Model-Based Discovery of Synthetic Agonists for the Zn2+-Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions

Article abstract of DOI:10.1021/acs.jmedchem.6b00648

The G-protein-coupled receptor 39 (GPR39) is a G-protein-coupled receptor activated by Zn²⁺. We used a homology model-based approach to identify small-molecule pharmacological tool compounds for the receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of ligand binding to other receptors with similar binding pockets to select iterative series of minilibraries. These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520 compounds were tested in vitro, making this method broadly applicable for tool compound development. The compounds of the initial library were inactive when tested alone, but lead compounds were identified using Zn²⁺ as an allosteric enhancer. Highly selective, highly potent Zn²⁺-independent GPR39 agonists were found in subsequent minilibraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin secretion.

Full text of DOI:10.1021/acs.jmedchem.6b00648

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Article
Model-based Discovery of Synthetic Agonists for
the Zn<sup<2+</sup>-sensing G Protein Coupled
Receptor 39 (GPR39) Reveals Novel Biological Functions
Thomas M Frimurer, Franziska Mende, Anne-Sofie Graae, Maja S. Engelstoft, Kristoffer L. Egerod,
Rie Nygaard, Lars-Ole Gerlach, Jakob Bondo Hansen, Thue W. Schwartz, and Birgitte Holst
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00648 • Publication Date (Web): 03 Jan 2017
Downloaded from http://pubs.acs.org on January 3, 2017
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Model-based Discovery of Synthetic Agonists for the Zn -sensing G Protein
Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions
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,
2,3,
⊥
,1,2
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Thomas M. Frimurer*
, Franziska Mende* , Anne-Sofie Graae , Maja S. Engelstoft , Kristoffer L.
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,2
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1,2
1,2,
⊥
Egerod , Rie Nygaard , Lars-Ole Gerlach , Jakob Bondo Hansen , Thue W. Schwartz and Birgitte Holst
1
. Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology; 2. Novo
Nordisk Foundation Center for Basic Metabolic Research; University of Copenhagen, Blegdamsvej 3, DK-
2200, Copenhagen, Denmark; 3. Novo Nordisk Foundation Center for Protein Research; University of
Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen, Denmark.
Keywords: GPR39 agonists, positive allosteric modulator, zinc-sensing receptor, antidiabetic treatment,
virtual ligand screening, academic drug discovery
⊥
Corresponding authors
E-mail: thomas.frimurer@sund.ku.dk; phone: +45 3029 2089 (T.M.F)
E-mail: holst@sund.ku.dk; phone: +45 2148 7699 (B.H.)
*
T.M.F. and F.M. contributed equally to the manuscript.
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Abstract
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The G-protein coupled receptor 39 (GPR39) is a G protein-coupled receptor activated by Zn . We used a
homology model-based approach to identify small-molecule pharmacological tool compounds for the
receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of
ligand binding to other receptors with similar binding pockets to select iterative series of mini-libraries.
These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520
compounds were tested in vitro, making this method broadly applicable for tool compound development.
The compounds of the initial library were inactive when tested alone, but lead compounds were identified
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using Zn as an allosteric enhancer. Highly selective, highly potent Zn -independent GPR39 agonists were
found in subsequent mini-libraries. These agonists identified GPR39 as a novel regulator of gastric
somatostatin secretion.
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Introduction
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TM G protein-coupled receptors (GPCRs) collectively constitute the largest family of mammalian
membrane proteins and are targets of a major fraction of both approved drugs and drugs under
1
development . However, the pharmaceutical companies follow rather common paths and focus their
discovery and development processes on a relatively small number of the many hundred types of available
receptor targets, which means that pharmacological ligands and tool compounds are only available for
relatively few GPCRs. In fact, a surprisingly large fraction of GPCRs remain orphans, i.e., receptors without a
2
known endogenous ligand and consequently without useful pharmacological tools . In many cases, animal
models in which the receptor has been genetically deleted or over-expressed may indicate certain potential
physiological roles. However, these approaches are far from ideal and can be misleading due to the
development of various types of genetic, cell biological and physiological compensatory mechanisms. Thus,
there is a great need for efficient approaches to identify small-molecule synthetic ligands for GPCRs as
useful tools to help determine the physiological importance of these receptors and to probe for their
3
pharmacological potential as future drug targets , without requirement to screen several thousand
compounds.
GPR39 is an example of a receptor that, due to a limited availability of suitable pharmacological tool
compounds, has not yet been assigned a clear physiological role. Consequently, it has not been validated as
a drug target, despite evidence indicating that it plays a major role in the control of many important
endocrine and metabolic functions. GPR39 is a member of the ghrelin family of receptors. All other
members of this receptor family bind ligands that are well-established peptide hormone and neuropeptide
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regulators of metabolism: neurotensin, neuromedin U and motilin, in addition to ghrelin itself . However,
no peptide ligand has yet been identified for GPR39. In contrast, GPR39, which couples to Gαq
,
Gαs and β-
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arrestin, was found to act as a Zn sensor that is activated by physiological concentrations of Zn
.
