Skip to main content
NCBI home page
ACS Pharmacology & Translational Science logoLink to ACS Pharmacology & Translational Science
. 2022 Jul 18;5(8):668–678. doi: 10.1021/acsptsci.2c00096

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors

Florence Gbahou †, Sergiy Levin ‡, Irina G Tikhonova §, Gloria Somalo Barranco †, Charlotte Izabelle †, Rachel Friedman Ohana ‡, Ralf Jockers †,*
PMCID: PMC9380205  PMID: 35983281

Abstract

graphic file with name pt2c00096_0007.jpg

The two human melatonin receptors MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [3H]melatonin and 2-[125I]iodomelatonin (2-[125I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time. However, developing such assays for the two melatonin receptors depends on the availability of fluorescent tracers, which has been challenging predominantly owing to their narrow ligand entry channel and small ligand binding pocket. Here, we generated a set of melatonergic fluorescent tracers and used NanoBRET to evaluate their engagement with MT1 and MT2 receptors that are genetically fused to an N-terminal luminogenic HiBiT-peptide. We identified several nonselective and subtype-selective tracers. Among the selective tracers, PBI-8238 exhibited high nanomolar affinity to MT1, and PBI-8192 exhibited low nanomolar affinity to MT2. The pharmacological profiles of both tracers were in good agreement with those obtained with the current standard 2-[125I]-MLT radioligand. Molecular docking and mutagenesis studies suggested the binding mode of PBI-8192 in MT2 and its selectivity over MT1. In conclusion, we describe the development of the first nonradioactive, real-time binding assays for melatonin receptors expressed at the cell surface of living cells that are likely to accelerate drug discovery for melatonin receptors.

Keywords: melatonin receptors, fluorescent ligand, tracer, NanoBRET binding assay

Introduction

Melatonin or N-acetyl-5-methoxytryptamine is a neurohormone synthesized by the pineal gland in a circadian manner with peak levels during the night. Because of its lipophilic properties, melatonin is rapidly distributed all over the body through passive diffusion.1,2 Melatonin triggers its biological effects by activating two receptors, MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family.3−5 Melatonin plays a well-defined role in circadian rhythms, sleep, retinal physiology, and seasonal reproduction in mammals.3,4,6 Drugs that target these receptors, such as ramelteon, agomelatine, and tasimelteon, are currently used to treat insomnia, depression, and circadian disorders, respectively.7 Future therapeutic indications include neurodegenerative diseases, pain management, and disorders related to glucose metabolism and cardiovascular functions. The detection and pharmacological characterization of melatonin receptors became possible owing to the development of two specific radioligands, [3H]melatonin ([3H]-MLT)8,9 and 2-[125I]iodomelatonin (2-[125I]-MLT),3,10 in the 1980s. Both radioligands are full agonists of melatonin receptors and show very similar pharmacological profiles.11,12 The vast majority of studies use 2-[125I]-MLT as a radioligand owing to its higher specific activity. Three other iodinated radioligands have been recently developed;13 two of them, [125I]DIV880) and [125I]S70254, are full and partial MT2-selective agonists, respectively.14 Attempts to develop [11C]- and [18F]-labeled melatonin receptor tracers for positron emission tomography studies failed as blocking studies showed no specific binding in nonhuman primate brains, most likely because of the high hydrophobicity of the probes.15 Several fluorescently labeled melatonin receptor ligands have been generated, some of which retained acceptable binding affinity for these receptors (see ref (16) for a review). Unfortunately, high nonspecific binding and intracellular uptake render these tracers unsuitable for fluorescence imaging and fluorescence-based binding studies. Furthermore, structure analyses reveal for both receptors narrow ligand entry channels and small orthosteric ligand binding pockets, presenting additional challenges for fluorescent tracer development.17−20

Here, we generated a series of 11 new fluorescent melatonin receptor ligands compatible with the recently developed HiBiT peptide-based NanoBRET ligand binding assay. This assay combines the high specificity of BRET governed by the proximity required for efficient energy transfer (<10 nM) with the bright luminescence of the HiBiT/LgBiT complementation reporter and the cell impermeability of LgBiT, which limits signal generation to the cell surface and eliminates background luminescence originating from unoccupied receptors accumulated in intracellular compartments. We identified several nonselective and subtype-selective NanoBRET tracers. Pharmacological profiles determined using PBI-8238, a selective MT1 tracer, and PBI-8192, a selective MT2 tracer, were in excellent agreement with reported values obtained using a standard 2-[125I]-MLT binding assay. Molecular docking and mutagenesis studies revealed the unique binding mode of the MT2-selective PBI-8192 tracer with the melatonin headgroup occupying the orthosteric binding site and the linker and fluorophore occupying an extended subpocket present in MT2.

Material and Methods

Reagents

S20928, S75436, and agomelatine were obtained from Institut de Recherche Servier (Croissy sur Seine, France). The UCM 924 compound was a generous gift from Dr. Gabriella Gobbi (McGill University, Montreal, Canada). MO-24 was obtained from Dr. Darius Zlotos (German University in Cairo, Cairo, Egypt). ZINC compounds were purchased from Enamine (Riga, Latvia). ZINC compounds were chosen based on their chemotype novelties, their low topological similarity to known melatonin receptor ligands, and their reported functional properties.21 Most of the selected ZINC compounds are MT2-selective agonists or inverse agonists. All of the other compounds were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) and Tocris (Bio-Techne, Noyal Châtillon sur Seiche, France). 2[125I]iodo-melatonin was purchased from PerkinElmer (PerkinElmer SAS, Villebon-sur-Yvette, France).

Expression Vectors

Expression vectors for HiBiT-GPCR fusion proteins were previously described.22,23 Briefly, cDNAs for human GPCRs obtained from Kazusa DNA Research Institute (Chiba, Japan) were subcloned into a modified pF5 CMV-neo Flexi vector (Promega, USA) using the Flexi cloning system (Promega, USA). The pF5 vector was modified to enable generation of GPCR constructs tagged at their N-termini with an IL6 secretion tag (MNSFSTSAFGPVAFSLGLLLVLPAAFPAP), followed by a VS linker, a HiBiT tag (VSGWRLFKKIS), and a 2 X GSSG linker.

Organic Synthesis

Structures and synthetic procedures are included in Supporting Information 1 (Experimental Procedures). For all fluorescent tracers, the red-emitting BODIPY NanoBRET 590 dye (Ex_576 nm; Em_589 nm) was chosen because it is a well-suited energy acceptor for the HiBiT/LgBiT complementation reporter.22

Cell Culture and Transfection

HEK293 cells were grown in complete medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 4.5 g/L glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1 mM glutamine) (Life Technologies, Villebon-sur-Yvette, France). Transient transfections were performed using JetPEI (Polyplus Transfection, Illkirch, France), according to the manufacturer’s instructions.

