Time-Resolved FRET Binding Assay to Investigate Hetero-Oligomer

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Time-Resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer Candide Hounsou, Jean-françois Margathe, Nadia Oueslati, Abderazak Belhocine, Elodie Dupuis, Cécile Thomas, Andre Mann, Brigitte Ilien, Didier Rognan, Eric Trinquet, Marcel Hibert, Jean-Philippe Pin, Dominique Bonnet, and Thierry Durroux ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb5007568 • Publication Date (Web): 28 Oct 2014 Downloaded from http://pubs.acs.org on November 3, 2014

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ACS Chemical Biology

Time-Resolved FRET binding assay to investigate heterooligomer binding properties: proof of concept with dopamine D1/D3 heterodimer Candide Hounsou1,‡, Jean-François Margathe2,‡, Nadia Oueslati1, Abderazak Belhocine1, Elodie Dupuis3, Cécile Thomas2, André Mann2, Brigitte Ilien4, Didier Rognan2, Eric Trinquet3, Marcel Hibert2, Jean- Philippe Pin1, Dominique Bonnet2,*, Thierry Durroux1,* 1

CNRS UMR 5203, and INSERM U661, and Université Montpellier I et II, Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France. 2 Laboratoire d’Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg, Labex Médalis, Faculté de Pharmacie, 74 route du Rhin, 67412 Illkirch, France. 3 Cisbio Bioassays, Parc Marcel Boiteux, BP84175, 30200 Codolet, France. 4 Unité Biotechnologie et Signalisation cellulaire, UMR 7242 CNRS/Université de Strasbourg, Labex Médalis, Ecole Supérieure de Biotechnologie de Strasbourg, 300 Bvd S. Brant, 67412 Illkirch, France. Keywords: G protein-coupled receptors, fluorescent probes, dopamine receptors, heteromer, dimerization

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ABSTRACT: G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and hetero-dimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. Based on the high-resolution structure of the dopamine D3 receptor (D3R), six fluorescent probes derived from a known D3R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D3/D2 receptors, and for other dopamine receptor subtypes. The highest affinity ligand 21 was then employed in the development of the D1/D3 heteromer assay. The TRFRET was monitored between a fluorescent tag donor carried by the D1 receptor (D1R) and a fluorescent acceptor D3R ligand 21. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.


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INTRODUCTION G-protein-coupled receptors (GPCRs) represent the largest family of cell surface membrane proteins encoded by the human genome. They are targeted by more than 30% of all drugs presently on the market.1 GPCRs have long been considered to exist and function as monomers within the cell plasma membrane. However, in recent decades, homo- and hetero-oligomerization of GPCRs have been described as a new way to modulate receptor functional activity. Indeed, the binding and coupling properties of oligomers may differ depending on the nature of the interacting partners, as described for the opioid receptors.2-4 Therefore, the existence of GPCR oligomers, which has been proven in native tissues,5-9 opens fascinating perspectives in physiology and therapy. Important questions arise regarding the specific role of hetero-oligomers vs. homo-oligomers and monomers, and the potential interest of these complexes as specific drug targets. Establishing the pharmacological properties of hetero-oligomers remains difficult since the binding strategies used so far do not discriminate between ligand binding on hetero-oligomers, homo-oligomers or monomers, which precludes a straightforward identification of specific ligands for hetero-oligomers. To overcome this difficulty, we have developed an original binding assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) that allows specific tracking of ligand binding to a target receptor involved in a supramolecular homo- or hetero-oligomer.10 In the last decades, FRET strategies have emerged as preferred techniques to investigate molecular interactions, since FRET is only observed if the distance between partners is short. They have successfully been used to study receptor oligomerization and more recently ligand/receptor interactions, providing an attractive alternative to radioactive ligand-based binding assays. Among FRET techniques, TR-FRET exhibits a significant advantage owing to its high signal-to-noise ratio. Indeed, lanthanide cryptates exhibit long lifetimes for their excited states and thus offer the possibility of introducing a time delay between fluorophore excitation and emission measurement long enough to allow short-lived fluorescence species to disappear. Such a property was crucial for selective assay development. In TR-FRET assays, ligands are usually coupled to conventional fluorescein-like or dy647-like fluorophores (acceptor species), whereas receptors are labeled with lanthanide cryptates (donor species) which, owing to their long fluorescence lifetimes, confer increased sensitivity to the assay. Such an approach has been successfully applied to study ligand-receptor11-14 or receptor-receptor interactions at the cell surface of living cells15, 16 or in native tissues.5 To date however, no TR-FRET based ligand binding assay has been developed to examine the binding properties of each subunit of a GPCR hetero-oligomer.

