Accepted Manuscript Design and validation of an HTRF ® cell-based assay targeting the ligand-gated ion channel 5-HT3A Emilie Blanc, Patrick Wagner, Fabrice Plaisier, Martine Schmitt, Thierry Durroux, Jean-Jacques Bourguignon, Michel Partiseti, Elodie Dupuis, Frederic Bihel PII: DOI: Reference:

S0003-2697(15)00246-8 http://dx.doi.org/10.1016/j.ab.2015.03.035 YABIO 12083

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

26 November 2014 16 March 2015 24 March 2015

Please cite this article as: E. Blanc, P. Wagner, F. Plaisier, M. Schmitt, T. Durroux, J-J. Bourguignon, M. Partiseti, E. Dupuis, F. Bihel, Design and validation of an HTRF ® cell-based assay targeting the ligand-gated ion channel 5HT3A, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.03.035

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Design and validation of an HTRF® cell-based assay targeting the ligand-gated ion channel 5-HT3A.

Emilie Blanc1-2, Patrick Wagner3, Fabrice Plaisier4, Martine Schmitt3, Thierry Durroux2, Jean-Jacques Bourguignon3, Michel Partiseti4, Elodie Dupuis1and Frederic Bihel3*

1

Cisbio Bioassays, Parc Marcel Boiteux, BP84175 30200 Codolet, France

2

Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, CNRS UMR 5203, INSERM

U661, Université Montpellier I et II, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France 3

Laboratoire d’Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74

route du Rhin, 67401 Illkirch, France 4

SANOFI R&D, Research center of Vitry/Alfortville, LGCR/LIT, Vitry-sur-Seine, France

Corresponding author: * (F.B.) Phone : +33 3 688 54 130; Fax : +33 3 688 54 310; Email : [email protected]

KEYWORDS. Ligand-gated ion channels, 5-HT3A-R, Tag-lite®, HTRF®, fluorescent probes

Subject category : Membranes and receptors Short title : HTRF cell-based assay targeting 5-HT3A

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ABSTRACT Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite® ligand-binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF®) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.

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Ligand-gated ion channels (LGICs) play critical physiological roles by mediating the intercellular communications in the nervous system [1]. LGICs are oligomeric transmembrane proteins forming a pore, with at least one orthosteric-binding site for neurotransmitters. Acting as agonists, neurotransmitters trigger the opening of the ion channel in a process called “gating”. The opening of the channel can also be regulated through the binding of modulators acting at allosteric sites. While the role of LGICs is wellestablished in the nervous system, these receptors have also been described as acting in the peripheral system. Because of this wide range of activity, LGICs are considered as attractive targets for new therapeutic agents. Whereas high-throughput screening assays are routinely used for target families such as G-protein coupled receptors (GPCRs) and enzymes, this type of investigation is less developed in the ion channel field [2, 3]. The “gold standard” for ion channels is a functional screening using patch clamp electrophysiology. This technology provides high quality data, but its low throughput appears to be a limitation in drug screening, even if several automated patch-clamp instruments are now commercially available. In order to reach high throughput screening on LGICs, radioligand binding assays have been developed and extensively used for drug screening on ion channels. However, this radioactivity-based technology has several drawbacks, including delivery and disposal of the radioactive material, and the relatively long half-life of the ligands. Moreover, this kind of assay requires filtration steps, which are time consuming and generate a large amount of radioactive wastes. Some of these drawbacks were overcome through the development of scintillation proximity assays (SPA) [4], but the issues related to radioactivity still remain. To avoid this problem, fluorescent-based techniques have been used to develop binding assays for LGICs: fluorescence resonance energy transfer (FRET) [5-8], fluorescence polarization (FP) [9], fluorescence correlation spectroscopy (FCS) [10], and total internal reflection fluorescence (TIRF) [11, 12]. Each of these technologies presents some advantages and disadvantages for HTS on LGICs [13]. More particularly, FRET-based assays suffer from interferences from background fluorescence. To overcome this limitation, a new Tag-lite® ligand-binding assay targeting LGICs on living cells was developed in this study. The technology combines the use of self-labeling protein tags [14] fused to channels of interest with homogeneous time-resolved fluorescence (HTRF®) as the detection readout. 3

Already validated with G protein coupled receptors (GPCRs) [15, 16] and receptor tyrosine kinases (RTKs) [17], the Tag-lite® technology was validated on ligand-gated ion channels. The Homogeneous Time-Resolved Fluorescence (HTRF®) detection method was used with a covalent labeling technology called HaloTag® previously developed [18]. For the proof of concept, we chose the homo-pentameric 5HT3A serotonin receptor (5HT3A-R), which belongs to the superfamily of Cys-loop receptors, responsible for fast synaptic neurotransmission in both central and peripheral nervous systems. HALOTag® sequence can easily be fused either to subunit N- or C-terminus which are extracellular, then labeled with the specific HALOTag® substrate coupled with the HTRF® donor (Fig.1). Materials and methods Preparation of the fluorescent ligands GR-flu and GR-DY GR-flu was prepared as described by Tairi et al [19]. GR-DY was prepared by reacting GR119566 with DY-647-NHS-ester (Dyomics, Jena, Germany) in the presence of diisopropylethylamine in DMSO, 3h at room temperature, and purifying by HPLC. Tag-lite® and HTRF® experiments Reagents The Tag-lite® buffer used was from Cisbio Bioassays (Codolet, France; ref. LABMED). The 96-well plates (ref. 655086), the 384-well small volume plates (ref. 784075) and the 96-well clear bottom plates (ref. 655090) were purchased from Greiner Bio-One. Specific chloroalkane-Lumi4-Tb was synthesized by Cisbio Bioassays and is commercialized as Halo-Lumi4-Tb (ref. SHALOTBC). Two ondansetron derivatives labeled with a green fluorescent probe (GR-Flu; Cisbio Bioassays) and a red fluorescent probe (GR-DY; Cisbio Bioassays) were used for the 5HT3A receptor binding assays. The various compounds used for competition assays were obtained from Tocris (Bristol, UK) and from University of Strasbourg.