NanoBRET Binding Assay

HEK293 cells were reverse transfected in 96-well plates using JetPEI (Polyplus Transfection, Illkirch, France). Briefly, 100 000 cells were directly added per well to the transfection reagent containing 5 ng of cDNA diluted into an empty vector (100 ng final) in a 96-well plate precoated with poly-ornithine (Sigma-Aldrich, Saint Quentin Fallavier, France). The next day, cells were washed with PBS, and the medium was replaced with Opti-Mem, followed by subsequent saturation. Competition or kinetic binding assays were carried out according to previously published protocols.22 Briefly, for saturation binding assays, increasing concentrations of tracer were incubated in the absence or presence of melatonin (30 μM) for 90 min, and then NanoLuc complementation was detected by the addition of the substrate LgBiT for 15 min before the plate was read. For kinetics, the substrate LgBiT was added first for 15 min followed by increasing concentrations of tracer before the plate was immediately read for at least 30 min. For competition binding assays, a fixed concentration of tracer was competed by increasing concentrations of unlabeled compounds for 90 min prior to the addition of the substrate LgBiT for an additional 15 min before the plate was read. Filtered luminescence was measured using a Microplate Reader equipped with a 450 nm filter (donor) and a 600 nm long-pass filter (acceptor). BRET was calculated by dividing the acceptor >600 nm light output by the donor 450 nm emission. Values were background corrected by subtracting the BRET values from samples treated with excess unmodified ligand followed by normalization. Data are expressed as the BRET ratio (acceptor/donor) and for competition binding assays are further normalized to the melatonin maximum effect. Results were analyzed by PRISM (GraphPad Software), and the data were fitted using nonlinear regression to determine Kd, Kon, Koff, or IC50 values. Ki values were calculated using the Cheng and Prussof formula: Ki = IC50/[1 + (L/Kd)], where L represents the ligand concentration and Kd the dissociation constant.

2-[125I]iodomelatonin Binding Assay

Membranes from HEK293 cells transiently expressing human MT1 or MT2 receptors were prepared as previously described.24 Binding assays were carried out as previously described in ref (25). Briefly, 2-[125I]-MLT saturation binding experiments were performed in the range of 1–300 pM, and specific binding was defined as binding displaced by 10 μM melatonin. Competition curves were performed by simultaneous incubation of 2-[125I]-MLT (50 pM) and increasing concentrations of the respective ligands. Assays were carried out in duplicate for 120 min at 37 °C, followed by rapid filtration through GF/C glass fiber filters (Whatman, Clifton, NJ). Filter-retained radioactivity was determined with a gamma-counter LB2111 (Berthold Technologies; Bad Wildbad, Germany). Ki values were calculated from IC50 values using the Cheng and Prussof formula: Ki = IC50/[1 + (L/Kd)], where L represents the ligand concentration and Kd the dissociation constant. Kd values were 367 and 125 pM for MT2 and HiBiT-MT2 receptors, respectively. IC50 values were calculated from GraphPad Prism software by fitting data on a nonlinear regression analysis.

Accumulative cAMP Assay

The cyclic AMP assay was performed as previously described (Gbahou, Cecon, et al., 2017).25 Briefly, HEK293 cells expressing human HiBiT-MT2 receptors were dispensed into a 384-well plate (4000 cells per well) and stimulated for 30 min at room temperature with 1 μM forskolin in the presence of increasing concentrations of MLT or PBI-8192 in PBS buffer supplemented with 1 mM 3-isobutyl-methylxanthine (IBMX, Sigma-Aldrich, St. Quentin Fallavier, France). Cells were then lysed, and cAMP levels were determined according to the manufacturer’s instruction (Cisbio Bioassays, Codolet, France) and measured using the Infinite F500 Tecan microplate reader. Data were fitted by nonlinear regression to determine EC50 values and normalized to forskolin-induced response (100%) and melatonin-induced maximum inhibition (0%) using GraphPad Prism software.

Molecular Docking

The crystal structures of the human MT1 and MT2 receptors (PDB codes 6ME3 and 6ME6) were used for molecular docking and optimization. PBI-8192 and iodomelatonin were docked into the MT2 and MT1 receptors using a standard precision docking protocol available in the Glide module of Schrödinger software (2020–1).26 The receptor structures were prepared using the protein preparation module, and the structures of ligands were assessed using the ligand preparation module of the Schrödinger software. 2-Phenylmelatonin of the X-ray complexes was selected as the center of the docking box. Docking poses were evaluated with the XP Glide docking score. The most frequent docking mode was selected as the most probable pose. Short minimization of docking complexes was carried out with the MacroModel module of the Schrödinger software. A default protocol of minimization in implicit solvent with distance-dependent electrostatic treatment was used to obtain the final complexes. The OPLS_2005 force field was used in all calculations. The 3D images were created in Maestro 2020–1.

Site-Directed Mutagenesis

The HiBiT-MT2 mutant was generated using the QuikChange II XL Site-Directed Mutagenesis kit protocol (Agilent Technologies, Les Ulis, France). The wild type plasmid pATG5131 human HiBiT-MT2 was used as a template, and the presence of the mutations was verified by DNA sequencing.

Data and Statistical Analyses

Data and statistical analyses were performed using GraphPad Prism software version 9.

Statistical analysis was performed by a Student’s t test when comparing two groups. Values of P < 0.05 were considered statistically significant.

Results

Development of the NanoBRET Ligand Binding Assay for Melatonin Receptors

To develop NanoBRET ligand binding assays utilizing the HiBiT/LgBiT complementation reporter as an energy donor, we generated DNA constructs expressing MT1 and MT2 genetically fused to an N-terminal HiBiT tag, a small 11-amino acid peptide that can produce bright luminescence upon high-affinity complementation with LgBiT, an 18 kDa subunit derived from NanoLuc (Figure 1). 2-[125I]-MLT competition binding experiments with 20 melatonergic compounds confirmed that the HiBiT tag did not modify the pharmacological properties of the MT2 receptor (correlation coefficient r2 = 0.92; p < 0.0001) (Figure S1 and Table S1). To generate fluorescent tracers that can serve as energy acceptors, the red-emitting NanoBRET 590 fluorophore22,23,27 was attached to different melatonergic scaffolds through either a short alkylamide chain or a 4-unit poly(ethylene glycol) (PEG4)-based linker (Figure 1). In total, 11 fluorescent tracers were synthesized using as scaffolds either the high-affinity melatonin receptor agonist melatonin or 4-azamelatonin (including its previously described ICOA-9 derivative25) or the melatonin receptor antagonist luzindole3 (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Schematic representation of an HiBiT-tagged receptor and design of fluorescent ligands for NanoBRET binding assay on melatonin MT1 and MT2 receptors. A short (alkylamide chain) or long (4-unit poly(ethylene glycol) (PEG4) linker was used to fuse different melatonergic scaffolds to the red-emitting BODIPY NanoBRET 590 fluorophore. R = linker attachment points on the template. Red balls on the linkers depict the attachment points of the fluorophore.

For saturation binding analyses, HEK293 cells expressing HiBiT-MT1 or HiBiT-MT2 were treated with increasing concentrations of the developed tracers in the absence or presence of competing unmodified melatonin. BRET was measured upon treatment with recombinant LgBiT and subsequent complementation with HiBiT. Binding specificity was determined through a decrease in BRET owing to competitive displacement of a bound tracer by an excess of unmodified melatonin (30 μM) (Figure 2 and Figure S2).