As a proof of concept, we selected dopaminergic receptors as GPCR models. Indeed, dopamine controls various physiological functions, including locomotor activity, learning and memory, motivation and reward through its binding to five distinct dopamine (D1 to D5) receptors.17 Dopaminergic dysfunctions have been implicated in the etiology of various neurological and psychiatric disorders such as Parkinson’s disease, schizophrenia, and drug



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addiction.18 The D3R is of particular interest as it has been identified as a potential target for drug discovery in the field of drug addiction. Recently, the D3R has been also demonstrated to interact directly with the D1R, both receptors interacting synergistically in the striatal membranes and co-transfected cells.4, 19 Targeting D3/D1 heterodimers may lead to functional responses that would differ from targeting D3R monomers, and therefore represent a potential and promising therapeutic strategy for disorders related to the dopaminergic system. Therefore, to accelerate the discovery of ligands acting specifically on the D3R involved in a D3/D1 heterodimer, we developed an assay based on FRET between a fluorescent ligand bound to the D3R and the D1 tagged receptor. This rapid and easy to handle TR-FRET based assay could be readily extended to other GPCR heteromers.

RESULTS AND DISCUSSION Design of Fluorophore-Tagged D3R Ligands. BP 897 was chosen as the prototype ligand since this partial agonist not only displays a nanomolar affinity for the D3R (Ki = 0.92 ± 0.2 nM) but also a chemical structure that is easy to derivatize.20 A series of fluorescent BP 897 compounds labeled with TR-FRET compatible dyes such as dy647 and fluorescein (FLUOR) was then envisaged (Figure 1). The influence of the length and nature of the spacer on both the affinity and the selectivity for dopamine receptors was carefully investigated. Based on the recently published high-resolution structure of the D3R,21 docking BP 897 into the receptor binding pocket enabled us to rapidly delineate the BP 897 positions where fluorophore attachment would not be detrimental to binding (Figure 2). The 4-(2-methoxyphenyl)piperazine moiety of BP 897 is predicted to bind to the orthosteric dopamine-binding site involving TMs 3, 5 and 6 whereas the naphthyl moiety binds to a remote pocket at the interface of TMs 2, 7 and the first extracellular loop (ecl1). This binding mode accounts for all known mutagenesis data on the D3R, and is fully compatible with a recent study22 pinpointing the important role of Gly94 (ecl1) in the selective binding to D3R subtypes. The observed selectivity of BP 897 for the D2R and D3R subtypes can be explained by the interaction of the ligand with key residues (Val86, Gly94, His349, Tyr365, Thr369) which undergo substitutions in other subtypes (e.g. Val86 replaced by Lys in D1R/D5R and Phe (D4R), insertion of Gly94 only in D2R/D3R, Tyr365 replaced by Lys (D1R), Arg (D5R) or Val (D4R), and His349 by Pro (D1R/D5R)) Clearly, the fluorophore could be introduced at the C-6’ position of the naphthyl moiety through a spacer located outside of the transmembrane cavity (Figure 2). In addition, unlike the human muscarinic M1 receptor and its fluorescent pirenzepine derivatives,23 the D3R-BP 897 model suggests that a long spacer between the pharmacophore and the fluorophore is not necessary to accommodate the steric hindrance of the dye from the binding pocket. However, to investigate this parameter, we decided to insert 3 to 12 atom-long spacers between BP 897 and dy647 or FLUOR.

Preparation of FLUOR and dy647 Labeled Ligands for TR-FRET Applications. As depicted in Scheme 1A, bifunctional spacers 8, 9 and 10 were synthesized by conversion of the hydroxyl group of compounds 5, 6