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Similarly to several other members of this receptor family, GPR39 displays some degree of constitutive
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activity . GPR39 is mainly expressed in endocrine and metabolic tissues , and based on studies in GPR39-
deficient and GPR39-overexpressing mice, this receptor has been implicated in the control of glucose-
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induced insulin secretion , pancreatic β-cell differentiation and control of lipolysis in white adipose
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tissue . Based on similar studies, GPR39 has also been implicated in gastric fluid secretion, gastric and GI-
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tract motility , endothelial function and cell survival and was recently found to have various functions in
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the central nervous system and depression
, hemostatic regulation and function as an extracellular pH
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regulator . A general problem associated with GPR39 - and similar real orphan receptors - is a limited
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availability of pharmacological tool compounds. In the case of GPR39, apart from Zn itself, only a small
2
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number of ligands are currently known
.
Most current drug discovery approaches depend on access to facilities capable of testing large libraries of
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chemical compounds, i.e., various types of high-throughput screening approaches . In this study, we used a
model based ‘site-directed drug discovery’ approach, which was originally introduced in the biotech
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industrial context , to identify small synthetic ligands for GPCRs. Through this approach, iterative series of
mini-libraries of compounds are selected from the collective pool of commercially available screening
compounds (Table S1), which are then probed in functional assays, which allow the use of both positive and
negative information for the selection of compounds for subsequent mini-libraries. Based on the notion
2
4–26
that many small agonists in GPCRs can act as both agonists and allosteric modulators
, we tested the
2
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compounds both alone and in the presence of a submaximal dose of Zn . Through this allosteric enhancer-
assisted approach, we were able to identify sub-micromolar GPR39-agonist hit and lead compounds, which
would not have been otherwise detected by functional assays. A complementary approach for discovering
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allosteric modulators for GPCRs has been described by Huang and co-workers . Importantly, selective,
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highly potent Zn -independent GPR39 agonists were identified in subsequent series of ligand based
optimized mini-libraries. Although expression analysis at an early stage demonstrated that GPR39 is
strongly expressed in the stomach, its physiological role in the GI tract remained unclear. The novel GPR39
agonists now identify GPR39 as an important regulator of gastric somatostatin secretion.
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Results
Structure-based discovery of agonists for the human GPR39 receptor
A large number of the currently available X-ray structures of 7TM GPCR-ligand complexes demonstrate that
although the ligands can bind in many different ways to the GPCRs, the vast majority of ligands bind to
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what originally was proposed as the ‘main ligand binding site’ of GPCRs , i.e., to the cavity between the
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outer segments of TM-II, -III, -IV, -V and -VI . To obtain agonist hit and lead compounds for GPR39 we used
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the so-called ‘Site-Directed Drug Discovery’ approach . In brief, the process involves the analysis of the
physicochemical properties of the binding pockets of GPCRs and the design of 3D pharmacophore search
queries that exploit i) information from the structure of the binding pocket of GPR39 derived from
comparative homology modeling based on the X-ray structures of structurally related GPCRs combined
with ii) 3D structural information from small-molecule compounds, drugs and pharmacological tool
compounds that are known to bind to ‘binding pocket-related’ receptors, i.e., receptors with binding
pockets with similar physicochemical properties (Figs. 1a and 1b).
The analysis of amino acids located at 22 selected positions in the main ligand-binding pocket of GPR39 (Fig.
1
a) identified other members of the ghrelin receptor family, i.e., the neurotensin receptors 1 and 2 (NTS
1
and NTS ), the motilin receptor (MtlR) and the ghrelin receptor (GhrR), as well as phylogenetically non-
2
related receptors, i.e., the chemokine receptors CCR1, CCR3 and CCR8, as being the closest ‘binding pocket-
related’ receptors (Fig. 1a). The availability of structural information of small-molecule ligands for GhrR,
CCR3 and CCR8, combined with structural information on how these ligands bind in molecular models of
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these receptors and how these binding sites correspond to interaction sites in GPR39 itself, were used as
the basis for a 3D pharmacophore search query. The pharmacophore model was initially applied for the
virtual screening of a GPCR-enriched chemical library of approx. 40 thousand compounds (Fig. 1b).
Subsequent diversity selection (Suppl. methods) resulted in a focused library containing 197 compounds
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(Library 1), which was tested for agonist activity at concentrations of 1 and 10 µM in HEK293 cells
transfected with the human GPR39 receptor to probe for Gα coupling, as measured by an inositol
q
phosphate accumulation assay. When tested alone, however, none of the compounds from Library 1 were
able to increase GPR39 signaling (data not shown).
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Use of Zn as an allosteric modulator to enhance hit and lead identification
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We previously have identified Zn as an agonist for GPR39. Two Histidine residues, namely His and His ,
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located in the N-terminal segment of the receptor are essential for the effect of the metal ion (Fig. 2) .