Figure 2.

Figure 2

Open in a new tab

Structure activity of fluorescent melatonergic ligands on MT1 and MT2 receptors in the NanoBRET binding assay. Different fluorescent tracers consisting of either ICOA-9 (A), luzindole (B), or 4-azamelatonin (C) scaffold attached to BODIPY NanoBRET 590 red-emitting fluorophore through a short or long linker. The attachment point is highlighted in orange. The resulting PBI ligands were evaluated for saturation binding to HiBiT-tagged MT1 or MT2 expressed on the cell surface of HEK293 cells using a NanoBRET ligand binding assay. The specific binding curves (red curves) represent the difference between the total (buffer) and nonspecific (binding in the presence of 30 μM competing melatonin) saturation curves. The values are means ± SEM of seven experiments for PBI-8192 and PBI-8210 on MT2 and PBI-8238 on MT1. For PBI-8320, n = 3, and for PBI-8232 and PBI-8322, n = 2 for each receptor, respectively. All experiments were performed in duplicate or triplicate. NS = nonspecific.

Tracers PBI-8192 and PBI-8210 were designed based on ICOA-9, a previously described fluorescent compound with a BODIPY-FL fluorophore attached at the C2 carbon position of 4-azamelatonin25 (Figure 2A). Both tracers exhibited selective, saturable monophasic binding to HiBiT-MT2 with dissociation constants (Kd) of 54 and 113 nM for PBI-8192 and PBI-8210, respectively (Figure 2A and Table S2). PBI-8238, which was generated by attaching NanoBRET 590 fluorophore to the C2 carbon of 4-azamelatonin via a short linker, exhibited specific and saturable monophasic binding to HiBiT-MT1 with a Kd of 412 nM. No specific binding was observed for MT2, qualifying PBI-8238 as an MT1-selective tracer (Figure 2B). The corresponding analogue with a longer linker, PBI-8232, showed no specific binding for either receptor (Figure 2B). Finally, PBI-8211 generated by the attachment of NanoBRET 590 fluorophore on the indolic nitrogen of 4-azamelatonin exhibited no binding to either receptor (Figure S2C).

The luzindole-based tracers PBI-8320 and PBI-8322 were obtained by attaching the NanoBRET 590 fluorophore to the para-position of its benzyl group (Figure 2C). Interestingly, PBI-8320 showed specific binding to both melatonin receptors with Kd values around 500 nM (Figure 2C and Table S2) while its analogue PBI-8322 containing a longer PEG4 linker did not show any specific binding to either receptor (Figure 2C). Similar MT2 selectivity was observed for the luzindole-based PBI-8240/PBI-8241 tracers, devoid of the CH2 linker present in luzindole between the indole and benzyl rings, with Kd values around 300–400 nM (Figure S2A and Table S2). Similarly, low-affinity tracer binding to both receptors was observed for the melatonin-based tracer PBI-8298 with the NanoBRET 590 fluorophore attached at the methoxy group (Figure S2B) while its analogue, the PBI-8297 containing a PEG4 linker, was inactive (Figure S2B).

Taken together, we developed NanoBRET tracers, which bind to MT1 and/or MT2 receptors with different selectivity. While the affinity of the MT2-selective tracers PBI-8192 and PBI-8210 was around 50 and 100 nM, the affinity of the MT1-selective tracer PBI-8238 was around 400 nM and for the best nonselective tracer (PBI-8320) around 500 nM. The longer and hydrophilic PEG4 linker tended to be less tolerated than the short alkylamide linker. This is in agreement with the hydrophobic characteristics of the narrow melatonin binding pockets of MT1 and MT2. Due to its low affinity, we discarded the nonselective tracer PBI-8320 from further studies and perused the characterization of the MT1-selective PBI-8238 tracer and the MT2-selective PBI-8192 tracer.

Characterization of the MT1-Selective PBI-8238 Tracer

The specificity of the PBI-8238 tracer was further validated by testing its binding to the closely related serotonin 5-HT2A and 5-HT2C receptors. Incubation of HEK293 cells expressing HiBiT-tagged 5-HT2A and 5-HT2C receptors with increasing concentration of PBI-8238 resulted in no specific NanoBRET signal (Figure 3A). Competition of 2-[125I]-MLT binding to MT1 by increasing concentrations of PBI-8238 was complete and occurred with a pKi of 5.22 ± 0.05 (n = 3) (Figure 3B). This value is about 15 times lower than the Kd of PBI-8238 binding measured in the NanoBRET assay indicating that the Kd of PBI-8238 is driven not only by its melatonergic headgroup but also by other parts of the tracer. NanoBRET competition binding experiments using PBI-8238 tracer with a series of melatonergic reference ligands confirmed this hypothesis as all ligands showed a right-shift affinity (pKi) of 1 to 1.5 Log compared to the pKi values obtained in the 2-[125I]-MLT binding assay (Figure 3C, D and Table S3). To derive pKi values representing a more direct measure of the affinity of PBI-8238 for the orthosteric melatonin binding pocket, we calculated “adjusted” NanoBRET pKi values by replacing the NanoBRET Kd of PBI-8238 by its Ki obtained in 2-[125I]-MLT competition binding experiments (Table S3). In both cases, pKi values of the selected melatonergic ligands correlated well with their pKi values determined by 2-[125I]-MLT competition binding (r2 = 0.92; p < 0.0001) (Figure 3D). Collectively, the PBI-8238 tracer is highly selective for MT1 with similar pharmacological properties as 2-[125I]-MLT, but its use is limited by its low affinity, in particular for ligands with estimated affinities in the high nanomolar and micromolar range.

Figure 3.

Figure 3

Open in a new tab

Pharmacological characteristics of the PBI-8238 tracer for MT1. (A) Specificity of PBI-8238 binding (1 μM) to HiBiT-tagged MLT (MT1, MT2) or 5-HT (5-HT2A, 5-HT2C) receptors expressed on the surface of HEK293 cells in the absence (total binding) or presence (nonspecific binding) of a saturating concentration of competing melatonin (30 μM). The histograms represent the mean ± SEM of four independent experiments performed in triplicate. (B) Competitive radioligand binding assay (50 pM 2-[125I]-MLT) for PBI-8238 and MLT using membranes of HEK293 cells expressing a nontagged MT1 receptor. (C) Competitive NanoBRET ligand binding assay (1 μM PBI-8238) for melatonergic reference compounds using intact HEK293 cells expressing a HiBiT-tagged MT1 receptor. Data are expressed as mean ± SEM from three to four independent experiments normalized to the melatonin maximum effect. (D) Correlation plot of pKi values derived from 2-[125I]-MLT and adjusted NanoBRET competition binding assays (see Table S3).

Characterization of the MT2-Selective PBI-8192 Tracer

The specificity of the PBI-8192 tracer was further confirmed by the absence of any specific BRET signal in HEK293 cells expressing the HiBiT-tagged 5-HT2A and 5-HT2C receptors (Figure 4A). The binding constant (Kd) of PBI-8192 for HiBiT-MT2 was determined in a kinetic binding assay. The association (Kon) and dissociation (Koff) constants were 8.60 × 106 M–1 min–1 and 0.307 min–1, respectively, leading to a kinetic Kd of 36 nM (Kd = Koff/Kon), which is similar to the Kd value derived from the equilibrium binding assay (54 nM) (Figure 4B).