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and 7 to the corresponding mesylate. These spacers differ in length but also in their structure (alkyl vs polyethylene glycol). The azido moiety was installed by nucleophilic substitution of either bromo (1 and 2) or tosylate groups (4) in the presence of NaN3. Compound 4 was readily obtained by the selective monoalkylation of diol 3 in the presence of tosyl chloride, KI and Ag2O.24 Then, prior to its connection to spacers, the naphthyl group of BP 897 was functionalized at C-6' position with a hydroxyl group (Scheme 1B). The synthesis of the 6-hydroxy substituted naphthyl 13 was performed via condensation of 6-hydroxynaphthoic acid with amino intermediate 12 obtained in a two-step Gabriel sequence: alkylation of piperazine 11 with 4-bromobutylphthalimide and hydrazinolysis. Next, short and long alkyl spacers 8 and 9 and PEG3 spacer 10 were incorporated on 13 by O-alkylation under basic conditions to provide the corresponding azido derivatives 14, 15 or 16. Under Staudinger conditions, the azido moiety was readily reduced to primary amine. To avoid a final purification step in order to eliminate the phosphine oxide, the reduction was advantageously performed in presence of a solid-supported triphenylphosphine, readily separated by simple filtration at the end of the reaction. The resulting amino derivatives 17, 18 or 19 were subsequently acylated by the Nhydroxysuccinimide ester of dy647 or of FLUOR to give fluorescent probes 20-25. The purity and identity of all final compounds were checked by analytical RP-HPLC, liquid chromatography mass spectrometry (LC-MS) and high-resolution mass spectrometry (HRMS).

TR-FRET-Based Ligand Binding Assay for Dopamine Receptors. To define the binding affinities of probes 20-25 for dopamine receptors, a TR-FRET based assay was developed. This fluorescent technology, based on the use of a terbium cryptate, Lumi4-Tb25 as a donor and dy647 or fluorescein as acceptor species, presents the main advantage of reducing the signal to noise ratio, thus increasing the sensitivity of the assay compared to classic FRET systems.14 The SNAP tagged D1-D5 receptors which were expressed at the surface of HEK293 cells, were covalently labeled with Lumi4-Tb via reaction between a substrate terbium cryptate benzyl guanine derivative (SNAP-Lumi4Tb) and the SNAP-tag suicide enzyme.26-30 In agreement with previous reports indicating that such a fusion did not affect the GPCR binding properties or functional responses,16 all five SNAP-tagged dopamine receptors were found to retain their functional activity (cAMP assays; see Supporting Information Figure S1). Saturation experiments using the strategy illustrated in Figure 3A were then performed with fluorescent probes 20-25. HEK293 cells were transiently transfected with SNAP-tagged dopamine receptors, labeled with the TR-FRET donor and incubated in the presence of increasing concentrations of the probe. Figure 3 depicts typical saturation experiments performed with 21 on the D3R (Figure 3B) and on all five dopamine receptor subtypes (Figure 3C). Ligand binding is defined by the TR-FRET ratio 665/620 in which the signals at 665 nm and 620 nm correspond to the FRET signal and the fluorescence of the donor, respectively. The



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specific TR-FRET signal was then plotted as function of fluorescent probe concentrations (Figure 3B and 3C), enabling determination of the dissociation constants (Table 1). Strikingly, despite the extent of chemical modification (polarity, bulkiness) introduced into BP 897, all fluorescent probes retained nanomolar affinity for the D3R (1-30 nM). As predicted by the three-dimensional model of the D3R-BP 897 complex, the naphthyl group of BP 897 is a suitable location for dye incorporation. Unlike the results observed with human muscarinic M1 receptor,31 the length of the spacer did not seem to be crucial for affinity, as similar dissociation constants were obtained when using either short three-atom (20 and 21; Kd values at 5 nM and 1.6 nM, respectively) or long twelve-atom (24 and 25; Kd values at 5.5 nM and 4.5 nM, respectively) spacers. Regardless of the length and the composition (alkyl vs PEG) of the spacers, the dyes (dy647 or FLUOR) did not interfere with the binding of the fluorescent probes to the D3R, except for 22 which displayed a lower affinity (Kd = 29.8 nM) than its dy647 counterpart 23. Most probes also exhibited nanomolar affinities (6 to 51 nM) for the D2 receptor (D2R). Again, 22 displayed the highest Kd value (183 nM). Unlike the results obtained for the D3R, the nature of the fluorophore was found to influence probe affinity (Table 1). Indeed, Kd values for dy647-labeled ligands (21, 23, 25) are higher than those determined for the FLUOR counterparts (20, 22, 24), thereby suggesting that the dyes may participate in the probe binding to D2R. It is noteworthy that the incorporation of the dyes onto BP 897 influenced D2/D3 binding selectivity of fluorescent probes 20-25. Indeed, the D2/D3 affinity ratio ranged from 4 to 10, the best selectivity being obtained with 20 bearing the shortest spacer and FLUOR dye as acceptor. For the D1R and D4R, the length and the nature of the spacer together with the nature of the acceptor fluorophore play an important role for the affinity. Hence, as with the parent BP 897 which showed a low affinity for the D1R (Ki = 3 µM), all fluorescent derivatives but 23 (Kd = 172 nM) also displayed low affinity for this receptor (Kd > 1 µM). The importance of the linker and the dye is even more pronounced for the D4 receptor (D4R). Whereas the binding affinity of BP 897 for D4R is close to 0.3 µM, the corresponding fluorescent probes 21, 22, 24 and 25 display lower affinities (> 0.6 µM). However, as described for the D1R, 23 is the only probe for which the affinity (Kd = 120 nM) was found to be higher than that of the parent BP 897. It is difficult to provide a clear molecular explanation for this observation since the second extracellular loop (ecl2) of D1R (30 residues) is much longer than that of the D3R (14 residues) and therefore cannot be modeled with precision. It is likely that the dy647 moiety, when combined with a sufficiently long linker, specifically interacts with one or several charged residues present at the ecl2 insert of D1R. The absence of binding for analog 25 could be simply due to a different conformation of the polyethylene glycol linker. Finally, all probes displayed a very low affinity (Kd > 1 µM) for the dopamine D5 receptor. Based on these results, probe 21 was selected for its nanomolar affinity at the D2 and D3 receptors, and for its very weak interaction at D1R. To evaluate the relevance of this probe in screening assays, TR-FRET competition experiments (Figure


