Identification of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor

Article abstract of DOI:10.1016/j.bmcl.2012.05.024

The discovery of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor is described. The characterization and redressing of the issues associated with these compounds is detailed. An efficient three-step synthesis and a binding assay were relied upon as the primary means of rapidly improving potency and ADMET properties for this class of inverse agonist compounds. Compound 10n bearing distributed polarity in the form of an imidazo-thiazole acetamide and a phenyl triazole is a unit lower in log P and has significantly improved binding affinity compared to the hit molecule 10a, providing support for further optimization of this series of compounds.

Full text of DOI:10.1016/j.bmcl.2012.05.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4281–4287
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of spirocyclic piperidine-azetidine inverse agonists
of the ghrelin receptor
⇑
Daniel W. Kung , Steven B. Coffey, Ryan M. Jones, Shawn Cabral, Wenhua Jiao, Michael Fichtner,
Philip A. Carpino, Colin R. Rose, Richard F. Hank, Michael G. Lopaze, Roger Swartz, Hou (Tommy) Chen,
Zachary Hendsch, Bruce Posner, Christopher F. Wielis, Brian Manning, Jeffrey Dubins, Ingrid A. Stock,
Sam Varma, Mary Campbell, Demetria DeBartola, Rachel Kosa-Maines, Stefanus J. Steyn, Kim F. McClure
⇑
Pfizer Worldwide Research and Development, Eastern Point Road, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor is described. The
characterization and redressing of the issues associated with these compounds is detailed. An efficient
three-step synthesis and a binding assay were relied upon as the primary means of rapidly improving
potency and ADMET properties for this class of inverse agonist compounds. Compound 10n bearing dis-
tributed polarity in the form of an imidazo-thiazole acetamide and a phenyl triazole is a unit lower in
logP and has significantly improved binding affinity compared to the hit molecule 10a, providing support
for further optimization of this series of compounds.
Received 8 March 2012
Revised 4 May 2012
Accepted 8 May 2012
Available online 17 May 2012
Keywords:
Ghrelin
GHS-R1a
Ó 2012 Elsevier Ltd. All rights reserved.
Antagonist
Inverse agonist
Spirocyclic
Piperidine-azetidine
Introduction
Based on the structures of the antagonists reported recently
(see 1,14 2,15 3,16 4,17 5,18 619 in Fig. 1) the characteristics of the
The development of small molecules targeting the growth hor-
mone secretagogue receptor 1a (GHS-R1a) goes back to the mid
1980s, predating the identification of the natural ligand, ghrelin.1
While small molecule agonists of this peptide G-protein coupled
receptor (GPCR) such as ibutamoren,2 capromorelin,3 and ulimore-
lin4 have reached the clinic, none has reached the market. More re-
cently interest has turned to the opposite functional profile.
Although some aspects of the switch to functional antagonists
are controversial,5 the change has been driven by a steady stream
of supporting evidence suggesting that ligand neutralization6,7 or
receptor deletion8,9 can have beneficial effects on body weight
and glucose homeostasis.10–12
Accompanying the move in functional profile to antagonists or
inverse agonists is a decision to target a centrally acting agent
capable of reaching receptors in the hypothalamus, or to target
an agent that is more peripherally restricted. The agonist capromo-
relin with its peptidic character and multiple hydrogen bond do-
nors is a peripherally restricted molecule that reaches its target
in the pituitary, but does not reach other central nervous tissue.13
key N-terminal amino acid residues20 of the endogenous peptide
are less apparent than for the early agonist structures. Likely, this
results from advances in the ability to rapidly screen this target
and to identify novel chemical matter. Herein we report the iden-
tification and initial optimization of a series of spirocyclic piperi-
dine-azetidines from an initial high throughput binding assay.
To identify hit compounds that possessed the potential to act as
antagonists or inverse agonists at the ghrelin receptor, a two-stage
screening sequence was established. Our primary screen was a
radioligand binding assay based on 125I-ghrelin and a scintillation
proximity assay (SPA) format.21 Although the ghrelin receptor is
unusually prone to agonists22 our plan to first assess ligand affinity
was driven by our desire to start with the most efficient substrate,
allowing maximum flexibility in the optimization process. This
strategy contrasts with high throughput screening efforts else-
where to identify lead matter using functional assays.19 Our initial
functional screening using a calcium mobilization FLIPR assay
identified the same limitations described by Pasternak18 and we
were unable to properly discriminate an agonist from an inverse
agonist. To fully characterize the functional profile of our binding
hits we used a GTP-c
-S assay.23 Interestingly, very few ‘silent
⇑
Corresponding authors.
