A Selective Antagonist Reveals a Potential Role of G Protein-Coupled

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Title Page

A selective antagonist reveals a potential role of G protein-coupled

receptor 55 in platelet and endothelial cell function

Julia Kargl*, Andrew J Brown, Liisa Andersen, Georg Dorn, Rudolf Schicho, Maria Waldhoer

and Akos Heinemann Downloaded from

Primary laboratory of origin:

Institute for Experimental and Clinical Pharmacology, Medical University of Graz, 8010 Graz, Austria jpet.aspetjournals.org

Affiliation:

Institute for Experimental and Clinical Pharmacology, Medical University of Graz, 8010 Graz, Austria at ASPET Journals on September 26, 2021

(J.K., L.A., G.D., R.S., M.W., A.H.); Screening and Compound Profiling, GlaxoSmithKline,

Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK (A.J.B); current address:

Hagedorn Research Institute, Novo Nordisk A/S, 2820-Gentofte, Denmark (M.W.)

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version. JPET #204180

Running Title Page

Characterization of a GPR55 antagonist

*Corresponding author:

Julia Kargl

Institute for Experimental and Clinical Pharmacology

Medical University of Graz

8010 Graz, Austria Downloaded from

Telefon: +43 316 380 - 7851

Fax: +43 316 380 - 9645

Email : [email protected] jpet.aspetjournals.org

Number of text pages: 35

Number of tables: 1 at ASPET Journals on September 26, 2021

Number of figures: 8

Number of references: 47

Number of words in Abstract: 186

Number of words in Introduction: 750

Number of words in Discussion: 1253

Abbreviations:

AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide];

AM281 [1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3- carboxamide]; CB1, cannabinoid 1 receptor; CB2, cannabinoid 2 receptor; CID16020046, 4-[4-(3- hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo [3,4-c] pyrazol-5-yl] benzoic acid;

CNS, central nervous system; DMEM, Dulbecco´s modified Eagle´s Medium; DMSO,

Dimethylsulfoxid; ERK, Extracellular-signal Regulated Kinase; FBS, Fetal Bovine Serum; GPCR, G protein coupled receptor; GPR55, G protein coupled eceptor 55; HA, haemaglutinin; HEK293, human

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embryonic kidney cells; HRP, Horseradish peroxidase; IgG, Immunglobulin G; LPI, L-α- lysophosphatidylinositol; MAPK, mitogen-activated protein kinases; PBS, Phosphate buffered saline;

PNGase, N-Glycosidase; SR141716A [5-(4-Chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-

(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; TBS, Tris-Buffered Saline; WIN55,212–2 [(R)-(_)-[2,3- dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1- naphthalenylmethanone]

Downloaded from

Recommended section assignment:

Cellular and Molecular jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Abstract

The G protein coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several

(patho)physiological functions, its role is still enigmatic mainly owing to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4- methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo [3,4-c] pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In HEK293 cells stably expressing human GPR55 (HEK-GPR55), the compound Downloaded from behaved as an antagonist on LPI-mediated Ca2+ release and extracellular signal-regulated kinases

(ERK1/2) activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2).

CID16020046 concentration-dependently inhibited LPI-induced activation of nuclear factor of jpet.aspetjournals.org activated T-cells (NFAT), nuclear factor kappa of activated B cells (NF-κB) and serum response element (SRE), translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells (HMVEC-L) and reversed LPI- at ASPET Journals on September 26, 2021 inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

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Introduction

Cannabinoids bind to and induce signaling via the cannabinoid 1 (CB1) and the cannabinoid 2 (CB2) receptors. Nevertheless, studies on cannabinoid receptor knock-out mice suggested additional cannabinoid-sensitive targets (Mackie and Stella, 2006;Brown, 2007). One receptor for small lipid mediators and synthetic cannabinoids is the G protein-coupled receptor 55 (GPR55). GPR55 is highly abundant in the CNS as well as in intestine, bone marrow, spleen, platelets, immune and endothelial cells (Balenga et al., 2011a; Henstridge et al., 2011; Waldeck-Weiemair et al., 2008; Sawzdargo et al.,

1999; Ryberg et al., 2007; Pietr et al., 2009; Rowley et al., 2011). Moreover, GPR55 was detected in a Downloaded from variety of cancer tissues and cancer cell lines (Andradas et al., 2011; Pineiro et al., 2011; Ford et al.,

2010; Huang et al., 2011; Perez-Gomez et al., 2012). Several endogenous GPR55 signaling pathways

have been described to date despite controversial findings concerning its agonists and antagonists jpet.aspetjournals.org

(Balenga et al., 2011b). One consensus between several groups is that GPR55 couples to Gα13 and/or

Gαq proteins in HEK293 cells that transiently or stably express GPR55 (Henstridge et al., 2009; Ryberg et al., 2007; Sharir and Abood, 2010; Lauckner et al., 2008; Henstridge et al., 2010; Schroder et al., at ASPET Journals on September 26, 2021

2010). In addition, GPR55 has been reported to activate small GTPases (Henstridge et al., 2009;

Balenga et al., 2011a; Ryberg et al., 2007) and to induce calcium release from intracellular stores

(Henstridge et al., 2009; Henstridge et al., 2010; Oka et al., 2007; Brown, et al., 2011). Further downstream, GPR55 activation has been shown to lead to the activation of several transcription factors, such as nuclear factor of activated T-cells (NFAT), nuclear factor kappa of activated B cells

(NF-κB), serum response element (SRE), cyclic AMP response element-binding protein (CREB) and activating transcription factor 2 (ATF2) (Kargl et al., 2012a; Henstridge et al., 2009; Henstridge et al.,

2010; Oka et al., 2010). In addition, MAP-kinases, such as p38 and extracellular signal-regulated kinases (ERK1/2), are activated upon GPR55 stimulation (Oka et al., 2010; Henstridge et al., 2010).

The lipid lysophosphatidylinositol (LPI) has been described as the first endogenous ligand for GPR55 by Oka et al. (Oka et al., 2007). In addition, several synthetic CB1 receptor inverse agonists/antagonists, such as AM251, AM281 and rimonabant (SR141716A) have been shown to activate GPR55 (Henstridge et al., 2009; Ryberg et al., 2007; Henstridge et al., 2010; Oka et al., 2007;

Brown et al., 2011; Kapur et al., 2009; Yin et al., 2009). Although several cannabinoid ligands can

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activate GPR55, the receptor lacks the classical “cannabinoid binding pocket” (Kotsikorou et al.,

2011).

Screening approaches have identified selective GPR55 agonists, such as GSK319197A or

GSK494581A (Kargl et al., 2012a; Brown et al., 2011), which generally appear to be inactive at CB1 and CB2 receptors, and hence are promising tools to elucidate the pharmacological, physiological and pathophysiological functions of GPR55. The first such chemical series of synthetic GPR55 agonists to be described were the benzoylpiperazines. Importantly, benzoylpiperazines were independently identified at GlaxoSmithKline (Brown et al., 2011; Kargl et al., 2012a) and by the National Institutes Downloaded from of Health Molecular Libraries Probe Identification program (Heynen-Genel et al., 2010b; Kotsikorou et al., 2011). The latter screen utilised β-arrestin fluorescent protein biosensors and identified further

agonists as well as antagonists (Heynen-Genel et al., 2010b; Heynen-Genel et al., 2010a). However, jpet.aspetjournals.org there is so far only limited characterization of these antagonists in peer-reviewed literature.

The lipid ligand LPI, the CB1 receptor inverse agonists/antagonists SR141716A, AM251 and AM281 and selective GPR55 agonists were described to activate transcription factors in human embryonic at ASPET Journals on September 26, 2021 kidney cells (HEK-293) transiently or stably expressing human GPR55 (Ryberg et al., 2007;

Henstridge et al., 2009; Henstridge et al., 2010; Brown et al., 2011; Kargl et al., 2012a). The phytocannabinoid cannabidiol (CBD) was reported to antagonize O-1602 mediated GPR55 activation in HEK-GPR55 cells and human osteoclasts (Ryberg et al., 2007; Whyte et al., 2009). In contrast,

CBD had no effect on Ca2+ mobilization and β-arrestin recruitment assays in HEK-GPR55 cells

(Kapur et al., 2009). In addition, Heynen-Genel et al. did not identify CBD as a GPR55 antagonist in a small molecule screen for GPR55 antagonists (Heynen-Genel et al., 2010a). Although CBD displays only low affinity for CB1 and CB2 receptors, CBD interacts with TRP receptors, such as TRPV2 or

TRPM8 (Qin et al., 2008; Pertwee et al., 2010). As such, CBD cannot be looked upon as a specific

GPR55 antagonist.

