Identification and pharmacological characterization of succinate receptor agonists

Article abstract of DOI:10.1111/bph.13738

Background and Purpose: The succinate receptor (formerly GPR91 or SUCNR1) is described as a metabolic sensor that may be involved in homeostasis. Notwithstanding its implication in important (patho)physiological processes, the function of succinate receptors has remained ill-defined because no pharmacological tools were available. We report on the discovery of the first family of potent synthetic agonists. Experimental Approach: We screened a library of succinate analogues and analysed their activity on succinate receptors. Also, we modelled a pharmacophore and a binding site for this receptor. New agonists were identified based on the information provided by these two approaches. Their activity was studied in various bioassays, including measurement of cAMP levels, [Ca²⁺]_i mobilization, TGF-α shedding and recruitment of arrestin 3. The in vivo effects of activating succinate receptors with these new agonists was evaluated on rat BP. Key Results: We identified cis-epoxysuccinic acid and cis-1,2-cyclopropanedicarboxylic acid as agonists with an efficacy similar to that of succinic acid. Interestingly, cis-epoxysuccinic acid was 10- to 20-fold more potent than succinic acid on succinate receptors. For example, cis-epoxysuccinic acid reduced cAMP levels with a pEC₅₀?=?5.57?±?0.02 (EC₅₀?=?2.7?μM), compared with succinate pEC₅₀?=?4.54?±?0.08 (EC₅₀?=?29?μM). The rank order of potency of the three agonists was the same in all in vitro assays. Both cis-epoxysuccinic and cis-1,2-cyclopropanedicarboxylic acid were as potent as succinate in increasing rat BP. Conclusions and Implications: We describe new agonists at succinate receptors that should facilitate further research on this understudied receptor.

Full text of DOI:10.1111/bph.13738

Title Page
Discovery and pharmacological characterization of succinate
receptor (SUCNR1/GPR91) agonists
Running title: cis-Cyclic dicarboxylic acids as SUCNR1 agonists
Pierre Geubelle1,2, Julie Gilissen1, 2, Sébastien Dilly2,3, Laurence Poma4, Nadine Dupuis1,
Céline Laschet1, Dayana Abboud1, Asuka Inoue5,6, François Jouret4, Bernard Pirotte2 and
Julien Hanson1,2*
1 Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases, University
of Liège, Avenue de l'Hôpital, 11, Liège, 4000, Belgium
2 Laboratory of Medicinal Chemistry, Centre for Interdisciplinary Research on Medicines
(CIRM), University of Liège, 15, Avenue Hippocrate, 4000 Liège, Belgium.
3 Laboratory of Molecular Modelling for (Bio)molecule engineering, Institute of Chemistry
and Biology of Membranes and Nano-objects, University of Bordeaux, Allée Geoffroy Saint
Hilaire, 33600 Pessac, France
4 Laboratory of Experimental Surgery, GIGA-Cardiovascular Sciences, University of Liège,
Liège, Belgium
5 Graduate School of Pharmaceutical Science, Tohoku University, Sendai, Miyagi, Japan
6 Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science
and Technology (PRESTO), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
* Correspondence: Dr. Julien Hanson, Laboratory of Molecular Pharmacology, GIGA-
Molecular Biology of Diseases, University of Liège, Quartier Hôpital, Avenue de l'Hôpital,
11, Liège, 4000, Belgium. j.hanson@ulg.ac.be
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doi: 10.1111/bph.13738
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Abstract
Background and Purpose
The succinate receptor (SUCNR1 or GPR91) has been described as a metabolic sensor that
may be involved in homeostasis. Notwithstanding its implication in important
(patho)physiological processes, the function of SUCNR1 has remained elusive because no
pharmacological tools were available. We report on the discovery of the first family of
synthetic potent agonists.
Experimental Approach
We screened a library of succinate analogues and analysed their activity on SUCNR1. In
addition, we modelled a pharmacophore and a binding site for the receptor. New agonists
were identified based on the information provided by these two approaches. Their activity
was studied in various bioassays, including measurement of cAMP levels, [Ca2+]i
mobilisation, TGF-α shedding and recruitment of arrestin 3. The in vivo impact of SUCNR1
activation by these new agonists was evaluated on rat blood pressure.
Key Results
We identified cis-epoxysuccinic acid and cis-1,2-cyclopropanedicarboxylic acid as agonists
with an efficacy similar to the one of succinic acid. Interestingly, cis-epoxysuccinic acid was
characterized by a 10 to 20 fold higher potency than succinate on the receptor. For example,
cis-epoxysuccinic acid reduced cAMP levels with a pEC50 = 5.57 ± 0.02 (EC50 = 2.7 µM) as
compared to succinate pEC50 = 4.54 ± 0.08 (EC50 = 29 µM). The rank order of potency of the
three agonists was the same in all bioassays tested cis-epoxysuccinic and cis-1,2-
cyclopropanedicarboxylic acid increased rat blood pressure to the same extent as succinate
did.
Conclusions and Implications
We provide new agonist tools for SUCNR1 that should facilitate further research on this
understudied receptor.
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Table of Links
TARGETS
GPCRsb
LIGANDS
Succinic Acid
Succinate Receptor
These Tables of Links list key protein targets and ligands in this article that are hyperlinked to corresponding
entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to
PHARMACOLOGY (Southan et al., 2016), and are permanently archived in The Concise Guide to
PHARMACOLOGY 2015/16 (a,b,c,d,eAlexander et al., 2015a,b,c,d,e).
