Lysophosphatidic Acid Receptor Agonism: Discovery of Potent Non-Lipid Benzofuran Ethanolamine Structures

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Title page

Lysophosphatidic acid receptor agonism: discovery of potent non- lipid benzofuran ethanolamine structures

Authors: Etienne Guillot, Jean-Christophe Le Bail, Pascal Paul, Valérie Fourgous, Pascale

Briand, Michel Partiseti, Bruno Cornet, Philip Janiak, Christophe Philippo Downloaded from

jpet.aspetjournals.org Principal affiliation: Sanofi R&D, 1 Av Pierre Brossolette, 91385 Chilly-Mazarin, France

at ASPET Journals on September 27, 2021 In detail affiliations

EG, JCLB, PB, PJ: Diabetes and Cardiovascular Unit, Sanofi R&D, 1 Av Pierre Brossolette,

91385 Chilly-Mazarin, France

CP: Global Research Portfolio and Project Management, Sanofi R&D, 1 Av Pierre Brossolette,

91385 Chilly-Mazarin, France

PP, VF: Translational Science Unit, Sanofi R&D, 1 Av Pierre Brossolette, 91385 Chilly-

Mazarin, France

BC: In-silico design, 1 Av Pierre Brossolette, 91385 Chilly-Mazarin, France

MP: Integrated Drug Discovery, Sanofi R&D, 13 quai Jules Guesde, 94400 Vitry-Sur-Seine,

France

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Running Title Page

a) Running title: New potent non-lipid lysophosphatidic acid receptor agonists

Downloaded from b) Corresponding author:

Dr. Etienne Guillot, PhD jpet.aspetjournals.org Sanofi R&D, 1, avenue Pierre Brossolette, 91385 Chilly-Mazarin, France

Tel: + 33.1.60.49.67.58-cell +33.6.07.25.90.86 at ASPET Journals on September 27, 2021 Email: [email protected]

c) Number of text pages: 41

Number of tables: 2 (Supplemental: 4)

Number of figures: 10 (Supplemental: 1)

Number of references: 44

Number of words in abstract: 247

Number of words in introduction: 737

Number of words in discussion: 1488

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d) Non-standard abbreviations:

LPA, lysophosphatidic acid; LPAR, LPA receptors; LPAn, LPA receptor n; hLPAn, human

LPAn; Sphingosine-1-phosphate receptor n, S1Pn; CpX, (2R)-2-(diethylamino)-2-(2,3- Downloaded from dimethylbenzofuran-7-yl)ethanol; CpY, (2R)-2-(diethylamino)-2-(2-ethyl-3-methyl- benzofuran-7-yl)ethanol; ATX, autotaxin; HuSMC, human Urethra Smooth Muscle Cells;

GPCR, G-protein coupled receptor; IUP, intra urethral pressure. jpet.aspetjournals.org

e) Recommended section: Drug discovery and translational medicine at ASPET Journals on September 27, 2021

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Abstract

Lysophosphatidic acid (LPA) is the natural ligand for two phylogenetically distinct families of receptors (LPA1-3, LPA4-6), whose pathways control a variety of physiological and pathophysiological responses. Identifying the benefit of balanced activation/repression of LPA receptors has always been a challenge due to the high lability of LPA together with the limited availability of selective and/or stable agonists. In the present study, we document the discovery Downloaded from of small benzofuran ethanolamine derivatives (called CpX and CpY) behaving as LPA1-3 agonists. Initially found as rabbit urethra contracting agents, their elusive receptors were jpet.aspetjournals.org identified from [35S]GTPγS-binding and β-arrestin2 recruitment investigations, then confirmed

3 by [ H]CpX binding studies (urethra, hLPA1-2 membranes). Both compounds induced a calcium

response in hLPA1-3 cells within a range of 0.4 to 1.5-log lower potency, as compared to LPA. at ASPET Journals on September 27, 2021

The contractions of rabbit urethra strips induced by these compounds perfectly matched binding affinities with values reaching the 2-digit nM level. The antagonist, KI16425, dose-dependently antagonized CpX-induced contractions in agreement with its affinity profile

(LPA1≥LPA3>>LPA2). The most potent agonist, CpY, doubled intra-urethral pressure in anesthetized female rats at 3 µg/kg i.v.. Alternatively, CpX was shown to inhibit human preadipocyte differentiation, a process totally reversed by KI16425. Together with original molecular docking data, these findings clearly established these molecules as potent agonists of LPA1-3 and consolidated the pivotal role of LPA1 in urethra/prostate contraction as well as in fat cell development. The discovery of these unique and less labile LPA1-3 agonists, would offer new avenues to investigate the roles of LPAR.

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Significance statement

We report the identification of benzofuran ethanolamine derivatives behaving as potent selective non-lipid LPA1-3 agonists and shown to alter urethra muscle contraction or preadipocyte differentiation. Unique at this level of potency, selectivity and especially stability compared to lysophosphatidic acid, they represent more appropriate tools for investigating the physiological roles of lysophosphatidic acid receptors and starting point for optimization of drug candidates for therapeutic applications. Downloaded from

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Introduction

Lysophosphatidic acid (LPA) is a bioactive phospholipid principally generated at lipoprotein or membrane levels through the enzymatic hydrolysis of lysophospholipids by autotaxin (ATX)

(Yung et al., 2014). LPA exists in different forms (16:0-, 18:2-, 18-1, 16:1-LPA being the most frequent), is ubiquitously distributed, present in interstitial fluids and circulates in plasma at concentrations that could reach µM levels. Over the last decades, extremely various Downloaded from physiological and pathophysiological roles have been evidenced for LPA (Choi et al., 2010) ranging from cellular proliferation to contraction of muscle tissues. To date, LPA has been jpet.aspetjournals.org shown to bind and activate six G-protein-coupled LPA receptors (LPAR): the closely related receptors LPA1-LPA2-LPA3 which belong to the endothelial differentiation gene family (EDG)

and LPA4-LPA5-LPA6, which belong to the phylogenetically distant non-EDG family (Yung et at ASPET Journals on September 27, 2021 al., 2014). Even if the nature of receptor activation may vary with the LPA form, the fine tuning of the various biological responses probably stems from heterogeneity of LPAR expression in tissues and species, and from the ability of individual receptors to couple to several distinct heterotrimeric G-proteins, each driving its own effector cascade. Additionally, the strong metabolic instability of LPA and its resultant very short half-life (Salous et al., 2013) together with its rapid de novo production by ATX (Saga et al., 2014) creates a serious risk of bias during functional characterization of exogenous LPA. Consequently, identification and understanding of the right LPAR combination controlling a specific biological response, remains challenging, even if specific genetic deletions have clarified some points (Choi et al., 2008). For all these reasons, search of more selective, potent and stable LPA-mimicking structures is needed to better explore the therapeutic potential of LPA/LPAR pathways. As of today, this search was hampered by the hydrophobic nature of the ligand pocket of LPAR (Chrencik et al., 2015).

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In the early 2000’s, the first LPAR antagonists such as KI16425 from Kirin were discovered

(Ohta et al., 2003). KI16425 keeps a short aliphatic tail and has been shown to display a selective antagonistic profile (LPA1≥LPA3>>LPA2) towards LPA responses. More recently, new non-lipid antagonist structures have demonstrated improved potency and sometimes better selectivity for single LPAR (Fells et al., 2008; Sakamoto et al., 2018; Kihara et al., 2015), like the specific LPA1 antagonists, SAR100842 or BMS-986020, being currently investigated in patients with systemic sclerosis or idiopathic pulmonary fibrosis. In contrast, limited progress has been made in appreciating the potential therapeutic value of selective LPAR agonists Downloaded from beyond a few preclinical studies (Choi et al., 2010) which have suggested for instance the potential of LPA mimetics to reduce the development of human nutritional obesity (Rancoule jpet.aspetjournals.org et al., 2014). The ability of LPA to increase intra-urethral pressure in rats (Terakado et al., 2016) also suggests that LPA mimetics could represent a therapeutic alternative to increase urethral pressure closure and avoid unintentional leaks of urine, the main problem of female stress at ASPET Journals on September 27, 2021 incontinence (Malallah et al., 2015). Several lipid-based agonists, derivatives of LPA, like

OMPT (Qian et al., 2003) or its alkyl forms (Qian et al., 2006), gained specificity for LPA3 but displayed only moderate improvement of metabolically stability. More recently, the glycidol derivative (S)-17 (UCM-05194) displayed selective LPA1 interaction (Gonzalez Gil et al.,

2019). Among few non-lipid structures, GRI977143 has been identified through virtual screening using a LPA1 pharmacophore and has been characterized as selective but weak LPA2 agonist (EC50~10 µM) (Kiss et al., 2012). Few specific non-lipid LPA3 modulators displaying activity close to µM range have been also patented (Shankar et al., 2003). To our knowledge, no potent, non-lipid LPA1-3 agonist has been reported. All in all, identification of new potent and more stable drug-like agonist compounds would be very useful to explore and consolidate on the potential therapeutic benefits of selective LPAR agonists.

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In the present document, we report the identification of benzofuran ethanolamine derivatives, initially discovered as orphan receptor smooth muscle contracting agents and behaving as selective and potent agonists of LPAR. Representatives of the chemical series, CpX and analogues, were profiled for cellular, binding and pharmacological responses in various biological models. Our results show that these molecules are strong binders of LPA1-2 and potent contractant of urethra, acting unambiguously on LPA1. Additional cellular calcium- based responses enlarged their agonistic pattern to LPA3. Additionally, these agonists behave as inhibitors of human pre-adipocyte differentiation by activating LPA1. These selective non- Downloaded from lipid LPA1-3 agonists represent excellent tools for deciphering LPA pathways and a unique starting point for optimization of drug candidates for new therapeutic applications. jpet.aspetjournals.org

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Material & Methods

Test compounds. The benzofuran ethanolamine derivatives (2R)-2-(diethylamino)-2-(2,3- dimethylbenzofuran-7-yl)ethanol alias CpX, its close analogue (2R)-2-(diethylamino)-2-(2- ethyl-3-methyl-benzofuran-7-yl)ethanol alias CpY (Fig.1) and several related structures CpZ1 to CpZ4 (Fig.7) were synthesized following described procedures by the chemistry department of Sanofi (France) as well as the LPA1/LPA3 specific antagonist Kirin KI16425 (Ohta et al.,

2003) and the specific LPA3 antagonists (Ceretek Cpd 701/Cpd 705, Shankar et al., 2003). Downloaded from

Radiolabeled [3H]CpX was synthesized by Amersham (30 Ci/mmol). The general synthetic route to benzylamine derivatives (CpX, CpY, CPZ3) described in this report includes three key jpet.aspetjournals.org sequences. First, a suitably substituted benzofuran derivative is prepared by cyclodehydratation in cold sulfuric acid. For instance, treatment of 3-(2-bromo-phenoxy)-2-butanone under those conditions yields to 2,3-dimethyl-7-bromo-benzofuran. Then, the bromide atom at the position at ASPET Journals on September 27, 2021

7 of benzofuran is converted to a chiral 1,2 ethanediol side chain found on intermediate CpZ4.

This second sequence is performed by a Stille cross-coupling reaction using tributylvinyltin followed by an enantioselective Sharpless dihydroxylation on vinyl benzofurans (Philippo et al., 2000). AD-mixα is used to deliver the more potent isomer. Finally, the amine is introduced by selective protection of the primary alcohol with a bulky group, such as tert- butyldimethylsilylchloride, followed by mesylation of the secondary alcohol and subsequent displacement by a secondary amine such as diethylamine. This last step results in an inversion of the chiral center. Finally, the protecting group is removed to allow access to compound CpX or CpY according to starting benzofuran moiety used. In the case of ethanolamine derivatives

(CpZ1, CpZ2), only the third sequence is slightly modified. The primary alcohol is not protected but converted by using tosyl chloride in a leaving group which is substituted by diethylamine.