Figure 4.

Figure 4

Open in a new tab

Pharmacological characteristics of the PBI-8192 tracer for the MT2 receptor. (A) Specificity of PBI-8192 binding (300 nM) to HiBiT-tagged MLT (MT1, MT2) or 5-HT (5-HT2A, 5-HT2C) receptors expressed on the surface of HEK293 cells in the absence (total binding) or presence (nonspecific binding) of a saturating concentration of competing melatonin (30 μM). The histograms represent mean ± SEM of two independent experiments performed in triplicate. (B) Representative PBI-8192 binding kinetics performed at different PBI-8192 concentrations in HEK293 cells expressing HiBiT-MT2 receptors. (C) Competitive radioligand binding assay (2-[125I]-MLT) for PBI-8192 and MLT using membranes of HEK293 cells expressing nontagged MT2 receptors. Data are expressed as mean ± SEM from three independent experiments normalized to the melatonin maximum effect. (D) Competitive NanoBRET ligand binding assay (100–300 nM PBI-8192) for melatonergic reference compounds using intact HEK293 cells expressing HiBiT-tagged MT2 receptors. Data are expressed as mean ± SEM from three independent experiments normalized to the melatonin maximum effect. (E) Correlation plot of pKi values for melatonergic reference compounds obtained in radioligand ([125I]-MLT) and NanoBRET (PBI-8192) binding assays. Values are from Table 1. (F) MLT- and PBI-8192-driven inhibition of cAMP production in forskolin (Fsk)-stimulated HEK293 cells expressing human HiBiT-MT2 receptors. Data are expressed as mean ± SEM from four independent experiments and are represented as a percentage of FSK-stimulated response.

In the 2-[125I]-MLT competition binding assay, PBI-8192 was able to fully compete with the 2-[125I]-MLT tracer on the nontagged (Figure 4C) and HiBiT-tagged MT2 receptor, indicating that both tracers are probing the melatonin binding pocket of MT2. The observed pKi of 7.06 ± 0.16 and 6.98 ± 0.27, respectively, closely matched the Kd values of PBI-8192 measured by NanoBRET, indicating that the indolic pharmacophore of PBI-8192 is the main molecular determinant for its affinity to MT2. We further used the PBI-8192 tracer in NanoBRET competition binding assays to determine the pKi of 20 melatonergic compounds, including reference compounds and new chemical scaffolds recently identified through virtual ligand screening28 (Figure 4D, Table 1, and Figure S3A, B). The correlation plot between pKi values obtained in the NanoBRET and 2-[125I]-MLT assays revealed an excellent correlation coefficient (r2 = 0.92; p < 0.0001) (Figure 4E, Table 1, and Figure S3C, D). High-affinity agonists tended to have lower pKi values in the NanoBRET assay vs the 2-[125I]-MLT assay. This tendency was not visible for antagonists and the low-affinity new chemical scaffolds (Table 1). Afterward, the propensity of PBI-8192 to activate the Gi/cAMP signaling pathway was evaluated in a functional test. PBI-8192 behaves as a full agonist on this pathway as it inhibits forskolin-stimulated cAMP production to a similar extend as the melatonin reference, although with a lower pEC50 of 6.77 ± 0.12 (Figure 4F).

Table 1. pKi Values of Melatonergic Reference Compounds Determined Using Either 2-[125I]-MLT or PBI-8192 in Radioligand or NanoBRET Binding Assays, Respectively.

  pKia
  HiBiT-MT2
compounds 2-[125I]-MLT NanoBRET
agonists    
MLT 9.84 ± 0.47* 9.10 ± 0.21
I-MLT 10.6 ± 0.49* 9.23 ± 0.12
agomelatine 10.1 ± 0.19* 9.06 ± 0.27
ramelteon 10.1 ± 0.27* 9.20 ± 0.06
IIK7 8.47 ± 0.36 7.95 ± 0.42
N-acetyl-5-OH-Trp 7.02 ± 0.17* 6.09 ± 0.18
UCM 924 8.27 ± 0.06** 7.57 ± 0.09
S75436 9.73 ± 0.36 8.92 ± 0.11
antagonists    
4P-PDOT 8.67 ± 0.67 7.91 ± 0.31
luzindole 7.45 ± 0.41 7.19 ± 0.21
S20928 6.78 ± 0.27 6.87 ± 0.32
bivalent ligands    
MO-24 (3e)b 6.87 ± 0.28 6.20 ± 0.17
new chemotypesc    
agonists    
ZINC000091496083 (1) 5.86 ± 0.01 5.93 ± 0.12
ZINC000151192780 (2) 5.41 ± 0.32 6.24 ± 0.14
ZINC000485552623 (3) 5.81 ± 0.20 ≥5
ZINC000354255673 (4) ≥5 ≥5
ZINC000790921948 (5) ≥5 ≥5
ZINC000580731466 (6) 5.82 ± 0.86 6.48 ± 0.31
inverse agonists    
ZINC555417447 (7) 6.02 ± 0.79 5.48 ± 0.25
ZINC157673384 (8) 6.02 ± 0.45 ≥5
Open in a new tab
a

pKi values are calculated from the competition curves shown in Figure S2. Results are given as mean ± SEM of at least three independent experiments performed in duplicate.

b

From Karamitri et al., Bioorg. Chem., 2019.

c

From Stein et al., Nature, 2020.45 The significance of differences in pKi between 2-[125I]-MLT and NanoBRET was determined by a Student’s two-tailed unpaired t test. *P < 0.5; **P < 0.001.

Taken together, PBI-8192 and 2-[125I]-MLT have similar binding properties to MT2. They are both full agonists and show similar pharmacological profiles based on a selection of 20 melatonin receptor ligands.

Molecular Docking and Experimental Validation of the PBI-8192 Binding Mode in Human MT2