E-mail addresses: daniel.w.kung@pfizer.com (D.W. Kung), kim.f.mcclure@
antagonists’24 were identified using this screening paradigm. The
vast majority of hits showed either partial agonist activity or
pfizer.com (K.F. McClure).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.024

4282
D. W. Kung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4281–4287
O
O
GSSFLSPEHQRVQQRKESKKPPAKLQPR
human ghrelin
N
O
O
F
Cl
N
O
N
O
1
N
O
HN
S
O
4
Cl
N
O
O
N
O
H2N
N
N
H
MK-0677 ibutamoren (GHS-R1a agonist)
NH2
O
O
O
O
2
S
O
H
O
N
N
H
5
N
O
O
N
N
H
N
H
O
O
N
N
N
H2N
N
H
O
Ph
CP-424391 capromorelin (GHS-R1a agonist)
N
F
O
H
N
O
N
NH
N
3
NH2
N
O
O
HN
HN
O
F
6
N
O
H2N
NH
N
O
O
TZP-101 ulimorelin (GHS-R1a agonist)
Figure 1. Ghrelin receptor agonists and antagonists.
inverse agonist properties. Priority for lead follow-up was given to
inverse agonists to ensure a non-agonist profile, and to compounds
with a low hydrogen bond donor count and physicochemical prop-
erties consistent with achieving CNS penetration.
mentary to absolute potency for assessing binding activity. To sim-
plify design of compounds and analysis of specific structure-
activity relationships, we focused primarily on making changes
to one end of the molecule at a time, with the expectation of sub-
sequent iterations of synthesis that would cross ‘good’ substituents
from the initial analogues. Recognizing that most of the hydropho-
bicity in the compound lay in the dimethylchroman group of the
lead compound 10a, the majority of our initial compounds shown
in Table 1 focused on changes to this region of the molecule,
although the flexible synthetic route enabled us to probe concur-
rently changes to both ends of the molecule.
Several active compounds that replaced the dimethylchroman
group in 10a with lower molecular weight or lower lipophilicity
groups were identified. The parent benzyl derivative 10b was syn-
thesized as a benchmark compound. General trends in SAR of the
azetidine-benzyl substituent included the following (some data
not shown). At the 2-position of the phenyl ring, small lipophilic
groups such as a chlorine atom (10c), provided enhanced ligand
efficiency relative to the hit compound; larger groups diminished
both potency and ligand efficiency. Substitutions at the 4-position
were preferred to those at the 3-position of the phenyl ring, based
on both potency and ligand efficiency. At the 4-position, electron
donating groups (e.g., isobutyoxy, 10d) provided enhanced potency
relative to electron withdrawing groups (e.g., oxadiazole, 10e). A
subset of compounds that spanned a range of structural types
and physical properties (as well as a range of ghrelin receptor bind-
ing potencies) were tested in high throughput ADMET assays to
establish a baseline understanding of structure-ADMET relation-
ships. Within the initial set of compounds tested in ADMET assays,
the oxadiazole 10e was noted because it demonstrated relatively
low HLM clearance and dofetilide binding.
Compound 10a (Table 1), identified from a screen of our corpo-
rate file, was an attractive hit because of its inverse agonist func-
tional profile and moderate binding potency. Its physical
properties (molecular weight 464, clogP 4.4) were modestly out-
side of our desired range for a hit that targeted CNS penetration,
but our expectations were tempered by the known difficulties of
finding low molecular weight ligands for peptidic GPCRs.25 The
structure of compound 10a, a spiro-piperidine-azetidine with the
two nitrogens functionalized respectively as an amide and a
tertiary amine, provided an opportunity for efficient synthesis of
analogues around the spiro-diamine core.26 Starting from tert-
butoxycarbonyl (Boc) protected diamine 7,27 we developed syn-
thetic conditions that enabled the parallel synthesis of analogues
as shown in Scheme 1. The identification of efficient synthetic con-
ditions utilizing readily available reagent classes (carboxylic acids
and aromatic aldehydes) that contain significant structural diver-
sity enabled us to rapidly query structure-activity relationships
with regard to potency, as well as with regard to absorption, distri-
bution, metabolism, excretion, and toxicity (ADMET) properties.