Based on the assumption that the zinc ion can act as an allosteric enhancer by stabilizing GPR39 in an active
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conformation and can thereby potentially increase the binding affinity and potency of other agonists , we
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re-tested Library 1 in the presence of submaximal doses of Zn . As shown in Fig. 1c, in the presence of 10
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µM Zn , compounds 1 to 5 were found to activate GPR39. Zn increased not only the potency but also the
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efficacy of the compounds. In the presence of Zn , the synthetic compounds generally reached a maximum
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efficacy approx. two-fold higher than that obtained with Zn alone (Fig. 1d). At a Zn concentration of
100 nM, no allosteric enhancement was observed for any of the compounds. However, by increasing the
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Zn concentration to 1 and 10 µM, both the potency and efficacy were dose-dependently improved, as is
2+
shown for 5 (TM-N973), which reached a maximal potency of 200 nM in the presence of Zn (Fig. 1d). Thus,
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using Zn as a positive allosteric modulator, it was possible to identify agonist hits for GPR39, which would
not have been detected on their own.
The synthetic small-molecule agonists bind to the main ligand-binding pocket of GPR39
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To test whether the identified compounds actually did bind to the presumed general ligand-binding pocket
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of GPR39, to which they were selected to bind, and to compare their binding mode with that of Zn , we
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probed compound 5 in a library of GPR39 mutants (Fig. 2). Ala substitutions of Glu (II:20, 2.60),
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Phe (III:08, 3.32), Glu (III:09, 3.33) and Glu (VII:06, 7.39) (The residue numbering refers to the generic GPCR
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numbering system described by Schwartz and Ballesteros , which is based on the actual location of the residues in
each trans-membrane helix) revealed that these key residues located in the presumed binding pocket are all
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essential for the ability of 5 to stimulate GPR39 signaling (tested in the presence of 10 µM Zn ) (Fig. 2).
These results are in agreement with the receptor-based pharmacophore model used for the initial virtual
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screening, where Glu (II:20, 2.60) and Glu (III:09, 3.33) were proposed to act as acceptor sites for
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hydrogen bond donor atoms. The central Glu (VII:09, 7.39) was modelled to associate with a hydrogen
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bond donor atom or a positively charged basic site on the ligand (Fig. 1b). Ala substitution of Arg (VI:20,
6
.55) increased the potency of 5 approximately fivefold (Suppl. data 1). This could not be directly predicted
from the pharmacophore model; however, one explanation could be that removal of the large side chain
located in the middle of the binding pocket allowed this particular type of ligand to interact better with
other residues in the binding pocket.
2+
17
Although the effect of 5 was highly dependent on the presence of Zn (Fig. 1d), Ala substitutions of His
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and His , which eliminated the effect of Zn when administered alone, surprisingly had a very limited
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effect on 5 ability to activate the GPR39 receptor in the presence of Zn (Fig. 2, upper panels). Thus, the
mutational mapping results are in agreement with the notion that 5 binds to the main ligand-binding
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pocket of GPR39 but indicate that Zn binds differently when acting as an agonist alone than when acting
as a positive allosteric modulator for novel synthetic agonists.
Design of iterative GPR39-focused mini-libraries to improve agonist drug-like properties and zinc
independency
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Having obtained appropriate agonist lead compounds through the initial library (Fig. 1c), we used a ligand-
based virtual screening to generate the following iterative series of mini-libraries of compounds to attempt
to broaden the number of active chemo-types, to optimize their potency and, in particular, to attempt to
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identify compounds that can activate GPR39 in the absence of Zn and present appropriate ADME
properties (steps 1-4 in Fig. 3a). In this process, we used structure-activity relationships (SARs) of both
active and inactive compounds to guide the design of the subsequent three focused mini-libraries
containing 176, 96 and 58 compounds, respectively (Supplementary tables, Table S2, S3, and S4), which
were obtained from the collective pool of commercially available synthetic compounds (Table S1). In the
design of the individual libraries, compounds with physicochemical properties similar to those of orally
active approved drugs, were prioritized over compounds containing undesired chemical functionalities,
3
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such as potential reactive groups , and novel chemotypes were prioritized by discarding compounds that
resembled small-molecules ligands known to be active on other protein or receptor targets (Compared to
more than 1.25 million chemicals associated with biological activities in the ChEMBL database). Notably,
compounds 3 and 4 contain a hydroxyphenyl hydrazone moiety, which has been recognized to be a
3
potential covalent modifier and a sequester of metal ions (when present in other chemotypes ) and
reported to be able to inactivate proteins and yield false signals across a variety of assays. While such
compounds may be valuable as ‘hits’ for the optimization and development of new ligands, the
hydroxyphenyl hydrazone moiety is certainly not preferred in more advanced tool compounds. Hence, we
deliberately rejected compounds containing this and other promiscuous moieties in the subsequent
libraries (Tables S2 – S4). Thus, in the search for improved and chemically attractive chemotypes, the zinc-
dependent agonists 4 and 5 from Library 1 (Fig. 1c) were used as seed structures in an initial “scaffold
hopping” similarity approach based on pharmacophore multiplets (Suppl. methods) to yield a second-
generation library collected from four chemical vendors (Library 2 - 176 compounds).