The reaction mixture mostly yields ethanolamines but is contaminated by a very small amount of benzylamines which is readily removed by chromatography on silica gel. Regioselectivity

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of this nucleophilic substitution evidenced generation of an epoxide as reaction intermediate.

All derivatives, but CpZ4, were converted into hydrochloride salts, recrystallized and fully characterized (Philippo et al.,1998a,1998b). Oleoyl-LPA (Cayman, ref 62215) was used in studies conducted by Eurofins Pharma Discovery/Cerep.

Tissue samples. Urethras from female pigs (Large White Landrace, 110 kg, Lebeaux, Gambais,

France), Sprague Dawley rats (150-300g, Iffa Credo, France), New-Zealand rabbits (3-4 kg) Downloaded from and beagle dogs (10-12 kg) (CEDS, Mezilles, France) were used in muscle contraction assays.

Brains from Sprague Dawley rats were also used. Human prostatic samples were obtained from

patients undergoing transvesical adenomectomy (Institut Mutualiste Montsouris, Paris) in the jpet.aspetjournals.org context of benign prostatic hypertrophy. Human adipose tissue was obtained from female patients undergoing liposuction procedures under local anesthesia (Clinique Alphand, Paris).

All subjects gave their informed consent for tissue sampling. at ASPET Journals on September 27, 2021

Cell samples: LPAR recombinant cells and HuSMC. To prepare membranes for binding studies, Chinese Hamster Ovary (CHO) dihydrofolate-reductase-negative cells were transfected with vectors derived from plasmid 658 carrying the supplementary 13-amino acid NH2- terminal C-myc (MEQKLISEEDLKL) and the cDNA encoding for the human LPA1-2, S1P3 and S1P4 receptors (Miloux and Lupker., 1994, Shire et al.,1996). Stably transformed cell lines were isolated as previously described (Miloux and Lupker, 1994; Serradeil-Le Gal et al., 2000).

They were grown in 10 mM HEPES, pH 7.4, minimal essential medium supplemented with 5% fetal calf serum and 8 g/l sodium bicarbonate and 300 μg/ml geneticin at 37°C in a humidified atmosphere containing 5% CO2. Wild-type CHO cells were routinely grown in a similar culture medium. Calcium mobilization was investigated in Chem-1 cells expressing LPA1, CHO-K1 cells expressing LPA2 or LPA3 and operated by Eurofins Pharma Discovery/Cerep, France

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(Catalogue reference 4374, 3171, 3158 respectively). Cells were loaded with a fluorescent probe (Fluo4/8) and responses monitored with a FLIPR® Tetra (Molecular Device). For the β- arrestin2 recruitment screening campaign, transformed African green monkey kidney fibroblast

(COS-7) cells were transiently co-transfected in 96 well plate format with plasmids (p312) encoding individual tested GPCR, including LPAR, and β-arrestin2-GFP, then refed with serum free medium 36 hours after transfection. Tested compounds were incubated for 20 minutes, fixed before GFP signal distribution being monitored by fluorescence microscopy. Human

Urethra Smooth Muscle Cells (HuSMC) were purchased from Clonetics-BioWhittaker Downloaded from

(Venders, Belgium) and were grown at 37°C in SmBM medium supplemented with smooth muscle cell growth factors (SingleQuotes, BioWhittaker), fetal calf serum (10%), and jpet.aspetjournals.org antibiotics. Culture medium was removed every other day, and cells were subcultured by treatment with 0.05% trypsin, 0.02% EDTA.

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Expression of LPA1-3. In human adipocytes and prostate, mRNA abundance was estimated by

TaqMan® analysis (Applied Biosystems) by using the following commercial probes:

Hs00173500_m1 (LPA1), Hs00173704_m1 (LPA2), Hs00173857_m1 (LPA3).

Membrane binding assays. Tissues or cells were homogenized on ice in TRIS HCl buffer (5 mM, pH 7.4) containing protease inhibitors (aprotinin 10 µg/mL, PMSF 0.2 mM, and benzamidine 0.83 mM). The resultant homogenate was centrifuged at 4350g for 10 min at 4°C, and then the supernatant was filtered and centrifuged at 48,400g for 1hour at 4°C. The pellet was frozen at -80°C until the binding experiment. Protein concentration was determined by the

Bradford method. Thawed membranes were diluted in Tris HCl buffer (50 mM pH 7.4) and incubated for 1 hour (i) with an increasing concentration of [3H]CpX for saturation experiments or (ii) for displacement studies with the indicated concentration of [3H]CpX and increasing

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concentrations of test compounds. Non-specific binding was determined in the presence of 10

µM cold CpX. The reactions were stopped by rapid filtration on PEI 0.5% pretreated filters

(GF/C or filtermat A) then filters were washed, and the signal determined by scintillation

(Perkin Elmer counters).

[35S]GTPγS binding-assay on brain slices. Sprague Dawley brains were quickly removed by dissection and frozen on dry ice. The tissues were cut on a HM500 cryostat (Microm, France)

to obtain 12 µm sections that were dehydrated at 4 °C under vacuum overnight before storage Downloaded from at -80°C. Sections were equilibrated 10min at 25°C in Tris-buffer (50 mM Tris-HCI, 3 mM

MgCl2 0.2 mM EGTA - 100 mM NaCl pH 7.4) then 15min at 25°C in Tris-buffer supplemented jpet.aspetjournals.org with GDP (2 mM). The sections were finally incubated 2h at 25°C in Tris-buffer-2mM GDP supplemented with [35S]GTPγS (0.05 nM) (Amersham). Agonist stimulated binding was measured in the presence of 1 µM CpX or CpY and nonspecific binding was determined in the at ASPET Journals on September 27, 2021 presence of non-labeled GTPγS (10 µM) (Sigma). Sections were washed twice with Tris-HCl

(50 mM) pH 7.0 at 4°C, dipped in water and air dried. Sections were exposed to autoradiography hyperfilm Beta-max (Amersham) in a x-Omatic closed cassette (Kodak).

Preadipocyte differentiation assay. Liposuctions were digested with collagenase, filtered and centrifuged. The pellet obtained was resuspended in NH4Cl (160 mM) to lyse contaminating red blood cells. Preadipocytes were then collected by centrifugation and filtered through a sterile 100 µm nylon mesh to remove cellular debris. Cells were frozen at -80°C until further use. For differentiation into adipocytes, thawed cells were grown 5 days in DMEM 1 g/l glucose

(Gibco), 10% fetal bovine serum (Gibco) until nearly confluent. Then, after trypsination and plating in a 24-well format, cells were allowed to differentiate with test compounds, 6 days in a medium (Zenbio, AM-1, containing 33 µM biotin, 17 µM pantothenate, 100 mM human insulin, 1 µM dexamethasone), complemented with IBMX (0.25 mM, Sigma). Fresh medium

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and test compounds were changed the 3rd day. Stock solutions of CpX and KI16425 (10 mM to

1 µM) were prepared in DMSO and diluted in medium (1000-fold). Controls were exposed to the same dilution of DMSO. Cells being differentiated were then harvested, suspended in

Laemmli buffer (Bio-Rad) and boiled for 10 min. Protein lysates were quantified by BCA assay

(Pierce) and 25 to 50 µg of protein mixture adjusted to 0.1 M DTT (Invitrogen), were submitted to electrophoresis on Criterion® Bis-Tris 4-12% Pre-Cast Gel (Bio-Rad). Separated proteins were transferred onto PVDF membranes (Bio-Rad), blocked in low-fat milk powder (10% w/v) in PBS-0.05% tween-20 and probed with the primary antibody: anti-perilipin (Progen; ref 29), Downloaded from anti-adiponectin (BD transduction laboratories; ref 611644), anti β-actin (Sigma; A5441). After washing, membranes were incubated with Protein A peroxidase (ZYMED) for perilipin and jpet.aspetjournals.org adiponectin and HRP-conjugated antibody for α-actin (Jackson Immuno Research). After final wash, immunoreactive bands were detected with ECL kit (Biorad) using hyperfilm ECL

(Amersham). Western blot bands were quantified by Genesys software (G:box Chemi-XL1, at ASPET Journals on September 27, 2021

Syngene, England) and normalized with beta-actin signal using Gene Tools software (Syngene,

England) in order to estimate CpX pEC50 and KI16425 pA2.

Contraction of isolated tissues. Smooth muscle strips were mounted in organ baths containing a modified Krebs solution, in the presence of 1 µM propranolol to block β-adrenoceptors. The

Krebs solution was maintained at 37°C and oxygenated with a mixture of 95% O2 and 5% CO2.

After a stabilization time of 60 min, tissues were contracted with 30 µM norepinephrine, followed, after 30 min of washing, by phenylephrine (100 µM). After additional 30 min of washing and a new stabilization period, a concentration-response curve of CpX was performed on each tissue. The contractile potency of structurally related analogues of CpX, as well as

KI16425 potency (1-3-10 µM) to shift CpX-induced contraction, were also investigated in rabbit urethra. Results are expressed as percentage of the response to phenylephrine.

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Protocol for measurement of urethral pressure in anesthetized rats. Female Sprague

Dawley rats (Charles River, France) weighing between 350-400g on the day of surgery and having had at least 3 litters were anesthetized with pentobarbital (15 mg/kg i.p.) and ketamine

(20 mg/kg i.p) for surgery. The abdominal artery and the femoral vein were catheterized respectively for blood pressure measurement and intravenous injection of test compounds or vehicle. After laparotomy, the urethra and the urinary bladder were exposed, and a catheter introduced by an incision at the level of the bladder neck and positioned into the urethra for a length by approximately 1.3 cm. The urethra was continuously infused with saline at a Downloaded from temperature of 37°C and at a rate of 0.5 mL/h. Arterial and urethral pressures were measured together with heart rate and continuously recorded and analyzed by dedicated software jpet.aspetjournals.org (MacLab). After a stabilization period of 20 min to record basal values, the first drug administration was operated along a 5-minute perfusion (1 ml of solution i.v.). Then, four successive doses of the drug were administered every 15 min. In case of the absence of an at ASPET Journals on September 27, 2021 urethral response, an intravenous injection of phenylephrine was given 10 min after the last dose of the compound. Absence of an urethral response to phenylephrine was an exclusion criterion for the animal. At the end of the experiment, animals were sacrificed by a lethal dose of pentobarbital. This study was performed in agreement with EU directives for the standard of care and use of laboratory animals and approved by the animal care and use committee of Sanofi

R&D. Pharmacokinetic complement: Blood samples were collected after intravenous administration of CpY (1 mg/kg i.v.) in rats. Plasma was extracted with acetonitrile, centrifuged and CpY concentrations determined by LC/MS-MS.

Data analysis. Results are expressed as mean ± standard deviation (SD). When applied (i) agonists binding affinities were determined by nonlinear regression using iterative fitting procedures (Sigma Plot software) and expressed as KD (affinity constant) and Bmax (maximum binding) during saturation experiments (B=Bmax X [L]/(KD + [L]) or alternatively, during

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displacement studies, by -log10(Ki) (pKi) determined by using the Cheng-Prussof formula Ki =

IC50 / (1+[L]/KD) where L is the concentration of ligand, (ii) agonist potency to contract tissues were determined by nonlinear regression using iterative fitting procedures (Sigma Plot software) and expressed as [-log10(EC50)], pEC50 (iii)antagonist apparent affinity was expressed as PKB determined from Schild plot analysis (slope statistically not different from 1) or pA2

(estimated affinity from one single antagonist concentration effect) determined by the formula

[-log10([B]/(CR-1))] where B is the concentration of the antagonist and CR is the ratio: IC50 with/IC50 without antagonist. In in vivo studies, statistical significances were determined by Downloaded from one-way repeated measures analysis of variance followed by a Dunnett pos-hoc test (SAS9.4 software). Urethral pressure was converted as % of baseline and doses increasing by 50% the jpet.aspetjournals.org baseline urethral pressure (DE50%) were estimated from linear regression (Sigma Plot software).