To better understand the molecular base for the interaction of PBI-8192 with the MT2 receptor, we computationally docked the PBI-8192 and I-MLT tracers into the orthosteric melatonin binding pocket of the MT2 crystal structure. The methoxy group and the alkylamide chain of PBI-8192 and I-MLT formed the expected hydrogen bonds with the N1754.50 (GPCR Ballesteros–Weinstein numbering is provided in the subscript29) and Q194 of the extracellular loop 2 (Q194ECL2) of the receptor (Figure 5A, B). The core of both tracers also displayed an aromatic stacking with phenylalanine 192 located in ECL2 (F192ECL2) within the orthosteric binding pocket (Figure 5A, B). Superimposition of I-MLT with PBI-8192 revealed that the pose adopted by PBI-8192 orients the fluorophore toward the previously described hydrophobic subpocket found within the MT2 receptor (Figure 5B, inset). This orientation seems to occur at the phenyl ring located at the equivalent C2 position of melatonin. The entry to this extra pocket has been described to be larger in MT2 than in MT1.18,20 The amino acid sequence and structural comparison of MT2 vs MT1 suggests that nonconserved V1283.40 and L2957.40 of MT2 could potentially be responsible for the enlarged entry to this subpocket in MT2. To experimentally verify our hypothesis, we replaced V1283.40 and L2957.40 by corresponding isoleucine and tyrosine found in MT1 in order to restrict binding of PBI-8192 to this hydrophobic pocket. Accordingly, we generated a MT2 double mutant by replacing leucine 295 in TM7 (L2957.40) with a tyrosine and valine 128 in TM3 (V1283.40) with an isoleucine. Binding of PBI-8192 tracer to the HiBiT-MT2-L295Y/V128I double mutant was undetectable in the NanoBRET assay despite successful expression of this mutant at the cell surface as monitored by the luminescence signal originating from HiBiT/LgBiT complementation (Figure 5C, inset). Importantly, the affinity of 2-[125I]-MLT for MT2 was not affected by the L295Y/V128I double mutations, 125 ± 19 vs 115 ± 60 pM, respectively, as shown in 2-[125I]-MLT saturation binding experiments (Figure 5D). These data support our docking results indicating that the pharmacophore headgroup of PBI-8192 occupies the orthosteric ligand binding pocket, and the linker and fluorophore part occupy the extended subpocket present in MT2. Selectivity of PBI-8192 for MT2 can be explained by the fact that this subpocket is more restricted in MT1.

Figure 5.

Figure 5

Open in a new tab

Determination of the PBI-8192 binding pocket within the human MT2 receptor. Docking of the PBI-8192 fluorescent tracer (A) or I-MLT (B) into the MT2 crystal structure (PDB: 6ME6). Inset shows an overlay of PBI-8192 and I-MLT docking. Mutation points within the crystal structure-based hMT2. Specific binding of increasing concentrations of PBI-8192 or 2-[125I]-MLT in NanoBRET (C) or radioligand (D) binding assays, respectively. Inset represents the HiBiT/LgBiT filtered luminescence signal at 460 nm for wild type and mutated HiBiT-hMT2 receptor. Data are represented as a mean ± SEM (C) or a representative experiment (D) of 2–4 experiments performed in duplicate.

Discussion

Here, we report the design and characterization of several fluorescent melatonin receptor ligands consisting of a red-emitting BODIPY NanoBRET 590 fluorophore that could be used as energy acceptors in the first fluorescence-based melatonin receptor NanoBRET ligand binding assays. Among them are PBI-8238, a MT1-selective tracer (Kd ∼ 400 nM), and PBI-8192, a MT2-selective tracer (Kd ∼ 50 nM). Pharmacological profiles generated for melatonin receptor reference ligands and new chemotypes matched well with the profiles generated using the classical 2-[125I]-MLT tracer. These new tracers and their implementation into NanoBRET-based ligand binding assays not only represent a nonradioactive alternative to the classical 2-[125I]-MLT tracer but, in addition, extend the list of existing assays toward real-time measurements of ligand binding by NanoBRET at the cell surface of living cells expressing HiBiT-tagged melatonin receptors. This is of particular interest for MT1, which is also expressed in intracellular compartments such as mitochondria.30

Recent advances in the design of fluorescent ligand binding assays combined with proximity-based approaches such as FRET and BRET greatly expanded the repertoire of pharmacological assays.31 NanoBRET-based ligand binding assays have become particularly popular due to their high sensitivity and specificity and the possibility to follow ligand–receptor interactions in living cells in real time.32 In addition, the HiBiT/LgBiT high-affinity complementation version of the NanoBRET ligand binding assay has the advantage to restrict the observation window to the plasma membrane due to the external addition of the LgBiT Nluc-derived fragment.22 This eliminates intracellular background signals that might otherwise arise from highly cell-permeable ligands such as melatonin and its derivatives25,33 reaching receptors expressed in intracellular compartments such as mitochondria in the case of melatonin receptors.30

The design of ligands with high affinity and specificity for the desired target is often the bottleneck in the development of such assays. Detailed structure–function knowledge is typically necessary to identify ligand positions that can be harnessed for linker/fluorophore functionalization. This is particularly challenging for small ligands but has been successfully accomplished for adenosine,34−37 histamine,38−40 and ß-adrenergic22,34 receptors. Melatonin receptors present a particularly difficult case in this respect. The natural ligand melatonin and synthetic high-affinity ligands are all relatively small,41,42 which reflects the small size of the orthosteric binding pocket and a narrow lateral ligand entry channel to reach this pocket from the membrane.17−20 This feature is very different from adenosine, histamine, and ß-adrenergic receptors, which all show a substantial opening of the ligand binding pocket toward the extracellular milieu that can accommodate the added linker/fluorophore without major space restrictions. In the MT2 receptor, there is an additional binding pocket extending toward the receptor core.18,20 MT2-selective ligands are typically designed to occupy this extended pocket, which is much smaller in MT1.43 Our molecular docking and mutagenesis studies indicate that the linker and the fluorophore of the MT2-selective PBI-8192 tracer fit well into this extended pocket while the melatonin pharmacophore fits into the orthosteric ligand binding site. The PBI-8298 tracer with the linker attached to the methoxy group of melatonin was designed to orient the fluorophore toward the lateral ligand entry channel as this methoxy group points toward the lateral channel in the crystal structures.17−20 PBI-8298, which contains the short linker, showed modest affinity for both MT1 and MT2 (Kd ∼ 1 μM) while its counterpart with a longer and more hydrophobic linker showed no specific binding.

The binding mode of the MT1-selective PBI-8238 tracer remains currently unknown. It is, however, remarkable that the addition of a benzene ring at the C2 position switches the molecule into the MT2-selective PBI-8192 tracer demonstrating that subtle changes at this position can determine melatonin receptor selectivity. In our molecular modeling studies of PBI-8192, the benzene ring appears to behave as a bend orienting the linker/fluorophore toward the extended binding pocket. In the absence of this benzene ring, the linker/fluorophore is not directed into this subpocket and might be oriented differently. One possible option might be a second, so-called “top”, ligand entry channel providing an opening toward the extracellular milieu. On the basis of the available crystal and cryo-EM structures, this channel remains currently speculative. Ligand dissociation studies with mutants in the lateral channel suggest that the lateral channel is more important, at least for ligand dissociation.18 Pharmacological studies with the only available hydrophilic melatonin receptor ligand, ICOA-13, support indeed a possible opening toward the extracellular milieu.25,44 Further structural studies and in particular molecular simulations will be necessary to capture the dynamics of this putative ligand entry channel to which the second extracellular loop significantly contributes44 to eventually propose a binding mode for the MT1-selective PBI-8238 tracer.