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Plasmid engineering The HALO-5HT3A plasmid was designed in house. The HaloTag® enzyme was inserted at the extracellular N-terminus extremity of the 5HT3A human receptor (NM_000869, NP_000860). The endogenous signal peptide was removed to avoid cleavage of HaloTag®, and replaced by a generic signal peptide (T8) inserted upstream HaloTag® sequence. Cell culture HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) glutaMAX supplemented with penicillin 50 U/mL, streptomycin 50 µg/mL, HEPES 2 mM, 1% non-essential amino acids, and 10% fetal calf serum. Transfection protocols Direct transient transfection performed on adherent cells. These transfections were performed in black 96-well plates. Prior to cell plating, wells were precoated with 50 µL poly-L-ornithine for 30 min at 37°C. Then, cells were added at the density of 100,000 cells per well and incubated at 37°C under 5% CO2 for 24 h. Transfection mixes were prepared using 50 µl optiMEM culture medium (GIBCO, Invitrogen) and 100 ng of the HALO-5HT3A plasmid (Cisbio Bioassays) per well. A ratio of ¼ between the plasmid and the lipofectamine 2000 (Invitrogen) was respected. These mixes were then preincubated for 20 min at room temperature, and added to the cells after medium removal. Plates were incubated at 37°C under 5% CO2 for 48 h, with a change of medium after 24 h of incubation. Direct transient transfections performed in batches to generate cells for freezing. HEK293 cells were grown in a T175cm2 flask at 37°C under 5% CO2. When 80% confluency was reached, cell medium was removed and replaced by 12 ml of fresh cell culture medium. In parallel, a transfection mixture containing 20 µg plasmid, 60 µl Lipofectamine 2000 and 8 mL OptiMEM was prepared and incubated for 20 min at room temperature before the addition of cells. The flask was then incubated at 37°C under 5% CO2 for 48 h.

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Covalent labelling of cells expressing HALO-tag® 5HT3A Adherent cell-based assays in 96-well plate. The medium was removed from the 96-well plates, and 200 nM of HALO-Lumi4-Tb diluted in the Tag-lite® labeling medium was added (50 µL per well). The labeled plates were incubated 1 h at 37°C under 5% CO2. The excess of HALO-Lumi4-Tb was then removed by washing each well 4 times with 100 µL of Tag-lite® labeling medium. TR-FRET signal was read in 100 µl per well of Tag-lite® labeling medium. Cell-based assays in suspension in small volume 384-well plate. After removal of the cell culture medium from the flask , 10 mL of Tag-lite® labeling medium containing 200 nM of HALO-Lumi4-Tb was added and incubated for 1 h at 37°C under 5% CO2. Cells were then detached and collected in a vial. The excess of HALO-Lumi4-Tb was removed by 3 centrifugation/washing steps (5 min at 1000 rpm) using 10 mL of Tag-lite® labeling medium. Pelleted cells were suspended in a cell culture medium containing 10% DMSO, dispensed at 3 million cells per vial, and slowly frozen to -80 °C in a box containing isopropanol. Prior to their use, the frozen cells were thawed quickly at 37°C, then diluted in Tag-lite® labeling medium and centrifuged. Finally, the cells were counted and suspended in the Tag-lite® labeling medium at the suitable cell density (10,000 cells per well) to read TR-FRET signal. Tag-lite® fluorescent ligand-binding assays Cell medium was removed before addition of the ligands. All the compounds were diluted in Tag-lite® labeling medium. To measure the total binding, 25 µL/well of Tag-lite® labeling medium (or 5 µL in 384well plate), plus 25 µL/well of an increasing concentration range of tested fluorescent ligand were added (or 5 µL in 384-well plate). To measure the non specific binding, 25 µL (or 5 µL in 384-well plate) of unlabeled ligand (5-HT at 100 µM (saturating concentration)) was added instead of Tag-lite®

labeling

medium. Specific signal was obtained by subtracting the non specific signal from the total binding signal. Plates were then incubated at RT for 2 h before signal detection.

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Tag-lite® competition binding assays Cell medium was removed before addition of the ligands. All the compounds were diluted in Tag-lite® labeling medium. 25 µL (or 5 µL in 384-well plate) of a fixed concentration of fluorescent ligand (Kd concentration) plus 25 µL of increasing concentrations of compounds to be tested (or 5 µL in 384-well plate) were added. Plates were then incubated at RT for 2 h before signal detection. Calcium flux measurements Cells were plated in 96-well clear bottom plates, precoated with 50 µL poly-L-ornithine for 30 min at 37°C. Cells were then added at the density of 100,000 cells per well and incubated at 37°C under 5% CO2 for 24 h. Flexstation buffer was prepared for a final volume of 100 mL: 10 mL HBSS 10X, 2 mL HEPES 1 M, 0.33 mL Na2CO3 1 M, 0.13 mL CaCl2 1 M, 0.1 mL MgSO4 1 M, 71 mg Probenecid, 1 mL NaOH 1 N, and 1 mL BSA 10%, pH 7.4. After Probenecid addition, this buffer was stored at 4°C. All the compounds were diluted in the Flexstation Buffer. Calcium flux was measured with the Fluo-4 dye at 1 µMf. Cell medium was removed, 75 µL of the dye were added, and then the mixture was incubated from 30 min to 1 h at 37°C. Prior to the experiment 50 µL/well of Flexstation buffer were added. Antagonist compounds were added 30 min before agonist injection. The excitation wavelength was set at 485 nm, and the emission at 538 nm (auto cutoff: 530 nm). The injection volume was 25 µL/well. Measurements started after 20 s of incubation, and were performed every 1.53 seconds. Signal detection and data analysis HTRF® is a homogeneous technology that combines a fluorescence resonance energy transfer (FRET) process with a time-resolved fluorescence detection used to probe biomolecular interactions. This combination, named TR-FRET, is possible through the use of long lifetime fluorescent FRET donors such as europium or terbium cryptates which are rare-earth lanthanides. The long life-time of the europium or terbium cryptates allows the measurement of FRET emission when all current fluorophores are switched off.