Molecular modelling. For GPCR transmembrane domain (TM) identification, reference alignments were made as published by Bissantz et al., 2004, with a representative panel of at ASPET Journals on September 27, 2021

GPCRs, giving residue type probabilities at each TM position. Then, the target sequence was slid over the reference alignment to obtain the best sum of probabilities for each TM. This method was applied successfully to assign all TMs of LPA1-3 except for TM5 for which low scores and very low differences between the first and second scores were obtained. In addition, proline was absent in the TM5 anchor position and we observed incomplete Baldwin invariants

(Baldwin et al., 1993) as well as no consensus sequence. We therefore noted a difference encountered in Xray structures of EDG-family members (no helix kink and a shift of ~5Å of the anchor residue) yielding different molecular environments for the helix portion and thus different sequences that would explain this low scoring. Therefore, because of this unsuccessful assignation, LPA1 antagonist Xray structure (pdb id: 4z34, Chrencik et al., 2015) and

LxMVxxYxxI invariant sequence position (Baldwin et al., 1993) were preferentially used to obtain a better, high-quality level TM 5 assignation. For an easier comparison between GPCR,

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Ballesteros-Weinstein numbering was used (Ballesteros and Weinstein, 1995). Starting with the activated conformation structure CNR1_HUMAN (PDB:5xr8) as a template, an automated model-building procedure was used, as proposed by Nilges and Brünger, 1991, in which the remaining backbone and side-chain atoms were only built using packing and stereochemical restraints, by using a furnished XPLOR script (Brünger et al., 1998; Nilges et al., 1988a, 1988b).

Ligand preparation was done with the LigPrep module for energy-minimization (with the

OPLS-2005 force field) and charge assignment (Schrodinger release 2019-4, LLC, New-York).

Docking calculations were performed using Glide SP (Halgren et al., 2004; Friesner et al., Downloaded from

2004). The docking region (grid) was centered on the template ligand in the model with default box sizes. Flexible ligands were docked by Glide into a rigid receptor structure by sampling of jpet.aspetjournals.org the conformational, orientational, and positional degrees of freedom of the ligand, generating many conformations for a ligand followed by a series of hierarchical filters to enable rapid evaluation of ligand poses. at ASPET Journals on September 27, 2021

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Results

Initial deciphering investigations and selectivity.

CpX and related compounds were initially discovered as rabbit urethra smooth muscle contracting agents, but associated receptors were not identified. First, modulators of classical pathways (intra/extra cellular) known to potentially alter smooth muscle contraction were tested Downloaded from but found inactive. For example, among the ~70 reference molecules tested, antagonists for α and β-adrenergic, histamine, dopamine, cholinergic and neurokinin receptors failed to reduce

CpX-induced rabbit urethra contraction (Supplemental Table 1). Only non-specific molecules jpet.aspetjournals.org inducing calcium depletion like caffeine impaired the contraction.

CpX was radiolabeled and binding sites were clearly identified in the urethra membranes (see at ASPET Journals on September 27, 2021 below) but also in membranes from other tissues including the brain. Using this binding assay, more than 650 molecules targeting different pathways were tested and found inactive in displacing [3H]CpX binding on pig urethra membranes (Supplemental Table 2). Additionally,

CpX (10 µM maximum) was tested in numerous screening campaigns as well as in receptor/enzyme selectivity profiles (including S1P receptors, LPA4-6 and CB1), but results never reached significant effect (<50% at 10 µM) (Supplemental Table 3).

Evidence for LPAR as candidates.

[35S]GTPγS binding-assay in rat brain sections: In the panel of the first assays used to identify the CpX receptor and decipher its pathway, [35S]GTPγS-binding in rat brain sections was studied after stimulation by CpX and CpY (1 µM). Representative images of sections are shown in Fig. 2. As compared to control sections, CpX and more intensely CpY, clearly enhanced the

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[35S]GTPγS signal in very specific areas, particularly those rich in white matter like the corpus callosum and the internal capsule.

β-arrestin2 recruitment-assay: In parallel, since numerous GPCR were known to internalize after agonist stimulation through an interaction with β-arrestin2, a screening model aimed at identifying GPCR responders to CpX exposure was developed (List of GPCR, Supplemental

Table 3). β-arrestin2 recruitment was monitored in COS-7 cells expressing a small collection of recombinant human hGPCR and GFP-tagged β-arrestin2. No recruitment was observed in control COS-7 cells exposed to 1 µM of CpX, as well as in almost all COS-7 expressing a Downloaded from

GPCR. In contrasts, a redistribution of the β-arrestin2 fluorescence was observed after a 10 to

20 min exposure to 0.5 µM of CpX in COS-7 cells expressing recombinant human LPA1 jpet.aspetjournals.org

(Fig.3). A similar redistribution was observed in human LPA2-expressing COS-7 cells.

Binding confirmation for LPA1 and LPA2: For receptor characterization, CpX was radiolabeled with tritium (Fig.1) and used to determine binding site constants in membranes at ASPET Journals on September 27, 2021 prepared from urethra, the initial tissue found to be contracted by CpX. Since the receptor was unknown, unlabeled CpX was used to define non-specific binding, after having confirmed the dynamic reversibility of the binding in association-dissociation studies. By using 10 nM

[3H]CpX, a specific binding signal was observed in urethra membranes (~80%, ~80 µg of protein per point) from different species but the pig urethra membrane preparation was selected for extended studies, given the easier access to tissue and larger membrane production capacity.

In pig urethra, CpX displayed a nanomolar affinity (KD=21 ± 5.4 nM, n=4) and maximal binding capacity at 1143 ± 605 fmol/mg of protein (Fig.4A). A similar affinity was obtained in human urethra cell (HuSMC) membranes (KD=17 ± 0.9 nM, n=3), with a very similar saturation curve shape (Fig.4B). To confirm the affinity of CpX for LPAR, membranes from CHO cells expressing recombinant LPA1 or LPA2 and two sphingosine-1-phosphate receptors (S1P3 and

3 S1P4) were investigated. Using 40 nM [ H]CpX, a concentration slightly higher that the KD

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level obtained in urethra, no significant specific binding was found in our conditions in membranes (100 to 200 µg per point) prepared from control CHO cell (<10%) or cells expressing either S1P3 (<10%) or S1P4 (<10%). In contrast, a high specific binding was observed in LPA1 (>80%) and LPA2 (>80%) membranes and a saturation study (Fig.4B) evidenced a nanomolar affinity constant for hLPA1 (KD= 60 ± 10 nM, n=3) and hLPA2 (KD=

68 ± 5.9 nM, n=3). Issues in our laboratory to express sufficiently LPA3 prevented the determination of CpX affinities on this receptor subtype. Alternatively, the ability of CpX, CpY and KI16425 to displace [3H]CpX-binding in pig urethra membrane preparations was tested, Downloaded from showing pKi values of 7.8, 8.5, 7.6, respectively (Table 1). No displacement (<20%) was observed with the selective LPA3 antagonist (Cpd 701/Cpd 705, published >13- to >40-fold jpet.aspetjournals.org selective, respectively vs LPA1-2) tested at 30µM. KI16425 affinity for hLPA1 (pKi=7.5), but not that for hLPA2 (pKi=6.1), perfectly matched its affinity for the pig urethra sites. In recombinant cell membranes, KI16425 displayed a ~20-fold higher affinity for LPA versus

1 at ASPET Journals on September 27, 2021

LPA2 (Table 1), as previously described (Ohta et al., 2003). A slightly lower affinity for LPA2 was observed for CpY.

Functional characterization.

Calcium mobilization in hLPA1-3 expressing cells: The calcium responses in hLPA1-3 expressing cells were operated by Eurofins Pharma Discovery/Cerep in well validated experimental conditions which guaranteed the absence of response in native host cells.

Accordingly, CpX and CpY had no effect in host cells (<10%) but induced a dose dependent calcium response in human receptor transfected cells, with an apparent range order of potency as follows hLPA3>hLPA2>hLPA1 (Figure 5). However, since the cell line, and construct, in

Eurofins/Cerep LPA1 model were different from LPA2-3 models, the range of potency of our agonists for hLPA1-3 are likely biased, as underlined by the marked difference of responses to the non-specific native ligand, Oleoyl-LPA, in hLPA1 (pEC50= 6.8) as compared to hLPA2

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(pEC50= 8.4) and hLPA3 (pEC50= 8.7) expressing cells. When compared head to head to

Oleoyl-LPA in each model, CpX appeared 7.5 less potent in hLPA1 (pEC50=5.9) and ~30-fold less potent in both hLPA2 (pEC50= 6.9) and hLPA3 (pEC50= 7.2) expressing cells. CpY was ~3- fold more potent than CpX, displaying only 2.7-fold lower potency in hLPA1 (pEC50=6.3) and

9-fold lower potency in both hLPA2 (pEC50= 7.5) and hLPA3 (pEC50= 7.7) expressing cells, compared to Oleoyl-LPA.

Contraction of smooth muscles: The contractile features of CpX were investigated in rabbit Downloaded from urethra strips. All strips were pre-contracted by 100 µM phenylephrine to qualify tissue contractility. CpX contracted urethra strips in an incremental manner to reach a plateau (~75-

85% of the effect of 100 µM phenylephrine) in the lower µM range (Fig.6A-C). In these jpet.aspetjournals.org conditions, CpX displayed a pEC50 at 8.0 ± 0.2 (n=6). A Schild-Plot analysis was conducted to characterize KI16425 antagonism. As depicted in Fig.6A, increasing KI16425 concentrations shifted the contracting responses of CpX to the right, allowing the at ASPET Journals on September 27, 2021 determination of a pKB of 7.35 (slope not different from 1). The contracting response of CpX was also explored in urethra strips from other species (Fig.6B), giving a similar contraction profile and efficacy values: pig (pEC50= 7.9 ± 0.1, n=8), dog (pEC50= 8.1 ± 0.1, n=7), rat

(pEC50=8.4 ± 0.3, n=7), human prostatic adenoma (pEC50= 7.8 ± 0.2, n=3). Accordingly, by testing a single concentration of KI16425 on CpX-induced contraction, the level of KI16425 potency was estimated in rat urethra (pA2= 7.5) and human prostatic adenoma (pA2= 7.34).

Additionally, LPA1 was mostly expressed in the human prostate (mRNA abundance of hLPA1-

2-3: 72/10/18%). Close analogues of CpX (CpZ1 to CpZ3), with variable binding affinity were also evaluated (Fig 6C) and a strong correlation was obtained (R²=0.97) between their abilities to displace [3H]CpX-binding in pig urethra membrane and to contract rabbit urethra

(Fig.7). All analogues behaved as weaker contractant as compared to 100 µM phenylephrine- response, reaching in this experiment, respectively, 82 ± 31% at 0.3µM CpX, 52 ± 5 % at 0.3

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µM CpY, 83 ± 16% at 30 µM CpZ1,78% ± 19% at 100 µM CpZ2, 64% ± 13% at 300 µM

CpZ3 of maximal phenylephrine contraction. Another analogue without amine derivative

(CpZ4) was considered inactive.