Apart from the fact that the PBI-8238 and PBI-8192 tracers show selectivity for each of the two melatonin receptor types, the overall selectivity of these tracers is also very good as tested in a large-scale screen against 50 other HiBiT-tagged GPCRs.23 On the basis of a selection of melatonin receptor reference agonists and antagonists and a series of MT2-selective agonists and inverse agonists with new chemotypes, we established that the competitive NanoBRET ligand binding assays using MT1-selective PBI-8238 tracer or MT2-selective PBI-8192 tracer reliably reproduce the pharmacological profile determined for these ligands with the classical 2-[125I]-MLT tracer. Notably, whereas pKi values obtained for reference antagonists matched very well between the PBI-8192 NanoBRET and the 2-[125I]-MLT binding assays, pKi values obtained for high-affinity agonists were about 1 log concentration lower in the NanoBRET assay vs the 2-[125I]-MLT assay. This difference is most likely due to the detection of the high-affinity ternary agonist/receptor/G protein complex by the 2-[125I]-MLT assay that can form in membrane preparations in the absence of free guanine nucleotide, as compared with the more transient ternary complexes in intact cells resulting in lower agonist binding affinities. A similar behavior was observed for the histamine H3 receptor in the NanoBRET assay.38 While the value for the live cells assays seems lower than the in vitro values using a radioligand, there is always the question of which values are more relevant to physiological conditions.

The PBI-8192 tracer competed not only with the classical melatonin receptor ligands but also with the new MT2-selective chemotypes. For these low-affinity agonists and inverse agonists, pKi values matched well between the NanoBRET and the 2-[125I]-MLT assay. For the MT1-selective PBI-8238 tracer, the pKi values did not show the agonist-specific shift toward lower affinities. Actually, for this tracer, pKi values for all compounds, irrespective of their functional nature (agonist/antagonist), were shifted toward lower values compared to those obtained with the 2-[125I]-MLT tracer.

Whether these systematically lower pKi values are related, at least in part, to differences in measurements in live cells vs membrane preparations remains a possibility to be further explored. Contact points of PBI-8238 with other parts of the binding pocket or the ligand entry channels that are not probed by 2-[125I]-MLT cannot be excluded either. A contribution of more extracellular parts like the HiBiT or HiBiT/LgBiT complex cannot be excluded formally but are unlikely to contribute as they are further away from the deep ligand binding pocket of MT1. The shift in pKi values restricts the use of the PBI-8238 tracer to molecules with affinities higher than 100 nM (referred to as the 2-[125I]-MLT tracer). A similar systematic shift of all ligand types has been observed for the histamine H4 receptor.38

In conclusion, our study reports the synthesis of several nonselective and subtype-selective fluorescent tracers for melatonin receptor NanoBRET ligand binding assays. Among them, the MT1-selective PBI-8238 tracer and the MT2-selective PBI-8192 tracer are the first tracers suitable for a nonradioactive melatonin receptor binding assay with the restriction of PBI-8238 for high-affinity ligands. These tools suggest new perspectives to study the pharmacology of melatonin receptors in real time in living cells and in a nonradioactive binding assay that could pave the way for the discovery of new drugs for these receptors.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00096.

  • Experimental procedures; pharmacological characterization of PBI-8192; saturation NanoBRET binding assay in HEK293 cells expressing HiBiT-tagged MT1 or MT2 receptors; competition curves of melatonergic reference compounds in 2-[125I]-MLT and PBI-8192 NanoBRET binding assays; pKi values of melatonergic reference compounds determined for nontagged human MT2 in 2-[125I]-MLT binding assay; pKd values of fluorescent tracers developed for melatonin human MT1 and MT2 receptors; pKi values of melatonergic reference ligands for MT1 determined in 2-[125I]-MLT and PBI-8238 NanoBRET binding assays (PDF)

Author Present Address

⊥ Firmenich SA, R&D North America, San Diego, CA 92121

Author Contributions

F.G., R.F.O., and R.J. designed the study; F.B., C.I., and G.S.B. conducted the pharmacological assays; S.L. planned and performed the organic synthesis of the molecules; I.G.T. performed the computational modeling experiments; F.G. and R.J. wrote the original draft; and F.G., S.L., I.G.T., R.F.O., and R.J. participated in the writing, reviewing, and editing. All coauthors approved the manuscript.

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), the Agence Nationale de la Recherche ((ANR-RA-COVID-19), (ANR-20-COV4-0001 to R.J.), (ANR-19-CE16-0025-01 to R.J.), and the “Who am I?” laboratory of excellence (ANR-11-LABX-0071; R.J.)) funded by the French Government through its “Investments for the Future” program operated by the French National Research Agency (ANR-11-IDEX-0005-01; R.J.). R.J. was supported by the Fondation de la Recherche Médicale (Equipe FRM DEQ20130326503) and La Ligue Contre le Cancer N/ref: RS19/75-127. G.S.B. was supported by a doctoral fellowship from the Fondation de la Recherche Médicale (FRM grant number ECO20170637544) obtained by R.J.

The authors declare no competing financial interest.

This paper was published ASAP on July 18, 2022, with the third author’s last name misspelled. The corrected version was reposted on July 19, 2022.

Special Issue

Published as part of the ACS Pharmacology & Translational Science virtual special issue " GPCR Signaling ".

Supplementary Material

pt2c00096_si_001.pdf (1.7MB, pdf)