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3D) were performed on D3 (Figure 3E) and D2 (Figure 3F) receptor-expressing cells, using 21 as a tracer and spiperone (antagonist), asenapine (antagonist) or bromocriptine (agonist) as competitors (See Supplementary information, Table S1).

Cell-surface D1R-D3R Heteromerization Study. To implement the TR-FRET based binding assay for dopamine D1/D3 heteromers, we defined experimental conditions leading to receptor heteromerization. Dopamine D1 and D3 receptors fused either to SNAP- or HALOTag were co-expressed. To this end, we developed polyclonal HEK 293 cell lines that stably express dopamine SNAP-D3, HALOTagD3 or HALOTag-D1 receptors, which in a second step were transiently transfected to express the second receptor. Experimental conditions were optimized in such a way that the D1 and D3 receptors were expressed in the same range (Figure 4A). The expression of each receptor is defined independently by the measurement of the fluorescence of the donor at 620 nm after receptor labeling with the cognate donor substrate. Co-expression of D1 and D3 receptors led to receptor hetero-oligomerization (Figure 4B) as evidenced by the existence of a significant FRET signal when receptors were labeled with compatible fluorophores to generate a TR-FRET signal (Figure 4B). It is noteworthy that the distances between the donor and the acceptor varied depending on the tag fused to the receptor and the substrate. Therefore, the amplitudes of the TR-FRET signal cannot be directly compared. To develop a screening assay for D1/D3 receptor hetero-oligomers, we took advantage of the high affinity of 21 for the D3R and of its negligible interaction with the D1 subtype to define its affinity for the D1/D3 heterooligomers. HEK 293 cells co-expressing SNAP-D1 and HALOTag-D3 receptors were labeled with SNAP-Lumi4Tb substrate and incubated in the presence of increasing concentrations of 21 (Figure 4C). TR-FRET intensity was plotted as a function of 21 concentrations. Analysis of the saturation curve resulting from FRET signals between 21 and the Lumi4-Tb donor carried by D1R (Figure 4C, filled circles) led to the determination of a dissociation constant of 0.46 + 0.08 nM. This dissociation constant is similar to those defined on SNAP-Lumi4-Tb labeled D3 receptors when they were expressed alone (Table 1) or co-expressed with HALOTag-D1 receptors (Figure 4C, open squares) (1.6 ± 0.2 nM and 1.52 ± 0.39 nM, respectively). This suggests that the interaction between D3 and D1 has a marginal effect on 21 affinity for the D3R binding site. By contrast, the labeling of D1 or D3 receptors with the SNAP-Lumi4-Tb substrate when SNAP-D1/HALOTagD3 (Figure 4C, filled circles) or HALOTag-D1/SNAP-D3 (Figure 4C, open squares) were expressed, resulted in a large variation in the TR-FRET signal amplitude. This may be explained by two main parameters. First, as 21 specifically binds to the D3R, the distance between the fluorophores carried by 21 and the D3R is probably shorter than the respective distance between 21 and the D1R, thereby leading to a higher FRET signal. Second, as depicted in Figure 4C, the TR-FRET signal originates exclusively from the D1/D3 hetero-oligomer or from D1/D3 het-