In vivo increase in intra-urethral pressure: Intra-urethral pressure (IUP) was measured in anesthetized female rat. The anesthesia procedure was qualified by the absence of significant effects on basal urethral pressure along the duration of the experiment (2h). Under these conditions, the baseline IUP measured was ~12 cmH2O (Supplemental Table 4). Successive Downloaded from intravenous perfusion of CpX dose-dependently increased IUP with a first significant dose established at 1 µg/kg and a maximal increase representing +88% from baseline reached at 30

µg/kg (Fig.8, Supplemental Table 4). CpY was more potent and increased significantly IUP jpet.aspetjournals.org from 0.3 µg/kg with a maximal increase of +102% at 3 µg/kg. As a comparator, the α1- adrenoceptor agonist phenylephrine was markedly less potent with a first significant dose at at ASPET Journals on September 27, 2021 30 µg/kg and a maximal increase of +74% at 100 µg/kg. The estimated doses increasing by

50% urethral pressure (DE50%) were 0.4µg/kg for CpY, 1.3 µg/kg for CpX and 35 µg/kg for phenylephrine. Compared to phenylephrine, which significantly increased mean arterial pressure (MAP) from 1 µg/kg, CpX was neutral on blood pressure up to 30 µg/kg where a modest reduction in MAP was observed (Supplemental Table 4). All compounds reduced heart rate at the highest doses. In addition, the pharmacokinetics of CpY, was investigated at 1 mg/kg i.v in rats. CpY was quantified at 0.5 µM post injection and above the limit of detection during 6h in plasma with a urethra/plasma AUC ratio of 6.1. The limit of detection established at 32 nM was above the affinity (5 nM) of CpY for LPA1.

Blockade of pre-adipocyte differentiation: In order to confirm more extensively the LPAR agonistic activity in a human biological system unrelated to muscle contraction, human preadipocytes were extracted from liposuction samples and allowed to differentiate into mature adipocytes as previously described (Halvorsen et al., 2001). Full maturation was monitored by

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the expression of the late differentiation markers perilipin and adiponectin (control without CpX in Fig.9). CpX added at the beginning of differentiation decreased the expression of both

markers in a concentration-dependent manner (1 nM to 10 µM), with an estimated pIC50=8. A similar effect was obtained with CpY (Supplemental Fig.1). CpX-inhibitory response was antagonized by increasing concentrations of KI16425 (estimated pA2 at 7.3 and 7.2, respectively), with a nearly total reversal at 10 µM. A trend toward an increase in adiponectin, but less in perilipin expression, was observed in the presence of KI16425 per se. As a complementary information, LPA1 was mostly expressed in fat cells as compared to LPA2 and Downloaded from even more to LPA3 (mRNA abundance of LPA1-2-3: 98.2/1.8/<0.1% in human preadipocytes and 98.8/0.9/0.3% in human adipocytes). jpet.aspetjournals.org

Molecular modelling: To better understand and compare the molecular interaction mode of the compounds, homology models of LPA1-3 have been built and CpX, CpY and the natural agonist

(18:1-LPA) have been docked onto the “classical” active cleft (meaning the same pocket as at ASPET Journals on September 27, 2021 retinal in rhodopsin reference structure). CNR1_HUMAN was used as a template (pdb id:

5xr8), because it: i) belongs to the same branch of the GPCR phylogenetic tree (Fredrickson et al., 2003); ii) has the same length and secondary structure (no disulfide bond located in the beginning of TM3 but one in the loop) of the very important 2nd external loop that covers the active site; iii) is in an activated conformation able to dock agonist compounds. By following the specific strategy (especially for TM5) described in the Material and Methods section, about

200 models were generated for each receptor by varying sidechain conformations and using the simulated annealing capacity of the homology procedure to sample active cleft shape and properties (Nilges and Brünger, 1991). After docking and analysis of clustering on LPA1, the best poses showed very similar interactions for CpX and CpY: a pi-cation interaction between the amine and W5.43 (Trp210), an ion bridge with D3.33 (Asp129), a hydrogen bond between the ethanol moiety and D3.33 (Asp129) and Q3.29 (Gln125) or Y2.57 (Tyr102) for some

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clusters and the benzofuran into the hydrophobic pocket made by W5.43 (Trp210), L6.55

(Leu278), L7.39 (Leu297), L3.36 (Leu132), A7.42 (Ala300), W6.48 (Trp271) (Table 2 and Fig.

10). The same interaction pattern and glide score repartition has been obtained for best docking poses in LPA2 and LPA3 (Table 2, Fig.10). When 18:1-LPA was docked in LPA1, its hydrophobic tail was shown to fill the same area occupied by our compounds. However, its polar phosphate group made an ionic bridge with K7.36 (Lys294) and its ester moiety made hydrogen bonds with Y2.57 (Tyr102) and Q3.29 (Gln125), both residues located closer to the edge of the site (Fig. 9). Additionally, this analysis applied to CpX derivatives likely provides Downloaded from interesting hypothesis for the structure-activity relationship (shown in Fig.7), namely the importance of (i) the pi-cation interaction (lack of amine in CpZ4), (ii) the hydrophobic jpet.aspetjournals.org benzofuran core (hydrophilic bulge in CpZ3) and of the exact orientation of ethanolamine vs benzofuran (CpZ2 and CpZ1) (iiii), the chirality, the CpX enantiomer being significantly less potent (pIC =6.7).

50 at ASPET Journals on September 27, 2021

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Discussion

Serendipity is sometimes at the origin of breakthrough discoveries. As an example, the present study relates the identification of the target of receptor-orphan chemical structures, initially characterized as potent urethra contracting agents (Philippo et al., 1998a, 1998b). These molecules originating from adrenergic-like agonist series, contracted rabbit urethra but unexpectedly could not be antagonized by the α1-adrenoceptor antagonist prazosin. In order to identify the elusive receptors, numerous reference molecules were screened (on urethra Downloaded from contraction and [3H]CpX binding) and CpX profiled on available panels of receptors, but all efforts failed, thus reinforcing the originality of the quest. After ultimate but unsuccessful jpet.aspetjournals.org attempts to purify the receptor, the clues for LPAR as candidates, arose fortuitously. The first hints came from [35S]GTPγS activation experiments performed in rat brain slices which revealed specific signals in white matter tracts, known as areas rich in myelin (Rozenblum et at ASPET Journals on September 27, 2021 al., 2014). Among a few possible receptors, LPA1 was suspected since an enrichment was observed in myelin-rich areas (Handford et al., 2001). Separately, a β-arrestin2 recruitment screening campaign, applied to a collection of GPCR overexpressing cells, pinpointed two hits responding to CpX: hLPA1 and hLPA2. With this hypothesis in mind, we confirmed that

3 [ H]CpX was a strong binder of LPA1 and LPA2. A similar 2-digit nanomolar affinity site was detected in membranes of hLPA1-expressing cells and of pig urethra. Binding displacement results obtained with specific antagonists demonstrated the predominant interaction with LPA1 in this tissue. The conclusion is supported by the selectivity profile of KI16425

(LPA1≥LPA3>>LPA2), the perfect matching of the affinities of KI16425 for urethra and hLPA1, and the absence of displacement with Ceretek LPA3 antagonists. From a functional perspective, the cellular calcium mobilization responses induced by CpX clearly showed an activation of

LPA1-2 but also LPA3, with a 1-log (LPA1) to 1.5-log (LPA2-3) lower potency as compared to

Oleoyl-LPA (0.4 to 1-log for CpY, respectively) thus presaging significant tissue contraction

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responses. Indeed, not only did CpX potently contract rabbit urethra, but it also induced very significant contractions in dog, rat, and pig urethras as well as in human prostate. In agreement with its LPA1 binding affinity, KI16425 potently antagonized CpX-induced rabbit urethra contraction, but also rat urethra and human prostatic adenoma contractions. Importantly, the implication of the LPA1 pathway was confirmed by the strong correlation between binding affinity and contracting potency obtained with benzofuran derivatives displaying a range of various affinities. To our knowledge, LPA-induced contractile responses have only been tested in rat urethra strips (Saga et al., 2014; Sakamoto et al., 2018) in which a pivotal role of LPA1 Downloaded from was pointed out only recently in Sakamoto’s study. Nevertheless, LPA was only active at high concentrations (≥ 1µM) suggesting a fast degradation. Following these observations, CpX jpet.aspetjournals.org clearly behaves as a more potent LPA1 agonist in isolated strips than LPA. Our data also extend to humans and other species the central role of LPA1 in urethra and prostate contractions.

Another notable finding is that CpX and even more, CpY, potently increased IUP in vivo in at ASPET Journals on September 27, 2021 anesthetized female rats at low concentrations which ranks it more efficacious than phenylephrine taken as a reference. As such, CpY with an absolute IUP increase of 16 cmH2O at 3µg/kg, appeared to be several orders of magnitude more potent in vivo than LPA for which

300 µg/kg was shown to increase IUP by 5 mmHg (which corresponds to 6.8 cmH2O) in a similar rat model (Terakado et al., 2016). This difference in efficacy likely results from a higher and longer LPA1 activation, due to a better plasma stability of CpY (hour-lasting half-life) compared to the minute-lasting half-life of LPA (Salous et al., 2013). Unlike phenylephrine, which increased MAP, only a reduction of MAP was observed at the highest doses of CpX was observed, thus supporting a lower incidence on cardiovascular function at doses increasing urethral pressure. Interestingly, LPA was shown to induce a transient MAP elevation by interacting with LPA4 and LPA6 (Kano et al., 2019), which likely explains the different hemodynamic profile of our selective LPA1-3 agonists. Like phenylephrine, our compounds

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decreased heart rate, but not through a baroreflex-mediated mechanism as blood pressure remained mainly unchanged. The mechanism responsible for this reduction in heart rate is not yet identified. For the first time, a marked increase in urethral tone was obtained by directly targeting LPA1 with a non-lipid agent. Accordingly, a decrease in urethral tone was demonstrated after injection of the specific ATX inhibitor ONO-8430506 (Saga et al., 2014), or after treatment with the new specific LPA1 antagonist ASP6462 (Sakamoto et al., 2018) suggesting an endogenous LPA/LPA1 tone. Interestingly, ASP6462 decreased urethral pressure during urine voiding and improved L-NAME-induced voiding dysfunction which differs from Downloaded from tamsulosin, underlining important differences between LPA1 and α1-adrenergic regulations

(Sakamoto et al., 2019). Although of significant concern for women, no drug therapy has been jpet.aspetjournals.org approved by health authorities so far and very few drugs are registered for pure stress incontinence. A trial has recently been conducted with the antidepressant serotonin/ norepinephrine reuptake inhibitor imipramine but ended up with inconclusive results (Kornholt at ASPET Journals on September 27, 2021 et al., 2019). Off-label use of several agents, like α1-adrenergic agonists are reported with variable efficacy and side effects which limit their use (Malallah et al., 2015). In that context, our new potent LPAR agonists should facilitate more dedicated investigations to clarify the therapeutic potential of LPA/LPAR pathway in stress incontinence.

Moreover, we further completed the characterization our agonists in human pre-adipocyte differentiation process. Indeed, through paracrine and autocrine signaling, the ATX-LPA axis, acting most likely via LPA1, was shown to switch the balance from differentiation to proliferation of pre-adipocytes, thus altering fat storage (Simon et al., 2005; Dusaulcy et al.,

2011). Accordingly, our agonists potently inhibited the maturation process, as shown by the reduced expression of the two late markers, perilipin and adiponectin. Conversely, KI16425 restored the inhibition of the maturation process. The presence of endogenous production of

LPA likely explains why KI16425 alone slightly increased differentiation level. These new

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findings confirm the tight control of adipocyte maturation by LPA1 and open new avenues for investigations in obesity, aiming at clarifying the global benefit/risk (fat storage reduction/ inflammation, insulin resistance) of activating the ATX-LPA axis (D’Souza et al., 2018).