To further elucidate the role of GPR55 in physiology and pathobiology, selective agonists and antagonists are urgently needed. Here we characterized the novel GPR55 antagonist CID16020046 in yeast cells and HEK293 cells stably expressing GPR55. In addition, we used CID16020046 as a tool to study the role of GPR55 in endothelial wound healing of primary human lung microvascular

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endothelial cells (HMVEC-Ls) and human platelet aggregation. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Methods

Drugs – CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo

[3,4-c] pyrazol-5-yl] benzoic acid) was obtained from Molport (Riga, Latvia) and dissolved in DMSO.

GSK319197A ([4-(3,4-Dichloro-phenyl)-piperazin-1-yl]-(4’-fluoro-4-methanesulfonyl-biphenyl-2-yl)- methanone) was provided by GlaxoSmithKline (Harlow, United Kingdom) and dissolved in DMSO.

LPI (L-α-lysophosphatidylinositol) was purchased from Sigma Aldrich (Vienna, Austria) and dissolved in H2O.

Downloaded from

Cell culture, transfections and stable cell lines – HEK293 cells were cultured in Dulbecco´s modified

Eagle´s Medium (DMEM) (Life Technology, Vienna, Austria) supplemented with 10% fetal bovine

serum (FBS) (Life Technology) at 37˚C in 5% CO2, humidified atmosphere. HEK293 cells stably jpet.aspetjournals.org expressing the human 3xHA-GPR55 (HEK-GPR55), human FLAG-CB1 (HEK-CB1) or human

FLAG-CB2 (HEK-CB2) were previously described (Kargl et al., 2012a). All cells were serum-starved in Opti-MEM (Life Technology) prior to all experiments. Transient transfections were performed at ASPET Journals on September 26, 2021 using Lipofectamine 2000 following the manufacturer’s instructions (Life Technology). Human lung microvascular endothelial cells (HMVEC-Ls) were purchased from Lonza and were maintained in

EGM-2 MV BulletKit medium (Lonza, Verviers, Belgium).

Yeast exoglucanase assay – Derivation of yeast strains and yeast reporter gene assays was described previously (Brown et al., 2011; Brown et al., 2003; Olesnicky et al., 1999). In brief, yeast strain

YIG151 contains FLAG-GPR55 and Gpa1/Gα13 chimeric G-protein α-subunit both integrated chromosomally. YIG151 cells were mixed with buffered growth media pH 7.0 containing 10 mM 3- aminotriazole and lacking histidine, and the Exg1p (exoglucanase) substrate fluorescein-D- glucopyranoside at 10 µM, preincubated with test antagonist or vehicle for 10 minutes, and incubated with test agonist in 384-well microtitre plates (50 μl/well; DMSO final concentration: 1%) for 21 hours. Fluorescein production was quantified using an Envision plate reader (Perkin Elmer,

Cambridge, United Kingdom) and curve-fitting performed using Graphpad PrismTM.

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Intracellular Ca2+ release assays

- 96-Well FlexStation II assay – Agonist-mediated intracellular Ca2+ release was measured in a 96-well plate format (FlexStation II; Molecular Devices, Sunnyvale, CA), as previously described (Sedej et al.,

2011). Briefly, HEK293, HEK-GPR55 and HEK-CB1 cells (40000 cells per well) and HMVEC

(10000 cells per well) were seeded in black 96-well plates. Prior to each experiment, HEK293 cells were starved for 4 hours in Opti-MEM and HMVEC-Ls for 2 hours in EBM incomplete medium.

Next, cells were loaded with the Ca2+ fluorophore (FLEX calcium assay kit, Molecular Devices) for 60 minutes. A subset of cells was pre-treated with vehicle or GPR55 antagonist CID16020046 for 10 Downloaded from minutes. Subsequently, cells were stimulated with increasing concentrations of the agonist diluted in assay buffer. Intracellular Ca2+ mobilization was measured immediately after agonist application and

2+ recorded in real time during 2 minutes at room temperature in a FlexStation-II System. Upon Ca jpet.aspetjournals.org release from intracellular stores, the Ca2+ fluorophore was excited with a wavelength of 485 nm and emitted fluorescent light with a wavelength of 525 nm. Cell numbers were determined previous to Ca2+ measurement in a FlexStation-II (Molecular Devices). Ultimately, relative fluorescence unit (RFU) at ASPET Journals on September 26, 2021 readings for each well of the microplate were obtained and normalized to cell number.

- Ca2+ flux flow cytometric assay – Changes in intracellular Ca2+ levels in HEK293, HEK-GPR55 and

HEK-CB1 cells following treatment with GPR55 agonists/antagonists were assessed by flow cytometry as previously described (Heinemann et al., 2003). Cells were starved in Opti-MEM for 4 hours, resuspended in 1 ml wash buffer (without Ca2+ and Mg2+) and incubated with 5 µmol/l of the acetoxymethyl ester of Fluo-3 in the presence of 2.5 mmol/l probenecid for 60 minutes at room temperature in the dark. Cells were washed and resuspended in assay buffer (with Ca2+ and Mg2+).

Flow cytometric analysis was performed on a FACScalibur flow cytometer. After baseline fluorescence had been recorded for one minute, cells were treated with desired concentrations of agonists and measured for another 4 minutes. The changes in intracellular Ca2+ levels were detected as an increase in fluorescence intensity of the [Ca2+]i-dependent signal of Fluo-3 at 526 nm and data were normalized to baseline fluorescence.

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Western Blot – ERK1/2 phosphorylation was detected as previously described (Kargl et al., 2012a). In brief, HEK293, HEK-GPR55, HEK-CB1 and HEK-CB2 cells were seeded in 6-well plates and confluent wells were serum-starved overnight. Then cells were incubated with pre-warmed Opti-MEM containing vehicle (H2O or DMSO, final concentration: 0,025%, Merck, Vienna, Austria), LPI (Sigma

Aldrich), WIN55-212,2 (Tocris, Avonmouth, United Kingdom) or CID16020046 (Molport), or combinations thereof for 25 minutes at 37°C. Cells were washed, snap-frozen in liquid nitrogen and lysed in IP-Buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 25 mM KCl, 1 mM CaCl2, 0.3% Triton

X-100, 92 mg/ml sucrose and protease inhibitors (Roche Applied Science, Vienna, Austria). Lysates Downloaded from were resolved by SDS-polyacrylamide gel electrophoresis (SDS/PAGE, Life Technology) and transferred to a PVDF membrane (Millipore, Vienna, Austria). Membranes were blocked in TBST

buffer (1 mM CaCl2, 136 mM NaCl, 2,5 mM KCl, 25 mM Tris-HCl, 0,1% (v/v) Tween 20) containing jpet.aspetjournals.org

5% milk, washed in TBST without milk and immunoblotted with rabbit anti-pERK1/2 (1:1000) or rabbit anti-tERK1/2 (1:1000) antibodies overnight at 4°C (New England Biolabs, Frankfurt,

Germany). Membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:4000, at ASPET Journals on September 26, 2021

Jackson ImmunoResearch, Suffolk, United Kingdom) for 2 hours at room temperature and proteins were visualized with ECL Western Blotting Substrate (Fisher Scientific, Vienna, Austria). At least three independent blots were analyzed for quantification of phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) levels using ImageJ software (NIH) and pERK1/2 was normalized to tERK1/2 levels.