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ero- and D3/D3 homo-oligomers (see Supporting Information, Figure S2) and D3 monomers when 21 binds to Snap-Lumi4-Tb labeled SNAP-D1/HALOTag-D3 or HALOTag-D1/SNAP-D3 receptors, respectively. To validate the assay, we next performed competition experiments using reference ligands selective for D1 (SKF38393, SCH23390) or D3 (7-OH-DPAT, GSK789472). HEK 293 cells co-expressing SNAP-D1 and HALOTag-D3 receptors (labeled with the SNAP-Lumi4-Tb substrate; Figure 4D) or expressing HALOTag-D3 receptors (labeled with the HALOTag-Lumi4-Tb substrate; Figure 4E) were incubated with 1 nM of probe 21 and increasing concentrations of competitor. The TR-FRET amplitudes were plotted as a function of competitor concentration to derive the drug inhibition constants from competition curve analysis. The Ki values of competitors for the D1/D3 heterodimers and for D3 monomers or homodimers are compared to literature values in Table 2. Regardless of the receptor selectivity of the competitors, their Ki values for D1/D3 hetero-oligomers coincide with those defined for D3R homo-oligomers, suggesting that in these conditions the interaction of D3 with the D1 receptor has no impact on its affinity for the ligands tested.

CONCLUSION In conclusion, we developed a new strategy based on TR-FRET binding assay that allows definition of ligand affinity toward D1/D3 receptor heteromers. D1 and D3 receptor co-expression leads to the formation of D1/D1 and D3/D3 homomers in addition to D1/D3 heteromers. Defining the binding properties for the heteromers alone was therefore challenging. The strategy we developed is based, on the one hand, on the development of a selective fluorescent ligand for the D3R and, on the other hand, on the TR-FRET signal between a fluorescent ligand bound to the D3R and a tagged D1R. This TR-FRET binding assay constitutes, to our knowledge, the first assay that allows discrimination between the binding of a given ligand to hetero-oligomers on the one hand and homooligomers or monomers on the other hand. These assays can easily be implemented for other receptor heteromers, which therefore opens up fascinating possibilities for identifying ligands that exhibit selectivity for heteromers.

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Methods Modeling. Two dimensional (2D) structures of BP 897 and compound 21 were drawn in Marvin Sketch v6.1 (ChemAxon Kft., HX1031 Budapest, Hungary) and converted in three dimensional (3D) coordinated by Corina v3.1.32 The correct protonation state at pH 7.4 was predicted using the Filter v2.1.1 program (OpenEye Sciencific Software, Santa Fe, NM 87508, U.S.A.). Hydrogen atoms were added to the X-ray structure of human D3R (Chain A:Tyr32-Lys221, Gly319-Ser400; PDB id 3pbl) by means of the SYBYL X-2.1 package (Tripos International, St. Louis, MO 63144-2319, U.S.A) Each ligand was then docked into D3R using standard settings of the program GOLD v5.2 (Cambridge Cristallographic Data Centre, Cambridge CB2 1EZ, United Kingdom). The active site was defined by any residue within a 15 Å-radius sphere centered on the D3R-bound eticlopride in the native X-ray structure. The best docking pose of 10 independent docking runs was selected in agreement with the known eticlopride-D3R X-ray structure22 and existing site-directed mutagenesis data.23 Finally, the selected docking pose corresponds to the highest docking score compatible with two interaction features: (i) close contacts of the naphthyl-amide moiety between the second transmembrane domain and the first extracellular loop22 and (ii) ionic bond between the protonable amine and Asp1103.32 conserved in all biogenic amine receptors..

Chemistry General methods. Reagents were obtained from commercial sources and used without any further purification. dy647-NHS was purchased from Dyomics GmbH (Jena, Germany) and 6-(fluorescein-5-carboxamido) hexanoic acid, succinimidyl ester (5-SFX), single isomer was obtained from Molecular Probes (ref. F6106). Thin-layer chromatography was performed on silica gel 60F254 plates. Merck silica gel (Kieselgel 60) was used for chromatography (230-400 mesh) columns. 1H NMR spectra were recorded at 300 or 400 MHz on a Bruker Advance spectrometer. Chemical shifts are reported in parts per million (ppm), coupling constants (J) are reported in hertz (Hz). Analytical reverse-phase high performance liquid chromatography (RP-HPLC) separations were performed on a C18 Ascentis Express (2.7 µm, 4.6 × 75 mm2) using a linear gradient (5% to 100% of solvent B in solvent A in 7.5 min, flow rate of 1.6 mL·min−1, detection at 220 nm, solvent A: water/0.1% TFA, solvent B: acetonitrile/0.1% TFA). Semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) separations were performed on a Waters XBridge RP-C18 column (5 µm, 19 × 100 mm) using a linear gradient (solvent B in solvent A, solvent A: water/0.1% TFA; solvent B: acetonitrile/0.1% TFA; flow rate of 20 mL.min-1; detection at 220 nm). Purified final compounds eluted as single and symmetrical peaks (thereby confirming a purity of ≥95%). High resolution mass spectra (HRMS) were acquired on a Bruker MicroTof mass spectrometer, using electrospray ionization (ESI) and a time-of-flight analyzer (TOF).