Abbreviations
AH: Acceptor Hydrogen Bound
BrSA: BromoSuccinic Acid
cCBDA: cis-CycloButane-1,2-Dicarboxylic acid
cCPDA: cis-CycloPropane-1,2-Dicarboxylic Acid
cESA: cis-EpoxySuccinic Acid
ClSA: ChloroSuccinic Acid
EMA: EthylMalonic Acid
GRK: G protein-coupled receptor kinase
MA: Malonic Acid
mDMSA: meso-DiMethylSuccinic Acid
MMA: MethylMalonic Acid
MOSA: MethoxySuccinic Acid
MSA: MethylSuccinic Acid
N: Negative Charge
OAA: Oxalocetic Acid
p-NPP: para-NitroPhenylPhosphate
PTX: Pertussis Toxin
SA: Succinic Acid
SAR: Structure-Activity Relationship
SDH: Succinate dehydrogenase
SUCNR1: Succinate Receptor 1
TACE: Transcatheter arterial chemoembolization
tCBDA: trans-CycloButane-1,2-Dicarboxylic acid
tCPDC: trans-CycloPropane-1,2-Dicarboxylic acid
TGF-α: Transforming Growth Factor-Alpha
WT: Wild Type
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Introduction
G Protein-coupled receptors or GPCR constitute the most broadly targeted proteins by drugs
in human medicine (Overington, Al-Lazikani & Hopkins, 2006). These receptors are
characterized by seven transmembrane domains and are implicated in nearly all physiological
processes. GPCRs signal through various intracellular partners that affect cell function. Four
main families of G proteins have been described, including Gi, Gs, Gq/11 and G12/13
(Wettschureck & Offermanns, 2005). Gi and Gs subunits are respectively able to negatively
and positively regulate the activity of AC and thus decrease and increase the cAMP levels in
cells. Following activation, the receptor is usually desensitized by phosphorylation of
intracellular sites by specific GPCR kinases called GRK (Lefkowitz & Shenoy, 2005).
Particular scaffold proteins named arrestins (for non-visual GPCR, -arrestin 1 and 2 or
arrestins 2 and 3) strengthen the desensitization and generally induce receptor internalization
(Lefkowitz & Shenoy, 2005). It was recently proposed that arrestins adopt receptor-
dependent conformation and activate specific signalling pathways in a manner similar to G
proteins (Lee et al., 2016; Nuber et al., 2016).
Succinate Receptor (SUCNR1 or GPR91) is a member of the rhodopsin-like GPCR family
and was initially identified as an orphan receptor (Wittenberger, Schaller & Hellebrand,
2001). In a landmark study, He et al. paired it with its natural ligand succinate (SA) (He et
al., 2004). SUCNR1 displays some homology with the purinergic receptor family, although it
does not bind nucleotide ligands (He et al., 2004). Few studies have addressed SUCNR1
signalling pathways. It is coupled to Gi and its activation negatively modulates cAMP levels
(Gilissen, Geubelle, Dupuis, Laschet, Pirotte & Hanson, 2015; He et al., 2004). In addition,
SUCNR1 activation promotes transient [Ca2+]i mobilization (Gilissen, Geubelle, Dupuis,
Laschet, Pirotte & Hanson, 2015; He et al., 2004). Although it has been suggested that Gq
was mediating this effect (He et al., 2004; Robben et al., 2009), more recent investigations in
native and heterologous systems could not detect Gq coupling and proposed the G dimer as
the protein responsible for PLC- activation (Gilissen, Geubelle, Dupuis, Laschet, Pirotte &
Hanson, 2015; Hakak et al., 2009; Hogberg et al., 2011; Sundström, Greasley, Engberg,
Wallander & Ryberg, 2013). Notwithstanding, there is also the possibility that coupling to Gq
is tissue-dependent. Upon activation, SUCNR1 induces PTX sensitive ERK phosphorylation
and is rapidly desensitized and/or internalized (Gilissen, Geubelle, Dupuis, Laschet, Pirotte &
Hanson, 2015; Hakak et al., 2009; He et al., 2004; Robben et al., 2009). It is currently not
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clear if arrestins or phosphorylation of the receptor take an active part in the process of
desensitization/internalization.
SA is an intermediate of the citric acid (or Krebs) cycle that takes place in the mitochondria.
In case of oxygen deprivation, SA may accumulate and be released in the extracellular space.
SA and its receptor have been linked to several (patho)physiological processes such as
hypertension (He et al., 2004; Toma et al., 2008), diabetes and obesity (McCreath et al.,
2015; Sadagopan et al., 2007; Toma et al., 2008), activation of the immune system (Rubic et
al., 2008), platelet aggregation (Hogberg et al., 2011; Spath, Hansen, Bokemeyer & Langer,
2012) and retinal angiogenesis (Sapieha et al., 2008).
Very few active ligands for SUCNR1 have been described. Maleate is a confirmed agonist
with lower potency (Gilissen, Geubelle, Dupuis, Laschet, Pirotte & Hanson, 2015; He et al.,
2004). In 2011, Bhuniya et al. published the discovery of potent antagonists from a high-
throughput screening campaign (Bhuniya et al., 2011). There is currently no full
pharmacological characterization of the compounds, although radiotracers based on these
scaffolds have been described (Klenc, Lipowska & Taylor, 2015). The paucity of
pharmacological tools for the receptor restricts current research and precludes a more
thorough understanding of SUCNR1 function.
The present study reports on the discovery and characterization of the first potent and highly
efficacious SUCNR1 agonists, namely cis-epoxysuccinic Acid (cESA) and cis-1, 2-
cyclopropanedicarboxylic acid (cCPDA). In addition, the identification of several other
SUCNR1 ligands led to the definition of a precise pharmacophore for agonistic activity on
SUCNR1. We refined the SA binding pocket by implementing a homology model and
validated it by site-directed mutagenesis. cESA showed higher potency compared to SA in
SUCNR1-mediated [Ca2+]i mobilization, arrestin binding, TGF-α shedding and depletion of
basal cAMP levels. In addition, cESA and cCPDA have no activity on the mitochondrial
SDH and can be utilized to specifically assess the impact of SUCNR1 activation without
interfering with citric acid cycle. Furthermore, cESA and cCPDA demonstrated activity in
vivo on rat blood pressure. Both agonists are commercially available, which may open new
possibilities for the characterization of SUCNR1 and its validation as a drug target.