Our initial approach to the design of compounds was to move
the physical properties of the hit compound toward lower lipophil-
icity and/or lower molecular weight chemical space, while also
probing potency, functional profile, and ADMET properties (with
a focus on stability in human liver microsomes (HLM) and on activ-
ity in a dofetilide binding assay as a proxy for hERG activity). Be-
cause one of our aims was to decrease molecular weight relative
to the hit, we employed ligand efficiency28 as a method comple-

D. W. Kung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4281–4287
4283
Table 1
Lead compounds and initial SAR
Compound
Human ghrelin
receptor
binding IC50
LE
ClogP
HLMc
Dofetilided Human
ghrelin
Funct.
Rat CLp mL/min/kg
(ElogDb) (RLM)
responsea,e (percent
pulmonary
a
receptor Kia
(nM)
(nM)
extraction)
10a
N
N
N
N
O
31
(71)
211
0.27 4.4
0.27 2.9
0.31 3.7
0.29 4.3
77
408
À22
O
N
O
O
10b
O
2980
N=1
À22
N=1
21
N
O
O
10c
O
231
86
47
88
445
20
À26
À18
Cl
N
O
O
10d
O
106
Cl
O
N
O
O
10e
N
N
N
N
1.8
(1.5)
O
1070
0.24
<8
46
52
À31
61 (22%)
N
N
O
N
O
O
10f
O
3.0
(1.9)
20
(32)
442
102
14
0.26
0.29
0.31
94
76
87
152
14
À32
À33
À40
N
N
N
O
O
10g
O
3.0
(2.2)
22
(42)
152
N
N
N
N
N
O
O
10h
O
3.9
(4.1)
54
(189)
11
83 (80%)
Cl
N
N
N
O
O
10i
N
3.2
(2.6)
31
(123)
407
0.28
0.29
64
114
À19
À21
447
N
O
N
O
O
10j
N
4.2
(3.4)
42
(135)
86
102
25
O
N
S
N
O
(continued on next page)

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D. W. Kung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4281–4287
Table 1 (continued)
Compound
Human ghrelin
receptor
binding IC50
LE
ClogP
(ElogDb) (RLM)
HLMc
Dofetilided Human
ghrelin
Funct.
Rat CLp mL/min/kg
responsea,e (percent
pulmonary
a
receptor Kia
(nM)
(nM)
extraction)
10k
N
3.8
(3.3)
63
(105)
120
0.28
0.29
14
À27
O
N
N
O
HN
10l
N
O
4.9
(4.4)
43
(107)
59
100
17
À17
238
Cl
O
N
N
O
O
a
b
c
Unless otherwise indicated cumulative in vitro characterization is the result of 3 or more determinations run in duplicate or triplicate.
Reference.29
Human and rat liver microsome intrinsic apparent clearance, lL/min/mg.
d
e
Dofetilide binding percent inhibition @ 10
lM.
Reference23, agonist mode maximum functional response (minimum asymptote) in GTP-
c
-S functional assay. A negative value indicates an inverse agonist.
such as 10h, which demonstrated the anticipated improvement in
potency and ligand efficiency at the expected expense of increased
lipophilicity and clearance. However, identifying specific structural
changes (para-heteroaryl, ortho-chloro) that enabled us to predict
relatively well the trade-offs between potency and clearance was
a significant breakthrough as we continued to further optimize
compounds in this series.
H
N
R1
R1
N
N
N
N
a
b,c
+
R1
O
N
Boc
7
8
10a-r
R2
9
Boc
O
On the piperidine amide side of the molecule, concerns about
possible metabolic pathways guided initial efforts focused on iden-
tifying structural alternatives to the masked hydroquinone func-
tionality. A structurally diverse set of amides was synthesized,
but only relatively small structural changes were found to retain
significant ghrelin receptor binding activity. A one-atom deletion
from phenoxyacetamide to phenylacetamide was tolerated (10i),
as was replacement of the dialkoxyphenyl group by selected bicyc-
loheteroaromatic rings (10j and 10k). Simple substitution of pyri-
dine for phenyl also provided adequate potency (10l; coupled
with the potency-enhancing ortho-chloro benzyl substitution).
These alternative heteroaryl acetamide derivatives in particular
provided a structural direction in which we could further modulate
overall lipophilicity of the compounds, as well as the electron-den-
sity in this aromatic ring. Importantly, from the initial iterations of
synthesis to develop SAR around both the amide and amine sub-
stituents on the spiro-piperidine-azetidine core, all of the com-
pounds tested in the functional assay displayed inverse agonist
profiles.