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General procedure for the synthesis of fluorescent probes 20-25. To a solution of 17, 18, or 19 (1.2 equiv) in DMSO (100 µL) were subsequently added 5-SFX or dy-647 NHS ester (10-20 mM in DMSO, 1-1.4 µmol, 1 equiv) and DIPEA (12 equiv). The mixture was stirred at room temperature for 1-3 h then purified by semipreparative RP-HPLC to afford the title compound. For 20: yellow solid (321 nmoles, 25%); HRMS (ESI) Calcd for C55H58N5O10 ([M+H]+) 948.41837, found 948.41754. For 21: blue solid (212 nmoles, 15%); HRMS (ESI) Calcd for C60H74N6O10S2 ([M+2H]2+) 551.24539, found 551.24402. For 22: yellow solid (299 nmoles, 30%); HRMS (ESI) Calcd for C64H76N5O10 ([M+H]+) 1074.55922, found 1074.55914. For 23: blue solid (255 nmoles, 25%); HRMS (ESI) Calcd for C69H92N6O10S2 ([M+2H]2+) 614.31582, found 614.31675. For 24: yellow solid (514 nmoles, 51%); HRMS (ESI) Calcd for C61H70N5O13 ([M+H]+) 1080.49701, found 1080.49614. For 25: blue solid (210 nmoles, 21%); HRMS (ESI) Calcd for C66H86N6O13S2 ([M+2H]2+) 617.28472, found 617.28456.

Biology Time-resolved FRET Binding Assays. The methods are extensively described 33. Briefly HEK293 cells transiently expressing SNAP-tagged or HALOtagged receptors were labeled with HALOTag-Lumi4-Tb and/or SNAPLumi4-Tb and/or SNAP-Red and/or HALOTag-Red substrates. Cell culture medium was first removed from the 96-well plates. 100 nM of labeling substrates, previously diluted in the Tag-lite labeling medium, were added (100 µL per well) and the plate further incubated 1 h at 37 °C under 5% CO2. The excesses of reagents were removed by 4 washes with 100 µL of Tag-lite labeling medium. Ligand binding experiments were then performed with plates containing 50 µL of Tag-lite labeling medium. 25 µL of unlabeled compound or Tag-lite labeling medium was added, followed by the addition of 25 µL of fluorescent ligand. Plates were then incubated at room temperature in the dark for 1h30 at least before signal detection (the duration to get equilibrium; see supplementary Figure S2). Affinities of the fluorescent ligands for the receptors were determined by incubating the cells at room temperature with increasing concentrations of the desired fluorescent ligand. For each fluorescent ligand concentration, the non-specific binding was determined in presence of an excess of unlabeled compound (10 µM SCH23390 for D1R and D5R; 10 µM Spiperone for D2R, D3R and D4R).

Data Analysis. Signal detection was performed on Infinite F500 (Tecan, Männedorf, Switzerland) for ligand binding assays. Time-resolved FRET readouts were recorded and analyzed as described previously.14 Briefly, fluorescent signal were measured at two wavelengths: 620 nm corresponding to the emission of Lumi4-Tb and 665 nm corresponding to the FRET signal. Results were expressed as the variation of 665/620 ratio infunction of the ligand concentration. For the competition curve, the results were expressed as the percentage of the FRET signal obtained in the absence of competitor. All binding data were analyzed with Prism 6 (GraphPad Software, Inc., San Diego, CA) using the one site-specific binding equation. All results are expressed as the Mean ± SEM of at least three independent experiments.