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In Library 2, the first two zinc-independent agonists, which presented low potency and low efficacy when
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tested without Zn , were identified, as exemplified by 6 (TM-N1034, library 2, Table S2). Compound 6 also
presented acceptable drug-like physicochemical properties (Table S2; Fig. 3b, upper panel). In the presence
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of increasing Zn concentrations, the potency of 6 could be increased by almost three orders of magnitude
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and presented an Emax that was approx. two-fold higher than that obtained with Zn alone (Fig. 3b, lower
panel).
To specifically attempt to improve the zinc-independent potential, we used 6 in a query based on similarity
and substructure searches (Suppl. methods) to acquire a third-generation library (Library 3 – 96 compounds,
Table S3). Among a number of zinc-dependent potent agonists, we discovered six purine-substituted
variants, exemplified by 7 (TM-N1230, library 3, Table S3), with improved zinc-independent activation of
the human (potency of 2.7 µM) and mouse (0.38 µM) GPR39 receptor compared with the 2,4-depyrimidin-
substituted lead compound, 6 (Fig. 3b). 7 was highly efficacious and had a potency of 18 nM in the
2
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presence of 10 µM Zn (Fig. 3b, middle panel, Suppl. data 8 and Table S3).
In the design of a fourth-generation library, we made use of the observation that substituents in the 2 and
4
positions of the right-side phenyl improved the potency compared with substituents in the 3 position,
which often resulted in partial or significant loss of activity (Table S3 and S4). Among the 58 purine-
substituted analogous constituting Library 4, we identified several highly efficacious GPR39 agonists
2
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displaying single-digit nanomolar potencies in the presence of Zn and, importantly, a number of agonists
2
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that exhibited high potency in the absence of Zn (Fig. 3b, Table S4). The most promising compound of this
class, 8 (TM-N1324), had a rigid structure and a reasonably low molecular weight (MW=397.08) and
presented generally improved drug-like physicochemical properties compared with the original zinc-
independent agonist lead, 6. Importantly, 8 activated human GPR39 with high efficacy and potencies of 280
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nM and 9 nM in the absence and presence of Zn , respectively (Fig. 3b). The compound had similar
potencies on murine GPR39, 180 nM and 5 nM (Suppl. data 8). Thus, the combined ligand-based and
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allostery-assisted approach has led to the discovery of several novel, potent and efficacious GPR39 agonist
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Further improvements in properties through SAR-guided synthesis
Having exhausted the existing commercially available chemical libraries, such as, for example, all of the
available preferred right-hand-side substituents from Library 2 and the left-hand-side purine moiety from
Library 3 and 4, we resorted to the synthesis of a final library of compounds in an attempt to further
improve the properties of this chemical series and create truly novel, patentable compounds. Based on the
collective knowledge of the SARs of the compounds in Libraries 2 to 4, we synthesized a fifth library (Library
5
– containing 30 compounds, Table 1) as described in the supplementary methods. In this library, we
maintained the purine moiety at the left-hand side, which enabled good agonist activity in Library 3 to 4,
exemplified by 10 (TM-N1231, Table S4) and combined it with a diverse set of chemical modifications in
position 4 of the right-hand side phenyl. These modifications included distinct primarily two-to-three-atom
linkers to aryl-substituted moieties, which had enabled the first observation of zinc-independent activity of
6
in Library 2 (Fig. 4a), which is key for activity when compared to 9. As shown in Table 1 and Fig. 4b,
approximately half of the compounds presented improved potency with respect to the zinc-independent
activation of GPR39 compared to 8, and eight compounds (13, 17, 18, 19, 26, 27, 28 and 39) had potencies
below 100 nM. Notably, the most potent compound 26 (TM-N1562) had a potency of 30 nM to activate
2
+
human GPR39 in the absence of Zn , i.e., an improvement of approx. nine-fold compared to that of
2
+
compound 8. 26 had a potency of 1.5 nM in the presence of Zn . It activated murine GPR39 with a similar
2+
potency of 2.6 nM in the presence of Zn (Suppl. data 8)
Both compounds 8 and 26 were further analyzed for their stimulation of cAMP signaling, as a measurement
2
+
for Gαs coupling. Both compounds, in the presence of 3 µM Zn , increased the intracellular cAMP level in a
dose-dependent manner with potencies of 17 nM and 12 nM, respectively (Fig. 4d, lower panel). When
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tested without Zn however, neither compound caused a marked increase in cAMP. Hence, in contrast to
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the Gαq coupling, none of the compounds induced efficient Zn independent Gαs coupling.
