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The development of novel ligands for G protein-coupled receptors (GPCRs), representing important drug targets, requires the determination of ligand-receptor affinities. This has primarily been done by radioligand competition binding experiments. However, the use of radioligands is disadvantageous with respect to safety concerns, legal handling regulations and waste disposal. Thus, alternative and radioactivity-free methods for studying ligand-receptor binding are needed. Over the last decades, techniques using fluorescent ligands have emerged as a promising option. A recently reported luminescence-based binding assay makes use of the phenomenon of BRET (bioluminescence resonance energy transfer). The N-terminus of the receptor of interest was tagged with a small luciferase, which generates bioluminescence upon the addition of its substrate (e.g. furimazine). When a fluorescent ligand binds to the receptor, BRET can occur due to the close proximity of the luciferase and the fluorescent ligand. The BRET signal correlates with the amount of receptor-bound ligand and, in contrast to radioligand binding assays, ligand binding can be measured in real time with high temporal resolution. The aim of this thesis was the development of BRET-based binding assays for different GPCRs as alternative methods to radioligand binding assays. First, a BRET-based binding assay was developed for the histamine H2 receptor (H2R). For this purpose, the H2R, N-terminally fused to the NanoLuc® (NLuc), was stably expressed in HEK293T cells. Out of three differently labeled fluorescent H2R ligands, which were investigated in BRET saturation binding experiments, the Py-1-labeled ligand 2.1 turned out to be the most favorable in terms of ligand affinity and signal-to-background (S/B) ratio. The receptor binding kinetics of 2.1 were studied in real time and competition binding experiments with 2.1 and several reported H2R ligands afforded H2R binding data in accordance with literature values. Furthermore, a BRET-based binding assay was developed for the M2 muscarinic acetylcholine receptor (M2R). Two TAMRA-labeled M2R ligands (3.1 and 3.2) were characterized by saturation binding, association and dissociation kinetic (in real time) and, for 3.1, competition binding experiments with standard MR ligands. As the fluorophore TAMRA is also suited for fluorescence anisotropy (FA) studies, the BRET-based M2R binding assay was compared with an FA-based real-time binding assay, also using 3.1 and 3.2 as probes. Both methods, representing different methodologies, yielded similar results (ligand binding affinities from saturation binding and competition binding studies). Kinetic data from both assays revealed a complex binding behavior for 3.1 and 3.2 (e.g. biphasic dissociation kinetics), being most pronounced for 3.2. In the case of the neuropeptide Y Y1 receptor (Y1R) or the neurotensin receptor 1 (NTS1R), both exhibiting long N-termini, application of the reported concept (N-terminal fusion of the GPCR to NLuc) failed to establish BRET-based binding assays for these receptors. Obviously, their longer N-termini led to an increased distance or an unfavorable position of the luciferase relative to the fluorescent ligand, precluding efficient BRET. Therefore, an alternative strategy was developed by inserting the luciferase into the second extracellular loop of the Y1R and the NTS1R. This novel approach resulted in feasible BRET binding assays. The same strategy, which is considered a widely applicable method, was applied to two other GPCRs with comparably short N-termini, the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R). When compared with the N-terminally NLuc-tagged AT1R and M1R, insertion of NLuc into ECL2 of the receptors led to ligand affinities in better accordance with literature data, and/or higher S/B ratios. Finally, a split luciferase-based assay was developed to assess the recruitment of the G protein coupled receptor kinase 2 (GRK2) to three