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ACKNOWLEDGMENT This work was supported by the Fonds Unique Interministériel and OSEO (Cell2lead program n° F1005035J/ATFUAA00LB/AAP9), the Centre National de la Recherche Scientifique, the Université de Strasbourg. The work was supported by the Fondation Recherche Médicale (FRM team DEQ20130326522) and the Fondation Bettencourt Schuller to JPP. This work was also made possible by the Plateforme de PharmacologieCriblage of Montpellier and the Region Languedoc-Roussillon. SUPPORTING INFORMATION General procedure for the preparation of compounds 4, 6-10, 12-19; Functional signaling assays with SNAPtagged dopamine receptors (Figure S1); Dopamine SNAP-D3 receptor homo-oligomerization (Figure S2); Saturation experiments with probe 21 on the D2R and D3R with different times of incubation (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors [email protected] and [email protected]; Author Contributions ‡These authors contributed equally ABBREVIATIONS AVP, arginine-vasopressin; D1R, human dopamine D1 receptor; D2R, human dopamine D2 receptor; D3R, human dopamine D3 receptor; D4R, human dopamine D4 receptor; D5R, human dopamine D5 receptor; eclL, extracellular loops; Fluor, Fluorescein; FRET, fluorescence resonance energy transfert; GPCR, G protein coupled receptor; HATU, 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HTS, high throughput screening; M1R, human muscarinic M1 receptor; TM, transmembrane domain; TR-FRET, time resolved FRET; 5-SFX, 6-(fluorescein-5-carboxamido) hexanoic acid, succinimidyl ester; V2R, vasopressin-arginin V2 receptor.

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Table 1. Binding affinities of compounds 20-25 for SNAP-tagged dopamine receptors as determined by time-resolved FRET ligand binding experiments.

Cpd

SPACER

Binding, (Kd, nM±SEM)

DYE D1R

BP 897

20

D2R

D3R

D4R

D5R

-

-

>1000

61± 0.2

0.92 ± 0.2

300

-

20

-CH2-CH2-

FLUOR

>1000

50.7 ± 3.1

5.0 ± 0.5

971 ± 167

>1000

21

-CH2-CH2-

dy647

>1000

8.4 ±2.4

1.9 ± 0.6

>1000

>1000

22

-(CH2-CH2-CH2)3-

FLUOR

>1000

183 ± 27

29.8 ± 2.1

>1000

>1000

23

-(CH2-CH2-CH2)3-

dy647

172± 10

23.1 ± 2.0

4.6 ± 0.8

120 ± 13

>1000

24

-(CH2-CH2-O)3-

FLUOR

>1000

40.6 ± 3.5

5.5 ± 0.6

650 ± 100

>1000

25

-(CH2-CH2-O)3-

dy647

>1000

23.8 ± 2.9

4.5 ± 0.5

>1000

>1000

Table 2. Ki values of reference compounds determined from D1R/D3R hetero-oligomer TR-FRET binding assays with probe 21 as tracer in comparison with Ki values from TR-FRET binding assays on D3R homo-oligomers and reference Ki values obtained from classical radioactive assays on D3R homo-oligomers.

Ligands

D1R Reference Ki (nM)

D1R-D3R heterooligomer, Ki (nM)a

D3R binding site, Ki (nM)a,b

D3R Reference Ki (nM)c

7-OH-DPAT

5300 + 50034

2.2 ± 1.2

1.7 ± 0.44

1.6 ± 0.4135

-

42 ± 20

45 ± 18

2536

> 100

> 1000

-

> 100

> 1000

480 ± 9935

GSK789472 37

SKF38393

61 -150

SCH23390

37

38

0.23 -0.35

38

a

Values are mean ± SEM of at least three independent experiments. bKi values from TR-FRET binding competition assays on the D3R homo-oligomers. cKi values from radioactive binding competition assays on the D3R in the literature.

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Scheme 1. Synthesis of (A) Spacers 8, 9 and 10 and (B) Fluorescent Compounds 20-25 for TR-FRET Applications a

a

Reagents: (i) TsCl, KI, Ag2O, 0 °C to rt, 14 h (61%); (ii) NaN3, DMF, 60 °C, 14 h (87-96%); (iii) MsCl, Et3N, CH2Cl2, 0 °C to rt, 14 h (85-86%); (iv) 4-bromobutylphthalimide, K2CO3, KI, CH3CN, reflux, 14 h; (v) NH2NH2, EtOH, reflux, 14 h then HCl 2N, reflux, 1 h (60% over 2 steps); (vi) 6-hydroxynaphthoic acid, HATU, Et3N, DMF, rt, 3 d (58%); (vii) K2CO3, DMF, 60 °C, 0.5 h then 8-10, 60 °C, 14 h (42-63%); (viii) PS-TPP, THF-H2O, rt, 2 d (57-65%); (xix) FLUOR-NHS or dy647-NHS, DIPEA, DMSO, rt, 2 h (15-51%).