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Drug-like properties of the discovered GPR39 agonists
Since chemical novelty was one of the selection criteria, both 8 and the in-house synthesized 26 were
clearly chemically distinct from known pharmacological ligands, as determined through comparisons with
more than 1.25 million ChEMBL chemicals associated with biological activities of all major target classes,
i.e., they both had Tanimoto coefficients less than 0.7 (Fig. 4c).
Compound 8 and 26 were also probed for off-target activities using a panel of 165 GPCRs, i.e., the Millipore
GPCR-profiler service (Suppl. data 2), and neither compound showed relevant agonist activities. Even when
tested in-house using signal transduction assays, the compounds did not exhibit agonist activities neither
on closely related receptors, such as the ghrelin, motilin and NMU2 receptors, nor on receptors with
similarities in the binding pocket, such as the chemokine CCR1 and CCR5 receptors (Suppl. data 3).
Furthermore, compound 8 was found to have promising in vitro ADME properties. Although an early
compound 7, exhibited low solubility, 8 proved to have reasonably good aqueous solubility (65 µM at pH
7
.0) (Suppl. data 4). The compound also demonstrated high membrane permeability, as measured by the
rate of transport across Caco-2 cells derived from a human colon carcinoma in the A-B direction (Papp A-B ~
-
6
25 x 10 cm/s), with good recoveries in both directions (Suppl. data 5). In addition, with respect to in vitro
metabolic clearance in human liver microsomes, 8 was superior to 7 (CLint of 58.3 vs. 23.7 and t1/2 of 58
min vs. 24 min, respectively) (Suppl. data 6), implying that the 2-chloro-4-fluoro-benzene substituents
improve stability by blocking potential metabolically liable oxidation sites and/or affect the molecular
conformation.
Importantly, the concentration of compound 8 in the plasma after oral administration of two different
doses (30 mg/kg and 60 mg/kg) reached micro molar concentrations after 30–240 minutes, which is
sufficient for maximal activation of the GPR39 receptor (Suppl. data 7).
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Probing the physiological functions of GPR39 using the novel synthetic agonist
Effect on insulin secretion - GPR39 is highly expressed in insulin-producing β-cells of the endocrine pancreas
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and has been proposed to be involved in a Zn -mediated autocrine feed-back control of insulin secretion
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,34
based on the impaired glucose tolerance and insulin secretion observed in GPR39-deficient mice
.
Furthermore, novel agonist ligands discovered by HTS efforts also suggest an improved insulin secretion
2
1
from artificial β-cell and in vivo studies . However, although 7 apparently weakly stimulates insulin
secretion from murine islets isolated on five independent days each including of 2 to 3 mice, a similar
response was observed in islets isolated from GPR39-deficient mice (Fig 5a,b). In contrast, no consistent
increase in insulin secretion was observed for 8 (Fig 5c,d). Because these compounds are clearly efficacious
with respect to stimulating GPR39 Gαq signaling in vitro it can be concluded that the role of GPR39 in the
control of β-cell function remains unclear.
8
GPR39 function in gastric endocrine cells - GPR39 is known to be highly expressed in the stomach , but no
function has yet been found for GPR39 in the ventricle. As part of the systematic characterization of
35
receptors controlling the secretion of ghrelin , we tested the effect of 8 and 26 on the release of ghrelin
from primary cultures of gastric mucosal cells. As shown in Fig. 6a, the GPR39 agonists at doses of 1 µM in
2
+
the presence of 10 ꢀM Zn inhibited basal ghrelin secretion to 79 and 61 % of the basal level, respectively,
3
5
i.e., which is similar to the efficiency obtained with other inhibitors in this system . This finding was
surprising in view of the fact that GPR39 is a Gαq-coupled receptor and would thus be expected not to
inhibit but rather stimulate hormone secretion. Because primary cultures of gastric mucosal cells also
contain somatostatin cells, we tested the effect of broad-spectra somatostatin receptor antagonists, which
completely eliminated the inhibitory effect of the two GPR39 agonists on the secretion of ghrelin and even
showed a trend toward a higher release in the presence of the antagonist (Fig. 6a). Measurements of
somatostatin confirmed that the GPR39 agonists 8 and 26 increased somatostatin release, i.e., by 53 and
82 %, respectively (Fig. 6d). The stimulatory effect of the two GPR39 agonists on somatostatin release was
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eliminated in cells isolated from GPR39-deficient mice (Fig. 6d and e). In accordance with the observation
that GPR39 activation increases somatostatin secretion and accordingly decreases the secretion of ghrelin,
we found that the fasting plasma levels of ghrelin were increased in GPR39-deficient mice (Fig. 6c). Finally
we tested that the GPR39 agonists acted through Gαq in a dose dependent manner also on the gastric cells
and found similar potency on the gastric cells compared what we observed on the heterologous expression
system (Fig. 6f). Thus, the novel GPR39 agonists enabled identification of novel important physiological
functions of GPR39 as a regulator of gastric somatostatin secretion (Fig 6g).