Reporter gene assay – Transcription factor luciferase assays were carried out as previously described

(Kargl et al., 2012a). Briefly, HEK293, HEK-GPR55 and HEK-CB1 cells were seeded in 96-well plates (40000 cells/well) and transiently transfected with the cis-reporter plasmids (PathDetect;

Stratagene, La Jolla, CA) for NFAT-luc (100-200 ng) SRE-luc (50 ng/well), NF-κB-luc (50ng/well) or

CREB (200ng/well) using Lipofectamine 2000. Twenty four hours post transfection cells were incubated with indicated ligand concentrations for 4 hours in serum-free media at 37°C. The cell number was determined in a FlexStation-II (Molecular Devices). Luciferase activity was visualized using the Steadylite Plus Kit (Packard Instrument Company, Meriden, CT) and was measured in a

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TopCounter (Top Count NXT; Packard) for 5 seconds. Luminescence values are given as relative light units (RLU). For reporter gene experiments, relative light units (RLU) were normalized to the cell number.

NFAT/NF-κB translocation – HEK-GPR55 cells were seeded in 6-cm dishes and transfected with 8

µg of enhanced green fluorescent protein (EGFP)-p65 plasmid (NF-κB p65-GFP) or NFATc3-GFP plasmid (Henstridge et al., 2009; Waldeck-Weiemair et al., 2008). 24 hours post transfection cells were seeded on coverslips and grown to 50% confluency in DMEM supplemented with 10% FBS. Downloaded from

Cells were serum-starved in OPTI-MEM for another 4 hours previous to treatments. Subsequently, cells were pre-treated with 2.5 µM GPR55 antagonist CID16020046 for 10 minutes and/or stimulated

with 2.5 µM LPI for 15 minutes, and the experiment was terminated by fixing cells with 3.7% jpet.aspetjournals.org formaldehyde. Fixed cells were washed twice with TBS (NaCl 135 mM, Tris-HCl 25 mM, CaCl2 1 mM, KCl 2.5 mM) and wet-mounted onto microscopy slides with Vectashield containing a DAPI dye to visualize nuclei (Roche). Images were taken with an Olympus inverted IX70 fluorescence at ASPET Journals on September 26, 2021 microscope equipped with a Hamamatsu Orca CCD camera.

Immunofluorescence microscopy – Cells were grown on poly-D-lysine (Sigma Aldrich) coated coverslips to 50% confluence, starved in Opti-MEM overnight and antibody feeding experiments were performed essentially as described (Kargl et al., 2012b). In brief, living cells were fed with anti-HA-11 antibody (1:1000, Covence, Berkeley, CA) for 30 minutes at 37˚C. Subsequently, cells were pre- stimulated with GPR55 antagonist CID16020046 for 10 minutes and/or stimulated with LPI for 45 minutes. Then cells were fixed in 3.7% formaldehyde, permeabilized in blotto (50 mM Tris-HCl, pH

7,5, 1 mM CaCl2, 0,3 % Triton X-100 and 3 % milk) and labelled with secondary antibodies

(AlexaFluor 488-conjugated IgG1 against the HA-tag (1:1000, Life Technology)) for 20 minutes.

Immunolabelled receptors were visualized by using an Olympus inverted IX70 fluorescence microscope.

RT-PCR and Real-Time PCR – PCR methods were conducted as previously described (Kargl et al.,

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2012c). HMVEC-L cells were frozen in liquid nitrogen and total RNA was extracted using

QIAshredder and RNeasy Kit (QIAGEN, Hilden, Germany), following the manufacturer´s instructions. RT-PCR was performed with 1 µg of total RNA and a high capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies) for cDNA transcription. The RT-PCR program was set at 25°C for 10 minutes, followed by 2 hours at 37°C with a terminal step at 85°C for

5 minutes. Quantitative PCR (qPCR) was performed using Fast SYBR® Green PCR Master Mix

(Applied Biosystems) following the manufacturer´s instructions. The following primers were used:

GPR55 (forward: 5′-CCTCCCATTCAAGATGGTCC-3′ reverse: 5′- Downloaded from

GACGCTTCCGTACATGCTGA-3′), CB1R (forward: 5′-CCTTCCTACCACTTCATCGGC-3′ reverse: 5′-CGTTGCGGCTATCTTTGCG-3′), CB2R (forward: 5′- GACCGCCATTGACCGATACC-

3′ reverse: 5′-GGACCCACATGATGCCCAG-3′) and GAPDH (forward: 5′- jpet.aspetjournals.org

ATGGGGAAGGTGAAGGTCG-3′ reverse: 5′-GGGGTCATTGATG-GCAACAATA-3′)). The specificity of the PCR products were assessed by melting curve analyses which showed only single amplified products, and agarose gel electrophoresis, which revealed fragments at the expected base at ASPET Journals on September 26, 2021 pair sizes. pcDNA3.1 plasmids encoding GPR55, CB1 or CB2 genes were used for standard curve calculations and absolute mRNA copy number was calculated.

Endothelial wound healing assay – HMVEC (50000 cells per chamber) were grown to confluence on

1% gelatin-pre-coated ECIS 8W1E polycarbonate arrays containing gold microelectrodes (Applied

Biophysics, Troy, NY). Cells were serum-starved for 2 hours before performing impedance measurements at multiple frequencies by using the Electric Cell-substrate Impedance Sensing (ECIS) system (Applied Biophysics). Confluent layers of endothelial cells were electrically wounded (20 seconds at 3000 μA and 100 kHz), resulting in severe electroporation and subsequent death of the cells situated on the electrodes. Impedance was continuously monitored to detect repopulation of the wound

(Keese et al., 2004).

Platelet aggregation – The study was approved by the Institutional Review Board of the Medical

University Graz. Blood was drawn from healthy volunteers after they signed an informed consent

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form. Platelet-rich and platelet-poor plasma was prepared from citrated whole blood by centrifugation and platelet aggregation was recorded using an APACT 4004 aggregometer (Haemochrom

Diagnostika, Essen, Germany) as described previously (Philipose et al., 2010). First, the ADP concentration (2.5-20 µM) which induced near half-maximal aggregation was determined for each donor and was used for further experiments. Samples were then incubated with LPI (300 nM-3 µM -10

µM) for 5 minutes at 37°C and platelet aggregation was induced by ADP. To determine the effect of the GPR55 antagonists CID16020046 on LPI-induced responses, samples were pre-treated with

CID16020046 (3 or 10 µM) or vehicle (DMSO diluted in saline) for 5 minutes at 37°C, and then Downloaded from incubated with LPI (10 µM) followed by activation of platelets with ADP. Platelet aggregation was expressed as percentage of maximum light transmission, with nonstimulated platelet-rich plasma being

0% and platelet-poor plasma 100%. jpet.aspetjournals.org

Statistical analysis – Statistical analyses were performed using t-tests or ANOVA for comparisons between multiple groups, followed by a Bonferroni’s post hoc analysis using Graphpad PrismTM. A p at ASPET Journals on September 26, 2021 value of < 0.05 was considered as statistically significant.

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Results

CID16020046 antagonizes human GPR55 in yeast

CID16020046 was originally described in PubChem to antagonize GPR55-mediated β-arrestin internalization (Data Source: Burnham Center for Chemical Genomics; Source Affiliation: Burnham

Institute for Medical Research, La Jolla, CA; Network: the National Institutes of Health Molecular

Libraries Probe Production Centers Network; Assay Provider: Dr. Mary Abood, California Pacific

Medical Center Research Institute). Yeast provide a useful host for functional expression of mammalian GPCRs, because they can be engineered to remove endogenous receptors, and to link Downloaded from activation of the heterologously expressed mammalian receptor to reporter genes (Dowell and Brown,

2002). In this way, GPR55 has previously been shown to respond to GPR55 agonists such as AM251,

LPI and GSK319197A in isolation from other GPCRs or secondary factors (Brown et al., 2011). Here, jpet.aspetjournals.org we utilized a yeast strain with significant basal (constitutive) exoglucanase activity due to expression of a FLAG-tagged version of human GPR55. CID16020046 (40 nM to 10 μM) acted as an inverse agonist, inhibiting GPR55 constitutive activity with IC50 0,15 μM (Fig.1, IC50 from Fig. 1B). at ASPET Journals on September 26, 2021

CID16020046 was tested for antagonist effects in combination with the agonists AM251,