Our studies have however some limitations: first we did not compare head to head our compounds and LPA in tissue materials. We initially attempted to displace [3H]CpX binding by LPA but failed due to a very rapid degradation, confirmed by using radiolabeled LPA.

Similar labile behavior of LPA was observed in tissue contraction conditions. Second, even if Downloaded from we could compare our compounds to Oleoyl-LPA in calcium mobilization assays, we could not estimate their absolute selectivity patterns for LPA1-3. Indeed, difference in cell line, level of

receptor expression, as well as non-physiological coupling, likely introduced bias in compound jpet.aspetjournals.org responses, well exemplified by the lower responses to LPA in LPA1 than in LPA2-

3overexpressing cells. at ASPET Journals on September 27, 2021 The structure-activity-relationships also indicates that moderate modifications of the structural backbone can finely tune their LPA1 efficacy, hopefully opening room for selectivity improvement towards LPA2 and LPA3. Since these molecules have no aliphatic chain, their interaction with LPAR could not be anticipated considering the interaction mode of LPA with the amphipathic binding pocket of LPA1 (Chrencik et al., 2015). Additionally, classical LPAR ligands contain a large nonpolar region and chiral hydroxyl, ester, and carboxylic acid groups.

In contrast, CpX and derivatives are basic molecules at physiological pH (pKa≥8.8) and are less lipophilic (logP ≤ 3.9). Interestingly, by using an optimized strategy for TM assignation applied to a GPCR modeling template close to LPA1-3 (CNR1) and suitable for agonist docking purpose, a high glide score and high-quality interaction pattern were obtained for CpX and CpY with LPA1. This specific interaction is notably characterized by a clear pi-cation interaction with a tryptophan residue, an ionic bridge with an acidic residue and the embedding of the benzofuran in a specific hydrophobic pocket. Nevertheless, these docking results cannot explain

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the difference in LPA1 affinity and calcium response as well in urethral response profile measured between these very similar compounds, CpX and CpY. The same interaction pattern was obtained with LPA2-3 in agreement with a very high level of residue identity. In contrast,

LPA yielded a completely different pattern of polar-based interactions. Indeed, its polar head interacts with residues at external edge of the site, far from CpX /CpY ionic bridges, while the hydrophobic tail of LPA and the benzofuran moiety of CpX/CpY stand in the same hydrophobic pocket. This analysis agrees with the docking of LPA into an inactivated LPA1 structure (pdb id: 4z34), published by Balogh et al., 2015. Downloaded from

Although, the exact interactions of our compounds with LPA1-3 would need crystallography investigations, these modelling explorations support that structurally different ligands can jpet.aspetjournals.org activate LPA1-3, likely through the interaction with the identified hydrophobic pocket.

To conclude, these LPAR agonists are unique at this level of potency, selectivity and especially stability compared to LPA, and represent more appropriate tools for investigating the at ASPET Journals on September 27, 2021 physiological and pathological roles of LPAR pathways.

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Acknowledgements

We would like to acknowledge all the numerous Sanofi R&D heads of services, scientists

(biologist and chemist) and especially Sonia Arbilla, Philippe Bovy, Itzchak Angel, Anne Marie

Galzin, Denis Martin, Pascual Ferrara, Olivier Curet, Annick Coste, François Lo-Presti, Gilles

Courtemanche, Pascal Shiltz, Mourad Kaghad, Anne Remaury, Stéfano Paléa, Philippe Lluel,

Bruno Chevallier, Martine Barras, Geneviève Comte, Valérie Poignez, Françoise Demange,

Marie-France Porquet, Olivier Crespin, Marie Claire Philippo, Thao Van Pham, many others Downloaded from and their respective teams who contributed significantly to the identification of LPAR as receptors for our agonists. We would also thanks Paul Schaeffer, Fiona Ducrey, Marco Meloni jpet.aspetjournals.org for proof reading of the manuscript.

at ASPET Journals on September 27, 2021

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Authorship contributions

Participated in research design: Guillot, Le Bail, Paul, Philippo

Conducted experiments: Le Bail, Paul, Briand, Fourgous, Partiseti, Cornet

Contributed new reagents design and synthesis: Philippo

Performed data analysis: Guillot, Le Bail, Paul, Philippo, Briand, Cornet, Partiseti

Wrote or contributed to the writing of the manuscript: Guillot, Cornet, Philippo, Partiseti, Le Downloaded from

Bail, Janiak

jpet.aspetjournals.org at ASPET Journals on September 27, 2021

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Legends for Figures

Fig. 1. Chemical structures of benzofuran ethanolamine derivatives CpX, [3H]CpX and CpY

Fig. 2. Determination of [35S] GTPγS-binding signal in control coronal sections of rat brain section or in section stimulated by 1 µM of CpX or CpY. Cc: corpus callosum, IC: internal capsule. Downloaded from

Fig.3. Representative images of CpX induced redistribution of GFP- β-arrestin2 in COS-7 cells expressing the LPA1 receptor. COS-7 cells were transiently transfected with GFP- β-arrestin2, jpet.aspetjournals.org empty plasmid (Top) or LPA1-plasmid (Below) and redistribution was monitored by fluorescence microscopy. CpX was tested at 1 µM (controls) or 0.5 µM (LPA1).

3 Fig.4. Saturation curves of H]CpX in membrane homogenates of pig urethra (A), human at ASPET Journals on September 27, 2021 urethra cells or CHO cells expressing recombinant LPA1 and LPA2 (B). Curves are representative of at least 3 independent experiments, from which affinity values were determined by nonlinear regression using iterative fitting procedures. Dotted line in B represents the saturation obtained with pig urethra membranes given in A. Figure values are mean of triplicates. KD, Bmax data are means +/- SD.

Fig.5. Calcium mobilization response of CpX, CpY and Oleoyl-LPA in Chem-1 cells expressing hLPA1 (A) and CHO-K1 cells expressing hLPA2 (B) or hLPA3 (C). Data are expressed as % of LPA maximal response and expressed as mean ± SD (n=3). Respective agonist potency is expressed as pEC50 +/- SD (n=3).

Fig.6. Effect of KI16425 on CpX-mediated contraction of rabbit urethra strips (A): ●controls, in the presence of 1µM KI16425 (▲), 3µM KI16425 (▼) or 10 µM KI16425 (♦). CpX pEC50 for control group was 8.0 ± 0.2 and pKB was measured at 7.35 for KI16425 [included Schild 38

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plot analysis, slope not statistically different from 1, 95% confidence interval (…)]. Data are transformed in % of 100 µM phenylephrine response (n=7). Comparative responses of tissues from different species to CpX, n=3-7 (B) and comparative response of rabbit urethra to a small series of benzofuran ethanolamine derivatives, n=4-8 (C). Data are expressed in % of respective maximal responses. Data are expressed as mean ± SD.

Fig.7. Correlation between pig urethra membrane affinity (pKi) and rabbit urethra contraction

(pEC50) for a small series of benzofuran ethanolamine derivatives. CpZ1-4 were selected to Downloaded from represent a large range of binding affinities.

Fig.8. Effect of agonists perfusion on intra urethra pressure (IUP). Anaesthetized female rats jpet.aspetjournals.org received successive increasing dose of CpX, CpY or phenylephrine (n=3-6). IUP (cmH2O) was measured before (baseline) or after the end of each incremental perfusion (raw data given in Supplemental Table 4) and expressed as % of respective baseline (mean ± SD). DE50% were at ASPET Journals on September 27, 2021 estimated from nonlinear regression using iterative fitting procedures.

Fig.9. Effect of KI16425 on CpX-inhibition of human preadipocyte differentiation.

Differentiation into mature adipocyte was confirmed by the expression of the two late markers perilipine and adiponectin (Control=C) separated by Western blot. β -actin was used as normalization protein. CpX alone or in the presence of KI16425 were added at the initiation of the differentiation process at indicated log [concentration]. This separation is representative of 2 independent experiments. Data were normalized by β-actin expression and represented as

% of respective raw control (bottom).

Fig.10. Analysis of ligand binding pocket of hLPA1-3 residues. Superimposition of docking poses of CpX (blue), CpY (green) and 18:1-LPA (orange) on LPA1 (A), LPA2 (B), LPA3 (C) are presented. The two agonists share a location with 18:1-LPA at the same hydrophobic pocket site but markedly differ in ionic and hydrogen bonds interaction mode.

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Tables

TABLE 1

Binding affinity (pKi) and contractile potency (pEC50) values

Ki values were calculated from the capacity of compounds to displace [3H]CpX binding to indicated membranes (IC50) by using Cheng Prussoff formula (n≥2). Contractile potency was Downloaded from measured in rabbit urethra strips and EC50 determined by non-linear sigmoid regression (n=6).

Data are expressed as –log10 (Ki or EC50) ± SD. jpet.aspetjournals.org

Binding affinities (pKi) Tissue contraction (pEC50)

hLPA1 hLPA2 Pig Urethra Rabbit urethra at ASPET Journals on September 27, 2021

KI16425 7.5(7.3-7.7) 6.1(5.9-6.4) 7.6(7.6-7.6) -

CpX 7.3±0.2 7.1±0.2 7.8±0.2 8.0±0.2

CpY 8.3±0.2 7.7±0.3 8.5±0.2 8.2±0.2

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TABLE 2

Description of the LPA1-3 residues interacting with CpX/CpY/Oleoyl-LPA for the best docking poses. *Specific ionic interactions for CpX/CpY. # Specific ionic interaction for

Oleoyl-LPA. Other interactions are shared. Downloaded from

Ballesteros-Weinstein hLPA1 hLPA2 LPA3 numbering jpet.aspetjournals.org Ionic pocket (N+) * 3.33 D129 D112 D110 5.43 W210 W193 W191

Ionic pocket (PO4-) # 7.36 K294 K278 R276 at ASPET Journals on September 27, 2021

Hydrogen Bond 2.57 Y102 Y85 Y83 3.29 Q125 Q108 Q106

Hydrophobic pocket 3.36 L132 L115 L113 6.48 W271 W254 W252 6.55 L278 L261 L259 7.39 L297 L281 L279 7.42 A300 A284 A282

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Figures

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Figure 1

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CC CC

IC 8.6 mm 8.6

INTERAURAL Figure 1 Control CpX 1 µM CpY 1 µM CC CC

5.7 mm

INTERAURAL

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at ASPET Journals on September 27, 2021

Figure 2

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T0 20 min

Empty plasmid Empty

T0 10 min 20 min

plasmid LPA1 hLPA1 plasmid

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Figure 3

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KD=21 ± 5.4 nM 1800 Bmax1143 ± 605 fmol/mg of proteins 1600

1400

1200 100

1000 80 60 800 40 600

Bound/Free 20 400 0 200 0 200 400 600 800 1000 1200 1400 1600 Bound (fmol/mg of proteins) Downloaded from [3H]CpXbound (fmol/mg ofproteins) 0 0 50 100 150 200 250 Free CpX (nM)

jpet.aspetjournals.org B

100

80 at ASPET Journals on September 27, 2021

40 %ofBmax

HuSMC: KD=17± 0.9 nM 20 LPA1: KD= 60 ± 10 nM LPA2: 68 ± 5.9 nM

0 0 20 40 60 80 100 120 140 Free CpX (nM)

Figure 4

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100 CpX: 5.9 ± 0.2 CpY: 6.3 ± 0.1