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Here, we chose to use the Terbium cryptate to label the HALOTag® substrate, because of the negligible emission around 520 nm and 665 nm and its energetic compatibility with both green and red compatible fluorescent dyes in GR-flu and GR-DY ligands. HTRF® experiments were performed on Pherastar FS reader (BMG Labtech, Offenburg, Germany), using dedicated HTRF® settings (excitation wavelength = 337nm; emission wavelength = 620nm for donor, 665nm for red acceptor, 520nm for green acceptor; integration delay (lag time before measurement) = 60µs; integration time (measurement) = 400µs). On HALO-5HT3A transfected cells, membrane expression measurements were represented by TR-FRET signal recorded at 620nm, which is the fluorescence signal of HALO-Lumi4Tb, while binding and competition assays, were represented by HTRF ratios recorded at 665nm and 620 nm or at 520 nm and 620 nm, which are the FRET signals of HALO-Lumi4Tb/GR-DY or HALO-Lumi4Tb/GR-flu.HTRF® ratios were obtained by dividing the acceptor signal (665 or 520 nm) by the donor signal (620 nm) and multiplying this value by 10,000. This ratiometric data reduction has been developed to normalize experiments and correct for well-to-well variability and signal quenching from assay components and media. Calcium flux measurements were performed on FlexStation III (Molecular Devices, Berkshire, United Kingdom), using standard settings. Data obtained were then analyzed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA). The mean and the SEM (Standard Error of the Mean) are given in the graphs. Kd values of the fluorescent ligands were obtained from saturation curves of the specific binding. Ki values of the compounds were determined from competitive binding experiments according to the IC50 values found and the Cheng and Prusoff equation [20] Signal-to-noise (S/N ratio) calculations were performed by dividing the mean of the maximum value by the mean of the minimum value obtained from the sigmoid fits. Patch-clamp experiments Transient transfection of HEK293 Cells

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HEK293 cells were transiently transfected onto poly-D-lysine (Sigma) coated coverslips in 24-well cell culture plates with 0.9 µg per well of the WT-5HT3A-R or HALO-5HT3A-R plasmid using the Lipidbased Fugene6 transfection reagent (Promega). Co-transfection with 0.1 µg per well of green fluorescence protein (GFP) enabled the selection of transfected cells under fluorescence optics. Electrophysiology Properties of the resultant current through the 5-HT3A receptor ion channel were investigated at 48 h and 72 h after transfection using the whole-cell configuration of the patch-clamp technique. Coverslips were placed into a small recording chamber which was constantly perfused (1-2 mL/min) with an external recording solution containing 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES (osmolarity set at 315 mOsm/L, pH 7.4). Heat-polished borosilicate glass pipettes (resistances of 3-6 MΩ) were filled with a prefiltered intracellular solution containing 130 mM CsCl, 4 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES (osmolarity set at 285 mOsm/L, pH 7.4). Voltage-clamp and data acquisition were performed using an Axopatch 200B (Axon Instruments, Union City, CA) amplifier coupled to pClamp control software (v10; Axon Instruments) via an analog to digital converter (Digidata 1440; Axon Instruments). Signals were prefiltered at a 5-10 kHz bandwidth and sampled at 2 kHz. All experiments were performed at room temperature on cells with seal resistances of 1 to 10 GΩ and at a holding potential of -40 mV. The whole-cell access resistance was typically 4-12 MΩ. After the whole cell configuration was completed, the external solution (control) was added first and the cell was stabilized for 5 min. Then the test compound was added from low concentrations to high concentrations cumulatively. Recorded inward currents are displayed as downward deflections. In all experiments, at least three patches of WT- and HALO-5HT3A constructs from at least two different co-transfections with GFP were examined. Agonist (5-HT) and antagonists (granisetron and azelastine) dissolved in external buffer were added via multibarreled pressure ejection pipettes controlled by electromagnetic switch valves (Warner Instruments, Holliston, MA) from pClamp 10 software through two-barrel theta glass tubing that had been pulled to a tip diameter of ~ 200 µm.

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Reagents Serotonin (5-hydroxytryptamine or 5-HT), granisetron hydrochloride and azelastine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). 5-HT and granisetron were aliquoted as stock solutions in external solution and azelastine in dimethyl sulfoxide (DMSO). Compounds were diluted from frozen stocks in the external solution on the day of recording so that the maximal DMSO concentration for azelastine was < 0.001 %. Data Handling and Analysis Data were analyzed using Clampfit (v10; Axon Instruments), Excel (Microsoft) and Microcal Origin (v9; OriginLab Corporation, Northampton, MA). All electrophysiological data are presented as mean ± standard deviation. Concentration-response curves were fitted using the four-parameter logistic equation: ‫=ݕ‬൦