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Discussion
In the study, we here used a structure-based approach to discover a number of highly potent and highly
efficacious pharmacological GPR39 agonist tool compounds. In the process, which was greatly facilitated by
2+
the use of the low-potency endogenous agonist Zn as an allosteric enhancer to detect the early hit series,
only 520 compounds were tested in in vitro signaling assays.
A large number of the currently available high-resolution X-ray structures of GPCR-ligand complexes
support the original hypothesis that although ligands bind in many different modes to their different target
receptors, they nevertheless mostly interact with residues located in a common, main ligand-binding
3
6,37
pocket between the extracellular segments of the transmembrane helices . Structure-based docking
screens using the actual ligand-binding sites of available high-resolution X-ray structures of GPCR-ligand
complexes have been highly efficient for the identification of novel and potent ligands, with hit-rates as
3
8–41
high as 20 to 70 %
. In this study we successfully used a structure-and-knowledge-based approach taking
advantages of both molecular models of the presumed binding sites for small molecules in molecular
models of the target receptor (GPR39) and structures of ligands binding to receptors with binding pockets
2
3,42
of similar physicochemical properties . Related approaches have been used in the pharmaceutical
43,44
industry to search for hits and leads in their large in-house chemical libraries . For example, Klarbunde
and coworkers used a similar approach in a combined retrospective and prospective screen to identify
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potent agonists for the complement component 3a receptor 1 (C3AR1) . Importantly, we propose that the
knowledge-based discovery process described in this manuscript is applicable in different settings, because
we selected and experimentally tested an iterative series of focused mini-libraries of compounds, which
were cherry-picked from the collective repertoire of chemicals available from commercial chemical vendors.
The approach complements the High-Throughput Screening (HTS) or “random” experimental testing of
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large compound collections, which is currently becoming available in a number of academic institutions .
Recently, GPR39 agonists were identified by Boehm and coworkers in the industrial setting through the
4
5
screening of 30,000 compounds selected from the Pfizer chemical collection . The computational approach
also allows for bias in the search strategy toward specific aims, such as finding novel chemotypes (‘scaffold-
jumping’) or enhancing certain physiochemical and/or pharmacological properties of a particular
chemotype – in this requiring only limited chemical synthesis.
2
+
The use of Zn as an allosteric enhancer in the search for agonists for GPR39 was based on the hypothesis
that the zinc ion would stabilize the receptor in an agonist-prone conformation and thereby facilitate the
2
+
discovery of small-molecule agonists. Despite its small size, Zn is a bona fide, albeit relatively low-potency
agonist for GPR39, as measured in several different signal transduction pathways, including Gαq, Gαs and
6
,19
arrestin mobilization . Over the last decade, it has become clear that many agonists, despite the
4
6
26,47–50
traditional pharmacological view , can act as allosteric enhancers for other agonists
. For GPR39, co-
2
+
administration with Zn was found to be essential for the identification of the initial hits from the virtual
screening because none of the compounds in Library 1 could activate the receptor alone. A complementary
2
7
approach to identify allosteric ligands for GPR68 and GPR65 has been described recently . Notably,
2+
Fjellström and co-workers at AstraZeneca recently discovered Zn modulated GPR39 agonists by a high
2
2
throughput screen, followed by a medicinal chemistry program . However, in the subsequent mini-libraries,
we were gradually able to discover compounds that were both potent and efficacious in the absence of
2
+
2+
Zn , and even more potent in the presence of Zn . Zinc increased both the potency and the efficacy of the
2
6
compounds in accordance with the classical properties of an ago-allosteric agonist .
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Interestingly, Zn has been described to act as an allosteric modulator for a number of GPCRs
and
can conceivably be used as an enhancer in the search for novel ligands for receptors other than GPR39.
Importantly, we believe that the allostery-enhanced approach is much more generally applicable. Due to
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2+
the chemical nature of the two types of ligands, we use the term ‘allosteric enhancer’ for Zn and ‘agonists’
2
+
for the novel synthetic compounds, although Zn may really be the natural, endogenous agonist for
6
,7
2+
GPR39 . We propose that the existing agonists, like Zn , should be used more broadly as an aid in the
search for ‘other agonists’ or allosteric modulators. The point is that allosteric modulators very often act as
agonists in their own right, and vice versa. Such modulators are generally referred to as PAM-agonists. It is
becoming increasingly clear that even rather large agonists may act as positive allosteric modulators for
47–50,55
other ligands, including even those with a very similar structure
. In this context it is interesting to
2
+
note that the endogenous partial agonist Zn binds differently compared to the more potent synthetic
agonists since elimination of the N-terminal zinc site (His17 and His19) does not affect the potency of the
zinc-dependent compounds, although a studie by Cohen et al. indicates that other residues in the
2
+
19
extracellular domain, such as e.g. Asp313, are able to modulate Zn binding .