With an initial understanding of the potency and ADMET SAR
within this series, we then sought to benchmark selected com-
pounds for in vivo pharmacokinetics. Our primary objective was
to understand the overall properties of this series of amido-piper-
idine-azetidines, rather than to characterize a single lead com-
pound. Thus, a subset of compounds representing the range of
productive structural modifications was selected for rat pharmaco-
kinetic studies properties using cassette dosing.30,31 Up to 5 com-
pounds were combined per cassette, ensuring at least 2.5 amu
differences between the compounds in the cassette. Animals were
dosed at 0.4 mg/kg for iv studies and 1 mg/kg for po studies.
Disturbingly, all compounds tested in rat showed high clearance
(in most cases, exceeding hepatic blood flow = 70 mL/min/kg; see
Table 1). Several hypotheses were put forth to explain the high rate
of clearance. Hypotheses that were subsequently disproved with
experimental data included: instability of compounds in rat
Scheme 1. Synthesis of piperidine-azetidine inverse agonists of the ghrelin
receptor. Reagents and conditions: (a) Na(OAc)3BH, 30–40%; (b) HCl, dioxane,
99%; (c) HBTU, DIEA, R2CO2H, DMF, 90%.
The initial SAR on the benzyl substituent suggested two distinct
directions for follow-up; both were pursued simultaneously. One
approach for compound design focused on the desirable ADMET
properties of the phenyl-oxadiazole 10e. The other approach built
on the observed increases in ligand efficiency or potency: ortho
substitution of the phenyl ring with a chlorine atom, and/or para
substitution of the phenyl ring with an alkoxy group.
The relatively good ADMET properties of the phenyl-oxadiazole
compound 10e were hypothesized to be due to the lower lipophil-
icity (clogP 1.8; elogD 1.5) and/or the electron withdrawing nature
of the oxadiazole substituent on the phenyl ring. A broad set of
compounds that further pursued this hypothesis was synthesized.
The first set of phenyl-heteroaryl derivatives focused on pyridine,
imidazole, pyrazole, and triazole isomers in the terminal ring as
representative compounds with varied overall lipophilicity as well
as different spatial placement of heteroatom(s). The variation in
ghrelin receptor binding potency across this set of compounds
was interpreted to be evidence of specific interactions between
this region of the molecule and the receptor. A subset of these
five- and six-membered heteroaryl derivatives did demonstrate
improved potency relative to the oxadiazole, and a partially over-
lapping subset also exhibited relatively good HLM stability. The
balance between potency and clearance exhibited by this group
of compounds, exemplified by the pyrazole and triazole derivatives
(10f and 10g), was appealing, although dofetilide binding re-
mained a concern.
A subsequent iteration of synthesis to combine the previously
identified potency-enhancing ortho-chloro substitution with a
selection of the phenyl-heteroaryl derivatives provided compounds

D. W. Kung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4281–4287
4285
Table 2
Compounds with improved overall profiles
Compound
Human ghrelin
receptor
binding IC50
LE
ClogP
(ElogDb) (RLM)
HLMc
Dofetilided Human
ghrelin
Funct.
Rat CLp mL/min/kg
responsea,e (percent
pulmonary
a
receptor Kia
(nM)
(nM)
extraction)
10m
N
2.6
(1.9)
18
(19)
N
N
25
0.31
0.33
0.28
0.30
20
40
56
11
69
À22
À35
À23
À23
48 (0%)
N
N
N
N
N
N
N
N
O
O
O
O
S
10n
10o
10p
N
N
N
3.4
(3.6)
34
(159)
Cl
N
N
4.6
298
90
7.0
206
14
41 (0%)
N
N
N
N
S
2.5
(1.5)
11
(<16)
N
N
N
S
N
2.1
(1.4)
14
(<16)
N
N
S
10q
N
3.0
(2.4)
204
321
0.27
0.25
23
63
32
23
27
À27
À28
17 (0%)
N
N
N
N
N
N
N
N
O
Cl
Cl
10r
N
2.6
(1.7)
<8
(<14)
N
N
N
O
a
b
c
Unless otherwise indicated cumulative in vitro characterization is the result of 3 or more determinations run in duplicate or triplicate.
Reference.29
Human and rat liver microsome intrinsic apparent clearance,
lL/min/mg.
d
e
Dofetilide binding percent inhibition @ 10 lM.