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Methods
Materials
The drug/molecular target nomenclature conforms to the BJP’s Concise Guide to
Pharmacology (Alexander et al., 2015). All chemicals used were from Sigma-Aldrich (St
Louis, Missouri, USA) unless otherwise stated. cis-epoxysuccinic acid, Citraconic acid,
acetylenedicarboxylic acid, (R)-methylsuccinic acid, (S)-methylsuccinic acid, (S)-malic acid,
(R)-malic acid, (S)-aspartic acid and (R)-aspartic acid were from Tokyo Chemical Industry
(Tokyo, Japan). (S)-Bromosuccinic acid and adipic acid were from Santa Cruz Biotechnology
(Dallas, Texas, USA). cis-1,2-Cyclopropanedicarbocylic acid was from Diverchim (Roissy,
France). trans-1,2-Cyclopropanedicarbocylic was from Enamine (Kiev, Ukraine). trans-1,2-
Cyclobutanedicarbocylic was from abcr (Karlsruhe, Germany). Antibiotics for cell culture
were from InvivoGen (San Diego, California, USA). Except for (S)-bromosuccinic acid
(Sigma-Aldrich), Halogeno succinic acids have been synthesized by us according to
published procedures (see Supplementary Fig. 1, supplementary methods and (Zurwerra et
al., 2012)). The pGloSensorTM-22F cAMP plasmid was obtained from Promega Corporation
(Fitchburg, Wisconsin, USA). Stable cell lines expressing WT human SUCNR1 and the
GloSensor system have been described previously (Gilissen, Geubelle, Dupuis, Laschet,
Pirotte & Hanson, 2015). pcDNA3.1 was from Thermo Fisher Scientific (Waltham,
Massachusetts, USA). HEK 293 cells were from ATCC (Manassas, Virginia, USA).
Cell culture
Cells were cultured at 5% CO2 and 37°C in DMEM adjusted to contain 10% FBS (Biochrom
AG, Berlin, Germany), 1% penicillin and streptomycin (Lonza, Verviers, Belgium), 1% L-
glutamine (Lonza, Verviers, Belgium).
Site-directed Mutagenesis
Wild-type (WT) human SUCNR1 with a Flag epitope at the N-terminal end has been cloned
into pcDNA3.1 vector bearing a neomycine resistance cassette. All mutagenesis was carried
out using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Massachusetts, USA)
according to the manufacturer instructions. Stable cell lines have been established for each
clone after selection with G418 (600 mg.L-1) and the expression at the membrane has been
verified by FACS measurements (See supplementary Fig. 2 and supplementary methods).
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GloSensor cAMP Assay
The assay has been conducted with a protocol previously described (Gilissen, Geubelle,
Dupuis, Laschet, Pirotte & Hanson, 2015). Briefly, HEK293 cells stably expressing cAMP
Glosensor with or without stable expression of human SUCNR1 were starved for 5 h with 1%
FBS, detached and incubated 1 h in the dark at RT in assay buffer HBSS (120 mM NaCl, 5.4
mM KCl, 0.8 mM MgSO4, 10 mM HEPES; pH 7.4, 10 mM glucose) containing IBMX (300
µM) and Luciferin (GloSensor reagent, Promega, Madison, Wisconsin, USA). Then, cells
were distributed into 96-well plates (150 000 cells per well, white lumitrac®, Greiner Bio-
One, Kremsmünster, Austria) containing the tested compound at different concentrations.
After 1 min agitation at 1200 rpm and 9 min additional incubation basal luminescence level
was recorded by using a microplate luminometer (Fluoroskan Ascent FL equipped with 2
dispensers, ascent software version 2.6, Thermo Fisher Scientific, Waltham, Massachusetts,
USA).
Intracellular Calcium mobilization assay
The assay has been conducted according to previous description (Gilissen, Geubelle, Dupuis,
Laschet, Pirotte & Hanson, 2015). Briefly, cells from a confluent T175 flask were detached
and incubated in assay buffer (HBSS: 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 10 mM
HEPES; pH 7.4, 10 mM glucose) containing 5 mM coelenterazine h (Regis Technologies,
Morton Grove, Illinois, USA) and 1.8 mM CaCl2 for 1 h in the dark at 37°C. Luminescence
was followed for 20s (40 measures; 500 ms integration) immediately upon ligand addition.
Measurements were acquired with a microplate luminometer (Fluoroskan Ascent FL
equipped with 2 dispensers, ascent software version 2.6, Thermo Fisher Scientific, Waltham,
Massachusetts, USA).
Arrestin complementation assay
Stable cell lines for the measurement of arrestin 3 recruitment by complementation of Firefly
Luciferase have been described previously (Gilissen, Geubelle, Dupuis, Laschet, Pirotte &
Hanson, 2015). Cells in suspension in the buffer (HBSS with 20 mM HEPES, pH 7.4, 10 mM
glucose) were incubated into 96-well plates (100 000 cells per well) containing the ligands at
different concentrations for 10 min at RT. Following injection of 50 µM luciferin (Synchem,
Felsberg, Germany), luminescence was recorded for 30 min using a high sensitivity
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luminometer (Centro XS³ LB 960, MicroWin 2000 software, equipped with 2 dispensers,
Berthold technologies, Bad Wilbad, Germany).
TGF-α shedding assay
The procedure and plasmids have been described previously (Inoue et al., 2012). Briefly,
expression vectors (a mixture of 2.5 µg AP-TGFα, 1 µg receptor and 0.5 µg promiscuous Gα
protein per 100 mm dish; 24h before the assay) were transfected in HEK293 cells using 12 µl
per 100 mm dish of x-tremegene 9 (Sigma-Aldrich, St Louis, Missouri, USA). Transfected
cells were pelleted by centrifugation and resuspended in PBS, followed by incubation for 10
min at room temperature. After centrifugation, cells were suspended in Hank’s Balanced Salt
Solution (HBSS) containing 5 mM HEPES (pH 7.4) and plated in 90 µl per well (40 000 cells
per well) in a 96-well plate and placed in a 37 °C incubator with 5% CO2. Thirty minutes
later, 10 µl per well of 10× concentration of compounds were added and incubated for 1 h at
37 °C under 5% CO2. Plates were centrifuged and conditioned medium (80 µl per well) was
transferred into another 96-well plate. Solution containing p-NPP (10 mM p-NPP, 40 mM
Tris-HCl (pH 9.5), 40 mM NaCl, 10 mM MgCl2) was added at 80 µl per well into both a
conditioned medium and a cell plate. Absorbance at 405 nm of both plates was read before
and after a 1 h incubation for 37 °C using a microplate reader (Infinite m200, TECAN,
Zurich, Switzerland). We calculated relative percentage of alkaline phosphatase activity in
conditioned medium = ∆OD405 CM / (∆OD405 CM + ∆OD405 Cell), where ∆OD405 CM and
∆OD405 Cell denote changes in OD405 in the conditioned medium and on the cell surface,
respectively, before and after a 1h incubation in the presence of p-NPP.