GSK522373A, and LPI. The half-maximal effective concentration of AM251 increased due to the presence of 10 μM CID16020046 by less than 2-fold (EC50 = 3.9 μM in the absence and 7.2 μM in the presence of CID16020046; Fig. 1A). However, the maximum asymptotes of agonist concentration- response curves to AM251 were significantly depressed by increasing concentrations of CID16020046

(Fig. 1A). GSK522373A is a benzoylpiperazine and close structural analogue of GSK319197A, and has been described previously as a GPR55 agonist (Brown et al., 2011). CID16020046 caused sequential rightwards shift of the agonist concentration-response curve to GSK522373A. The extent of

2 this curve-shift fitted closely to a linear regression model (r = 1.00) with Hill slope of 0.73 (Schild analysis; Fig. 1D), giving an estimated pA2 = 7.3. Again, CID16020046 depressed the maximum effect of GSK522373A (Fig. 1C). LPI was tested up to a limiting concentration of 10 μM, since higher concentrations are toxic to yeast cells (Brown et al., 2011), and hence accurate EC50 values could not be determined. CID16020046 reduced or even abolished the agonist effect of 10 μM LPI on GPR55-

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expressing yeast cells. In conclusion, CID16020046 is an inverse agonist and antagonist of human

GPR55, able to block the effects of multiple chemical classes of GPR55 agonist. The chemical structure of CID16020046 is shown in Fig. 1F.

LPI-induced Ca2+ signaling is inhibited by CID16020046

Activation of GPR55 by several ligands can promote intracellular Ca2+ release (Oka et al., 2007;

Henstridge et al., 2010; Henstridge et al., 2009; Lauckner et al., 2008). We tested whether LPI and

GSK319197A can induce intracellular Ca2+ release in HEK-GPR55 cells using two different Downloaded from experimental approaches, with adherent cells using the FLEX system, and with cells in suspension by flow cytometry. We observed intracellular Ca2+ release in HEK-GPR55 cells upon stimulation with

increasing concentrations of LPI (Fig. 2A and D) and GSK319197A (Fig. 2D) in a concentration- jpet.aspetjournals.org dependent manner. To investigate the effect of CID16020046 on GPR55-mediated release of Ca2+ from intracellular stores, we pre-treated HEK-GPR55 cells with increasing concentrations of

CID16020046 for 15 minutes before exposure to 10 µM LPI (Fig. 2A and G) or 1 µM GSK319197A at ASPET Journals on September 26, 2021

(Fig. 2G) (IC50 values are shown in Table 1). CID16020046 inhibited LPI- and GSK319197A- induced

2+ 2+ Ca mobilization (Fig. 2A and G). The CB1 agonist WIN55,212-2 induced intracellular Ca release in

HEK-CB1 cells, but 1 µM WIN55,212-2 induced Ca2+ release in HEK-CB1 cells was not altered in the presence of increasing concentrations of CID16020046 (Fig. 2B and E). CID16020046 alone failed to induce intracellular Ca2+ release in HEK-GPR55 and HEK-CB1 cells (Fig. 2A, B and D). No effect of any ligand on intracellular Ca2+ levels was observed in control HEK293 cells at the concentrations tested (Fig. 2C and F). These data indicate that CID16020046 acts as a GPR55 antagonist, but has no effect in HEK293 and HEK-CB1 cells.

CID16020046 inhibits GPR55-mediated ERK1/2 phosphorylation

GPR55 activation has been shown to induce ERK1/2 phosphorylation in several cellular systems

(Henstridge et al., 2010; Kargl et al., 2012a; Whyte et al., 2009; Andradas et al., 2011; Perez-Gomez et al., 2012). In order to test whether CID16020046 acts as a selective GPR55 antagonist, we measured

ERK1/2 phosphorylation in HEK293, HEK-GPR55, HEK-CB1 and HEK-CB2 cells in the absence or

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presence of CID16020046. As previously described (Kargl et al., 2012a), stimulation with 2.5 µM LPI for 25 minutes induced ERK1/2 phosphorylation in HEK-GPR55 cells (Fig. 3A). 2.5 µM of the

GPR55 antagonist CID16020046 significantly inhibited the LPI-induced ERK1/2 phosphorylation.

Treatment with vehicle and CID16020046 alone showed no ERK1/2 phosphorylation over background

(Fig. 3A). 2.5 µM of WIN55,212-2 induced ERK1/2 phosphorylation in HEK-CB1 and HEK-CB2 cells, but phospho-ERK1/2 signal was not altered when combined with 2.5 µM CID16020046 (Fig.

3B).

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Effects of CID16020046 on GPR55-mediated transcription factor activation

Further, we investigated whether GPR55-mediated transcription factor activation can be modulated by

CID16020046. Previously, we have shown that various transcription factors, i.e. NFAT, SRE, NF-κB jpet.aspetjournals.org and CREB can be activated via GPR55 (Henstridge et al., 2009; Henstridge et al., 2010; Kargl et al.,

2012a). Here we demonstrate that both the endogenous GPR55 agonist LPI and the selective GPR55 agonist GSK319197A, can induce NFAT (Fig. 4A) and NF-κB (Fig. 4E) activation as well as SRE at ASPET Journals on September 26, 2021 induction (Fig. 4C) in HEK-GPR55 cells. In contrast, CID16020046 alone did not induce GPR55 mediated transcription factor activation (Fig. 4A, C and E). Pre-treatment with CID16020046 led to a concentration-dependent decrease in GPR55-mediated NFAT activation (Fig. 4B), NF-κB activation

(Fig. 4F) and SRE induction (Fig. 4D) in response to 1 μM LPI or GSK319197A (IC50 values are shown in Table 1). These data indicate that CID16020046 can inhibit GPR55-mediated transcription factor activation in HEK-GPR55 cells.

We next tested whether CID16020046 could antagonize CB1-mediated CREB activation. Stimulation of HEK-CB1 with WIN55,212-2 led to concentration-dependent CREB activation (Fig. 4G). Pre- incubation of HEK-CB1 cells with increasing concentrations of CID16020046 did not result in decreased CB1-mediated CREB activation after stimulation with 1 µM WIN55,212-2 (Fig. 4G). No effect of CID16020046 alone was observed in HEK-CB1 cells (Fig. 4G) and empty HEK293 cells

(data not shown).

In order to visualize the nuclear translocation of transcription factors in response to activation of cells with LPI, we transiently expressed NFATc3-GFP or NF-κB-p65-GFP in HEK-GPR55 cells. In

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untreated and vehicle-treated cells, NFATc3-GFP and NF-κB-p65-GFP localized predominantly in the cytosol (Fig. 5A and G), indicating that NFAT and NF-κB signaling cascades are not activated in the absence of GPR55 agonists. Upon stimulation with 2.5 µM LPI (Fig. 5B and H) or vehicle + 2.5 µM

LPI (Fig. 5E and K), NFATc3-GFP and NF-κB-p65-GFP rapidly translocated into the nucleus.

NFATc3-GFP and NF-κB-p65-GFP were observed in the cytosol upon incubation with 2.5 µM

CID16020046 prior to 2.5 µM LPI stimulation (Fig. 5F and L), indicating that CID16020046 blocks the GPR55-mediated NFAT and NF-κB activation. These data demonstrate that CID16020046 antagonizes GPR55-mediated activation and nuclear translocation of transcription factors, but has no Downloaded from effect on CB1-mediated CREB activation.

CID16020046 inhibits GPR55 internalization jpet.aspetjournals.org

GPR55 has been described to rapidly internalize upon agonist stimulation in several cell models

(Henstridge et al., 2010; Kargl et al., 2012a; Kargl et al., 2012b; Kapur et al., 2009; Henstridge et al.,

2009). In general, GPCR antagonists inhibit receptor internalization. Here we set out to investigate if at ASPET Journals on September 26, 2021

CID16020046 has the ability to inhibit LPI-induced GPR55 internalization. Antibody feeding experiments in live HEK-GPR55 cells showed that GPR55 is predominantly located on the cell surface under non-stimulated conditions (Fig. 6A). As expected, GPR55 internalized following treatment with

2.5 µM of LPI or vehicle + 2.5 µM of LPI for 45 minutes (Fig. 6B and E). Pre-treatment with 2.5 µM

CID16020046 for 10 minutes inhibited LPI-induced GPR55 internalization (Fig. 6F). Vehicle

(DMSO) or CID16020046 alone did not induce GPR55 internalization (Fig. 6C and D).