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Legends of the Figures Figure 1. Chemical structure of BP 897. Figure 2. Proposed docking mode of BP 897 to the human dopamine D3 receptor. (A) Top view from the extracellular side of BP 897 (sticks) in the transmembrane cavity of the D3R (tan ribbons). The seven transmembrane domains (TM1-7) as well as the three extracellular loops (ecl1-3) are indicated. BP 897 atoms are colored according to the following scheme: carbon, cyan; nitrogen, blue; oxygen, red. (B) Two-dimensional diagram of the binding mode. Ionic and hydrogen bonds between protein and ligand are drawn as dashed lines. Hydrophobic contacts are represented by means of green spline sections highlighting the hydrophobic parts of the ligand and the label of the contacting amino acid. Breaks in the spline sections indicate exit channels from the binding cavity. The C-6’ position of the naphthyl group in BP 897 is indicated by a black arrow. The figure was rendered with PoseView.35 Figure 3. Time-resolved FRET based assays with tracer 21 and SNAP-tagged dopamine receptors. (A) Principle of the saturation assay. (B) Saturation experiments with probe 21 on the D3R. HEK 293 cells expressing D3Rs were labeled with SNAP-Lumi4-Tb substrate and incubated in a second step in the presence of increasing concentrations of 21 and in the presence (non-specific binding) or the absence (total binding) of an excess of unlabeled spiperone (10 µM). Ligand binding is defined by the TR-FRET ratio 665/620, in which signals correspond to the FRET signal measured at 665 nm and fluorescence of the donor at 620 nm. The Specific FRET signal corresponds to the difference between total and non-specific FRET signals. (C) Saturation experiments performed on HEK 293 cells expressing each of the dopamine receptors. Specific TR-FRET signals are plotted as a function of 21 concentration. (D) Principle of the competition binding assay. HEK 293 cells expressing dopamine D3 (E) or D2 (F) receptors labeled with SNAP-Lumi4-Tb were incubated in the presence of 21 (1 nM) and of increasing concentrations of various competitors. The Specific TR-FRET signal is plotted as a function of competitor concentration. Saturation and competition curves correspond to representative results of at least three independent experiments performed in triplicate. Kis were calculated from IC50 values with the Cheng Prusoff equation39.

Figure 4. Hetero-oligomerization of dopamine D1 and D3 receptors. (A) The expression of dopamine D1 and D3 receptors when SNAP-D1 and HALOTag-D3 or HALOTag-D1 and SNAP-D3 receptors are co-expressed in HEK 293 cells defined by measurement of the fluorescence measured at 620 nm (fluorescence of the donor) after receptor labeling with either SNAP-Lumi4-Tb or HALOTag-Lumi4-Tb substrates. (B) TR-FRET signal resulting from hetero-oligomerization of labeled D1 and D3 receptors. Values are mean ± SEM for at least ten independent experiments. (C) Saturation assays with probe 21. HEK cell expressing dopamine SNAP-D1 and HALOTag-D3 or HALOTag-D1 and SNAP-D3 were incubated in the presence of SNAP-Lumi4-Tb substrate leading to the labeling of the SNAP-receptor (*). In a second step, labeled cells were incubated in the presence of increasing concentrations of 21 with or without an excess of spiperone (10 µM). Specific TR-FRET is plotted as a function of 21 concentration. (D) and (E) show the competition assay on HEK cell expressing dopamine SNAP-D1 and HALOTag-D3 receptor (D) or only HALOTag-D3 receptor (E). Cells were incubated in the presence of SNAP-Lumi4-Tb substrate (D) and/or HALOTag-Lumi4-Tb, and in a second step were incubated in the presence of 21 (1 nM) and increasing concentrations of competitor. TR-FRET signals are plotted as a function of competitor concentration. Kis were calculated from IC50 values with the Cheng Prusoff equation39. The Specific TR-FRET signal is plotted as a function of competitor concentration. Competition curves correspond to the average of at least three independent experiments performed in triplicate. Kis were calculated from IC50 values with the Cheng Prusoff equation39.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

20


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SYNOPSIS TOC

FRET Tb

D3R Structure-based design

Ligand synthesis

D1R

TR-FRET binding assay


Time-Resolved FRET Binding Assay to Investigate Hetero-Oligomer

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