SDH activity
SDH activity was measured with the Succinate Dehydrogenase Activity Colorimetric Assay
Kit (BioVision, San Francisco, California, USA) following the manufacturer instructions.
HEK293 cells were lysed with SDH Assay Buffer (100µL for 106 cells) to isolate
mitochondrial SDH. 20µL of lysate and 1µL of disodium salts of the compounds (OAA,
cESA and cCPDA) dissolved in PBS were added in 96 well plate 5 min before adding the
probe. The absorbance was measured using a microplate reader (Infinite m200, TECAN,
Zurich, Switzerland).
Compliance with requirements for studies using animals
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The studies reported in this paper comply with the ARRIVE guidelines (McGrath & Lilley,
2015). The University of Liège Animal Ethic committee approved all animal procedures,
including experimental design and statistical determination of group size (Approval number
#1651). The protocol and procedures are compliant with Belgian and European regulations
on protection of animals used for scientific purposes (Arrêté Royal du 29 mai 2013 and EU
Directive 2010/63/UE). In total, 32 male Wistar rats (250-350g, 3 months of age) originating
from University of Liège animal facility (Federal authorization for breeding animals
LA1610002) were kept in a pathogen free (SPF) facility at least two per ventilated cages in a
controlled temperature and regular light/dark cycle. The non-invasive tail-cuff method used
to measure blood pressure has been extensively described as an adequate method for the
estimation of blood pressure and routinely used in our lab (Bialy et al., 2015; Dogne, Rath,
Jouret, Caron, Dessy & Flamion, 2016).
Rat blood pressure measurements
One week before the experiment, rats were acclimated to the experimental procedure and
contention devices to prevent stress. They were randomized according to weight in four
groups of 8 animals to receive vehicle (NaCl 0.9% m/v) or saline solutions of disodium salts
of the compounds (SA, cESA, cCPDC and tCPDC). BP was measured by the tail-cuff
method using CODA system (Kent Scientific Corporation; NIBP-CODA8-PACK). The
animals were injected via the tail vein. Immediately after injection, systolic, diastolic and
mean BP were recorded 10 times every 20 seconds after an acclimation of 5 runs (CODA
software). The method being non-invasive, no surgical procedure, anaesthesia, analgesia,
euthanasia has been implemented. General welfare assessment was conducted prior to and
following the experiments.
Randomization and blinding
The whole cohort of rats was constituted of animals of similar age and weight. They were
attributed a group randomly (number generated by computer program). The homogeneity of
weight in each group was not significantly different. The scientist in charge of preparing the
solutions was different than the one operating the blood pressure measuring device. The
solutions and their vessels did not differ in volume, colour or shape. The operator of the
blood pressure device was not aware of the nature of the injected solution. The results were
analysed by a third person that did not know which groups of animals received the solutions
containing active ligands.
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Pharmacophore Model
The pharmacophore model was built using the program phase 3.3. implemented in the
Maestro 9.2 software package (Schrödinger, LLC, New-York, New-York, USA, 2011) and
based on the results obtained from the primary SAR study. The 3D structures were initially
built using the program Ligprep 2.5 of Maestro 9.2 (Schrödinger, LLC, New-York, New-
York, USA, 2011). Structural conformers were generated with the thorough sampling option.
The top-ranked hypothetical pharmacophore including two hydrogen bond donors (AH) and
two negative charges (N) was selected for the subsequent virtual screening. Excluded
volumes were also added according the superimposition of inactive ligands.
Binding site and docking
The SUCNR1 receptor model was built by means of the SYBYL 8.0 molecular modelling
package (SYBYL, version 8.0; Tripos Inc.: St. Louis, Missouri, 2008). First, the human
sequence of the SUCNR1 receptor obtained from the Universal Protein Resource (code entry:
Q9BXA5) were aligned with the sequence of the human β2 adrenergic GPCR (code entry
P07550) by using the FUGUE module (Shi, Blundell & Mizuguchi, 2001). The next step was
the copy of a set of constraints derived from the crystal structure of the human β2 adrenergic
GPCR to the corresponding residues of the sequences to be modelled using the
ORCHESTRAR protein structure modelling module (Dilly & Liegeois, 2011). Since SA is a
SUCNR1 agonist, the SUCNR1 receptor model was built in its active form from a crystal
structure of the activated turkey β2 adrenergic receptor (PDB code 3P0G) (Rasmussen et al.,
2011).
The binding mode of SA into the SUCNR1 receptor was then investigated by molecular
docking using the GOLD 5.2 program (Jones, Willett, Glen, Leach & Taylor, 1997). The
binding pocked was defined from the one proposed by He et al. (He et al., 2004). The
structure of the resulting SA-SUCNR1 complex was finally refined by a 2ns molecular
dynamics simulation using the MMFF94 force field implemented in SYBYL 8.0 (Halgren,
1996), a temperature of 310 K, and a time step of 1 fs.
Data and statistical analysis
The data and statistical analysis comply with the recommendations on experimental design
and analysis in pharmacology (Curtis et al., 2015). Statistical analysis and plotting of
concentration-response curves were performed with GraphPad Prism version 5.0. The EC50
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were calculated by the software following nonlinear regression (curve fit) with 4 parameters.
In the screening experiments and in vitro determination all the compounds have been tested at
least in three independent experiments (n  3). Determinations of assay performance have
previously demonstrated the robustness and variability of the procedure, which is sufficient
for this number of independent experiments. For the in vitro cAMP determinations, the
results have been normalized to the activity of SA to be able to compare the activity of
compounds from different experiments, the absolute (but not the relative) response being
influenced by the number of receptors expressed by the cells, with slight variations from one
day to another. No statistical analysis has been performed in datasets of less than 5
independent experiments. For the evaluation of significant differences in the in vivo
determination of BP, a one way ANOVA followed by Bonferroni's multiple comparison test
was performed. The post-hoc tests have been run only when F achieved P<0.05. There was
no significant variance in homogeneity. P values less than 0.05 between means were
considered as statistically significant and are marked with * in the figures.