‫ܣ‬1 − ‫ܣ‬2 ൪ + ‫ܣ‬2 ‫ ݔ‬௣ ቀ ቁ 1+ ‫ݔ‬ ଴

Where y is the inward current amplitude recorded in the presence of agonist (5-HT) at concentration x, x0 is the concentration of agonist evoking a half maximal response (EC50), p is the Hill slope characterizing the slope of the curve at its midpoint. A1 and A2 are the estimated minimum and maximum responses, respectively. For calculations of antagonists potency, peak amplitudes induced by 5-HT at a concentration closed to the EC80 in the presence of Granisetron or Azelastine were normalized to those in the absence of antagonist for each cell. A similar equation, with antagonists (granisetron and azelastine) concentration replacing x and the concentration of antagonist (IC50) producing a 50% block of the response replacing EC50 was used to analyze concentration-inhibition data. The apparent antagonist dissociation constant (Kb) values were calculated from concentration-inhibition curves using a generalized form of the ChengPrusoff equation [21]. RESULTS AND DISCUSSION Expression of HALO-5HT3A at the cell surface 10

To detect cell membrane expression of HALO-5HT3A encoded by the Tag-lite® plasmid, transitory transfected HEK293 cells expressing HALO-5HT3A were labeled with a Lumi4-Terbium specific HaloTag® substrate. The resulting time-resolved (TR) fluorescence signal was compared between adherent cells (96-well plate) and labeled frozen cells in suspension (small volume 384-well plate). Similar signal amplitudes were observed for adherent and suspension cells. The measured signals were very specific and showed significant cell membrane expression of HALO-5HT3A in both assay formats (Fig.2). Pharmacological properties of HALO-5HT3A channels Tag-lite® technology requires the use of a HaloTag® enzyme fused to the N-terminal end of the 5HT3A-R. Therefore, it had to be checked that such fusion does not affect the functional response of the ligand-gated ion channel. First, the calcium flux entrance through the 5HT3A receptor was evaluated using a calcium fluorescent probe after the addition of the physiological agonist serotonin (5-HT). As shown in figure 3A, Ca2+ influx from HALO-5HT3A-R and WT-5HT3A-R were similar and the potencies obtained for 5-HT activation (EC50 = 360 nM for HALO-5HT3A-R and EC50 = 400 nM for WT-5HT3A-R) were consistent with the literature [22]. Moreover, the functional properties of the HALO-5HT3A-R were tested by the whole cell patch clamp technique in comparison with WT-5HT3A-R. At a holding potential of -40 mV, the local application of 5HT (0.3-300 µM) for 1 s elicited typical concentration-dependent inward currents from cells expressing either WT-5HT3A-R or HALO-5HT3A-R with similar characteristics of activation and desensitization (Fig. 3B). The values of the agonist concentration for half-maximum response (EC50) for 5-HT and Hill coefficients (nH) (EC50 = 2.7 µM ± 0.13; nH = 1.59 ± 0.11; n = 5 for WT-5HT3A-R and EC50 = 2.1 µM ± 0.21; nH = 1.47 ± 0.19; n = 5 for HALO-5HT3A-R) were equivalent (Fig. 3C) and correlated well with pharmacological data available from human 5-HT3A receptor literature [23, 24]. Taken together, these results demonstrate that the HALO-5HT3A-R is pharmacologically indistinguishable from WT-5HT3A-R after 5-HT activation, and appears to be functionally silent as the orthosteric ligand-binding site is preserved as well as the potency of 5-HT. 11

In the case of 5HT3A, we were able to fuse a HaloTag® enzyme to the extracellular N-terminal end of the channel. However, some LGICs such as P2X4 or P2X7 show both N- and C-terminus in the intracellular medium. In this case, using the bulky HaloTag may disturb the activity of these LGICs as it has to be inserted on extracellular loops, so smaller tags such as myc or HA may be required to maintain channel activity. Development of fluorescent-specific 5HT3A ligands Vogel et al. have already reported some very efficient fluorescent 5HT3A ligands based on the antagonist GR119566 [19, 25, 26]. Based on this, we successfully resynthesized GR-flu (GR119566 labeled with fluorescein). As Lumi4-Tb can also be associated with red acceptors, we synthesized GR-DY derivatized from GR119566 with Dy-647, the best HTRF® compatible Red acceptor. Tag-lite® binding assays on adherent HALO-5HT3A cells Adherent HEK293 transitory transfected cells expressing HALO-5HT3A channels were used to carry out the binding assays in 96-well microplates. After labeling of cells with HALO-Lumi4-Tb substrate, ligand binding properties of the fluorescent ligands were determined by saturation experiments using GR-flu and GR-DY ligands. The HTRF® signal, expressed as the HTRF® ratio, followed a standard binding curve upon addition of increasing concentrations of fluorescent ligands. The nonspecific signal was determined using an excess of unlabeled GR119566 (100 µM). A Kd value of 18 ± 0.9 nM determined for GR-DY was similar to that previously obtained for other fluorescent derivatives (not optimized for HTRF® and Tag-lite® technologies). High total binding amplitude and small nonspecific binding signal (less than 20%) consequently led to a significant S/N (superior to 5). In contrast, although exhibiting a Kd value of about 10 ± 0.4 nM (Fig. 5B), GR-flu led to a strong nonspecific signal in comparison with the total binding signal and consequently to a weak S/N (inferior to 2.5). On this basis, GR-DY appeared to be the most suitable fluorescent ligand for this assay. Next, well-known 5HT3A ligands were selected, including agonist and antagonist compounds (serotonin and ondansetron) to complete the validation of the assay with the GR-DY ligand. These compounds were 12