The main purpose of generating synthetic agonists for GPR39 was to obtain pharmacological tool
compounds that could be used to uncover physiological functions of the GPR39 receptor, which analogous
to orphan receptors in general, have otherwise been exclusively based on genetic data. Studies in GPR39-
9
,34
deficient mice had also indicated that the receptor may be important for insulin secretion . However, this
hypothesis could not be confirmed using the developed GPR39 agonists. Lack of acute insulinotropic effects
2
2
in rodents has also been observed by Fjellström and co-workers . Although we did observe effects on
insulin secretion in some studies, this response was not consistent and not dependent on GPR39. In this
connection, it should be noted that the effects of other GPR39 agonists on insulin and GLP-1 secretion
2
0,21
discovered by Peukert and coworkers at Novartis when searching for inhibitors of Hedgehog , have not
2
1,45
yet been shown to be dependent on GPR39 in control experiments performed using knockout mice
.
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Importantly, novel physiological functions of GPR39 could be revealed using the new GPR39 agonists.
8
Almost 10 years ago, it was shown that GPR39 is highly expressed in the stomach , but no function had
been connected to this expression prior to this study. In the present study, we found that GPR39 stimulates
gastric somatostatin secretion in a GPR39-dependent manner and thereby indirectly inhibits ghrelin
secretion. This modulation of the ghrelin secretion observed in primary gastric mucosal cells cultures was
supported by data from the GPR39 KO mice where plasma ghrelin was increased and under standard
condition also food intake was increased. Because somatostatin in the stomach dampens or inhibits both
endocrine and exocrine secretion in general, it will be interesting to study the potential indirect effects of
GPR39 agonists on, for example, secretion of gastrin, gastric acid and pepsinogen. Notably, a massive
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accumulation of gastric juice was originally described in GPR39-knockout mice , which may now be
connected to a lack of somatostatin stimulation in GPR39-deficient mice. The discovered compounds are
2
+
all zinc-dependent and display at least 10-fold higher potency in the presence of Zn than without. It could
2
+
be argued that the Zn dependency is a favorable property if we aim at functionality in tissues where the
2
+
Zn concentration is high such as in the pancreas. However, as described in the present manuscript, the
most important functions are related to adipocyte function and gastric hormone secretion. Neither of these
2+
tissues has been characterized with high Zn concentrations.
Conclusion – The present study demonstrates that it is possible to generate novel pharmacological tool
compounds that can be used to elucidate the physiological importance of a GPCR based on a combination
of structure-based computational chemistry and the use of a low-potency ago-allosteric enhancer. GPR39 is
an example of a receptor, where the knockout models have not revealed the physiological role and where
the therapeutic potential remains unknown. This applied approach complements the use of high-
throughput physical screening of large libraries of chemical compounds as it instead exploits the equally
large collective pool of commercially available compounds but, as exemplified in the present case, only
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leads to physical testing of a small number of compounds. However, as also indicated, a limited synthesis
step may be used to further improve, for example, the compound’s ADME properties when the commercial
libraries are exhausted.
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Methods
Homology models. A pair wise sequence alignment between the human GPR39 receptor (accession
5
6
number Q43194) and the sequence of bovine rhodopsin was constructed using ClustalW . The alignment
was manually refined to ensure proper alignment of conserved class A GPCR motifs. An ensemble of 50
5
7
three-dimensional comparative GPR39 models was constructed with MODELLER using bovine rhodopsin
58
(PDB ID 1GZM, 2.65 Å resolution) as a structural template . The N-terminal domain, the extra- and
intracellular loops, and the C-terminal domain were excluded. Using a computational protocol as described
23,42
previously , we initially applied a physicogenetic methodology to identify binding pocket-related GPCRs
and associated ligands to construct a five-feature pocket-based GPR39 pharmacophore search query
(further details are presented in Suppl. methods). The pharmacophore model was initially used for the
virtual screening of our in-house GPCR-enriched chemical library to generate library 1 using UNITY and
5
9
associated dbtools in the Tripos software package . Prior to the selection of compounds for experimental
testing, the hit list was filtered using automated scripts to remove accepted compounds in high-internal-
energy conformations and compounds violating a number of physiochemical properties.
Design of iterative focused chemical mini-libraries (Library 2 - 4) using virtual ligand screening - GPR39
agonists identified in the first-generation library were subsequently used as seed structures to search the
combined collection of commercial chemical vendor libraries (Table S1) stored in UNITY databases and
59
prepared for virtual screening using the Tripos software package to generate three focused mini-libraries
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(Libraries 2 – 4) acquired from five vendors (ChemDiv, Enamine, InterBioScreen, ChemBridge and Life-
Chemical). To improve the potency, efficacy and chemical properties of the novel GPR39 agonists, each
iteration/library used structural information obtained from positive as well as negative results to guide the
selection of compounds for the next-generation libraries. In this process, compounds were further
prioritized based on their predicted “drug-like” properties and novelty (details are provided in Suppl.
