A small molecule antagonist of ghrelin O-acyltransferase (GOAT)

Article abstract of DOI:10.1039/c1cc11817j

Using our recently disclosed fluorescence-based assay to monitor acyltransferase activity, the first non-peptidic, small molecule antagonists of ghrelin O-acyltransferase (GOAT), a potential anti-obesity and anti-diabetes target, have been discovered. Eac

Full text of DOI:10.1039/c1cc11817j

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COMMUNICATION
A small molecule antagonist of ghrelin O-acyltransferase (GOAT)w
Amanda L. Garner and Kim D. Janda*
Received 30th March 2011, Accepted 26th April 2011
DOI: 10.1039/c1cc11817j
Using our recently disclosed fluorescence-based assay to monitor
acyltransferase activity, the first non-peptidic, small molecule
antagonists of ghrelin O-acyltransferase (GOAT), a potential
anti-obesity and anti-diabetes target, have been discovered. Each
exhibits micromolar inhibition of the enzyme, and may be useful
probes for future study of the ghrelin-GOAT system.
demonstrated that this enzyme plays an important role in
metabolism, similar to ghrelin.9–11
To date, only two examples of GOAT antagonists have been
reported, and both are peptide-based (Fig. 1b). Compound 1,
discovered by Goldstein and Brown, is based on the
ghrelin(1–5) pentapeptide with
a diaminopropionic acid
residue in place of Ser-3 for added stability of the octanoyl
side chain.12 Only in vitro assay data has been published for
this compound, and an IC50 value of B1.5 mM was reported.12
Mechanism-inspired compound 2, also known as GO-CoA-
Tat, was reported by Cole and co-workers, and is based on the
premise that GOAT might use a ternary complex mechanism
to link octanoyl-CoA and des-acyl ghrelin.11 Both in vitro
and in vivo data have been reported for this compound.
Impressively, administration of GO-CoA-Tat in mice was
found to reduce weight gain and improve glucose tolerance.11
Although these examples provide evidence for the potential
impact of GOAT antagonism, the chemical design of
compounds 1 and 2 leaves much to be improved upon as both
are peptide based. As such, the discovery of more drug-like
GOAT antagonists that can be readily synthesized and
modified for use as both chemical probes and lead therapeutic
candidates against GOAT remains an important goal. Herein,
we disclose the first small molecule-based GOAT antagonists.
Using our high-throughput screening assay for GOAT, we
have examined a small, focused library of compounds and
Overweight and obesity currently affect nearly 1 billion people
worldwide, and this number is expected to grow in the continuing
years.1 Because of the rising number of cases of overweight
and obesity in both adults and children, much effort has been
invested in discovering new therapies, both surgical and non-
surgical, for these disorders. Although nonsurgical treatments
such as drugs are preferred, these efforts have met with limited
success. Thus, there is a crucial need to validate new therapeutic
targets for the development of effective therapies for overweight
and obesity.
Recently, the ghrelin–ghrelin O-acyltransferase system has
been implicated as a potential target for pharmacologic
modulation of obesity.2,3 Ghrelin is a peptide hormone that
acts as a stimulator of growth hormone secretion and is the
only circulating peptide shown to potently enhance feeding
and weight gain and to regulate energy homeostasis following
central and systemic administration.2 Despite findings suggesting
its potential role in overweight and obesity, recent studies have
provided evidence that ghrelin acts more as a central neuro-
modulator that controls whole-body metabolism, and thus, may
be more relevant in diabetic phenotypes.4,5 Intertwined with
ghrelin’s intriguing biological activities is also its unique acylated
structure, namely a 28-amino acid peptide with an n-octanoylated
post-translational modification at Ser-3 (Fig. 1a).6 Importantly,
ghrelin’s biological functions are solely dependent on this
modification and it is the only known peptide to contain
this unique post-translational modification. The enzyme
responsible for this acylation, ghrelin O-acyltransferase or
GOAT, was recently discovered.7,8 Of significance, murine
genetic models for loss or gain of GOAT function have also
Departments of Chemistry and Immunology and Microbial Science,
The Skaggs Institute for Chemical Biology and The Worm Institute
for Research and Medicine, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
E-mail: kdjanda@scripps.edu; Fax: (+1) 858-784-2595;
Tel: (+1) 858-784-2515
w Electronic supplementary information (ESI) available: Details for
assay conditions and synthesis of, characterization of and inhibition
curves for compounds 3a and 3b. See DOI: 10.1039/c1cc11817j
Fig. 1 (a) GOAT-catalyzed n-octanoyl transfer to ghrelin. (b) Structures
of reported GOAT antagonists. * = Diaminopropionic acid. Ahx =
aminohexanoate.