tested in competition with the 5HT3A red ligand GR-DY (Figure 6) in order to check that Ki values found matched those resulting from radioactive binding assays found in the literature (5-HT, Ki = 123 nM; Ondansetron, Ki = 4.9 nM). As expected, Figure 6 shows a strong decrease of HTRF® signal upon addition of increasing concentrations of the competitors. Ki values obtained for 5-HT and Ondansetron (468 ± 38 nM and 1.2 ± 0.12 nM) compounds were in good agreement with those reported in the literature [22]. Therefore this homogeneous binding assay using Tag-lite® and HTRF® technologies has been proven to be a new screening solution for identifying compounds targeting the 5HT3A receptor. Tag-lite® binding assays on HALO-5HT3A cells in suspension as miniaturized format Once the proof of concept had been established that both red ligand GR-DY and HALO-5HT3A ion channels were suitable for use in a binding assay, the test was miniaturized by using pre labeled cells in suspension. Binding properties of the GR-DY ligand were checked by saturation experiments using the red ligand GR-DY on HEK293 cells transiently expressing HALO-5HT3A ion channels. Cells were pre labeled

with HALO-Lumi4Tb substrate and frozen at -80°C until needed. To carry out the Tag-lite®

binding assays, the cells were thawed just before use and dispensed in small volume 384-well plates with the fluorescent ligand and the set of compounds to be tested. Figure 7 shows the results obtained for 10,000 cells per well. The calculated Kd value was 17 ± 0.7 nM, similar to the previous result obtained on adherent cells. The nonspecific signal represents less than 20% of the total binding at a saturating dose of red ligand, giving a suitable S/N (about 6) (Fig. 7A). Then similar competition experiments to those on adherent cells were performed, in order to check the Ki values of known competitive antagonist and agonist (Fig. 7B). Very similar results were obtained. The S/N (about 6) was still excellent and suitable for high throughput screening. These results proved that frozen prelabeled transitory transfected HEK 293 cells are a valid ready-to-use cellular reagent for high-throughput screening experiments in 5HT3A investigations. Once the feasibility of miniaturizing the Tag-lite® binding assays for 5HT3A ion channel investigations had been demonstrated, a pilot screen with a sub-set of molecules from an “in house” chemical library was performed. First, the assays were run at a concentration of 10 µM for all the

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compounds using frozen labeled cells in suspension in small volume 384-well plates. Next, to determine Ki values, competition experiments were performed with several compounds chosen from the panel of molecules tested which had an inhibition percentage higher than 80% at 10 µM. Among the hits, the results on well-known 5HT3A compounds were validated as references (Table 1). A few compounds which had never been described as binding 5HT3A ion channels were identified. Only one of them was chosen to illustrate our experiments. This compound is the drug azelastine (marketed as Allergodil®). The azelastine compound is a potent second-generation histamine H1 receptor antagonist, mainly indicated for the local treatment of seasonal allergic rhinitis symptoms. To our knowledge, azelastine has never been reported as a potent 5-HT3A receptor binder. Given that the inhibition percentage was 93% ± 5 at 10 µM for competitive binding with GR-DY on HALO-5HT3A-R, further investigations using HTRF® technology were performed to determine the Ki value, which proved to be 174 nM ± 10 (Fig. 8A). This result showed that azelastine can displace a specific binder of 5-HT3A receptor on the orthosteric ligandbinding site. To further validate this result, competition experiments were performed with whole cell patch clamp using HEK293 cells transfected with WT-5HT3A-R. The selective 5-HT3A receptor antagonist granisetron was compared with the supposed binding of azelastine. Granisetron is a well-known antagonist that exerts its action by competition with 5-HT for binding to the orthosteric ligand-binding site [27]. The blocking properties were evaluated by recording responses upon application of 5-HT at a concentration closed to EC80 (7 µM) determined previously ((Fig.3C) after preincubation (2-5 min) with increasing concentrations of granisetron or azelastine (Fig.8B). In agreement with published data, granisetron inhibited 5-HTinduced currents with an IC50 value of 0.2 ± 0.1 nM (nH = 1.13 ± 0.14) corresponding to a Kb value of 0.06 ± 0.02 nM [23, 28]. If we assume a similar nature of antagonism, azelastine exerted a less potent blocking effect than granisetron, with an estimated IC50 value of 5.15 nM (nH = 1.09) corresponding to a Kb value of 1.31 ± 0.6 nM. Taken together, these results represent a first confirmation that azelastine can bind and act as antagonist on 5HT3A-R.

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CONCLUSIONS As fluorescent based assays remain the most frequently used method for the primary screening of large chemical libraries, this work has demonstrated that homogeneous time-resolved fluorescence (HTRF®) technology can be used in the ion channel drug discovery field. This miniaturizable assay is reliable, rapid, and simple to run, meeting the requirements of HTS assays. Using the 5-HT3A receptor as a system model, it was proved that the Tag-lite® assay is highly efficient in identifying new ligand 5-HT3A receptor in a high throughput screening format. It had already been shown that the Tag-lite® technology could be successfully applied to a large set of GPCRs or RTKs, and today this study gives a proof of concept that it can also be adapted to the LGIC family. ACKNOWLEDGEMENTS The HTRF® screening experiments were performed on the ARPEGE (Pharmacology Screening Interactome) platform facility at the Institut de Génomique Fonctionnelle (Montpellier, France). Through the project “Cell2Lead”, this work was financially supported by the French government (Fond Unique Interministériel), OSEO, FEDER, Region Languedoc-Roussillon, Alsace Biovalley, Eurobiomed, CNRS, INSERM, University of Montpellier I and II, (Fond Unique Interministériel “Cell2Lead”), University of Strasbourg, Cisbio Bioassays, and Sanofi Aventis R&D. The team thanks all those involved. REFERENCES [1] D. Lemoine, R.T. Jiang, A. Taly, T. Chataigneau, A. Specht and T. Grutter, Ligand-Gated Ion Channels: New Insights into Neurological Disorders and Ligand Recognition, Chem Rev 112 (2012) 6285-6318 [2] G.C. Terstappen, R. Roncarati, J. Dunlop and R. Peri, Screening technologies for ion channel drug discovery, Future Med Chem 2 (2010) 715-730 [3] W. Zheng, R.H. Spencer and L. Kiss, High throughput assay technologies for ion channel drug discovery, Assay Drug Dev Techn 2 (2004) 543-552 [4] J.F. Glickman, A. Schmid and S. Ferrand, Scintillation proximity assays in high-throughput screening, Assay Drug Dev Techn 6 (2008) 433-455 [5] E. Ilegems, H. Pick, C. Deluz, S. Kellenberger and H. Vogel, Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor, Chembiochem 6 (2005) 2180-2185 [6] G. Milligan, Applications of bioluminescence- and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors, Eur J Pharm Sci 21 (2004) 397-405