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Results
Screening of a library of succinic acid analogues
In the search for novel SUCNR1 agonists, we tested a small library of 32 analogues of
succinic acid (Fig. 1A). The library was built using three parameters: 1) the substituent borne
by the backbone, 2) the size of the carbon backbone and 3) the influence of negative or
positive charges. For ease of the presentation, we refer to the acidic form of the tested
analogues even if the carboxylic acid moieties of the interacting molecules are deprotonated
at physiological pH. The chemical properties and formulas of all tested compounds in this
report are summarized in Table 1. Each compound was tested at a single concentration of 500
µM on a HEK293 cell line stably expressing SUCNR1 together with the cAMP GloSensor
(Gilissen, Geubelle, Dupuis, Laschet, Pirotte & Hanson, 2015). We evaluated their ability to
inhibit the basal cAMP levels according to a procedure established in the laboratory
(Gilissen, Geubelle, Dupuis, Laschet, Pirotte & Hanson, 2015). None of the evaluated
analogues was more active than SA at 500 µM (Fig. 1A). Compounds characterized by a
backbone of more than 4 as well as less than 3 carbon atoms were completely inactive (Fig.
1A and Table 1). Compounds with no negative charges were also unresponsive at 500 µM.
Succinamic acid and monomethylsuccinate, both having a single negative charge at
physiological pH (Table 1), showed partial response (Fig. 1A). Besides, we evaluated all the
compounds as antagonists to make sure that inactive analogues were not binding to the
receptor without activating it. We did not detect antagonistic activity of inactive compounds
toward SUCNR1 (Fig. 1B). We performed complete concentration-response curves on
compounds that displayed at 500 µM an activity higher than 10% (Normalized to SA activity
set at 100%, Fig. 1A). We chose 10% because we determined it was a significant level
compared to the background of the assay (Gilissen, Geubelle, Dupuis, Laschet, Pirotte &
Hanson, 2015). None of the tested compounds showed significant activity on cells not
transfected with SUCNR1.
Impact of the nature and stereochemistry of substituents
We analysed the impact of the nature of substituents and their stereochemistry on potency
and maximal efficacy. In the screening, methylsuccinic acid (MSA) showed some activity at
500µM (Fig. 1A). The methyl substituent introduces a chiral centre and two stereoisomers
exist. The compound evaluated in the screening was a racemic mixture of two stereoisomers.
Thus, we performed concentration-response curves on both R and S enantiomers (Fig. 2A).
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Interestingly, these two analogues were characterized by an important difference in their
activity, (S)-MSA being inactive in the range of tested concentrations. We followed a similar
strategy for the evaluation of bromosuccinic acid (BrSA). In this case, we observed an
opposite response with regard to stereochemistry compared to MSA. The (S)-BrSA
enantiomer could not reach its maximal efficacy in the range of tested concentrations whereas
the R enantiomer was inactive at the same concentrations (Fig. 2B). We reasoned that the size
of the bromine atom could preclude an efficient interaction in the binding pocket of the
receptor. Therefore, we synthesized (R)- and (S)-chlorosuccinic acid (ClSA), the chlorine
atom being smaller than the bromine atom (for the synthesis route, see supplementary Fig.
S1). The S derivative was also the most potent, being an agonist with potency and efficacy
close to the one of SA in our assay (SA pEC50 = 4.54 ± 0.08, EC50 = 29 µM; S-ClSA pEC50 =
4.14 ± 0.04, EC50 = 72 µM, Fig. 2B). We investigated other types of substituents such as the
hydroxy group (malic acid, Fig. 2C) that is able to establish hydrogen bond (as a donor). In a
similar fashion than with the BrSA, the (S)-malic (or (L)-malic) acid was the only one
showing an agonist behaviour although weaker compared to SA. We also addressed the
impact of positively charged substituents such as amines by evaluating aspartic acid (Fig.
2D). Both S and R isomers were inactive (Fig. 2D). Oxaloacetic acid (OAA) is characterized
by a carbonyl group on the succinic acid backbone (see Table 1) that is able to form hydrogen
bonds as an acceptor. This compound behaved as an agonist with similar efficacy but lower
potency compared to succinic acid (SA pEC50 = 4.85 ± 0.08, EC50 = 14 µM; OAA pEC50 =
4.15 ± 0.04, EC50 = 70 µM, Fig. 2D).
cis-Conformation of the negative charges is an essential feature for SUCNR1 agonism
In the screening results (Fig. 1A), we noticed that although maleic acid showed activity as
expected, fumaric acid, a very close derivative, was inactive at this concentration. We
confirmed with full concentration-response curves that fumaric acid was lacking activity on
the receptor even at the highest concentration tested whereas maleic acid was an agonist with
lower potency but similar efficacy compared to SA (Maleic acid pEC50 = 4.24 ± 0.07, EC50 =
57 µM, Fig. 3A). Surprisingly, the only difference between the two derivatives is the cis
(maleate) or trans (fumarate) configuration of the carboxylic acids. We reasoned that the
orientation of the negative charges had to be in close vicinity to interact with two adjacent
positively charged amino acids in the binding pocket. We tested this hypothesis with meso-
dimethylsuccinic acid (mDMSA) that has two bulky substituents (Table 1) that orient the
carboxylic acids in a pseudo trans configuration. mDMSA was inactive in the range of tested
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concentrations (Fig. 3B). We evaluated malonic acid (MA) and its substituted analogues
methyl (MMA) and ethylmalonic acid (EMA) (see Table 1 for chemical structures).