References

  1. Yu H.; Dickson E. J.; Jung S. R.; Koh D. S.; Hille B. High membrane permeability for melatonin. J. Gen Physiol 2016, 147 (1), 63–76. 10.1085/jgp.201511526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Reiter R. J.; Tan D. X.; Kim S. J.; Cruz M. H. Delivery of pineal melatonin to the brain and SCN: role of canaliculi, cerebrospinal fluid, tanycytes and Virchow-Robin perivascular spaces. Brain Struct Funct 2014, 219 (6), 1873–1887. 10.1007/s00429-014-0719-7. [DOI] [PubMed] [Google Scholar]
  3. Dubocovich M. L.; Delagrange P.; Krause D. N.; Sugden D.; Cardinali D. P.; Olcese J. International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors. Pharmacol Rev. 2010, 62 (3), 343–380. 10.1124/pr.110.002832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Jockers R.; Delagrange P.; Dubocovich M. L.; Markus R. P.; Renault N.; Tosini G.; Cecon E.; Zlotos D. P. Update on Melatonin Receptors. IUPHAR Review. Br. J. Pharmacol. 2016, 173 (18), 2702–2725. 10.1111/bph.13536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cecon E.; Oishi A.; Jockers R. Melatonin receptors: molecular pharmacology and signalling in the context of system bias. Br. J. Pharmacol. 2018, 175 (16), 3263–3280. 10.1111/bph.13950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Oishi A.; Gbahou F.; Jockers R. Melatonin receptors, brain functions, and therapies. Handb Clin Neurol 2021, 179, 345–356. 10.1016/B978-0-12-819975-6.00022-4. [DOI] [PubMed] [Google Scholar]
  7. Liu J.; Clough S. J.; Hutchinson A. J.; Adamah-Biassi E. B.; Popovska-Gorevski M.; Dubocovich M. L. MT1 and MT2Melatonin Receptors: A Therapeutic Perspective. Annu. Rev. Pharmacol Toxicol 2016, 56, 361–83. 10.1146/annurev-pharmtox-010814-124742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cardinali D. P.; Vacas M. I.; Boyer E. E. Specific binding of melatonin in bovine brain. Endocrinology 1979, 105 (2), 437–41. 10.1210/endo-105-2-437. [DOI] [PubMed] [Google Scholar]
  9. Niles L. P. [3H] melatonin binding in membrane and cytosol fractions from rat and calf brain. J. Pineal Res. 1987, 4 (1), 89–98. 10.1111/j.1600-079X.1987.tb00844.x. [DOI] [PubMed] [Google Scholar]
  10. Vakkuri O.; Leppaluoto J.; Vuolteenaho O. Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as a tracer. Acta Endrocrinol. 1984, 106, 152–157. 10.1530/acta.0.1060152. [DOI] [PubMed] [Google Scholar]
  11. Browning C.; Beresford I.; Fraser N.; Giles H. Pharmacological characterization of human recombinant melatonin mt(1) and MT2 receptors. Br. J. Pharmacol. 2000, 129 (5), 877–886. 10.1038/sj.bjp.0703130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Legros C.; Devavry S.; Caignard S.; Tessier C.; Delagrange P.; Ouvry C.; Boutin J. A.; Nosjean O. Melatonin MT1 and MT2 receptors display different molecular pharmacologies only in the G-protein coupled state. Br. J. Pharmacol. 2014, 171 (1), 186–201. 10.1111/bph.12457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Legros C.; Matthey U.; Grelak T.; Pedragona-Moreau S.; Hassler W.; Yous S.; Thomas E.; Suzenet F.; Folleas B.; Lefoulon F.; Berthelot P.; Caignard D. H.; Guillaumet G.; Delagrange P.; Brayer J. L.; Nosjean O.; Boutin J. A. New Radioligands for Describing the Molecular Pharmacology of MT1 and MT2 Melatonin Receptors. Int. J. Mol. Sci. 2013, 14 (5), 8948–62. 10.3390/ijms14058948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Legros C.; Brasseur C.; Delagrange P.; Ducrot P.; Nosjean O.; Boutin J. A. Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors. J. Pharmacol Exp Ther 2016, 356 (3), 681–692. 10.1124/jpet.115.229989. [DOI] [PubMed] [Google Scholar]
  15. Bdair H.; Singleton T. A.; Ross K.; Jolly D.; Kang M. S.; Aliaga A.; Tuznik M.; Kaur T.; Yous S.; Soucy J. P.; Massarweh G.; Scott P. J. H.; Koeppe R.; Spadoni G.; Bedini A.; Rudko D. A.; Gobbi G.; Benkelfat C.; Rosa-Neto P.; Brooks A. F.; Kostikov A. Radiosynthesis and In Vivo Evaluation of Four Positron Emission Tomography Tracer Candidates for Imaging of Melatonin Receptors. ACS Chem. Neurosci. 2022, 13, 1382. 10.1021/acschemneuro.1c00678. [DOI] [PubMed] [Google Scholar]
  16. Borgarelli C.; Klingl Y. E.; Escamilla-Ayala A.; Munck S.; Van Den Bosch L.; De Borggraeve W. M.; Ismalaj E. Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins. Chemistry 2021, 27 (34), 8605–8641. 10.1002/chem.202100296. [DOI] [PubMed] [Google Scholar]
  17. Stauch B.; Johansson L. C.; McCorvy J. D.; Patel N.; Han G. W.; Huang X. P.; Gati C.; Batyuk A.; Slocum S. T.; Ishchenko A.; Brehm W.; White T. A.; Michaelian N.; Madsen C.; Zhu L.; Grant T. D.; Grandner J. M.; Shiriaeva A.; Olsen R. H. J.; Tribo A. R.; Yous S.; Stevens R. C.; Weierstall U.; Katritch V.; Roth B. L.; Liu W.; Cherezov V. Structural basis of ligand recognition at the human MT1 melatonin receptor. Nature 2019, 569 (7755), 284–288. 10.1038/s41586-019-1141-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johansson L. C.; Stauch B.; McCorvy J. D.; Han G. W.; Patel N.; Huang X. P.; Batyuk A.; Gati C.; Slocum S. T.; Li C.; Grandner J. M.; Hao S.; Olsen R. H. J.; Tribo A. R.; Zaare S.; Zhu L.; Zatsepin N. A.; Weierstall U.; Yous S.; Stevens R. C.; Liu W.; Roth B. L.; Katritch V.; Cherezov V. XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity. Nature 2019, 569 (7755), 289–292. 10.1038/s41586-019-1144-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Okamoto H. H.; Miyauchi H.; Inoue A.; Raimondi F.; Tsujimoto H.; Kusakizako T.; Shihoya W.; Yamashita K.; Suno R.; Nomura N.; Kobayashi T.; Iwata S.; Nishizawa T.; Nureki O. Cryo-EM structure of the human MT1-Gi signaling complex. Nat. Struct Mol. Biol. 2021, 28 (8), 694–701. 10.1038/s41594-021-00634-1. [DOI] [PubMed] [Google Scholar]
  20. Wang Q.; Lu Q.; Guo Q.; Teng M.; Gong Q.; Li X.; Du Y.; Liu Z.; Tao Y. Structural basis of the ligand binding and signaling mechanism of melatonin receptors. Nat. Commun. 2022, 13 (1), 454. 10.1038/s41467-022-28111-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Viault G.; Poupart S.; Mourlevat S.; Lagaraine C.; Devavry S.; Lefoulon F.; Bozon V.; Dufourny L.; Delagrange P.; Guillaumet G.; Suzenet F. Design, Synthesis and Biological Evaluation of Fluorescent Ligands for MT1 and/or MT2Melatonin Receptors. RSC Adv. 2016, 6, 62508–62521. 10.1039/C6RA10812A. [DOI] [Google Scholar]
