous receptor activation. Such a differential propensity of
cell line was used that served as the source for the mem- J-2156 and SRIF to cause receptor internalization does not
branes of the [35S]GTP␥S binding assay, J-2156 and SRIF appear likely as an explanation for the superagonism of
cause a complete inhibition of forskolin-stimulated adenylyl J-2156 since a recent publication has reported that the extent
cyclase activity. In many cases of heterologously expressed of h sst4 internalization in CHO cells upon stimulation with
receptors, G-protein activation assays offer an advantage SRIF-14 was so small that it could not be observed by confo-
over second messenger-based assays with respect to the dis- cal microscopy (Smalley et al., 2001). Furthermore, the su-
crimination of partial versus full agonists. Cyclic AMP-based peragonism of J-2156 is detected in membrane preparations,
assays in particular appear to be prone to the receptor re- in which receptor internalization is not expected to play a
serve phenomenon, which can mask partial agonist activity role. This same reason also argues against a lack of de-
(Adham et al., 1992; Umland et al., 2001). Umland et al. sensitization by J-2156 compared with SRIF-14. Although
(2001), for example, have reported for the human ␣2C-adren- SRIF-14 has been described to desensitize the h sst4 receptor
ergic receptor that a 20-fold greater receptor occupancy was in intact CHO cells (Smalley et al., 1998), we did not observe
required for half-maximal agonist-induced [35S]GTP␥S bind- a reduction in the maximal response of J-2156 in the pres-
ing to membranes compared with the inhibition of cyclic ence of increasing concentrations of SRIF-14. This, however,
AMP formation. The reason for this susceptibility of cyclic is what would be expected to occur if SRIF-14 were to desen-
AMP-based assays to receptor reserves appears to lie in the sitize the h sst4 in membranes. Maybe SRIF-14 and J-2156
relative amounts of receptor, G-proteins and adenylyl cy- stabilize distinct receptor conformations of the h sst4 that
clase, with the cyclase representing the stoichiometrically differ significantly in their ability to interact productively
limiting component (Kim et al., 1994). In our experiments, with G-proteins. This has been suggested as a possible mech-
the potency of J-2156 is 240-fold higher for the inhibition of anistic difference between partial and full GPCR agonists
adenylyl cyclase than for the stimulation of [35S]GTP␥S bind- (Kenakin, 1996a,b; Tucek, 1997; Seifert et al., 2001). The use
ing. We also found higher potencies for SRIF-14 and SRIF-28 of subtype-specific G-protein antisera would allow one to
in the cyclic AMP assay compared with the [35S]GTP␥S bind- study which G-protein ␣-subunits physically associates with
ing assay, the fold shift being greater for the more efficacious sst4 and whether the extent of interaction between the re-
SRIF form (110- and 12-fold for SRIF-28 and SRIF-14, re- ceptor and a specific G-protein is agonist-dependent. In pre-
spectively). These agonist potency shifts are consistent with vious studies, it has been shown by immunoblotting that
the existence of a receptor reserve in the cyclic AMP assay or G␣i1, G␣o, and G␣z are not present at detectable levels in
a greater receptor reserve in the cyclic AMP assay compared CHO-K1 membranes and that G␣i2 and G␣q/11 are present at
with the [35S]GTP␥S binding assay. The presence of a recep- much higher levels than G␣i3 (Dell’Acqua et al., 1993; Gettys
tor reserve is predicted to produce a greater gain in apparent et al., 1994; Gu and Schonbrunn, 1997). Pertussis toxin com-
potency for highly efficacious agonists compared with partial pletely abolishes the [35S]GTP␥S binding stimulated by
agonists because lower levels of receptor occupancy are re- SRIF-14 or J-2156, indicating that sst4 couples to the G␣i/o
quired to elicit the maximal response at the level of the class of G proteins in CHO-K1 cells. Taken into account the
effector enzyme. Our interpretation of the results implies differential expression profiles of the G-protein ␣-subunits in
that an efficacy readout in the form of a cyclic AMP assay CHO-K1 cells, the agonist-mediated increase of [35S]GTP␥S
may provide a misleading picture in terms of agonist effica- binding in membranes of CHO-h sst4 cells most likely in-
cies when using recombinant cell lines with large receptor volves G␣i2 and/or G␣I3. Furthermore, one could hypothesize
expression levels.
that the binding domains of peptides and small molecule
In an attempt to obtain evidence that SRIF-14 and J-2156 agonists may differ to a sufficiently large degree as to give
act at a common site on the h sst4, we established dose- rise to the observed differences in agonist efficacies. To ex-
response curves for J-2156 in the presence of different con- plore this possibility further, site-directed mutagenesis and
centrations of SRIF-14. The presence of SRIF-14 has two molecular modeling will be needed to pinpoint the critical
effects on the dose-response curves of J-2156. It causes a residues in the h sst4 involved in the binding of the endoge-
larger extent of [35S]GTP␥S binding at ineffective concentra- nous peptides and J-2156.
tions of the compound, and it concentration dependently
In conclusion, we describe a novel compound, J-2156, that
shifts the dose-response curves of J-2156 to the right. Impor- belongs to a new class of somatostatin receptor ligands, i.e.,
tantly, however, there are no significant differences in the sulfonamido-peptidomimetics. J-2156 possesses high affinity
maximal response toward J-2156 in the absence or presence and selectivity for the h sst4 over any of the other four human
of SRIF-14. In other words, SRIF-14 displays the typical somatostatin receptor subtypes. Although the affinity of
behavior of a partial agonist in its effects on the dose-re- J-2156 at the h sst4 is comparable with that of SRIF-14 and
sponse curve of J-2156. A Schild analysis of the data results SRIF-28, its functional properties are unique in demonstrat-
in a regression line with a slope of 1.3 Ϯ 0.2. The 95% ing superagonism at the h sst4.

338
Engstro¨ m et al.
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Acknowledgments
We thank Einari Aavik for providing the h sst1 and h sst4 cDNAs
and Liisa Huovinen, Sari Kalliokoski, and Pa¨ivi Pohjola for excellent
technical assistance.
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