80 LPA: 6.8 ± 0.2

%of LPA maximal response

1e-9 1e-8 1e-7 1e-6 1e-5 Downloaded from

B Compounds (M)

120 CpX: 6.9 ± 0.1 CpY: 7.5 ± 0.1 jpet.aspetjournals.org LPA: 8.4 ± 0.2 100

40 at ASPET Journals on September 27, 2021

%of LPA maximal response

0 1e-10 1e-9 1e-8 1e-7 1e-6 1e-5

C Compounds (M)

CpX: 7.2 ± 0.1 120 CpY: 7.7 ± 0.1 LPA: 8.7 ± 0.1 100

%of LPA maximal response

0 1e-10 1e-9 1e-8 1e-7 1e-6 Compounds (M) Figure 5

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40 3

20 1 Log[CR-1]

0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5

0 Log [KI16425, M] %of 100µM phenylephrine contraction 1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 CpX (M) Downloaded from

Rabbit urethra Human prostate B Rat urethra Rat urethra + KI16425 (3 µM) Pig urethra Human prostate + KI16425 (10µM) Dog urethra 140 jpet.aspetjournals.org 120

100

pKB=7.35 80 Slope 1.03

60 at ASPET Journals on September 27, 2021 40

%ofrespective maximal responses 0 1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 CpX (M)

C 140 CpY 120 CpX

CpZ1 100 CpZ2 80 CpZ3 60

%of respective maximal responses 0 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 Compounds (M) Figure 6

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O N O H 9

) O OH 50 CpX N 8 CpY O

H2N CpZ1 O N O N 7 OH O H R²=0.97 6 Downloaded from CpZ OH 3 CpZ O O O 2 N O Figure5 7

Rabbiturethra contration (pEC CpZ Inactive 4 4 4 5 6 7 8 9 jpet.aspetjournals.org Pig urethra binding affinity (pKi)

at ASPET Journals on September 27, 2021

Figure 7

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CpX CpY PHE 120

100 0.4 µg/kg 80

%of baseline 20

0 1.3 µg/kg 35 µg/kg

-20 0.1 1 10 100 Downloaded from Perfused dose (µg/kg i.v.)

jpet.aspetjournals.org

at ASPET Journals on September 27, 2021

Figure 8

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CpX CpX CpX

C -9 -8 -7 -6 -5 C -9 -8 -7 -6 -5 C -9 -8 -7 -6 -5

C -7 -6 -5

Perilipin Adiponectin Beta Actin

Perilipin Adiponectin 140 140 120 120 100 100 80 80

60 60 Downloaded from 40 40 20 20

0 0 % of respective raw baseline baseline raw of% respective 1e-9 1e-8 1e-7 1e-6 1e-5 1e-9 1e-8 1e-7 1e-6 1e-5 CpX( M) CpX( M) jpet.aspetjournals.org Control KI16425: 1e-6 M KI16425: 1e-7 M KI16425: 1e-5 M

at ASPET Journals on September 27, 2021

Figure 9

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A Downloaded from

B jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Figure 10

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Lysophosphatidic acid receptor agonism: discovery of potent non- lipid benzofuran ethanolamine structures

Authors: Etienne Guillot, Jean-Christophe Le Bail, Pascal Paul, Valérie Fourgous, Pascale

Briand, Michel Partiseti, Bruno Cornet, Philip Janiak, Christophe Philippo

------

SUPPLEMENTAL Fig.1. Effect CpX and CpY on human preadipocyte differentiation.

Differentiation into mature adipocyte was confirmed by the expression of the two late markers perilipin and adiponectin separated by Western blot (C=Control). β -actin was used as normalization protein. Compounds were added at the initiation of the differentiation process at indicated log [concentration]. This separation is representative of 2 independent experiments.

CpX CpY

C -9 -8 -7 -6 C -9 -8 -7 -6

Perilipin Beta Actin Adiponectin

SUPPLEMENTAL TABLE 1

List of reference compounds screened against urethra contraction induced by CpX. Unless

indicated (doses) are in µM. No mark indicates no effect, * indicates a mild effect on CpX-

induced response (<20%), # indicates ~50 % of CpX-induced response alteration, $ indicates

total blockade of CpX-induced response.

Adrenergic: [Prazosin (10), Alfuzosin (1), Tamsulosin (0.1) Phentolamine (1), 5-Methyl-urapidil (1),

Phenoxybenzamine (0.1), Chloroethylchlonidine (50), HV 723(1)] α1, [Yohimbine (1), Idazoxan (1)] α2, [Buffer without propranolol (1)] β, Histaminergic: [Cetirizine (1)] H1, [Ranitidine (1)]H2, Clobenpropit (1)] H3,

Mepyramine (30), Dopaminergic: [SCH 23390(10)] D1, [Sulpiride (10)] D2, Haloperidol (10), Cholinergic:

* Gallamine (10), Muscarinic: Atropine (1), Histaminergic: [Mizolastine (30)] H1 , Purinergic: PPADS (100),

Nicotinic: Hexamethonium (10), d-Tubocurarine (10), Suramin (100), Serotoninergic: [Ketanserine (1)] 5HT1-

5HT2, [Tropisetron (50)] 5HT3-5HT4, Methysergide (1), Neurokinin: [RP 67580 (0.1), SR140933(1)] NK1,

[SR48968 (0.1)] NK2, Opioids: Naloxone (10), Oxytocin, Vasopressin: Atosiban (10), Thromboxane: SQ

29548 (30), Prostaglandin: Indomethacin (10), Leukotriene: [Bayer U9773 (3)] LTD4, Chloride channels:

Niflumic acid* (10), Mefenamic acid* (10), R(+)-IAA-94 (30), Sodium channel: Tetrodotoxin (10), Sodium channel: Amiloride (10), L-type calcium channel: Diltiazem* (10), Nitrendipine (1), Phosphodiesterase:

* Papaverine (10), IP3 receptor: Heparin (5mg/ml), 8-Bromo-guanosine-3',5'-monophosphate (30), PPADS (100),

Phosphokinase C: Staurosporine (0.01), GF109203X (1), Tyrosine kinase: Genistein (30), Phospholipase:

* * AEBSF (300), Phosphodiesterase: Theophylline (10), [Rolipram (1)] PDE4, [Sildenafil (1)] PDE5, Protein G modulator: Pertussis toxin (200 µg/ml), Cholera toxin (10 µg/ml), 5-lipoxygenase pathway:

Nordihydroguaiaretic acid (10), Noradrenaline release: Cocaine (100), Doxepin (30), No-donor: Na

Nitroprusside (100), Ca2+-ATPase: cyclopiazonic acid (30), Thapsigargin (1), Ca2+ release: Dantrolene (50),

Depletion of calcium stores: Caffeine$ (10000), Calmodulin: Trifluoperazine* (100) Caffeine binding site:

DBHC# (100), Econazole# (50), DIDS# (100), Ryanodine receptor: Ryanodine# (100), Ruthenium red (30) SUPPLEMENTAL TABLE 2

List of reference compounds screened (3.33 µM) against [3H]CpX (10 nM) binding in pig

urethra membranes(100 µg). None was found significantly active (>20 % signal inhibition).

Ruthenium red, Ryanodine, Heparin, Prazosin, Caffein, Nitrendipine, 1-7 Dimethyl Xanthine, Pentyfilline,

DBHC, IP3, Clonidine, Efaroxan, Idazoxan, Mizolastine, Mefenic acid, Niflunic acid, Cyprohepatadine,

Eliprodil, NS49, Phenylephrine, Bradikinin, Endothelin-2, Trifluoroperazine, Diltiazem, FK506, Rapamicin,

Cyclin-ADP ribose, Verapamil, MK801, SR120819A (NPY1), PD160170 NPY1), SR141716 (CB1),

SR144528 (CB2), Amiloride, Amiodarone, Calmodulin, Urotensin II, Apovencaminol, Verapamil, MK801,

Ouabain, A61603, Neuromedin U, HOE642, Veratridine, Tyramine, Vinpocetine, Octopamine, Tryptamine, m-CPP, Apomorphine R+, Apomorphine S-, Sphingosine-1P, phenylethylamine, 7-(beta-

Chloroethyl)theophylline, 1,3-Diethyl-8-phenylxanthine, Theophylline, 1,7-Dimethylxanthine, Theobromine,

3-Isobutyl-1-methylxanthine, R(-)-N6-(2-Phenylisopropyl)adenosine, Caffeine, 8-(p-Sulfophenyl)theophylline,

5'-N-Ethylcarboxamidoadenosine, Adenosine, N6-Cyclopentyladenosine, 3-n-Propylxanthine, 2-

Chloroadenosine, 2-Methylthioadenosine triphosphate tetrasodium, 5'-N-Methyl carboxamidoadenosine, 1-

Methylisoguanosine, Aminophylline, Adenosine amine congener, 1-Allyl-3,7-dimethyl-8-p- sulfophenylxanthine, AB-MECA, Alloxazine, N6-Benzyladenosine, N6-Benzyl-5'-N- ethylcarboxamidoadenosine, 8-Cyclopentyl-1,3-dipropylxanthine, 8-Cyclopentyl-1,3-dimethylxanthine, 5'-(N-

Cyclopropyl)carboxamidoadenosine, CGS-21680 hydrochloride, 2-Chloro- N6-cyclopentyladenosine, 2-

Chloro-adenosine triphosphate tetrasodium, Chlorpropamide, Chlorzoxazone, Chlomezanone, 8-(3-

Chlorostyryl)caffeine, CGS-15943, Dipyridamole, Disopyramide phosphate, Phenytoin sodium, Danazol,

DPMA, Debrisoquin sulfate, P1,P4-Di(adenosine-5')tetraphosphate triammonium, Diclofenac sodium, erythro-

9-(2-Hydroxy-3-nonyl)adenine hydrochloride, Furosemide, (±)-Ibuprofen, Iofetamine hydrochloride, IB-

MECA, Lidocaine hydrochloride, Loxapine succinate, N6-Methyladenosine, Metolazone, alpha, beta- Methylene adenosine 5'-triphosphate dilithium, 2-Methylthioadenosine diphosphate trisodium, beta, gamma-

Methylene-L-adenosine- 5'-triphosphate tetrasodium, Metrifudil, S-(4-Nitrobenzyl)-6-thioinosine, S-(4-

Nitrobenzyl)-6-thioguanosine, N6-Cyclopentyl-9-methyladenine, Isoxanthopterin, Neopterin, 2-

Phenylaminoadenosine, N6-2-Phenylethyladenosine, N6-Phenyladenosine, Phenylbutazone, Podophyllotoxin,

Primidone, PPADS, Quinine sulfate, Reserpine, Spironolactone, Sulindac, Suramin hexasodium, Tracazolate,

Tetracaine hydrochloride, Tolazamide, Tolbutamide, (±)-Vanillylmandelic acid, Valproic acid sodium,

Xanthine amine congener, Albuterol hemisulfate, Alprenolol hydrochloride, (±)-Atenolol, Agmatine sulfate,

AGN 192403 hydrochloride, Clonidine hydrochloride, p-Aminoclonidine hydrochloride, (±)-threo-DOPS,

DSP-4 hydrochloride, Benextramine tetrahydrochloride, MHPG sulfate potassium, 6-Fluoronorepinephrine hydrochloride, Xylamine hydrochloride, Benoxathian hydrochloride, MHPG piperazine, WB-4101 hydrochloride, Phenoxybenzamine hydrochloride, Bretylium tosylate, BU224 hydrochloride, B-HT 933 dihydrochloride, B-HT 920 dihydrochloride, BRL 37344 sodium, CGS-12066A dimaleate, Cimetidine, (±)-