Consistent with the idea that promoting a conformation where the negative charges are closer
will produce compounds with increased potency, MMA (pEC50 = 3.77 ± 0.05, EC50 = 169
µM, Fig. 3B) was more potent than MA (pEC50 > 3.15, EC50 > 700µM, Fig. 3B). EMA was
inactive in the range of tested concentrations (Fig. 3B). With all these results, we developed
an in silico model of the SUCNR1 receptor. We achieved a docking of SA into the binding
pocket proposed by He et al. (Fig. 3C) (He et al., 2004). The binding results predicted that SA
negatively charged oxygen atoms (carboxylate functions) would establish ionic interactions
with positively charged nitrogen atoms (guanidinium functions) of arginines 252 (R2526.55
,
superscripts indicates the residue topology according to the Ballesteros-Weinstein numbering
system (Ballesteros & Weinstein, 1995)) and 281 (R2817.39) (Fig. 3C). In our model, arginine
99 (R993.29) and histidine 103 (H1033.33), although involved in the interaction with SA, were
not critical. To confirm our model, we generated mutants for the 4 AA presumably involved
in SA binding. We generated stable cell lines expressing the mutants and evaluated the ability
of the different receptors to be activated by SA (Fig. 3D). R252A and R281A mutants were
unresponsive to SA at concentrations up to 100 mM whereas R99A and H103A could be
activated by SA although at very high concentrations (SA pEC50 on R99A < 1.50, EC50 on
R99A > 30 mM ; SA pEC50 on H103A < 0.85, EC50 on H103A > 140 mM, Fig. 3D).
cis-Cyclic derivatives of succinic acid are SUCNR1 agonists
Based on the active and inactive compounds that we identified so far, we developed a
pharmacophore for activity at SUCNR1 (Fig. 4A). The yellow spheres represent exclusion
volume. Pink and red spheres indicate the hydrogen bond acceptor and negative sites,
respectively. We screened the ZINC library and obtained as hits cyclic compounds such as
cCPDA (Fig. 4B). We evaluated this compound with our cAMP assay on SUCNR1 and
found that cCPDA was an agonist of similar potency and efficacy compared to SA (cCPDA
pEC50 = 3.31 ± 0.02, EC50 = 49 µM, Fig. 4C). Consistent with our binding site model, the
trans analogue of 1, 2-cyclopropanedicarboxylic acid was inactive (Fig. 4C). We evaluated
the cis-1, 2-cyclobutanedicarboxylic (cCBDA) acid and measured an activity for the cis
isomer (pEC50 < 3.60, EC50 > 250 µM, Fig. 4C), although it did not reach maximal efficacy in
the range on tested concentrations, whereas the trans isomer was inactive (Fig. 4C). The cis
and trans 1, 2-cyclopentanedicarboxylic acid were both inactive (data not shown). Given the
good potency obtained with OAA (pEC50 = 4.15 ± 0.04, EC50 = 70 µM, Fig. 2D), we
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reasoned that a hydrogen bond acceptor in the cycle could have beneficial effect on the
activity of the cyclic dicarboxylic acids. This led to the evaluation of cis-epoxysuccinic acid
(cESA) on SUCNR1. This compound displayed a 100% efficacy and was more potent than
SA (cESA pEC50 = 5.57 ± 0.02, EC50 = 2.7 µM, Fig. 4D).
cis-epoxysuccinic acid and cis-1, 2-cyclopropanedicarboxylic acid are agonists for all known
SUCNR1 pathways and do not interfere with succinate dehydrogenase
We performed a complete validation of cESA and cCPDA pharmacology on SUCNR1. First,
we analysed the agonist behaviour of the compounds on a recently described TACE induced
TGF shedding assay (Inoue et al., 2012). We detected activation of the receptor with the
transient transfection of the alpha subunit of the chimeric Gqi1 that couples to Gi1 receptors
and induces the Gq pathway. In this assay, cESA was the most potent agonist and cCPDA
displayed a similar potency and efficacy compared to SA (cESA pEC50 = 4.38 ± 0.04, EC50 =
42 µM; cCPDA pEC50 = 3.05 ± 0.04, EC50 = 887 ± 80 µM; SA pEC50 = 3.46 ± 0.03, EC50 =
350 µM, Fig. 5A). A similar experiment with the chimeric Gqi3 that couples to Gi3 receptors
and induces the Gq pathway gave similar results (data not shown). Next, we used a firefly
luciferase complementation assay (Gilissen, Geubelle, Dupuis, Laschet, Pirotte & Hanson,
2015) to evaluate the arrestin 3 recruitment to the receptor. cESA and SA, but not cCPDA,
activated the receptor with similar efficacies at the highest concentrations tested (cESA pEC50
= 4.13 ± 0.04, EC50 = 74 µM; SA pEC50 = 3.06 ± 0.07, EC50 = 865 µM; cCPDA pEC50 = 2.97
± 0.07, EC50 = 1076 µM, Fig. 5B). SUCNR1 is able to elicit [Ca2+]i mobilization and the
three tested compounds elicited an activation of this pathway in the range of tested
concentrations (cESA pEC50 = 3.72 ± 0.01, EC50 = 191 µM; cCPDA pEC50 = 2.98 ± 0.01,
EC50 = 1040 µM; SA pEC50 = 3.23 ± 0.01, EC50 = 581 µM, Fig. 5C). We tested the activity of
the two synthetic agonists on SDH activity. At concentrations up to 500 µM neither cESA
nor cCPDA had an effect on SDH activity (Fig. 5D). OAA was used as a positive control for
SDH inhibition. None of the agonists displayed activity on cells not expressing SUCNR1.
cis-1, 2-cyclopropanedicarboxylic acid and cis-epoxysuccinic acid are active in vivo
We finally tested SUCNR1 agonists in an in vivo model. We noninvasively measured BP in
rats following intravenous perfusion of SA and observed a significant increase in BP, as
previously reported (He et al., 2004; Vargas, Toma, Kang, Meer & Peti-Peterdi, 2009) (Fig.
6). We observed a similar increase in response to the injection of cCPDA (10 mg.kg-1) and
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SA (10 mg.kg-1). At the dose of 1 mg.kg-1, cESA injection was followed by an increase in BP
that was not statistically different from the one induced by SA or cCPDA (Fig. 6).
Discussion
Although the human genome sequence has been published in 2001 (Venter et al., 2001),
many proteins continue to be understudied, presumably because of a lack of research tools
such as small molecule ligands (Edwards, Isserlin, Bader, Frye, Willson & Yu, 2011).