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[7] A.J. Pope, U.M. Haupts and K.J. Moore, Homogeneous fluorescence readouts for miniaturized highthroughput screening: theory and practice, Drug Discov Today 4 (1999) 350-362 [8] P. Vallotton, A.P. Tairi, T. Wohland, K. Friedrich-Benet, H. Pick, R. Hovius and H. Vogel, Mapping the antagonist binding site of the serotonin type 3 receptor by fluorescence resonance energy transfer, Biochemistry-Us 40 (2001) 12237-12242 [9] M. Allen, J. Reeves and G. Mellor, High throughput fluorescence polarization: A homogeneous alternative to radioligand binding for cell surface receptors, J Biomol Screen 5 (2000) 63-69 [10] A. Pramanik, Ligand-receptor interactions in live cells by fluorescence correlation spectroscopy, Curr Pharm Biotechno 5 (2004) 205-212 [11] R. Hovius, E.L. Schmid, A.P. Tairi, H. Blasey, A.R. Bernard, K. Lundstrom and H. Vogel, Fluorescence techniques for fundamental and applied studies of membrane protein receptors: The 5-HT3 serotonin receptor, J Recept Signal Tr R 19 (1999) 533-545 [12] E.L. Schmid, A.P. Tairi, R. Hovius and H. Vogel, Screening ligands for membrane protein receptors by total internal reflection fluorescence: The 5-HT3 serotonin receptor, Anal Chem 70 (1998) 1331-1338 [13] L.A.A. de Jong, D.R.A. Uges, J.P. Franke and R. Bischoff, Receptor-ligand binding assays: Technologies and applications, J Chromatogr B 829 (2005) 1-25 [14] A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel and K. Johnsson, A general method for the covalent labeling of fusion proteins with small molecules in vivo, Nat Biotechnol 21 (2003) 86-89 [15] D. Maurel, L. Comps-Agrar, C. Brock, M.L. Rives, E. Bourrier, M.A. Ayoub, H. Bazin, N. Tinel, T. Durroux, L. Prezeau, E. Trinquet and J.P. Pin, Cell-surface protein-protein interaction analysis with timeresolved FRET and snap-tag technologies: application to GPCR oligomerization, Nat Methods 5 (2008) 561-567 [16] J.M. Zwier, T. Roux, M. Cottet, T. Durroux, S. Douzon, S. Bdioui, N. Gregor, E. Bourrier, N. Oueslati, L. Nicolas, N. Tinel, C. Boisseau, P. Yverneau, F. Charrier-Savournin, M. Fink and E. Trinquet, A Fluorescent Ligand-Binding Alternative Using Tag-lite (R) Technology, J Biomol Screen 15 (2010) 1248-1259 [17] S.Y. Li, C.Y. Guo, X.Q. Sun, Y.Z. Li, H.L. Zhao, D.M. Zhan, M.B. Lan and Y. Tang, Synthesis and biological evaluation of quinazoline and quinoline bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential epidermal growth factor receptor(EGFR) tyrosine kinase inhibitors and EPR bio-probe agents, Eur J Med Chem 49 (2012) 271-278 [18] G.V. Los, L.P. Encell, M.G. McDougall, D.D. Hartzell, N. Karassina, C. Zimprich, M.G. Wood, R. Learish, R.F. Ohane, M. Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G. Vidugiris, J. Zhu, A. Darzins, D.H. Klaubert, R.F. Bulleit and K.V. Wood, HatoTag: A novel protein labeling technology for cell imaging and protein analysis, Acs Chem Biol 3 (2008) 373-382 [19] A.P. Tairi, R. Hovius, H. Pick, H. Blasey, A. Bernard, A. Surprenant, K. Lundstrom and H. Vogel, Ligand binding to the serotonin 5HT(3) receptor studied with a novel fluorescent ligand, Biochemistry-Us 37 (1998) 15850-15864 [20] Y. Cheng and W.H. Prusoff, Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochem Pharmacol 22 (1973) 3099-108 [21] D.A. Craig, The Cheng-Prusoff Relationship - Something Lost in the Translation, Trends Pharmacol Sci 14 (1993) 89-91 [22] J. Walstab, S. Combrink, M. Bruss, M. Gothert, B. Niesler and H. Bonisch, Aequorin luminescencebased assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization, Anal Biochem 368 (2007) 185-192 [23] D. Belelli, J.M. Balcarek, A.G. Hope, J.A. Peters, J.J. Lambert and T.P. Blackburn, Cloning and functional expression of a human 5-hydroxytryptamine type 3A(S) receptor subunit, Mol Pharmacol 48 (1995) 1054-1062