Chlorpheniramine maleate, (±)-CGP-12177A hydrochloride, Clobenpropit dihydrobromide, Cirazoline hydrochloride, CGP 20712A methanesulfonate, Dimaprit dihydrochloride, Diphenhydramine hydrochloride,

Dobutamine hydrochloride, (-)-Epinephrine bitartrate, (-)-Ephedrine hydrochloride, (-)-Ephedrine,N-methyl-,

Guanabenz acetate, L-Histidine hydrochloride, Histamine dihydrochloride, Histamine, R(-)-alpha-methyl-, dihydrochloride, Histamine, N-alpha-methyl-, dihydrochloride, Histamine, 1-methyl-, dihydrochloride,

Hydrochlorothiazide, (±)-Isoproterenol hydrochloride, p-Iodoclonidine hydrochloride, ICI 118,551 hydrochloride, Imetit dihydrobromide, Metanephrine hydrochloride, (-)-alpha-Methylnorepinephrine,

Methoxamine hydrochloride, (±)-Normetanephrine hydrochloride, L(-)-Norepinephrine bitartrate, L(-)-

Norephedrine, Nisoxetine hydrochloride, Nylidrin hydrochloride, Naftopidil dihydrochloride, (±)-Octopamine hydrochloride, Oxymetazoline hydrochloride, Prazosin hydrochloride, (±)-Pindobind, Prazobind, Pindolol, (±)-

Propranolol hydrochloride, Pyrilamine maleate, Phentolamine mesylate, Phenylephrine hydrochloride, (+)-

Pseudoephedrine, Protriptyline hydrochloride, Promethazine hydrochloride, Ranitidine hydrochloride,

Rauwolscine hydrochloride, SKF 91488 dihydrochloride, Triprolidine hydrochloride, Thioperamide maleate, Tripelennamine hydrochloride, S(-)-Timolol maleate, Urapidil hydrochloride, UK 14,304, Xylazine hydrochloride, Yohimbine hydrochloride, YS-035 hydrochloride, N-Aminodeanol chloride, Atropine sulfate,

Amiloride hydrochloride, Amiodarone hydrochloride, 4-Aminopyridine, Aminobenztropine, Arecaidine propargyl ester hydrobromide, N-Acetylprocainamide hydrochloride, Ambenonium dichloride, A-85380 dihydrochloride, Bethanechol chloride, Benztropine mesylate, Bepridil hydrochloride, (±)-Bay K 8644, 2,3-

Butanedione monoxime, Arecoline hydrobromide, "10-(alpha-Diethylaminopropionyl)-phenothiazine hydrochloride", (+)-cis-Dioxolane, McN-A-343, OXA-22, Carbachol, 4-DAMP methiodide, Diltiazem hydrochloride, R(+)-Butylindazone, Diazoxide, Dihydro-beta-erythroidine hydrobromide, 1,1-Dimethyl-4- phenyl-piperazinium iodide, Decamethonium dibromide, (-)-Eseroline fumarate, Flunarizine dihydrochloride,

FPL 64176, Gallamine triethiodide, Glibenclamide, Glipizide, Hexahydro-sila-difenidol hydrochloride,

Hexahydro-sila-difenidol hydrochloride, p-fluoro analog, Hexamethonium dichloride, (+)-Himbacine,

Ipratropium bromide, R(+)-IAA-94, Lidocaine, N-ethyl bromide quaternarysalt, Linopirdine, (-)-N-

Methylphysostigmine, Methoctramine tetrahydrochloride, Mecamylamine hydrochloride, (±)-Methoxy verapamil hydrochloride, Methyl carbamylcholine chloride, Minoxidil, Methyl furtrethonium iodide,

Neostigmine bromide, Nifedipine, Nicardipine hydrochloride, (±)-Niguldipine hydrochloride, Nitrendipine,

Nimodipine, 5-Nitro-2-(3-phenylpropylamino) benzoic acid, NS-1619, Oxotremorine methiodide, (+)-

Pilocarpine hydrochloride, Pirenzepine dihydrochloride, Procainamide hydrochloride, Pyridostigmine bromide,

Pralidoxime iodide, Pinacidil, (-)-Physostigmine, N-Phenylanthranilic acid, Phenamil methanesulfonate,

Quinidine sulfate, (-)-Scopolamine hydrobromide, (-)-Scopolamine, n-Butyl-, bromide, Succinylcholine chloride, Tetraethylammonium chloride, TMB-8 hydrochloride, Telenzepine dihydrochloride, Trihexyphenidyl hydrochloride, Triamterene, Taxol, Tropicamide, (±)-Vesamicol hydrochloride, (±)-Verapamil hydrochloride,

Amantadine hydrochloride, Agroclavine, A-77636 hydrochloride, Bupropion hydrochloride, (+)-

Bromocriptine methanesulfonate, Benserazide hydrochloride, R(+)-6-Bromo-APB hydrobromide, (±)-

Butaclamol hydrochloride, S-(-)-Carbidopa, (±)-Chloro-APB hydrobromide, Chlorpromazine hydrochloride, Clozapine, 4'-Chloro-3-alpha-(diphenylmethoxy)tropane hydrochloride, 6,7-ADTN hydrobromide, R(-)-

Apocodeine hydrochloride, R(-)-Apomorphine hydrochloride, 4-Hydroxy-3-methoxyphenylacetic acid, 4-

Hydroxyphenethylamine hydrochloride, Dopamine hydrochloride, 3-Methoxy-4-hydroxyphenethylamine hydrochloride, 4-Methoxy-3-hydroxyphenethylamine, N-Methyldopamine hydrochloride, R(-)-

Propylnorapomorphine hydrochloride, R(-)-2,10,11-Trihydroxyaporphine hybrobromide, "R(-)-2,10,11-

Trihydroxy-N-propylnoraporphine hydrobromide", Dipropyldopamine hydrobromide, R(-)-Norapomorphine hydrobromide, R(-)-N-Allyl norapomorphine hydrobromide, Amfonelic acid, (+)-Bulbocapnine hydrochloride,

(±)-SKF-38393 hydrochloride, Spiperone hydrochloride, GBR-12909 dihydrochloride, R(+)-SCH-23390 hydrochloride, Domperidone, Dihydroergocristine methanesulfonate, Dihydroergotamine methanesulfonate,

Dihydroergocriptine, S(-)-DS 121 hydrochloride, Ergocristine, Ergometrine maleate, Fluspirilene,

Fluphenazine dihydrochloride, cis(Z)-Flupentixol dihydrochloride, Haloperidol, (±)-7-Hydroxy-DPAT hydrobromide, Indatraline hydrochloride, JL-18, R(+)-Lisuride hydrogen maleate, L-745,870 hydrochloride,

Metoclopramide hydrochloride, Mesulergine hydrochloride, Nomifensine maleate, (±)-Octoclothepin maleate,

(6R)-5,6,7,8-Tetrahydro-L-biopterin hydrochloride, Pimozide, R(+)-3PPP hydrochloride, S(-)-3PPP hydrochloride, (±)-PPHT hydrochloride, Prochlorperazine dimaleate, Pergolide methanesulfonate, S(+)-PD

128,907 hydrochloride, (-)-Quinpirole hydrochloride, Quinelorane dihydrochloride, Risperidone, S(-)-

Raclopride L-tartrate, RBI-257 maleate, (±)-Sulpiride, (±)-6-Chloro-PB hydrobromide, R(-)-SCH-12679 maleate, (±)-SKF 38393, N-allyl-, hydrobromide, R(+)-SKF-82957 hydrobromide, R(+)-SKF-81297 hydrobromide, Trifluperidol hydrochloride, Thiothixene hydrochloride, Trifluoperazine dihydrochloride,

Thioridazine hydrochloride, R(+)-Terguride, U-101958 maleate, U-99194A maleate, (±)-2-Amino-4- phosphonobutyric acid, L-Aspartic acid, 2-Amino-3-phosphonopropionic acid, 2-Amino-5- phosphonopentanoic acid, (±)-HA-966, AMAA zwitterion, trans-(±)-ACPD, (±)-N-Allylnormetazocine hydrochloride, Arcaine sulfate, 1-Amino-1-cyclohexanecarboxylic acid hydrochloride, Aniracetam, (±)-1-

Aminocyclobutane-cis-1,3-dicarboxylic acid, AMOA, cis-Azetidine-2,4-dicarboxylic acid, trans-Azetidine-

2,4-dicarboxylic acid, AIDA, (±)-CPP, 7-Chlorokynurenic acid, beta-CFT naphthalene sulfonate, Capsaicin, L- Cysteine hydrochloride, D-Cycloserine, (+)-Cyclazocine, Calcimycin, Carbetapentane citrate, R(-)PPAP hydrochloride, Capsazepine, Cyclothiazide, 2-Chloro-2-deoxy-D-glucose, L-Cysteine, N-Acetyl-, S(+)-4-

Carboxyphenylglycine, CNQX disodium, Dextromethorphan hydrobromide, DNQX, Dextrorphan D-tartrate,

6,7-Dichloroquinoxaline-2,3-dione, 5,7-Dichlorokynurenic acid, 1,10-Diaminodecane, 5-Fluoroindole-2- carboxylic acid, AMPA hydrobromide, (±)-2-Amino-7-phosphonoheptanoic acid, Kainic acid, L-Glutamic acid hydrochloride, L-Glutamine, D-gamma-Glutamylaminomethanesulfonic acid, L-Glutamic acid, N- phthaloyl-, GYKI 52466 hydrochloride, (2S,4R)-4-Methylglutamic acid, (±)-3-Hydroxy-phenylglycine, (±)-

Ibotenic acid, Ifenprodil tartrate, S(-)-IBZM, Kynurenic acid, N-Methyl-D-aspartic acid, (+)-MK-801 hydrogen maleate, MDL 26,630 trihydrochloride, (±)-alpha-Methyl-4-carboxyphenylglycine, Memantine hydrochloride, 3-Methoxy-morphanin hydrochloride, MDL 105,519, (+)-Normetazocine, NS 102, NBQX disodium, (±)-cis-Piperidine-2,3-dicarboxylic acid, O-Phospho-L-serine, Pentamidine isethionate, L-trans-

Pyrollidine-2,4-dicarboxylic acid, Propofol, Phenylbenzene-omega-phosphono-alpha-amino acid,

Phthalamoyl-L-glutamic acid trisodium, (+)-Quisqualic acid, Quinolinic acid, Rimcazole dihydrochloride,

Riluzole, Spermine tetrahydrochloride, D-Serine, (-)-cis-(1S,2R)-U-50488 tartrate, Hydrouracil, (±)-cis-5- fluoro-6-hydroxy-, Uracil, (±)-5-trifluoromethyl-5,6-dihydro-, S(-)-Willardiine, NG-Nitro-L-arginine, NG-

Nitro-L-arginine methyl ester hydrochloride, Rp-cAMPS triethylamine, Sp-cAMPS triethylamine,

Aminoguanidine hemisulfate, L-Arginine, AG 1478, AG 1295, AMT hydrochloride, nor-Binaltorphimine dihydrochloride, (±)-Bremazocine hydrochloride, 8-Bromo-cAMP sodium, 8-Bromo-cGMP sodium,

BW373U86 hydrochloride, Calmidazolium chloride, 8-(4-Chlorophenylthio)-cAMP sodium, Ceramide,