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Fig. 2 cat-ELCCA for screening GOAT activity. (a) General assay approach. (b) Anticipated assay outcome in the presence of GOAT agonists
and antagonists. HRP = horseradish peroxidase. THPTA = tris[(3-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine.
have discovered two small molecules with potential as GOAT-
targeted probes.
(see Supporting Informationw).12 Thus, cat-ELCCA can be
used to screen for antagonists of GOAT. However, because no
agonists of GOAT activity have been reported, we were unable
to validate this potential application.
Our group has recently reported a conceptually new enzyme
assay based on cat-ELISA, which we have termed catalytic assay
using enzyme-linked click chemistry assay, or cat-ELCCA.13
In cat-ELCCA, an enzyme-linked click chemistry substrate is
utilized to arm the assay with catalytic signal amplification
necessary for ultrasensitive detection. Using this assay technol-
ogy, we developed the first fluorescence-based, potentially
high-throughput screening system for GOAT (Fig. 2a).13 The
key step of our assay technology involves the use of azido-
modified horseradish peroxidase (HRP), which covalently
modifies the octynoyl ester of the immobilized ghrelin substrate
using click chemistry (see Supporting Information for more
detailw). The HRP-triazole product is then detected using amplex
red to provide catalytic fluorescence signal amplification of the
n-octynoyl ester-HRP triazole product concentration.
To target protein–protein interactions, our laboratory
proposed the concept of ‘‘credit card’’ libraries.16,17 More
specifically, these libraries are based upon flat, rigid scaffolds
elaborated with chemical diversity elements. The underlying
concept behind the design of such molecules lies in the fact that
the ‘‘hot spots’’ at the interfaces between proteins can be viewed
as aromatic, slot-like regions, or ‘‘card readers’’. Thus, a small
molecule ‘‘credit card’’ with the correct code or favorable
properties to disrupt this space should trigger an event at the
protein–protein interface. To demonstrate this concept, a library
of compounds based on a naphthalene core was synthesized
via the Ugi multicomponent reaction, and lead compounds
targeting the Myc–Max protein–protein interaction16 and
formation of the HIV-1 gp41 fusogenic core18 were identified.
Because protein–protein and protein–peptide interactions
are also likely to be involved in GOAT-catalyzed n-octanoylation
of ghrelin, we were interested in designing a new series of
compounds based on our previously reported naphthalene
scaffold for modulation of GOAT. Using the Ugi multi-
component reaction, a small, focused library of naphthalene-
derived compounds (3) was synthesized using aliphatic
carboxylic acids (R1), varying amines (R2) and benzylisonitrile
as diversity elements (Fig. 3a) (see Supporting Information for
more detailw). The compounds were then analyzed for
agonism or antagonism of GOAT using cat-ELCCA. All
compounds were initially examined at 50 mM, and to our
delight, two compounds were discovered from this small
library as potential GOAT antagonists, compounds 3a and
3b (Fig. 3b). No agonists were discovered from this series of
compounds. Importantly, upon further examination of com-
pounds 3a and 3b, each was found to exhibit dose-dependent
antagonism of GOAT with measured IC50 values of 7.54 mM
and 13.1 mM, respectively (see Supporting Information for
inhibition curvesw). Thus, using cat-ELCCA we have success-
fully discovered the first non-peptidic small molecule antago-
nists of GOAT. It is important to note here that although
Using our cat-ELCCA for GOAT activity design, both
agonists and antagonists of GOAT can theoretically be
identified (Fig. 2b), which is critical since the exact role of
GOAT in obesity and metabolism is currently unknown.
Additionally, while most fluorescence-based high-throughput
screening assays are subject to compound interference,14,15
cat-ELCCA does not suffer from such limitations. More
specifically, because compound screening and fluorescence
measurement are conducted in separate steps, fluorescent
compounds will not interfere with the assay read-out
since all compounds are removed prior to detection. Thus,
cat-ELCCA should provide reproducible and reliable screening
results and we were optimistic that our assay could be used to
screen for modulators of GOAT activity.
Because we had not previously validated the potential of
cat-ELCCA for screening, compound 1 was evaluated as a test
compound since an IC50 value for this molecule has been
reported and it is more synthetically tractable than compound
2. Varying concentrations of compound 1 were added during
the acyl transfer reaction (step 1, Fig. 2a) and the resulting
GOAT activity was assayed. Importantly, peptide 1 exhibited
dose-dependent inhibition with an IC50 value of 1.1 mM,
which is in agreement with that reported in the literature
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Chem. Commun., 2011, 47, 7512–7514 7513