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[24] A.J. Thompson, Recent developments in 5-HT3 receptor pharmacology, Trends Pharmacol Sci 34 (2013) 100-109 [25] H. Pick, A.K. Preuss, M. Mayer, T. Wohland, R. Hovius and H. Vogel, Monitoring expression and clustering of the ionotropic 5HT(3) receptor in plasma membranes of live biological cells, BiochemistryUs 42 (2003) 877-884 [26] P. Vallotton, R. Hovius, H. Pick and H. Vogel, In vitro and in vivo ligand binding to the 5HT(3) serotonin receptor characterised by time-resolved fluorescence spectroscopy, Chembiochem 2 (2001) 205-211 [27] D. Yan and M.M. White, Spatial orientation of the antagonist granisetron in the ligand-binding site of the 5-HT3 receptor, Mol Pharmacol 68 (2005) 365-371 [28] C. Schreiter, R. Hovius, M. Costioli, H. Pick, S. Kellenberger, L. Schild and H. Vogel, Characterization of the ligand-binding site of the serotonin 5-HT3 receptor - The role of glutamate residues 97, 224, and 235, J Biol Chem 278 (2003) 22709-22716 [29] T. Fukuda, M. Setoguchi, K. Inaba, H. Shoji and T. Tahara, The Antiemetic Profile of Y-25130, a New Selective 5-Ht3 Receptor Antagonist, Eur J Pharmacol 196 (1991) 299-305 [30] A.G. Hope, J.A. Peters, A.M. Brown, J.J. Lambert and T.P. Blackburn, Characterization of a human 5hydroxytryptamine(3) receptor type A (h5-HT(3)R-A(s)) subunit stably expressed in HEK 293 cells, Brit J Pharmacol 118 (1996) 1237-1245 [31] Y. Rival, R. Hoffmann, B. Didier, V. Rybaltchenko, J.J. Bourguignon and C.G. Wermuth, 5-HT3 antagonists derived from aminopyridazine-type muscarinic M1 agonists, J Med Chem 41 (1998) 311-317 [32] E. Morelli, S. Gemma, R. Budriesi, G. Campiani, E. Novellino, C. Fattorusso, B. Catalanotti, S.S. Coccone, S. Ros, G. Borrelli, V. Kumar, M. Persico, I. Fiorini, V. Nacci, P. Ioan, A. Chiarini, M. Hamon, A. Cagnotto, T. Mennini, C. Fracasso, M. Colovic, S. Caccia and S. Butini, Specific Targeting of Peripheral Serotonin 5-HT3 Receptors. Synthesis, Biological Investigation, and Structure-Activity Relationships, J Med Chem 52 (2009) 3548-3562

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Figure 1: Principle of Tag-lite® technology to label HALO-5HT3A receptors at the cell surface.

18

40000

20000

Ad he r

N eg at iv e

en tc Su el sp ls en si on ce lls

0 co nt ro l

TR-FRET signal (620nm)

60000

Figure 2. Cell membrane expression of HALO-5HT3A. HEK293 cells transiently transfected with 5HT3A channels were incubated with a saturating concentration of HaloTag® specific substrate coupled with Lumi4-Tb at 200 nM. A strong significant time-resolved signal (TR-FRET signal) was observed. Negative control was determined using untransfected HEK293 cells incubated with the same solution of HALO-Lumi4-Tb substrate as for adherent and suspension cells. Specific signal can be determined by subtracting the nonspecific signal from total signal. Each result represents the data from one experiment, representative of the three performed in triplicate. Values represent the mean ± SEM of the three independent experiments.

19

A

Max-Min (RFU)

3

HALO-5HT3A WT 5HT3A Negative control

2

1

0 -11

-10

-9

-8

-7

-6

-5

-4

-3

log [5-HT] M

B

C

Figure 3. Pharmacological properties of the HALO-5HT3A-R in comparison with WT-5HT3A-R. (A) HEK293 cells transiently expressing the HALO-5HT3A-R and WT-5HT3A-R were incubated with increasing concentrations of serotonin as agonist compound. Significant signal for activation and calcium entrance was detected for HALO-5HT3A-R (black circles) and WT-5HT3A-R (black triangles) in comparison with negative control (clear circles). Negative control was determined by adding the same concentration range on untransfected cells as on transitory transfected cells. Potencies obtained on HALO-

20

5HT3A-R (EC50 = 465 nM) and WT-5HT3A-R (EC50 = 395 nM) are well correlated with pharmacological data available from literature. (B) Representative current-responses of individual HEK293 cells transiently expressing HALO-5HT3A-R and WT-5HT3A-R following the application of 5-HT (0.3-300 µM) for 1 s. Currents were recorded at a holding potential of -40 mV in whole cell configuration. Bars indicate the application of 5-HT (1 s). (C) Superimposition of the concentration-response curves of 5-HT-activated currents for the WT-5HT3A-R (white squares) and HALO-5HT3A-R (black circles). Data were normalized to peak current activated by 100 µM 5-HT for each cell and fitted to the logistic equation. Parameters derived from these curves are: EC50 = 2.7 ± 0.13 µM and nH = 1.59 ± 0.11 for WT-5HT3A-R; EC50 = 2.1 ± 0.21 µM and nH = 1.47 ± 0.19 for HALO-5HT3A-R. Each data point represents mean ± SD from 5 cells.