Chelerythrine chloride, Carboxy-PTIO potassium, Cyclosporin A, Dantrolene sodium, 5,5-Dimethyl-1- pyrroline-N-oxide, Daidzein, 2,4-Diamino-6-hydroxypyrimidine, D609 potassium, Diphenyleneiodonium chloride, 4,5-Dianilinophthalimide, Erbstatin analog, Forskolin, Genistein, Guanosine-5'-O-(2- thiodiphosphate) trilithium, GR-89696 fumarate, HA-1004 hydrochloride, H-7 dihydrochloride, H-8 dihydrochloride, H-9 dihydrochloride, ICI 204,448 hydrochloride, L-N5-(1-Iminoethyl)-ornithine hydrochloride, L- N6-(1-Iminoethyl)lysine hydrochloride, KN-62, Loperamide hydrochloride, Levallorphan tartrate, LY-294,002 hydrochloride, NG-Monomethyl-L-arginine acetate, MDL 12,330A hydrochloride, S-

Methylisothiourea hemisulfate, Naltrindole hydrochloride, Noscapine, (-)-Norcodeine, Naloxonazine,

Naltriben methanesulfonate, NPC-15437 dihydrochloride, 7-Nitroindazole, Naloxone benzoylhydrazone,

NADPH tetrasodium, Naloxone hydrochloride, Naltrexone hydrochloride, Nalbuphine hydrochloride,

Olomoucine, ODQ, PD 098,059, SC-10, Sphingosine, SQ 22536, Sepiapterin, Tamoxifen citrate, Tamoxifen,

4-Hydroxy-, (Z)-, Tamoxifen, 3-hydroxy-, citrate, (E)-, Thiocitrulline, 1-(-2-Trifluoromethylphenyl)imidazole,

Thio-NADP sodium, Tyrphostin B42, Tyrphostin A9, (±) trans-U-50488 methanesulfonate, U-62066, U-

73122, U-73343, W-7 hydrochloride, R(+)-WIN 55,212-2 mesylate, Zaprinast, Amitriptyline hydrochloride,

Azathioprine, Amoxapine, Alaproclate hydrochloride, Aminorex, Acetohexamide, Acyclovir, Buspirone hydrochloride, BMY 7378 dihydrochloride, (±)-p-Chlorophenylalanine, Cyproheptadine hydrochloride,

Carisoprodol, Carbamazepine, Clofibrate, 5-Carboxamidotryptamine maleate, Clofilium tosylate,

Clomipramine hydrochloride, Cysteamine hydrochloride, 1-(m-Chlorophenyl)-biguanide hydrochloride,

Chloroquine phosphate, Cinanserin, CI-988 N-methyl-D-glucamine, (±)-DOI hydrochloride, Doxepin hydrochloride, Desipramine hydrochloride, 5,7-Dihydroxytryptamine creatinine sulfate, Fluoxetine hydrochloride, 5-Hydroxyindolacetic acid, 5-Hydroxy-L-tryptophan, Iproniazid phosphate, Imipramine hydrochloride, LY-53,857 maleate, Lorglumide sodium, LY-278,584 maleate, L-703,606 oxalate, Proglumide,

Benzotript, 2-Methyl-5-hydroxytryptamine maleate, alpha-Methyl-5-hydroxytryptamine maleate, Melatonin,

Methiothepin mesylate, Metergoline, p-MPPI hydrochloride, Nortriptyline hydrochloride, NAN-190 hydrobromide, 5-(Nonyloxy)-tryptamine hydrogen oxalate, 1-Phenylbiguanide, Pirenperone, Pindobind-

5HT1A, Pregnenolone sulfate sodium, PD 142,898 N-methyl-D-glucamine, Quipazine, N-methyl-, dimaleate,

Quipazine, 6-nitro-, maleate, Ritanserin, (±)-8-Hydroxy-DPAT hydrobromide, 1-(1-Naphthyl)piperazine hydrochloride, 5-Methoxy DMT oxalate, TFMPP hydrochloride, Ketanserin tartrate, Quipazine dimaleate, 1-

(2-Methoxyphenyl)piperazine hydrochloride, PAPP, Serotonin hydrochloride, 1-(3-Chlorophenyl)piperazine dihydrochloride, Mianserin hydrochloride, Spiroxatrine, SQ 29,548, SDZ-205,557 hydrochloride, SC 53116 hydrochloride, SB 206553 hydrochloride, 3-Tropanyl-3,5-dichlorobenzoate, 3-Tropanyl-indole-3-carboxylate hydrochloride, Tryptamine hydrochloride, L-Tryptophan, Trazodone hydrochloride, S(-)-UH-301 hydrochloride, R(+)-UH-301 hydrochloride, WIN 51,708, WAY-100635 maleate, Zimelidine dihydrochloride,

9-Amino-1,2,3,4-tetrahydroacridine hydrochloride, 5-Aminovaleric acid hydrochloride, Allopurinol, (±)-p-

Aminoglutethimide, 3'-Azido-3'-deoxythymidine, Acetazolamide, 3-Aminopropyl-(methyl)phosphinic acid hydrochloride, cis-4-Aminocrotonic acid, gamma-Acetylinic GABA, (±)-Baclofen, S(-)-p-Bromotetramisole oxalate, (-)-Bicuculline methbromide, 1(S), 9(R), Benzamide, 2-Cyclooctyl-2-hydroxyethylamine hydrochloride, 4'-Chlorodiazepam, Captopril, Chlorothiazide, DL-alpha-Methyl-p-tyrosine, Papaverine hydrochloride, Pargyline hydrochloride, 3-Iodo-L-tyrosine, L-alpha-Methyl-p-tyrosine, Diacylglycerol kinase inhibitor I, (±)-2,3-Dichloro-alpha-methylbenzylamine hydrochloride, 3,5-Dinitrocatechol, DL-alpha-

Difluoromethylornithine hydrochloride, N-Methyl-beta-carboline-3-carboxamide, Etazolate hydrochloride,

(±)-Etomoxir sodium, Fusaric acid, FGIN I-27, Isoguvacine hydrochloride, (±)-Nipecotic acid, "4,5,6,7-

Tetrahydroisoxazolo[4,5-c]pyridin-3-ol hydrobromide", Guvacine hydrochloride, Thiomuscimol hydrobromide, Piperidine-4-sulphonic acid, Isonipecotic acid, GABA, Muscimol hydrobromide,

Hydroxylamine hydrochloride, Hemicholinium-3, 2-Hydroxysaclofen, (+)-Hydrastine, (±)-cis-4-

Hydroxynipecotic acid zwitterion, Indomethacin, 1,5-Isoquinolinediol, Kojic amine hydrobromide,

Ketoconazole, SKF-525A hydrochloride, R(-)-Deprenyl hydrochloride, Clorgyline hydrochloride, 6-Methoxy-

1,2,3,4-tetrahydro-9H-pyrido[3,4b] indole, L-alpha-Methyl DOPA, 3-methyl-GABA bis-naphthalene sulfonate, Nialamide, NO-711 hydrochloride, Aminopterin, 3-Phenylpropargylamine hydrochloride,

Tranylcypromine hydrochloride, Picrotoxin, Phaclofen, PK 11195, Pentoxifylline, Propentofylline, Quinacrine dihydrochloride, Ro 16-6491 hydrochloride, Ro 41-0960, Ro 15-4513, Ro 05-3663, Ro 20-1724, Rolipram,

SR-95531, Semicarbazide hydrochloride, Sulfaphenazole, Sanguinarine chloride, Tubulozole hydrochloride,

THIP hydrochloride, Trimethoprim, (±)-gamma-Vinyl GABA

SUPPLEMENTAL TABLE 3

List of receptors or enzymes screened for interaction with CpX.

CpX was tested at 10 µM in high throughput screenings [binding, cellular, enzymatic

developed for specific screening of each target (native or recombinant)] or at 1µM in β-

arrestin2 recruitment screening.

Hight throughput screening [* indicates a mild effect of CpX (<50%)]

Receptors: GPR7, GPR34, GPR35, GPR39, GPR48, GPR55, GPR75, GPR83, GPR84, GPR92, GPR97,

GPR100, GPR119, GPCR135, GPR146, GPR150, HPRAJ70, P2Y1, P2Y5, P2Y6, P2Y12, TLR3, TLR7, Insulin,

GIP, SST2, SST4, SST5, AdipoR1, ADGRG3, OX1, RXPF1, RXPF3, RXPF4, GAL1, GAL2, GAL3, B1, GLP-1,

NPY2, MC3, FFA1, FFA2, GPER, BB2, BB3, NPFF2, NMU2, VPAC2, AM2, NTS1, APJ, NK1, IL-13, IL-18,

CB1, CB2, S1P1, S1P3, TGR5, RAR-α, LXR-α, α1-A, α2-A*, mGluR7, LPA4, LPA5, LPA6, H3, D1, D2, D4, M2,

* M4, 5-HT1A, 5-HT1C, 5-HT2, 5-HT7, Sigma*, Opioid k. Ionic Channels: Kv4.3, Nav1.7, Na channel (site2) .

Enzymes: Endothelial lipase, AMPK, Nicotinamide N-methyltransferase, FAS, DPP4

β-arrestin2 recruitment screening [* indicates a recruitment]

Receptors: GPR1, GPR2, GPR3, GPR4, GPR6, GPR7, GPR8, GPR12, GPR7, GPR11, GPR12, GPR14, GPR15,

GPR16, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR23 (LPA4), GPR26, GPR27,GPR30, GPR31,

GPR32, GPR34, GPR35, GPR37, GPR38, GPR39, GPR40, GPR41, GPR43, GPR45, GPR48, GPR49, GPR50,

GPR52, GPR55, GPR57, GPR58, GPR61, GPR62, GPR63,GPR68, GPR72,GPR75, GPR77, GPR80, GPR82,

GPR84,GPR86, GPR87, GPR92 (LPA5), GPR91, GPR103, GPR105 HPRAJ70, P2Y1, P2Y5, P2Y10, P2Y12,

TGIP, A1, A3, SST2, SST4, SST5, OX1, OX2, MCH1, MCH2, GAL3, GLP-1, GLP-2, NPY2, NPY4, NPY5, MC3,

* * NPFF1,NPFF2, NMU2, NTR1, APJ, NK4, PGD2,RDC1, TAR1, hGH, S1P1, S1P3,S1P4, LPA1 , LPA2 , LPA4,

LPA5, G2A, ORL1, mGluR7, H3, H4, 5-HT2

SUPPLEMENTAL TABLE 4

Effect of agonist perfusion on intra urethral pressure (IUP), mean arterial pressure (MAP) and

heart rate (HR). Anaesthetized female rats received successive increasing doses of CpX, CpY

or phenylephrine. IUP (in cmH2O), MAP (in mmHg) and heart rate (in Beats Per Minute,

BPM) were measured before (baseline) or after the end of each incremental perfusion and

expressed as mean ± SD (n=3-6). *p<0.05, #p<0.01 versus baseline, One-way Anova with

repeated measures, Dunnett’s test versus baseline (PHE=Phenylephrine).

Perfused doses (g/kg i.v.)

Baseline 0.1 0.3 1 3 10 30 100

IUP 12.2  1.6 - - 17.8  4.4# 19.3  3.8# 21.1  2.6# 22.5  2# -

CpX MAP 111  19 - - 111  22 99  22 85  13 77  15* -

HR 297  59 - - 302  80 268  59 189  44# 167  37# -

IUP 12.9  2.6 15.3  3.7 17.5  4.2# 21.8  4.7# 25.5  3.8# - - -

CpY MAP 117  9 117  8 118  13 120  13 115  16 - - -

HR 302  14 298  15 285  41 281  45 214  47# - - -

IUP 12.9  2.1 - - - 13.1  4.0 15.4  2.0 18.4  3.0# 22.3  2.2#

PHE MAP 113  15 - - - 129  15 150  14# 161  12# 178  14#

HR 334  71 - - - 331  59 301  58 269  71* 264  82*