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Although our discovered credit card-based compounds are
not the most potent antagonists of GOAT, they are more
drug-like than the presently known peptide-based inhibitors.
Additionally, based on the ease of chemical synthesis (only
1 step), structure–activity relationship studies may readily be
employed to identify more potent GOAT antagonists based on
our scaffold. Future cellular-based assay studies and ghrelin-
GOAT-based phenotypic studies will be reported in due
course.
This work was supported by The Skaggs Institute for
Chemical Biology (K.D.J.) and by the NIH (postdoctoral
fellowship F32-DK083179 to A.L.G.). We are grateful to
Mr. Greg McElhaney for the synthesis of compounds 3.
Notes and references
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Fig. 3 (a) Synthesis of potential GOAT agonists or antagonists using
the Ugi multicomponent coupling reaction and (b) structures of lead
naphthalene-based compounds identified using cat-ELCCA.
these compounds were tested as racemates, one enantiomer
may be more potent than the other. Examination of stereo-
chemical impact is currently ongoing.
Based on the structures of compounds 3a and 3b, it is
plausible to suggest that these compounds block octynoyl-
CoA from binding to GOAT since each contains a long
alkyl chain similar to that found in octanoyl-CoA. In the cell,
acyl-CoA substrates are transported as covalent adducts with
acyl-CoA binding proteins. To transfer octanoyl-CoA to
ghrelin, GOAT must interact with octanoyl-CoA binding
protein, thus, establishing a potentially important protein–
protein interaction. Since our crude membrane-bound GOAT
extract does not contain octanoyl-CoA binding protein, it will
be interesting to examine this possible mechanism in cells.
However, it cannot be ruled out that compounds 3a and 3b
are simply blocking the octanoyl-CoA binding site of GOAT.
In summary, using our recently disclosed fluorescence-based
assay for GOAT activity, cat-ELCCA, we have discovered the
first non-peptidic, small molecule antagonists of GOAT,
a
promising anti-obesity and anti-diabetes drug target.
Importantly, the identification of such small molecule-based
compounds should aid in furthering our understanding of the
impact of GOAT modulation in the ghrelin-GOAT system.
18 Y. Xu, H. Lu, J. P. Kennedy, X. Yan, L. A. McAllister,
N. Yamamoto, J. A. Moss, G. E. Boldt, S. Jiang and
K. D. Janda, J. Comb. Chem., 2006, 8, 531–539.
c
7514 Chem. Commun., 2011, 47, 7512–7514
This journal is The Royal Society of Chemistry 2011