  22. Boursier M. E.; Levin S.; Zimmerman K.; Machleidt T.; Hurst R.; Butler B. L.; Eggers C. T.; Kirkland T. A.; Wood K. V.; Friedman Ohana R. The luminescent HiBiT peptide enables selective quantitation of G protein-coupled receptor ligand engagement and internalization in living cells. J. Biol. Chem. 2020, 295 (15), 5124–5135. 10.1074/jbc.RA119.011952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Killoran M. P.; Levin S.; Boursier M. E.; Zimmerman K.; Hurst R.; Hall M. P.; Machleidt T.; Kirkland T. A.; Friedman Ohana R. An Integrated Approach toward NanoBRET Tracers for Analysis of GPCR Ligand Engagement. Molecules 2021, 26 (10), 2857. 10.3390/molecules26102857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ayoub M. A.; Levoye A.; Delagrange P.; Jockers R. Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers. Mol. Pharmacol. 2004, 66 (2), 312–21. 10.1124/mol.104.000398. [DOI] [PubMed] [Google Scholar]
  25. Gbahou F.; Cecon E.; Viault G.; Gerbier R.; Jean-Alphonse F.; Karamitri A.; Guillaumet G.; Delagrange P.; Friedlander R. M.; Vilardaga J. P.; Suzenet F.; Jockers R. Design and validation of the first cell-impermeant melatonin receptor agonist. Br. J. Pharmacol. 2017, 174 (14), 2409–2421. 10.1111/bph.13856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schrödinger. Maestro; Schrödinger, LLC: New York, 2020. [Google Scholar]
  27. Machleidt T.; Woodroofe C. C.; Schwinn M. K.; Mendez J.; Robers M. B.; Zimmerman K.; Otto P.; Daniels D. L.; Kirkland T. A.; Wood K. V. NanoBRET--A Novel BRET Platform for the Analysis of Protein-Protein Interactions. ACS Chem. Biol. 2015, 10 (8), 1797–804. 10.1021/acschembio.5b00143. [DOI] [PubMed] [Google Scholar]
  28. Stein R. M.; Kang H. J.; McCorvy J. D.; Glatfelter G. C.; Jones A. J.; Che T.; Slocum S.; Huang X. P.; Savych O.; Moroz Y. S.; Stauch B.; Johansson L. C.; Cherezov V.; Kenakin T.; Irwin J. J.; Shoichet B. K.; Roth B. L.; Dubocovich M. L. Virtual discovery of melatonin receptor ligands to modulate circadian rhythms. Nature 2020, 579 (7800), 609–614. 10.1038/s41586-020-2027-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ballesteros J. A.; Weinstein H.. Chapter 19. Integrated methods for the construction of three-dimensional models and computational probing of structure–function relations in G protein-coupled receptors. In Methods in Neurosciences; Sealfon S. C., Ed.; Academic Press: Cambridge, MA, 1995; Vol. 25, pp 366–428. [Google Scholar]
  30. Suofu Y.; Li W.; Jean-Alphonse F. G.; Jia J.; Khattar N. K.; Li J.; Baranov S. V.; Leronni D.; Mihalik A. C.; He Y.; Cecon E.; Wehbi V. L.; Kim J.; Heath B. E.; Baranova O. V.; Wang X.; Gable M. J.; Kretz E. S.; Di Benedetto G.; Lezon T. R.; Ferrando L. M.; Larkin T. M.; Sullivan M.; Yablonska S.; Wang J.; Minnigh M. B.; Guillaumet G.; Suzenet F.; Richardson R. M.; Poloyac S. M.; Stolz D. B.; Jockers R.; Witt-Enderby P. A.; Carlisle D. L.; Vilardaga J. P.; Friedlander R. M. Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (38), E7997–E8006. 10.1073/pnas.1705768114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stoddart L. A.; White C. W.; Nguyen K.; Hill S. J.; Pfleger K. D. Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding. Br. J. Pharmacol. 2016, 173 (20), 3028–37. 10.1111/bph.13316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stoddart L. A.; Kilpatrick L. E.; Hill S. J. NanoBRET Approaches to Study Ligand Binding to GPCRs and RTKs. Trends Pharmacol. Sci. 2018, 39 (2), 136–147. 10.1016/j.tips.2017.10.006. [DOI] [PubMed] [Google Scholar]
  33. Thireau J.; Marteaux J.; Delagrange P.; Lefoulon F.; Dufourny L.; Guillaumet G.; Suzenet F. Original Design of Fluorescent Ligands by Fusing BODIPY and Melatonin Neurohormone. ACS Med. Chem. Lett. 2014, 5 (2), 158–61. 10.1021/ml4004155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stoddart L. A.; Johnstone E. K. M.; Wheal A. J.; Goulding J.; Robers M. B.; Machleidt T.; Wood K. V.; Hill S. J.; Pfleger K. D. G. Application of BRET to monitor ligand binding to GPCRs. Nat. Methods 2015, 12 (7), 661–663. 10.1038/nmeth.3398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Bouzo-Lorenzo M.; Stoddart L. A.; Xia L.; IJzerman I. J.; Heitman L. H.; Briddon S. J.; Hill S. J. A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor. Purinergic Signal 2019, 15 (2), 139–153. 10.1007/s11302-019-09650-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Comeo E.; Kindon N. D.; Soave M.; Stoddart L. A.; Kilpatrick L. E.; Scammells P. J.; Hill S. J.; Kellam B. Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A2A Receptor. J. Med. Chem. 2020, 63 (5), 2656–2672. 10.1021/acs.jmedchem.9b01856. [DOI] [PubMed] [Google Scholar]
  37. Comeo E.; Trinh P.; Nguyen A. T.; Nowell C. J.; Kindon N. D.; Soave M.; Stoddart L. A.; White J. M.; Hill S. J.; Kellam B.; Halls M. L.; May L. T.; Scammells P. J. Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells. J. Med. Chem. 2021, 64 (10), 6670–6695. 10.1021/acs.jmedchem.0c02067. [DOI] [PubMed] [Google Scholar]
  38. Mocking T. A. M.; Verweij E. W. E.; Vischer H. F.; Leurs R. Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H3 and H4 Receptors on Living Cells. Mol. Pharmacol. 2018, 94 (6), 1371–1381. 10.1124/mol.118.113373. [DOI] [PubMed] [Google Scholar]
  39. Gratz L.; Tropmann K.; Bresinsky M.; Muller C.; Bernhardt G.; Pockes S. NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells. Sci. Rep 2020, 10 (1), 13288. 10.1038/s41598-020-70332-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rosier N.; Gratz L.; Schihada H.; Möller J.; Işbilir A.; Humphrys L. J.; Nagl M.; Seibel U.; Lohse M. J.; Pockes S. A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor. J. Med. Chem. 2021, 64 (15), 11695–11708. 10.1021/acs.jmedchem.1c01089. [DOI] [PubMed] [Google Scholar]
  41. Zlotos D. P.; Jockers R.; Cecon E.; Rivara S.; Witt-Enderby P. A. MT1 and MT2Melatonin Receptors: Ligands, Models, Oligomers, and Therapeutic Potential. J. Med. Chem. 2014, 57 (8), 3161–3185. 10.1021/jm401343c. [DOI] [PubMed] [Google Scholar]
  42. Liu L.; Jockers R. Structure-Based Virtual Screening Accelerates GPCR Drug Discovery. Trends Pharmacol. Sci. 2020, 41 (6), 382–384. 10.1016/j.tips.2020.04.001. [DOI] [PubMed] [Google Scholar]
  43. Boutin J. A.; Witt-Enderby P. A.; Sotriffer C.; Zlotos D. P. Melatonin receptor ligands: A pharmaco-chemical perspective. J. Pineal Res. 2020, 69 (3), e12672. 10.1111/jpi.12672. [DOI] [PubMed] [Google Scholar]
  44. Cecon E.; Liu L.; Jockers R. Melatonin receptor structures shed new light on melatonin research. J. Pineal Res. 2019, 67 (4), e12606. 10.1111/jpi.12606. [DOI] [PubMed] [Google Scholar]
  45. Karamitri A.; Sadek M. S.; Journe A. S.; Gbahou F.; Gerbier R.; Osman M. B.; Habib S. A. M.; Jockers R.; Zlotos D. P. O-linked melatonin dimers as bivalent ligands targeting dimeric melatonin receptors. Bioorganic Chemistry 2019, 85, 349–356. 10.1016/j.bioorg.2019.01.004. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pt2c00096_si_001.pdf (1.7MB, pdf)

Articles from ACS Pharmacology & Translational Science are provided here courtesy of American Chemical Society

RESOURCES