Secondary pharmacology (selectivity) profile of CID16020046

CID16020046 was tested in a panel of routinely run assays to determine its activity against a broad spectrum of GPCRs, ion channels, enzymes including kinases, and nuclear receptors, thirty-six targets in total (Supplementary Table 1). The most potent activities detected were for inhibition of phosphodiesterases, PDE3A and PDE4B (pIC50 = 5 ± 0.01 and 4.8 ± 0.05 respectively; mean ± SD, n=2). CID16020046 was observed to have weak activities close to the top concentrations tested in several other assays: these were for inhibition of acetylcholineesterase (pIC50 = 4.4 ± 0.11),

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antagonism of the mu-opioid receptor (pIC50 = 4.6 ± 0.01) and blockade of KCNH2, the hERG channel pIC50 = 4.6; n=1). In all other assays, CID16020046 was inactive up to the highest concentration tested of 25 μM (i.e. pXC50 <4.6) to 100 μM (i.e. pXC50 <4.0). Taken together, these data support the conclusion that CID16020046 is selective for GPR55 and give confidence that effects of CID16020046 observed in complex primary cell systems and tissues may be mediated via GPR55.

Functional characterization of CID16020046 in primary human lung microvascular endothelial cells

(HMVEC-Ls) Downloaded from

We next investigated whether inhibition of GPR55 in HMVEC-Ls with CID16020046 may have an effect on cellular function that involves GPR55. It was previously described that human dermal

microvascular endothelial cells express GPR55 and that this receptor might be involved in jpet.aspetjournals.org angiogenesis and endothelial wound healing capacity (Zhang et al., 2010). Here, we tested whether another primary human endothelial cell line, human lung microvascular endothelial cells (HMVEC-L) express GPR55 and whether this receptor may have a similar physiological relevance in this cell line. at ASPET Journals on September 26, 2021

In fact, HMVEC-L expressed both GPR55 (Fig. 7A), and CB1 receptors, but only very little CB2 receptor mRNA was detected (Fig. 7A). We then tested whether LPI can induce Ca2+ mobilization and enhance endothelial wound healing in HMVEC-Ls and whether these effects were blocked by pre- treatment with CID16020046.

10 µM of LPI induced intracellular Ca2+ release in HMVEC-L (Fig. 7B) and this effect was inhibited upon pre-treatment with 25 and 50 µM CID16020046 (Fig. 7C). Further, we observed that enhanced endothelial wound healing following electric wounding of the monolayer was augmented by 0.1 µM of

LPI (Fig. 7D) as compared to vehicle (H2O, Fig. 7D) indicating that GPR55 activation may be involved in migration of cells and hence in wound closure. Pre-treatment with 1 µM of CID16020046

(Fig. 7E) abolished the LPI-induced stimulation of wound healing in HMVEC-Ls when compared to vehicle control (Fig. 7E). In summary, these data show that CID16020046 is a specific inhibitor of

GPR55 function in HMVEC-L cells, which endogenously express this receptor.

CID16020046 reverses the inhibitory effect of LPI on ADP-induced platelet aggregation

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Human platelets were previously shown to express the mRNA for GPR55 (Henstridge et al., 2011;

Rowley, et al., 2011). Therefore, we tested whether GPR55 is involved and LPI is capable of modifying platelet function. Up to concentrations of 10 µM, LPI itself did not induce platelet aggregation (n=4, data not shown). To investigate modulatory effects of LPI on platelet aggregation in response to a known inducer, platelets were stimulated with concentrations of ADP (2.5-20 µM) that gave half-maximal aggregatory responses. Under these conditions, LPI significantly attenuated the

ADP-induced platelet aggregation by about 20-25% (Fig. 8A and C). This effect was completely reversed by the GPR55 antagonist CID16020046 (10 µM; Fig. 8B and C). At this concentration, the Downloaded from antagonist by its own did not significantly alter ADP-induced platelet aggregation (data not shown, n=4). This is a preliminary indication that GPR55 may be involved in the regulation of platelet

function. jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Discussion

CID16020046 originates from a high-throughput screen of approximately 300,000 compounds for molecules able to block GPR55-mediated internalization of a β-arrestin-GFP biosensor

(http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=16020046). This screen was performed by the Abood group at the Burnham Institute as part of the NIH MLSCN (Molecular Library

Screening Center Network) program. In this screen, multiple chemical series of both GPR55 agonists and antagonists were identified (Heynen-Genel et al., 2010b; Heynen-Genel et al., 2010a).

CID16020046 does not appear to be one of the GPR55 antagonists chosen by the Abood group for Downloaded from more detailed analysis (Heynen-Genel et al., 2010b; Heynen-Genel et al., 2010a), but nonetheless was of interest to us because it coincided with a chemical series that had been identified independently in a

separate high-throughput screen performed at GlaxoSmithKline, which used yeast as expression host jpet.aspetjournals.org to provide a gene-reporter assay (data not shown) (Brown et al., 2011). Here, we present further corroboration of the activity of CID16020046 as a GPR55 antagonist, performed in a third lab

(Medical University of Graz) working independently. We also provide data from GlaxoSmithKline to at ASPET Journals on September 26, 2021 show that CID16020046 also antagonizes GPR55 in the yeast assay, as expected.

Independent confirmation of newly identified GPR55 ligands is of paramount importance because the literature around this receptor contains many inconsistencies and contradictions. Several compounds have been described as antagonists and used to support a hypothesis of functional expression of

GPR55, even though these compounds do not consistently block the effect of acknowledged GPR55 agonists such as AM251 and LPI in many cell systems. Furthermore, many of the tools used in these studies are not selective, having known effects at other targets. For example, the reported GPR55 antagonist CBD interacts with TRP receptors (Qin et al., 2008; Pertwee et al., 2010) whereas we show here that CID16020046 is inactive at the TRPV4 channel. The complexity of GPR55 pharmacology and the lack of selective ligands have made it challenging in the extreme to convincingly determine the physiological role of GPR55. This area has been reviewed extensively by others and will not be elaborated further here (Henstridge, 2012; Balenga et al., 2011b; Sharir and Abood, 2010; Pertwee et al., 2010).

We show that CID16020046 antagonizes agonist-mediated GPR55 activation in yeast cells (Fig. 1)

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and that this compound inhibits GPR55-mediated Ca2+ release from intracellular stores (Fig. 2; Table

1) as well as transcription factor activation (Fig. 4 and 5; Table 1) in HEK293 cells stably expressing

GPR55. CID16020046 is selective for GPR55 over CB1, since it had no effect on WIN55,212-2-

2+ induced Ca release and did not affect CB1-mediated CREB activation (Fig. 2 and 4). LPI induced

ERK1/2 phosphorylation was blocked upon pre-treatment with CID16020046 (Fig. 3), while the antagonist had no effect on CB1- and CB2-mediated ERK1/2 phosphorylation (Fig. 3). In addition, internalization of GPR55 was inhibited by CID16020046 pre-treatment (Fig. 6). Importantly,

CID16020046 had similar antagonistic effects on both the physiological ligand LPI and the synthetic Downloaded from agonists GSK319197A and GSK522373A. The calculated IC50 values for the antagonist effect of

CID16020046 at GPR55 were 0.21 μM (Ca2+ mobilization; 10 μM LPI), 0.48 μM (NFAT activation; 1

μM LPI), and 0.71μM (NF-κB activation; 1 μM LPI) (see Table 1). Although these are not expected to jpet.aspetjournals.org be numerically identical, they are consistent with both the calculated pA2 value (7.3, equating to 54 nM) from the yeast assay using GSK522373A as agonist, and the IC50 of inverse agonism in yeast

(0.145 μM). Blockade of SRE induction appears somewhat an outlier, with IC50 values for at ASPET Journals on September 26, 2021

CID16020046 of 2.0 μM (1 μM LPI). It seems reasonable to expect a functional potency for

CID16020046 in blocking LPI-evoked GPR55-mediated effects in the approximate range 0.1-1 μM.