Despite extensive knowledge on the function and pharmacology of some receptors, and
despite their potential as drug targets, the majority of the GPCR family is actually
understudied (Roth & Kroeze, 2015). Thus, an important number of GPCRs are poorly
characterized or even devoid of known ligands and labelled as "orphans". Recently, Roth and
Kroeze proposed that it was the availability of good ligands that made some GPCR popular
and not the other way around (Roth & Kroeze, 2015). The receptor for SA, SUCNR1,
belongs to this category of attractive drug target (Gilissen, Jouret, Pirotte & Hanson, 2016)
whose pharmacology is poorly defined. Notwithstanding its demonstrated implication in
immune responses and inflammation (Littlewood-Evans et al., 2016; Rubic et al., 2008),
retinal angiogenesis (Sapieha et al., 2008) and regulation of renin release (Peti-Peterdi,
Gevorgyan, Lam & Riquier-Brison, 2013; Toma et al., 2008), surprisingly few research tools
are available and only scarce information about its molecular structure has been published.
In the initial report describing its pairing with SA, He et al. evaluated 200 carboxylic acids,
of which SA was the more potent (He et al., 2004). They disclosed the activity for a couple of
them including the weaker agonists maleate and methylmalonate. They identified one
important feature for agonist activity, the mandatory dicarboxylic nature of active molecules
(He et al., 2004). Here, we aimed to go several step further and propose a full structure-
activity relationship analysis. Compared to previous work, our investigation adds novel
essential features required for activation of SUCNR1. In summary, succinate derivatives with
an agonist profile must have: i) 2 negative charges at physiological pH; ii) a distance from 3
to 5 carbon atoms between these two negatively charged atoms and iii) the possibility for the
molecule to adopt a cis conformation of the two charges. The pharmacophore for agonist
ligands is schematically summarized on Fig. 6B. This refined pharmacophore led to the
establishment of an improved model for the SA binding pocket. It was initially proposed that
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four AA were critical for SA binding to the receptor (He et al., 2004). However, the
published data showed only the results at one concentration of SA (200µM) on the mutant
(He et al., 2004). Having defined the three criteria for SUCNR1 activation, we speculated that
the most important interactions should be between two positively charged AA located in
close proximity and on the same side inside the binding pocket. We performed docking
simulation in an improved binding pocket model (Fig. 3B) and identified R2526.55 and
R2817.39 as the probable candidates. Mutagenesis experiments confirmed that these two AA
are indeed critical for the interaction whereas R993.29 and H1033.33 were important but not
crucial. Interestingly, residues 3.33, 6.55 and 7.39 are topologically involved in agonist
binding in several other class A GPCRs (Venkatakrishnan, Deupi, Lebon, Tate, Schertler &
Babu, 2013). A better delineation of the SA binding mode is important for future modelling
of the receptor to identify new ligands. Subsequently, we identified cis-cyclic dicarboxylic
acids as potent agonists, in particular cESA that is characterized by a 10- to 20-fold improved
potency compared to SA in various assays. The presence of an oxygen atom in its cycle,
together with the fact that OAA is an agonist with good potency suggest that a putative AA
able to establish an hydrogen bond is ideally positioned inside the binding pocket.
In all bioassays that were used, the ligands showed the same rank order of potencies. This
observation suggests that the ligands are not biased for the measured response although more
thorough experiments would be required to analyse biased signalling (or its absence).
Interestingly, the potency of the ligands was lower compared to cAMP related pathways (Fig.
5B). This is consistent with previous reports by us (Gilissen, Geubelle, Dupuis, Laschet,
Pirotte & Hanson, 2015) and others (Southern et al., 2013). The luciferase complementation
assay utilized to monitor arrestin recruitment is very different from the physiological
environment of the receptor, which may impact the observed potency. The discrepancy
between the assays could also be the consequence of a receptor reserve for the Gi mediated
inhibition of AC. This raises the interesting possibility that in native tissues, in a given
(patho)physiological context, the activation of some pathway might occur only in certain
conditions of very high SA concentrations whereas the other pathways (such as Gi-related
ones) are activated at lower SA concentrations due to receptor reserve and amplification.
The other synthetic ligands that have been described as antagonists are reported as being able
to inhibit succinate mediated [Ca2+]i mobilization in CHO-K1 cells overexpressing human
SUCNR1 in the nanomolar range for the best compounds (2c and 4c) (Bhuniya et al., 2011;
Gilissen, Jouret, Pirotte & Hanson, 2016). Their selectivity has been evaluated and they
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represent valuable tools to characterize the receptor (Klenc, Lipowska & Taylor, 2015).
Interestingly and in stark contrast with the compounds described here, these ligands have no
clear structural relationship to succinate. In addition, they display no negative charges at
physiological pH. It would be interesting to test whether these antagonists can block the
action of cESA and cCPDA with the same potency. Although the compounds reported by
Bhuniya et al. are not available commercially, the novel agonists that we have identified will
permit to investigate more precisely the mechanism of action and the binding mode
(competitive vs. irreversible orthosteric or negative allosteric modulators...) of the SUCNR1
antagonists.
We also addressed two important characteristics in the perspective of the use of our
compounds as pharmacological tools: specificity and in vivo efficacy. Historically, several
SA analogues such as oxaloacetate, malonate, or L-malate have been described as
mitochondrial complex II (or SDH) inhibitors. Although they show agonist activity on
SUCNR1, they would not be useful as tools because they also disrupt cellular respiration and
could lead to artefacts. Similarly, there are some limitations in using SA to address SUCNR1
roles, the endogenous ligand being also an important cellular metabolite. Our results clearly
show that cESA and cCPDA are devoid of activity on SDH activity and suggest that they can
be used to specifically address the consequences of SUCNR1 activation in cells, native
tissues and organs, although we cannot exclude other off-target effects. Another important
aspect for chemical tools is their usefulness in vivo. More specifically, cESA has an "epoxy"
(see Table 1) function that may be metabolically unstable. SA has been shown to increase
blood pressure through SUCNR1 and release of renin in mice (He et al., 2004; Vargas, Toma,
Kang, Meer & Peti-Peterdi, 2009). Therefore, we assessed in rats the ability of our compound
to recapitulate the effects of SA in vivo after intravenous injection. Both compounds were
able to significantly increase BP with similar amplitude compared to SA. Although it is
tempting to speculate that it is the consequence of an activation of SUCNR1, additional
experiments are needed (with antagonist or knock-out animals) to substantiate the direct link
between cESA- and cCPDA- mediated increase in blood pressure and SUCNR1. The
oxoglutarate receptor (OXGR1) and the purinergic P2Y1 receptor are the two most closely
SUCNR1 related receptors (Gilissen, Jouret, Pirotte & Hanson, 2016). Although SA has been
shown previously to be unable to activate these receptors in various assays (He et al., 2004),
future investigation should address the activity of SA and other SUCNR1 ligands with regard
to these receptors.