different receptors. Besides the possibility of generating concentration-response curves, the novel assay allowed analyzing GRK2 recruitment in a time-dependent manner. It revealed different recruitment kinetics for the NTS1R, which showed a more sustained interaction with GRK2, when compared with the M1R and the M5R, both showing a short-lasting interaction with GRK2. This suggested a potential classification of GPCRs based on their differential interaction with GRKs., Ein wichtiges Element bei der Entwicklung neuer Liganden für G Protein-gekoppelte Rezeptoren (GPCRs), welche bedeutsame Zielstrukturen für Arzneistoffe darstellen, ist die Bestimmung der Ligand-Rezeptoraffinitäten. Klassischerweise erfolgen die Affinitätsbestimmungen in Radioligandkompetitionsassays. Allerdings hat die Verwendung von radioaktiv markierten Substanzen Nachteile z.B. in Bezug auf Arbeitssicherheit oder Abfallentsorgung. Daher besteht ein großer Bedarf an alternativen Methoden ohne Einsatz von Radioaktivität zur Untersuchung der Ligand-Rezeptor-Bindung. In den letzten Jahrzehnten stellten sich besonders fluoreszenzbasierte Methoden als vielversprechende Option heraus. So nutzt ein erst kürzlich beschriebener Bindungsassay das Phänomen des Biolumineszenz-Resonanzenergietransfers (BRET). Der N-Terminus des untersuchten Rezeptors wird hierbei mit einer kleinen Luciferase markiert, die nach Zugabe ihres Substrats (z.B. Furimazin) in der Lage ist, Biolumineszenz zu erzeugen. Wenn ein Fluoreszenzligand an den Rezeptor bindet, kommt es aufgrund der unmittelbaren Nähe der Luciferase zum Fluorophor des Liganden zu einem Energieübertrag mittels BRET. Das BRET-Signal korreliert dabei mit der Menge des rezeptorgebundenen Liganden. Im Gegensatz zu Radioligand-Bindungsassays kann der Prozess der Rezeptorbindung in Echtzeit mit hoher zeitlicher Auflösung untersucht werden. Ziel dieser Arbeit war die Entwicklung von derartigen BRET-basierten Bindungsassays für verschiedene GPCRs als Alternative zu den klassischen Radioligand-Bindungsassays. Zunächst wurde ein BRET-basierter Bindungsassay für den Histamin H2-Rezeptor (H2R) entwickelt. Dazu wurde der Rezeptor, an dessen N-Terminus die NanoLuc® (NLuc) angehängt wurde, stabil in HEK293T-Zellen exprimiert. Von den drei untersuchten Fluoreszenzliganden, die unterschiedliche Fluoreszenzlabel trugen, erwies sich der Py-1-markierte Ligand 2.1 als der beste Kompromiss bezüglich der Rezeptoraffinität und des Signal-Hintergrund-Verhältnisses. Die Bindungskinetik von 2.1 wurde in Echtzeit untersucht und für verschiedene beschriebene H2R-Liganden konnten in Verdrängungsexperimenten mit 2.1 Bindungskonstanten bestimmt werden, die mit Literaturdaten übereinstimmten. Des Weiteren wurde ein BRET-basierter Bindungsassay für den muskarinischen Acetylcholin-Rezeptor M2 (M2R) entwickelt. Zwei TAMRA-markierte Liganden (3.1 und 3.2) wurden durch Sättigungs- und kinetische Experimente charakterisiert. Zudem wurden durch Verdrängungsexperimente mit 3.1 auch die Affinitäten verschiedener Standardliganden des M2R bestimmt. Da der Fluorophor TAMRA auch für Fluoreszenzanisotropie (FA)-basierte Studien geeignet ist, wurden die Resultate des BRET-Assays auch mit einem FA-basierten Echtzeit-Bindungsassay verglichen, ebenfalls unter Verwendung der Liganden 3.1 und 3.2. Beide Methoden, obwohl sie auf unterschiedlichen Phänomenen beruhen, produzierten vergleichbare Ergebnisse. Zudem wurde in beiden Assays ein komplexes Bindungsverhalten für 3.1 und in größerem Ausmaß für 3.2 beobachtet (z.B. biphasische Dissoziationskinetik). Für den Neuropeptid Y Y1-Rezeptor (Y1R) und den Neurotensin-1-Rezeptor (NTS1R), welche beide lange N-Termini aufweisen, konnte das beschriebene Konzept, d.h. Fusion der NanoLuc® an den N-Terminus, nicht angewendet werden. Es wurde kein spezifisches BRET-Signal detektiert, da ihre längeren extrazellulären N-Termini offensichtlich in einem zu großen Abstand oder einer ungünstigen Orientierung der Luciferase zum Fluoreszenzliganden resultierten. Daher wurde im Laufe der Arbeit eine