Reference23, Agonist mode maximum functional response (minimum asymptote) in GTP-
c
-S functional assay. A negative value indicates an inverse agonist.
plasma, selective partitioning of compounds into blood from plas-
ma, biliary excretion, and renal excretion.
Though we did not experimentally test the possibility, we be-
lieved that if the observed pulmonary clearance were due to lung
P450 enzymes,33 we should emphasize structural changes that
were likely to diminish CYP-mediated metabolism. We thus
focused on electron rich phenyl rings and on relatively high overall
lipophilicity, and crossed the general SAR findings from the two
ends of the molecule – specifically, replacing alkoxyphenyl rings
with heteroaryl derivatives, believing that the heteroaryl groups
would enable tuning of both electronics and lipophilicity. Signifi-
cant effort across multiple iterations of compound design and syn-
thesis was dedicated to identifying heteroaryl derivatives that
delivered the desired potency and ADMET properties. Ultimately,
a set of lead compounds with desirable potency, HLM stability,
Pulmonary extraction to explain the high in vivo clearance was
the hypothesis best supported by our data. In our standard rat
intravenous PK protocol, compounds were dosed via the jugular
vein, with samples taken via the carotid artery. One hundred per-
cent of the intravenous dose passes through the lungs before
reaching systemic circulation. A modified protocol utilized intra-
arterial dosing (and sampling) via the carotid artery, thus by-pass-
ing pulmonary first pass extraction. The comparison of AUCs for
compound exposure between the intravenous and intra-arterial
dose routes provided a measure of pulmonary extraction, as re-
ported in Table 1.32

4286
D. W. Kung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4281–4287
18. Pasternak, A.; Goble, S. D.; de Jesus, R. K.; Hreniuk, D. L.; Chung, C. C.; Tota, M.
R.; Mazur, P.; Feighner, S. D.; Howard, A. D.; Mills, S. G.; Yang, L. Bioorg. Med.
Chem. Lett. 2009, 19, 6237.
19. Yu, M.; Lizarzaburu, M.; Beckmann, H.; Connors, R.; Dai, K.; Haller, K.; Li, C.;
Liang, L.; Lindstrom, M.; Ma, J.; Motani, A.; Wanska, M.; Zhang, A.; Li, L.;
Medina, J. C. Bioorg. Med. Chem. Lett. 2010, 20, 1758.
20. Bednarek, M. A.; Feighner, S. D.; Pong, S.-S.; McKee, K. K.; Hreniuk, D. L.; Silva,
M. V.; Warren, V. A.; Howard, A. D.; Van der Ploeg, L. H. Y.; Heck, J. V. J. Med.
Chem. 2000, 43, 4370.
and dofetilide binding were identified. Further, selected com-
pounds evaluated for rat in vivo clearance and pulmonary extrac-
tion provided support for the hypothesis that replacing the
alkoxyphenyl rings with heteroaryl derivatives would improve
in vivo PK properties. The most promising compounds from this
series (Table 2) contained a fused bicyclic imidazo-acetamide
group on the piperidine, and a heterobiaryl derivative on the azeti-
dine. The rat in vivo PK properties of compound 10n were further
characterized; it demonstrated oral bioavailability of 43% (30 mg/
kg dose in Sprague–Dawley rat), and it exhibited reasonable pene-
tration into the brain ([free brain] = 324 nM, [free plasma] = 560
nM @ 30 min, 30 mg/kg, po).
In summary, we exploited an efficient three-step synthetic pro-
cess that allowed variation at each end of a piperidine-azetidine
core structure from readily accessible carboxylic acid and aromatic
aldehyde starting materials. The ability to rapidly explore SAR
hypotheses, around ghrelin receptor binding potency, in vitro AD-
MET, and in vivo rat PK, was critical to the identification of specific
structural changes that enabled the improvement of potency and
of ADMET properties. Decreased lipophilicity and replacement of
alkoxyphenyl rings with heteroaryl derivatives were the key
structural changes that drove this series toward more drug-like
properties. A hypothesis of pulmonary extraction as a primary
source of extrahepatic clearance among early compounds in this
series was tested by the use of jugular vein and carotid artery
cannulated rats.
21. Ghrelin SPA binding assays were performed in 384-well plates in a final
volume of 90 lL containing 250 ng human GHS-R1a (HEK293 tetracycline-
inducible cell line expressing the human growth secretagogue receptor 1a;
prepared as membranes) coupled to 0.5 mg SPA beads (wheat germ agglutinin
coated, GE Healthcare, RPNQ0060) and 50 pM [125I]-ghrelin (Perkin Elmer Life
Sciences, NEX-388), plus varying concentrations of test compound or vehicle.