methods). Library 2 - The zinc-dependent agonists 4 (TM-N279) and 5 (Library 1) was used as seed
structures for ligand-based virtual screening using Pharmacophore Multiplets that have been demonstrated
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to be effective for lead and scaffold-hopping
. Pharmacophore multiplets, produced from the steric
features, hydrophobic centroids, aromatic rings, hydrogen acceptors, hydrogen donors, cations and anions
features, were decomposed into its constituent three feature pharmacophore distance multiplets and
encoded into binary fingerprints (so called Triplets), to search the pre-computed chemical vendor libraries
(Table S1). The high-ranking chemical hit lists were subsequently filtered. Several classes of accepted
molecules were rejected based on the criteria described in the supplementary information. Finally, the
compound was manually inspected which is a common accepted practice for cherry-picking compounds for
experimental testing. Library 3 was based on similarity (using 2D UNITY fingerprints) and substructure
searches around the zinc-independent agonists 6 (Library 2), while Library 4 was based on similarity
searches of the purine-substituted zinc-independent agonists 7. A Tanimoto Coefficient, TC > 0.70, was
applied in the search for similar vendor compounds. In cases where only few compounds were identified, a
lower similarity threshold was used. The compound selection criteria for compounds in Libraries 3 and 4
were similar to the criteria used for Library 2. All of the computations were performed on 40-core 3.00-GHz
Linux workstations.
Chemical synthesis. InterBioScreen Ltd (http://www.ibscreen.com/) synthesized the compounds in Library
5 (Table 1) under an agreement with the University of Copenhagen according to Synthesis Schemes 1 to 3
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(Suppl. methods). It is important to mention that all chemical structures were confirmed using proton (1H)
NMR spectra at 300/400 MHz and liquid chromatography−mass spectrometry (LC-MS) analysis confirmed a
minimum purity of 95% for all compounds (spectra of compounds are listed in Supporting data).
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In vitro ADME and chemical receptor profiling of key compounds. The solubility (Suppl. data 4), Caco-2
membrane permeability (Suppl. data 5) and liver microsomal stability (Suppl. data 6) of two key compounds
7
and 8 were assessed in mouse and human using Cyprotex Discovery Ltd., a fully integrated R&D CRO. To
evaluate the potential off-targets effects, FLIPR assays were conducted to profile the agonist and
antagonist activities of 8 and 26 with a panel of 165 GPCRs using the Millipore GPCR profiler service (Suppl.
data 2). We also tested the activity of 8 on a series of structurally and functionally related receptors (CCR1,
CCR5, GhrR, NMUR2, MtlR, GPBA, NK1R, LPA2, ETB, and D2R) (Suppl. data 3).
In vitro Screening of the compound mini-libraries. The activities of the focused chemical libraries were
tested through an inositol phosphate (IP) accumulation assay. HEK293 cells were transiently transfected
with GPR39 cDNA in the pCMV-Tag 2B vector via the calcium phosphate method and then frozen at -150°C.
Transfected frozen HEK293 cells were rapidly thawed and transferred to cell culture medium containing 5
3
µCi/ml myo-[2- H]inositol. A total of 40,000 cells per well were seeded in poly-D-lysine-coated 96-well
plates. After 24 h incubation, the IP accumulation assay was performed as decribed in the Suppl. methods.
The EC50 values were determined by nonlinear regression using GraphPad Prism v6.03 for Windows,
GraphPad Software, La Jolla California USA.
Physiological analyses.
Ex vivo ghrelin and somatostatin secretion studies. The day after the preparation of gastric mucosal cells,
cells were incubated with the ligands in triplicates for 4 h, and the supernatant was collected. Acyl-ghrelin
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was measured using a “Rat/Mouse Ghrelin (active) ELISA” (Millipore #EZRGRA-90K). Somatostatin was
measured using an in house somatostatin RIA. The response of the cells in terms of ghrelin/somatostatin
secretion to the administered ligands was expressed as a percentage of the secreted ghrelin/somatostatin
from cells exposed to medium alone.
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Acknowledgement
We are grateful for the expert technical assistance provided by Susanne Hummelgaard. The Novo Nordisk
Foundation Center for Basic Metabolic Research (http://www.metabol.ku.dk) is supported by an
unconditional grant from the Novo Nordisk Foundation to University of Copenhagen. F.M. received support
for a PhD scholarship from the Faculty of Health and Medical Sciences, University of Copenhagen. M.S.E.
was supported by a postdoc fellowship from the Danish Diabetes Academy supported by the Novo Nordisk
Foundation.
Competing financial interests
The authors declare no competing financial interests.
Author information
Present addresses: Rie Nygaard, Stanford University, Chemistry Department, 380 Roth Way, CA-94350
Stanford; Lars-Ole Gerlach, Novo Nordisk A/S, 2760 Måløv, Denmark.
⊥
Corresponding authors: E-mail: thomas.frimurer@sund.ku.dk; phone: +45 3029 2089 (T.M.F)
E-mail: holst@sund.ku.dk; phone: +45 2148 7699 (B.H.)
Supporting Information Available: Including description of molecular modeling, preparation of chemical
databases, virtual screening, chemical synthesis, in vitro assays, counter screens and ex vivo experimental
and analytical procedures.
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