alternative Strategie entwickelt, indem die Luciferase in die zweite extrazelluläre Schleife des entsprechenden Rezeptors eingefügt wurde. Dieser neuartige Ansatz ermöglichte die Detektion eines spezifischen BRET-Signals und führte dadurch zu robusten BRET-Bindungsassays. Um die breitere Anwendbarkeit zu demonstrieren, wurde die gleiche Strategie auf zwei GPCRs mit kürzeren N-Termini übertragen, den Angiotensin II-Typ 1-Rezeptor (AT1R) und den muskarinischen Acetylcholin-Rezeptor M1 (M1R). Verglichen mit den N-terminal markierten Rezeptoren, führte die Insertion der Luciferase in die zweite extrazelluläre Schleife zu Bindungskonstanten in besserer Übereinstimmung mit Radioligand-Bindungsdaten bzw. zu höheren Signal-Hintergrund-Verhältnissen. Schließlich wurde ein Split-Luciferase-Komplementationsassay entwickelt, um die Rekrutierung der G Protein-gekoppelten Rezeptorkinase 2 (GRK2) an unterschiedliche GPCRs zu untersuchen. Dieser Assay ermöglichte neben der Erstellung von Konzentrations-Wirkungskurven auch die zeitaufgelöste Analyse der GRK2-Rekrutierung. Hierbei zeigte sich, dass die Interaktion der GRK2 mit dem M1R und M5R deutlich kurzlebiger war im Vergleich zum NTS1R. Diese Unterschiede deuteten darauf hin, dass GPCRs auch basierend auf ihrer Interaktion mit GRKs klassifiziert werden können.

Luminescence-based techniques play an increasingly important role in all areas of biochemical research, including investigations on G protein-coupled receptors (GPCRs). One quite recent and popular addition has been made by introducing bioluminescence resonance energy transfer (BRET)-based binding assays for GPCRs, which are based on the fusion of nanoluciferase (Nluc) to the N-terminus of the receptor and the occurring energy transfer via BRET to a bound fluorescent ligand. However, being based on BRET, the technique is strongly dependent on the distance/orientation between the luciferase and the fluorescent ligand. Here we describe an alternative strategy to establish BRET-based binding assays for GPCRs, where the N-terminal fusion of Nluc did not result in functioning test systems with our fluorescent ligands (e.g., for the neuropeptide Y Y1 receptor (Y1R) and the neurotensin receptor type 1 (NTS1R)). Instead, we introduced Nluc into their second extracellular loop and we obtained binding data for the fluorescent ligands and reported standard ligands (in saturation and competition binding experiments, respectively) comparable to data from the literature. The strategy was transferred to the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R), which led to affinity estimates comparable to data from radioligand binding experiments. Additionally, an analysis of the binding kinetics of all fluorescent ligands at their respective target was performed using the newly described receptor/Nluc-constructs.

Fig. S1 Saturation binding of UR-CG072 binding to live CHO-K1 cells. The CHO-K1 cells (30000 cells/well) were incubated for 5 h with two-fold serial dilutions of UR-CG072 in the range of 0–8 nM and with (nonspecific binding, ●) or without (total binding, ∆) 3.7 µM scopolamine. The background-corrected cells’ fluorescence intensities were determined by the cell detection algorithm as described in Materials and Methods and are presented as individual replicates from an experiment performed in duplicate. Four images from different fields of view were taken from a single well.

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTP&gamma, S, [&gamma, 32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z&rsquo, factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTP&gamma, S binding assay.

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D2long and D3 receptors and measure time-resolved β-arrestin2 recruitment to the D2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no β-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the β-arrestin recruitment to the D2longR and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/β-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.