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In conclusion, this is the first study, to the best of our knowledge, reporting on a synthetic
agonist for SUCNR1/GPR91 that is more potent that the endogenous ligand, SA. The clinical
relevance of these results is the possibility that SUCNR1 might represent an innovative drug
target, for instance in hypertension-related diseases. However, it needs thorough preclinical
validation first. We expect that the emergence of a properly characterized tool, which is
readily commercially available, will spur new research on the understudied SUCNR1 that
should advance its validation as a drug target.
Author Contributions
JH designed and supervised the study. PG, JG, ND, CL and DA performed the in vitro
experiments and acquired the data. SD modelled the pharmacophore and receptor, identified
privileged structures and docked succinate. LP and FJ designed and performed the in vivo
experiments. PG, FJ, BP and JH analysed the data, interpreted the results, created the figures
and wrote the paper. AI designed and set up the shedding assay and provided research tools.
Acknowledgements
This work was supported y the Fonds pour la echerche Scientifique (F. .S.-F S)
Incentive Grant for Scientific esearch (F.4510.14), University of i ge (Cr dit de
D marrage-Fonds Sp ciaux) and on Fredericq Foundation. JH and CL are F.R.S.- FNRS
Research Associate and Ph.D. fellow, respectively. ND is a FRIA PhD fellow. AI was funded
by JST, PRESTO. The authors wish to thank Céline Piron for excellent technical assistance
and Vincent Seutin for critical reading of the manuscript. We thank the GIGA Imaging
Platform for technical support in FACS analysis.
Conflicts of Interest Statement
The authors declare no conflicts of interest.
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Table 1. Chemical structure of tested compounds
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Figure 1. Screening of library of succinic acid analogs
We selected diverse compounds that share some characteristics with SA. The compounds
were grouped on three criteria: 1) nature of substituent 2) length of carbon backbone and 3)
charge of the molecule. See Table 1 for a complete list of molecular structures. A. The
different compounds were tested at 500µM on SUCNR1. The agonist nature was evaluated
by measuring the levels of cAMP in the presence of compounds compared with Vehicle
control and succinate response. No compounds showed activity on cell line without
SUCNR1. B. 100µM of compounds were added prior to the addition of 500µM SA to
evaluate their antagonistic activity. Results are given as percentage of SA activity for ease of
comparison and as Mean ± SEM of 3 independent experiments.
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Figure 2. Impact of the nature and stereochemistry of substituents
Some of the compounds showing activity in screening at one concentration were evaluated
with full concentration-response curves. Concentration-response curve for the effect on
cAMP of A. (R)- and (S)-methylsuccinic acid; B. Bromo- and chlorosuccinic acid; C. Malic
acid; D. Aspartic and oxaloacetic acid. In all experiments, SA has been used as a reference
compound and the data normalized accordingly. Data are expressed as Mean ± SEM of
independent 3 experiments. E. Model for the interaction of substituents with the binding
pocket.
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Figure 3. cis-Conformation of the negative charges is an essential feature for SUCNR1
agonism. A. Maleic acid is a full agonist for SUCNR1, albeit with a lower potency compared
with SA. Fumaric acid is completely inactive B. Malonic, methylmalonic, ethylmalonic and
meso-dimethylsuccinic acid concentration-response curves on basal cAMP levels. C.
Homology modeling of SA binding pocket. SA is positioned in a pseudo cis conformation. D.
Evaluation of the effect of SA on basal cAMP level in several HEK293 cell lines stably
transfected by GloSensor system and SUCNR1 mutants (H103A, R99A, R252A, R281A).
Data are presented as Mean ± SEM of three independent experiments.
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Figure 4. cis-Cyclic derivatives of succinic acid are SUCNR1 agonists
A. Model for the SUCNR1 agonist pharmacophore. Yellow spheres represent exclusion
volume and red spheres negative charges. B. cis-1, 2-cyclopropanedicarboxylic acid fit with
the pharmacophore model. C. Evaluation of cis and trans cyclic compounds on the inhibition
of basal cAMP levels. D. cis-epoxysuccinic acid concentration-response curve on the
inhibition of basal cAMP level. E&F. Kinetic measurement of the inhibition of cAMP levels
followed in HEK293 cells stably expressing cAMP Glosensor and SUCNR1 upon addition of
the SUCNR1 agonists cis-1, 2-cyclopropanedicarboxylic (E) and cis-1, 2-
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cyclopropanedicarboxylic (F). Succinic acid has been used as a positive control. Data are
expressed as Mean ± SEM of 3 independent experiments.
Figure 5. cis-epoxysuccinic acid and cis-1, 2-cyclopropanedicarboxylic acid are agonists
for all known SUCNR1 pathways and do not interfere with succinate dehydrogenase
Concentration-response curves for SA, cis-epoxysuccinic acid and cis-1, 2-
cyclopropanedicarboxylic acid on A. TGF- shedding; B. Arrestin 3 recruitment; C. [Ca2+]i
mobilization and D. Succinate dehydrogenase activity. Data are presented as Mean ± SEM of
6 independent experiments.
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Figure 6. cis-epoxysuccinic acid and cis-1, 2-cyclopropanedicarboxylic acid are active in
vivo
A. Repeated noninvasive measures of blood pressure in rats (n=8 in each group) injected
intravenously with a saline solution of various test compounds. These experiments have been
performed at least three times for each condition on different animal cohorts. Data are
expressed as the difference between mean blood pressure before injection and 15 min post-
injection. Data are presented as Mean ± SD. * P value < 0.05 based on 1 way ANOVA. B.
Proposed pharmacophore for succinate derivatives agonists.
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