The effect of CID16020046 on the shape of agonist concentration-response curves (depression of the maximum asymptote) is suggestive of a non-competitive mechanism, which may be confirmed once validated radioligands become available.

To illustrate the power of the newly available GPR55-selective antagonist CID16020046, we show a preliminary investigation into the putative signaling properties of GPR55 in human cells endogenously expressing the receptor, including primary cells: firstly in microvascular lung endothelial cells

(HMVEC-Ls), and secondly in human platelets. GPR55 mRNA levels are expressed in HMVEC-Ls and LPI (10 µM) caused intracellular Ca2+ release from HMVEC-L intracellular stores, and

CID16020046 could block this effect. However, higher concentrations of CID16020046 were required to block HMVEC-L Ca2+ release (25 and 50 µM, 10 µM was ineffective) than had been observed to

2+ block Ca release in the recombinant assay (IC50 = 0.21), and in this concentration range

CID16020046 may have non-specific effects, for example at phosphodiesterases (Supplemental Table

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1). Therefore, further experiments will be required to confirm that GPR55 in HMVEC-Ls couples to

Ca2+ release. However, we found much lower concentrations of LPI to be effective in enhancing endothelial wound healing in the HMVEC-L model. Moreover, the degree of enhancement attributable to LPI was largely abolished by 1 µM CID16020046, a concentration in keeping with its potency in recombinant assays. Taken together, these data support functional expression of GPR55 by HMVEC-L cells. We also provide preliminary evidence that ADP-induced platelet aggregation may be attenuated by 10 µM LPI and that this effect is reversed by CID16020046 (3 and 10 μM). Therefore, it appears that platelets may be another cell type in which GPR55, and its ligand LPI, play a role. Downloaded from

Accumulating evidence suggests that GPR55 plays a role in diverse physiological and pathological processes (Henstridge et al., 2011). For instance, it has been reported that GPR55 and CB2 receptors

are co-expressed and crosstalk at the level of small GTPases in human neutrophils. GPR55 thereby jpet.aspetjournals.org enhances the migratory capacity of neutrophils, while it limits their bactericidal functions, such as reactive oxygen species (ROS) production and degranulation (Balenga et al., 2011a). It has also been reported that GPR55 is highly expressed in malignant human tumors and its expression level is at ASPET Journals on September 26, 2021 directly correlated to the aggressiveness of the tumors (Andradas et al., 2011; Henstridge et al., 2011;

Perez-Gomez et al., 2012). GPR55 was shown to be expressed in human osteoclasts and its activation could stimulate osteoclast polarization and resorption in vitro (Whyte et al., 2009). In addition, GPR55 knock-out mice were resistant to inflammatory and neuropathic pain (Staton et al., 2008) and GPR55 may play a role in inflammatory responses of microglial cells (Pietr et al., 2009). None of these studies were able to access CID16020046 or the other new GPR55 antagonists to validate and support their findings, but the prospect in future is for a much more well-founded understanding of GPR55 function, and especially whether medicines targeting GPR55 could be therapeutically beneficial. CID16020046 and at least one of the GPR55 antagonists characterized by the Abood group, ML-193, are commercially available from Molport. Since ML-193 and CID16020046 are chemically distinct, a powerful approach will be to use them in combination. If for example LPI-evoked effects in primary cells and tissues were blocked by both these agents, and at concentrations consistent with the potency of each antagonist observed in recombinant assays, this would constitute strong evidence that GPR55 is indeed functionally important. We have not so far accessed ML-193, but the reproducibility of both

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synthetic agonists (benzylpiperazines; (Brown et al., 2011)) and antagonists (CID16020046) arising out of the MLSCN screen, give confidence of its usefulness.

In summary, CID16020046 is a selective GPR55 antagonist, which is commercially available and allows a range of applications to study GPR55 pharmacology and signaling properties. In combination with the other selective agonist and antagonist tools recently described, it offers the prospect of a hugely improved understanding of the physiological and pathophysiological role of this complex and controversial receptor. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Acknowledgments

We thank Veronika Pommer, Martina Ofner, Viktoria Konya and Wolfgang Platzer for technical support.

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Authorship Contributions

Participated in research design: Kargl, Heinemann; Conducted experiments: Kargl, Brown, Andersen,

Dorn; Performed data analysis: Kargl, Brown, Heinemann; Wrote or contributed to the writing of the manuscript: Kargl, Brown, Schicho, Waldhoer, Heinemann

The authors declare no conflicts of interest. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Footnotes

This work was supported by funds from the Austrian Science Fund [P18723 to MW, P22521 to AH and P22771 and P25633 to RS], the Jubilaeumsfonds of the Austrian National Bank [Grants No.

12552 to MW,13487 and 14263 to AH, and 14429 to RS] and the Lanyar Stiftung (to MW), the

START-Funding Program of the Medical University of Graz, the PhD program ‘Molecular Medicine’ of the Medical University of Graz and an EMBO short term fellowship (all JK). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Legends for Figures

Figure 1: CID16020046 antagonizes human GPR55 in yeast. Yeast cells expressing human GPR55

(YIG151) were incubated with the agonists AM251 (A), GSK522373A (C) and LPI (E), in the absence or presence of increasing concentration of CID16020046 (0.04 µM, 0.16 µM, 0.6 µM, 2.5 µM and 10

µM). (B): Inverse agonism of CID16020046 (inhibition of the basal gene reporter activity due to

GPR55 constitutive activity). (D): Schild analysis of the concentration-response curves to

GSK522373A. Yeast growth media were supplemented with the fluorogenic substrate fluorescein-D- glucopyranoside, and production of fluorescein after 24 hours was measured in an Envision plate Downloaded from reader. The chemical structure of CID16020046 is shown (F).

2+ Figure 2: CID16020046 inhibits GPR55 mediated Ca -signaling. HEK-GPR55 (A), HEK-CB1 (B) jpet.aspetjournals.org and HEK293 (C) cells were loaded with a Ca2+ fluorophore for 1 hour and cells were subsequently stimulated with increasing concentrations of LPI, WIN55,212-2 or CID160220046. Intracellular Ca2+ release was measured in HEK-GPR55 (A) and HEK-CB1 (B) cells after stimulation with receptor at ASPET Journals on September 26, 2021 agonists using a FLEX station. No intracellular Ca2+ release was observed in HEK293 cells (C). Pre- treatment of HEK-GPR55 cells with increasing concentrations of CID16020046 inhibited agonist induced intracellular Ca2+ release (A). No antagonistic effect of CID16020046 was observed in HEK-

CB1 cells (B). CID16020046 alone had no effect on HEK293, HEK-GPR55 and HEK-CB1 cells (A-

C). Data were normalized to cell numbers and expressed as relative fluorescence units (n = 3).

Intracellular Ca2+ flux was determined using fluorescence cytometry (D-G). Cells were loaded with the

Ca2+ sensitive dye Fluo-3 AM and stimulated with increasing concentrations of agonists. Ca2+ flux was observed in HEK-GPR55 cells upon treatment with LPI and GSK319197A, but no effect was observed after stimulation with CID16020046 (D). WIN55,212-2 induced Ca2+ flux in HEK-CB1 cells, but this effect was not blocked by the pre-treatment with CID16020046 (E). LPI and GSK319197A did not induce Ca2+ flux in HEK293 cells (F). Increasing concentrations of CID16020046 blocked LPI and

GSK319197A induced Ca2+ flux in HEK-GPR55 cells (G). Results are represented as fold increase as means ± SEM of at least three independent experiments.

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Figure 3: ERK1/2 activation is blocked in HEK-GPR55 cells upon CID16020046 pre-treatment.