21

Figure 4. Chemical structure of the fluorescent ligands. The 5HT3 antagonist GR119566 (R = H) was labeled at R with fluorescein (GR-flu) or DY-647 (GR-DY)

22

B

A

2000

HTRF Ratio (520/620)

HTRF Ratio (665/620)

14000 12000 10000 8000 6000 4000 2000

1500 1000 500 0

0 0

20

40

60

[GR-DY] nM

80

100

0

20

40

60

80

100

[GR-flu] nM

Figure 5. Tag-lite® binding assays on adherent HEK293 cells. HEK 293 cells transiently transfected with HALO-5HT3A plasmid were labeled with a saturating concentration of HALO-Lumi4-Tb substrate (200 nM), then incubated with increasing concentrations of fluorescent ligands. (A) A strong Homogeneous Time-Resolved Fluorescence (HTRF®) signal was observed and expressed as the HTRF® ratio 665/620 nm. Using GR-DY fluorescent ligand, a strong total binding (clear circles) was measured. Non specific binding (black triangles) was determined by adding 100 µM of unlabeled GR119566. The specific binding signal (black circles) was obtained after subtracting total binding from non-specific binding, and a Kd of 18 ± 0.9 nM was estimated by fitting the specific binding using a GraphPad Prism 5 binding hyperbola equation. (B) The same experiments were carried out with GR-flu. A low HTRF® signal was observed and expressed as the HTRF® ratio 520/620 nm. Kd = 10 ± 0.4 nM. Each result represents the data from one experiment representative of the three performed in triplicate. Values represent ± SEM of three independent experiments.

23

HTRF Ratio (665/620)

10000 8000 6000 4000 2000

Ondansetron 5-HT

0 -12

-10

-8

-6

-4

log [compounds] M

Figure 6. HTRF® competition assays on adherent HEK293 cells. Competition assays adding 15 nM of GR-DY and increasing concentrations of competitors were performed on HALO-5HT3A transitory transfected cells. Ondansetron (GR119566) competed with the GR-DY with an IC50 of 2.7 ± 0.12 nM and 5-HT with an IC50 of 1000 ± 38 nM when fitting the data with a sigmoid model. Calculation with the Cheng-Prusoff equation [20] resulted in a Ki of 1.2 ± 0.12 nM for Ondansetron and 468 ± 38 nM for 5HT. Each result represents the data from one experiment representative of the three performed in triplicate. Values represent ± SEM of three independent experiments.

24

13000

80

100

-3

60

-4

40

GR-DY (nM)

-5

20

-6

0

1000 -7

0

Ondansetron 5-HT -8

3000

5000

-9

6000

9000

-1 0

9000

-1 1

12000

-1 2

HTRF Ratio (665/620)

HTRF Ratio (665/620)

B

A

15000

log [compounds] M

Figure 7. Ligand-binding assay for 5HT3A using cells in suspension. (A) Transitory transfected HEK293 cells expressing the HALO-5HT3A ion channel were incubated with increasing concentrations of GR-DY ligand. A robust HTRF® signal was observed. The graph shows significant total (clear circles) and specific (black circles) binding to the 5HT3A ion channel. Nonspecific binding (black triangles) was measured by adding 100 µM of unlabeled GR119566 to the wells. The nonspecific signal was subtracted from total binding signal to obtain the specific binding curve. A Kd of 17 ± 0.7nM was determined for GR-DY ligand using GraphPad equation hyperbola. (B) Competition assay by adding 20 nM of GR-DY and increasing concentrations of competitors were performed. Ondansetron and 5-HT compete with the GRDY ligand. Calculations with the Cheng-Prusoff equation [20] result in a Ki of 7.3 ± 0.3 nM for Ondansetron and 729 ± 24 nM for 5-HT. Each figure represents the data from one experiment representative of the three performed in triplicate. Values represent ± SEM of three independent experiments.

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B

Figure 8. Competition assays for azelastine on the 5HT3A receptor. (A) Competition for azelastine with GR-DY was evaluated by HTRF® assays on cells in suspension. Transitory transfected HEK293 cells expressing HALO-5HT3A-R were incubated with increasing concentrations of azelastine (dark circles) as competitor. GR-DY ligand was added at 20 nM. Azelastine competed with the GR-DY ligand giving an IC50 of 379 ± 10 nM. Calculations with the Cheng-Prusoff equation [20] resulted in a Ki of 174 ± 10 nM. (B) Inhibition of 5-HT efficacy by granisetron (white squares) and azelastine (black triangles) was measured on individual HEK293 cells transiently expressing WT-5HT3A-R, using the whole cell patch clamp technique. After incubation with increasing concentrations (0.001 to 100 nM) of granisetron or azelastine, the maximal and steady-state response upon the addition of 5-HT for 2 s at the EC80 (7 µM) was recorded. For each cell, currents were normalized to the control current response to 5-HT at EC80 before the incubation with compounds and fitted to the logistic equation. Parameters were calculated for the antagonist granisetron: IC50 = 0.2 ± 0.1 nM, nH = 1.13 ± 0.14 and for azelastine: IC50 = 5.15, nH = 1.09. Calculations with the Cheng-Prusoff equation [20] resulted in Kb values of 0.06 ± 0.02 nM for granisetron and 1.31 ± 0.6 nM for azelastine. Each data point represents mean ± SD from 3 cells.

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Table 1. Comparison of Ki values obtained for reference antagonist compounds with Tag-lite® binding technology on cells in suspension (small volume 384 well plates) and radioactive binding assays (literature data).

Compounds

Tag-lite® binding assay (Ki ±SEM , nM)

Radioactive binding assay (Ki, nM)

Refs

Y-25130

3.6±0.7

2.9

[29]

Ondansetron

7.3 ± 0.3

5.7

[22]

Granisetron

3.2 ± 0.7

1.4

[30]

Azasetron

4.1 ± 0.2

4.3

[22]

MDL 72222

33.4 ± 1.1

54

[31]

Quipazine

6.8 ± 1.2

1.0

[32]

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GRAPHICAL ABSTRACT

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Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy invol...
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