Controls were included on each assay plate to define 0% effect (ZPE) where no
compound was included in the binding reaction and 100% effect (HPE) where
2 lM unlabeled ghrelin was added to maximally displace the radioligand. All
reagents were diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 0.2% BSA,
EDTA-free protease inhibitors mix, pH 7.4) and reactions were incubated for
8 h at room temperature to allow binding to reach equilibrium. The amount of
receptor–ligand complex was determined by liquid scintillation counting using
a
1450 Microbeta Trilux (Wallac). Data analysis was performed using
proprietary software package. Briefly, the percent effect for each compound
dose (Sample) was calculated from raw data as follows: Effect = 100–
100 Â ((Sample-HPE)/(ZPE À HPE)) where HPE and ZPE values are averages of
16 wells each. The compound effect values were then plotted versus
a
%
%
concentration and the IC50 was determined using a standard 4-parameter fit
algorithm.
22. Holst, B.; Mokrosinski, J.; Lang, M.; Brandt, E.; Nygaard, R.; Frimurer, T. M.;
Beck-Sickinger, A. G.; Schwartz, T. W. J. Biol. Chem. 2007, 282, 15799.
23. To measure the ability of test compounds to modulate the activity of human
GHS-R1a (agonize, antagonize, partially agonize, inversely agonize), a DELFIA
GTP-binding assay (Perkin Elmer, AD0260 and AD0261) was performed. The
assay monitors the ligand-dependent exchange of GDP for GTP. GPCR
activation results in an increase in fluorescence as receptor-bound GDP is
replaced by Europium-labeled GTP. Antagonist binding prevents GDP-GTP
exchange whereas binding of an inverse agonist pushes the receptor to the
GDP bound (inactive) state, both resulting in decreased fluorescence. Ghrelin
References and notes
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functional assays were performed in a final volume of 39.5 lL containing
720 ng human GHS-R1a (HEK293 tetracycline-inducible cell line expressing
the human growth secretagogue receptor 1a, prepared as membranes), 9 nM
GTP-Europium and varying concentrations of test compound or vehicle. To test
for receptor antagonism (antagonist mode), membranes were incubated in the
presence of agonist ghrelin (Anaspec, 24158) at the EC80 concentration, plus
test compound or vehicle. To test for receptor agonists (agonist mode),
membranes were incubated in the absence of ghrelin. Briefly, test compounds
were prepared in DMSO at room temperature in 384-well plates (Matrix, 4340)
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HEPES pH 7.4, 3.7 mM MgCl2, 250 lM EGTA, 125 nM GDP) with and without
EC80 concentration of ghrelin peptide. Controls were included on each assay
plate to define 0% effect (ZPE) where no compound was included in the binding
reaction and 100% effect (HPE) where either 10
lM ghrelin was added to
determine maximal agonist activity or the EC80 concentration of ghrelin was
omitted to determine maximal antagonist activity. Samples were then
transferred to 384-well filter plates (Pall, 5071) containing hGHS-R1a
membrane and 0.35 mg/mL saponin (Perkin Elmer, AD0261) in basal buffer.
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were shielded from light and incubated for 90 minutes further at room
temperature with gentle shaking. The reactions were suctioned dry with
vacuum, washed three times with ice cold 1Â GTP Wash Solution (Perkin
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%Effect = 100–100 Â ((Sample-HPE)/(ZPE–HPE)) where HPE and ZPE values are
averages of 16 wells each. The compound % effect values were then plotted vs.
compound concentration and the Ki was determined for antagonists and
inverse agonists as follows: KB = IC50/(1 + ([radioligand]/Kd)) where IC50 was
determined from a standard 4-parameter fit algorithm, the [radioligand] = EC80
concentration ghrelin, and Kd is experimentally determined in each run by
performing a ghrelin titration. Similarly, for compounds tested in agonist mode
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and ZPE controls. The % effect values for each compound/dose are then plotted
to generate a dose response curve for each compound/batch tested. The agonist
compounds will have an increasing curve where % effect will increase with
dose. Antagonist compounds will appear inactive. Inverse agonist compounds
will have decreasing curves with the % effect values becoming more negative
with increasing concentration of compound. The agonist activity of the
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