HEK-GPR55 (A), HEK-CB1 and HEK-CB2 (B) cells were serum-starved for 4 hours and stimulated with either vehicle, 2.5 µM LPI, 2.5 µM CID16020046, 2.5 µM WIN55,212-2 and combinations thereof for 25 minutes. LPI treatment induced ERK1/2 phosphorylation in HEK-GPR55 cells and this signal was inhibited upon pre-treatment with CID16020046 (A). In contrast, HEK-CB1 and HEK-CB2 cells showed ERK1/2 activation after stimulation with WIN55,212-2 which was not altered by

CID16020046 (B). CID16020046 alone had no effect on ERK1/2 phosphorylation in HEK-GPR55 cells (A). Representative blots of three independent experiments are shown. ***p<0.001 as indicated. Downloaded from

Figure 4: CID16020046 inhibits GPR55 mediated transcription factor activation. HEK-GPR55

(A-F) and HEK-CB1 (G) cells were transfected with NFAT (A and B), SRE (C and D), NF-κB (E and jpet.aspetjournals.org

F) or CREB (G) transcription factor-luciferase-reporter plasmids. 24-hours post transfection, cells were stimulated with either increasing concentrations of LPI or GSK319197A (A, C and E) or with

1µM LPI or GSK319197A in combination with increasing concentrations of the GPR55 antagonist at ASPET Journals on September 26, 2021

CID16020046 (B, D and F) for 4 hours in serum-free medium. NFAT, SRE and NF-κB activation was observed in HEK-GPR55 cells after stimulation with LPI or GSK319197A (A, C and E). Increasing concentrations of CID16020046 (B, D and F) inhibited LPI and GSK319197A induced GPR55 transcription factor activation. WIN55,212-2 induced CB1-mediated CREB activation, but CB1- mediated signaling was not inhibited in the presence of CID16020046 (G). CID16020046 alone did not induce transcription factor activation in HEK-GPR55 and HEK-CB1 cells. Data are means ± SEM from one of three independent experiments performed in triplicates. Data were normalized and expressed as percent of maximum activation which was set as 100%, RLU (relative light units).

Figure 5: CID16020046 inhibits NFAT and NF-κB transcription factor translocation. HEK-

GPR55 cells were left untreated (A and G), stimulated with 2.5 µM LPI (B and H) for 15 minutes or in addition pre-incubated with vehicle (DMSO) (E and K) or 2.5 µM CID16020046 (F and L) for 10 minutes. Thereafter, nuclear translocation of NFAT-GFP and NF-κB-p65-GFP was visualized. LPI induced the translocation of NFATc3-GFP and NF-κB-p65-GFP into the nucleus, whereas

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CID16020046 inhibited this response. Cell nuclei were stained with DAPI (blue). Representative cells of three independent experiments are shown. Scale bars = 10 µM.

Figure 6: CID16020046 blocks LPI mediated GPR55 internalization. GPR55 was located on the cell surface without agonist treatment (A) and internalized following LPI stimulation in HEK-GPR55 cells (B). Vehicle (DMSO) (C) and CID16020046 (D) alone did not induce GPR55 internalization.

Pre-treatment of HEK-GPR55 cells with vehicle before stimulation with 2.5 µM LPI induced GPR55 internalization, whereas pre-treatment with 2.5 µM of CID16020046 kept the receptor on the cell Downloaded from surface. Scale bar = 10 μm.

2+ Figure 7: CID16020046 reduces LPI-induced intracellular Ca release and wound healing of jpet.aspetjournals.org

HMVEC-L. HMVEC-L express transcripts of GPR55 and CB1 receptors (A). 10 µM LPI induced intracellular Ca2+ release in HMVEC-Ls (B), and this effect was reduced after pre-incubation with 25

µM and 50 µM GPR55 antagonist CID16020046 (C). HMVEC-L treated with 0.1 µM of LPI showed at ASPET Journals on September 26, 2021 enhanced wound healing as compared to control treatment after electronic wounding (D). Pre- stimulation of HMVEC-Ls for 10 minutes with CID16020046 reduced wound healing capacity of

HMVEC-L in response to 0.1 µM LPI (E). Data show mean + SEM impedance (Ω) of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001 as indicated.

Figure 8: CID16020046 reverses the inhibitory effect of LPI on ADP-induced platelet aggregation. Typical tracings of the inhibitory effect of LPI on platelet aggregation, in the absence or presence of CID16020046 (10 µM), is shown in A and B, respectively. Platelets were stimulated with

ADP (2.5-20 µM) to give near half-maximal aggregation. Statistical analysis of above experiments are summarized in C. Data are mean + SEM, n= 4-8 with samples from different donors. *p<0.05 as indicated.

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Tables

Table 1: IC50 of CID16020046 in HEK-GPR55 cells (in µM)

CID16020046 + 10µM LPI

Ca2+ mobilization 0.21 ± 0.09

CID16020046 + 1µM LPI CID16020046 + 1µM GSK319197A

NFAT activation 0.48 ± 0.03 0.31 ± 0.05

SRE induction 1.99 ± 0.16 1.48 ± 0.48

NF-κB activation 0.71 ± 0.08 0.64 ± 0.27 Downloaded from

Ca2+ mobilization and Reporter gene assay data are means ± SEM (n= 3–4). NFAT, nuclear factor of

activated T cells; SRE, serum response element; NF-κB, nuclear factor kappa of activated B cells. jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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10µM CID16020046 A 2.5µM CID16020046 B 0.62µM CID16020046 ) 4 at ASPET Journals on September 26, 2021 5 ) 10 0.16µM CID16020046 5 0.04µM CID16020046 8 Vehicle 3

6 2 4 1 2 Fluorescence Units (x 10 Fluorescence Units (x 10 0 0 -8 -7 -6 -5 -4 -8 -7 -6 -5 Log [AM251], M Log [CID16020046] (M) C D

) 5

5 Schild Analysis for GSK522373A 2.0 4 1.5 3 1.0 2 0.5

1 Log (DR-1) 0.0

Fluorescence Units (x 10 0 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 -0.5 log[CID16020046] Log [GSK522373A], M E F

) 6 5

Fluorescence Units (x 10 0 -8 -7 -6 -5 Log [LPI], M JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org

Fig 2

A B C at ASPET Journals on September 26, 2021

D E F

G JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version.

Fig 3 A LPI + Downloaded from Vehicle + LPI + Vehicle CID16020046 Untreated LPI Vehicle CID16020046

p-ERK jpet.aspetjournals.org HEK-GPR55

t-ERK at ASPET Journals on September 26, 2021

B + WIN55,212-2+ Vehicle Vehicle WIN + Vehicle CID16020046

p-ERK HEK-CB1 t-ERK

p-ERK

HEK-CB2

t-ERK JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version.

Fig 4 A B Downloaded from jpet.aspetjournals.org

C D at ASPET Journals on September 26, 2021

E F

G JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

Fig 5

Untreated LPI Veh CID16020046 Veh + LPI CID16020046 + LPI A B C D E F NFAT-GFP NFAT-GFP G H I J K L NfkB-GFP NfkB-GFP JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

Fig 6 HA-GPR55 HA-GPR55

A D

B E

C F LPI untreated LPI Veh CID16020046 CID16020046+LPI Veh+LPI JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

Fig 7

A B C

D E JPET Fast Forward. Published on May 2, 2013 as DOI: 10.1124/jpet.113.204180 This article has not been copyedited and formatted. The final version may differ from this version.

Fig 8 Downloaded from A jpet.aspetjournals.org at ASPET Journals on September 26, 2021

C JPET #204180

A selective antagonist reveals a potential role of G protein-coupled

receptor 55 in platelet and endothelial cell function

Julia Kargl*, Andrew J Brown, Liisa Andersen, Georg Dorn, Rudolf Schicho, Maria Waldhoer

and Akos Heinemann JPET #204180

Supplementary Table 1

Class Gene Name Target Common Technology Biological Reagent Mode pXC50 Name

Direct detection of substrate/product ACHE Acetylcholineesterase Purified enzyme (SigmaAldrich) Inhibitor 4.4 ± 0.11 (Mass Spectrometry)

Solubilised cell extract from Sf9 FLINT (diacetyldichloro‐ insect cells transduced with PTGS1 Cycloxygenase‐2 Inhibitor <4 fluorescein) FLAG‐tagged COX‐2 baculovirus

Enzyme Microsomes prepared from FLINT (coupled MAOB Monoamineoxidase‐B insect cells transduced with Inhibitor <4 (n=1) fluorogenic reaction) MAO‐B baculovirus

Luminescent Cambrex Enzyme purified from yeast PDE4B Phosphodiesterase 4B assay (coupled cAMP Inhibitor 5 ± 0.01 transfected with PDE4B hydrolysis)

Scintillation ‐proximity PDE3A Phosphodiesterase 3A assay (SPA); [3H]‐AMP‐ Purified recombinant enzyme Inhibitor 4.8 ± 0.05 specific beads

Membranes prepared from Agonist <4 [35S]GTPS‐binding HTR1B Serotonin‐1B receptor CHO cells stably expressing (LEADseeker SPA) 5HT1B Antagonist <4

Aequorin luminescence Frozen HEK293F cells stably Agonist <4.6 HTR2A Serotonin‐2A receptor of intracellular calcium expressing aequorin and concentration 5HT2A Antagonist <4.6

Aequorin luminescence Agonist <4.6 Frozen CHO‐K1 cells stably HTR2C Serotonin‐2C receptor of intracellular calcium expressing aequorin and 5HT2C concentration Antagonist <4.6

CHO cells stably expressing A2a ADORA2A Adenosine A2a receptor TR‐FRET cAMP assay Agonist <4 receptor GPCR Aequorin luminescence Frozen HEK293 cells stably ADRA1B  Adrenoceptor of intracellular calcium expressing aequorin and Antagonist <4.6 concentration transduced with 1B BacMam

CHO‐K1 cells stably expressing ADRA2C 2C Adrenoceptor TR‐FRET cAMP assay Agonist <4 2C receptor

Agonist <4 CHO cells stably expressing 2 ADRB2 2 Adrenoceptor TR‐FRET cAMP assay adrenoceptor Antagonist <4

Yeast reporter gene Yeast (S.cerevisiae) cells stably CNR2 Cannabinoid CB2 receptor Agonist <4 assay expressing CB2 receptor

Membranes prepared from [35S]GTPS‐binding HEK293F cells stably expressing DRD1 Dopamine D1 receptor Antagonist <4 (LEADseeker SPA) D2 receptor and rat Go alpha subunit JPET #204180

Membranes prepared from Agonist <4 [35S]GTPS‐binding HEK293F cells stably expressing DRD2 Dopamine D2 receptor (LEADseeker SPA) human D2 receptor and rat Go alpha subunit Antagonist <4

Aequorin luminescence Frozen CHO cells stably HRH1 Histamine H1 receptor of intracellular calcium expressing aequorin and H1 Antagonist <4.6 concentration receptor

FLIPR fluorescence of Agonist <4.3 Muscarinic M1 acetylcholine Frozen CHO cells stably CHRM1 intracellular calcium receptor expressing M1 receptor concentration Antagonist <4.3

FLIPR fluorescence of Agonist <4.3 Muscarinic M2 acetylcholine Frozen CHO cells stably CHRM2 intracellular calcium receptor expressing human M1 receptor concentration Antagonist <4.3

Aequorin luminescence U2OS cells transduced with TACR1 Tachykinin NK1 receptor of intracellular calcium Antagonist <4.6 NK1 Bacmam concentration

Membranes prepared from Agonist <4 [35S]GTPS‐binding OPRK1 opioid receptor CHO‐K1 cells transduced with (LEADseeker SPA) OR Bacmam Antagonist <4

Membranes prepared from Agonist <4 [35S]GTPS‐binding OPRM1 opioid receptor CHO cells transduced with OR (LEADseeker SPA) Bacmam Antagonist 4.6 ± 0.01

FLIPR fluorescence of CHO cells stably expressing V1a AVPR1A Vasopressin V1a receptor intracellular calcium Antagonist <4.3 receptor concentration

FLIPR fluorescence of Opener <4.3 CHO cells stably expressing HTR3A Serotonin‐3 receptor intracellular calcium 5HT3 concentration Blocker <4.3

FLIPR fluorescence of Frozen HEK293 cells stably Opener <4.3 1 nicotonic acetylcholine CHRNA1 intracellular calcium expressing  AChR receptor concentration subunits Blocker <4.3

Frozen HEK293 cells stably FLIPR fluorescence of expressing a, IC CACNA1C CaV1.2(L‐type) channel intracellular calcium Blocker <4.3 transduced after thawing with concentration human 1C and IK1 Bacmams

Functional CHO cells stably expressing KCNH2 hERG electrophysiology Blocker 4.6 (n=1) KCNH2 (IonWorks Barracuda)

Functional Kv7.1/MinK potassium CHO cells stably expressing KCNQ1/KCNE1 electrophysiology Blocker <4.6 channel KCNQ1 and minK (IonWorks Quattro)

Functional CHO cells stably expressing KCNA5 Kv1.5 channel electrophysiology Blocker <4.3 (n=1) Kv1.5 (IonWorks Quattro) JPET #204180

Functional HEK293 cells stably expressing SCN5A Nav1.5 channel electrophysiology Blocker <4 Nav1.5 (IonWorks Quattro)

NMDA (ionotropic FLIPR fluorescence of U2OS cells transduced with GRIN1/GRIN2B Glutamate channel) intracellular calcium Blocker <4.3 NR1A and NR2B Bacmams receptor concentration IC (cont.) FLIPR fluorescence of Opener <4.6 Transient receptor potential HEK MSRII cells transduced TRPV4 intracellular calcium cation channel, TRPV4 with TRPV4 Bacmam concentration Blocker <4.6

FLAG‐His tagged Aurora B IMAP Fluorescence protein purified from AURKB Aurora B kinase Inhibitor <4.5 Polarisation baculovirus‐transfected Sf9 cell lysate

Kinase lymphocyte‐specific protein IMAP Fluorescence LCK Purified His‐tagged LCK protein Inhibitor <4.5 tyrosine kinase Polarisation

His‐tagged PI3K protein purified from lysate of PIK3CG Phosphoinositol‐3‐kinase‐ TR‐FRET Inhibitor <4.5 (n=1) baculovirus‐transfected Sf9 cells

Invitrogen LS180 cells stably Invitrogen liveBLAzer kit: AHR Aryl Hydrocarbon Receptor transfected with a CYP1A1‐ Activator <4 FRET (Envision) NR lactamase gene‐reporter

Frozen HepG2 cells pre‐ Luciferase gene‐reporter NR1L2 Pregnane‐X‐receptor transfected with human PXR Activator <4.3 luminescence (Viewlux) and CYP3A‐luciferase reporter

Legend: Secondary Pharmacology (selectivity) profile of CID16020046

Method: CID16020046 was dissolved in DMSO to 10mM and purity and identity confirmed by LC- MS. Eleven-point dilution series (1:2, 1:3 and 1:4) were prepared and 50-500nl aliquots dispensed for assay in 384-well plates. Each assay in this panel has been performed biweekly for a minimum of 3 months to assure consistency, using established technologies listed in the Table. Each assay plate contained 16 no-effect controls (usually vehicle-treated) and 16 high-effect controls (treated with a maximal concentration of a known active agent) and data were only accepted when a Z’ parameter (Zhang, et al.,1999) exceeded predefined limits (typically Z’>0.4, after exclusion of statistically- defined extreme outliers (Coma, et al., 2009)). Also, each experiment contained a minimum of three internal standards, being compounds known to modulate the specific target/mode; data from the entire

experiment were only accepted when pXC50 values were within ± 0.5 log units of the respective average assay value for that compound over the previous 3 months of testing, for at least two-thirds of standard compounds. All targets tested were human and were selected based on association to overt physiological effects (Bowes, et al., 2012). Data were obtained from concentration-response curves using a 4-parameter logistic fitting module in Microsoft Excel™ within IDBS ActivityBase™.

Activity is reported as pXC50 which is the negative logarithm of the concentration for which a half

maximal effect is achieved. pXC50 values shown are pEC50 values for agonist/opener/activator and JPET #204180

pIC50 values for antagonist/blocker/inhibitor. pXC50 values are presented as mean ± SD. All data is from n=2 except where indicated. Where no activity of CID16020046 was observed, pXC50 values are presented as “<”. Hence, CID16020046 was inactive (failed to meet criteria for curve-fitting) up to the top concentration tested in that assay, i.e. pXC50 <4 indicates inactive up to 100 M. Abbreviations used: GPCR: G protein-coupled receptor; IC: Ion Channel; NR: Nuclear Receptor; FLINT: fluorescence intensity; TR-FRET: time-resolved fluorescence energy transfer.