A general method for quantifying ligand binding to unmodified receptors using Gaussia luciferase
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100366
ISSN1083-351X
AutoresAndrás Dávid Tóth, Dániel Garger, Susanne Prokop, Eszter Soltész‐Katona, Péter Várnai, András Balla, Gábor Turu, László Hunyady,
Tópico(s)Molecular Communication and Nanonetworks
ResumoReliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer–based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development. Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer–based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development. The superfamily of plasma membrane (PM) receptors consists of a diverse range of signaling proteins, such as G protein–coupled receptors (GPCRs), tyrosine kinase receptors, enzyme-linked receptors, nutrient receptors, or ion channels. In addition to their important role in sensing environmental signals, their altered function is commonly reported in a wide array of pathological conditions. Accordingly, most of our currently prescribed drugs target cell surface receptors (1Santos R. Ursu O. Gaulton A. Bento A.P. Donadi R.S. Bologa C.G. Karlsson A. Al-Lazikani B. Hersey A. Oprea T.I. Overington J.P. A comprehensive map of molecular drug targets.Nat. Rev. Drug Discov. 2017; 16: 19-34Crossref PubMed Scopus (857) Google Scholar). A growing number of evidences indicates that characteristics of drug‒receptor interaction may profoundly shape the pharmacological outcome. In addition to affinity and efficacy, kinetic ligand parameters were also shown to be decisive factors for the clinical action of drugs. Association and dissociation rate constants (kon and koff) of drugs, by defining ligand residence time, have been linked to various drug properties, such as duration of action, efficacy, or occurrence of side effects (2Copeland R.A. Pompliano D.L. Meek T.D. Drug–target residence time and its implications for lead optimization.Nat. Rev. Drug Discov. 2006; 5: 730-739Crossref PubMed Scopus (998) Google Scholar, 3de Witte W.E.A. Danhof M. van der Graaf P.H. de Lange E.C.M. In vivo target residence time and kinetic selectivity: The association rate constant as determinant.Trends Pharmacol. Sci. 2016; 37: 831-842Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 4Klein Herenbrink C. Sykes D.A. 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Although various techniques with different advantageous properties are available, they all have serious drawbacks (8Stoddart L.A. Kilpatrick L.E. Hill S.J. NanoBRET approaches to study ligand binding to GPCRs and RTKs.Trends Pharmacol. Sci. 2018; 39: 136-147Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). For example, traditional radioligand binding measurements are ponderous and burdensome, especially in case of kinetic binding measurements, and the strict rules required for handling radioactive compounds limit their use. In contrast, fluorescence-based approaches are not hindered by the practical limitations surrounding the use of radioactivity but often suffer from low signal-to-noise ratio due to the autofluorescence of biological samples. By overcoming these limitations, resonance energy transfer–based measurements have revolutionized the application of fluorescent ligands in binding measurements (9Castro M. Nikolaev V.O. Palm D. Lohse M.J. Vilardaga J.-P. Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16084-16089Crossref PubMed Scopus (137) Google Scholar, 10Lohse M.J. Nuber S. Hoffmann C. Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling.Pharmacol. Rev. 2012; 64: 299-336Crossref PubMed Scopus (230) Google Scholar, 11Emami-Nemini A. Roux T. Leblay M. Bourrier E. Lamarque L. Trinquet E. Lohse M.J. Time-resolved fluorescence ligand binding for G protein–coupled receptors.Nat. Protoc. 2013; 8: 1307-1320Crossref PubMed Scopus (48) Google Scholar, 12Stoddart 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: 661-663Crossref PubMed Scopus (147) Google Scholar, 13Stoddart L.A. White C.W. Nguyen K. Hill S.J. Pfleger K.D.G. Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.Br. J. Pharmacol. 2016; 173: 3028-3037Crossref PubMed Scopus (68) Google Scholar). In these assays, resonance energy transfer is detected between a fluorescent or luminescent donor attached to a receptor and a fluorophore conjugated to a receptor ligand. Since the efficiency of resonance energy transfer strongly depends on the molecular proximity, a condition that is met during ligand binding, the signal-to-noise ratio is greatly enhanced. Time-resolved Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) have been successfully used to detect ligand binding of various receptors for equilibrium and kinetic measurements as well. However, these methods require the covalent labeling of the target protein, which may substantially alter the receptor function. Furthermore, the high cost of some substrates may limit their use in high-throughput applications. Therefore, there is a continuous need for new and surpassing assays to assess ligand‒receptor binding. Here we report a novel BRET-based approach to measure ligand binding of cell surface receptors. The method is cost-effective, does not need receptor modification, and is also applicable in high-throughput drug screenings. Since N-terminal receptor tagging with naturally nonsecreted and high-molecular-weight luciferases (such as Renilla luciferase) induces endoplasmic retention and impaired PM expression of several receptors, it was not possible to study receptor‒ligand binding with BRET for a long time (8Stoddart L.A. Kilpatrick L.E. Hill S.J. NanoBRET approaches to study ligand binding to GPCRs and RTKs.Trends Pharmacol. Sci. 2018; 39: 136-147Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This limitation has been overcome by the development of the small luciferase NanoLuciferase (NanoLuc) (14Hall M.P. Unch J. Binkowski B.F. Valley M.P. Butler B.L. Wood M.G. Otto P. Zimmerman K. Vidugiris G. Machleidt T. Robers M.B. Benink H.A. Eggers C.T. Slater M.R. Meisenheimer P.L. et al.Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.ACS Chem. Biol. 2012; 7: 1848-1857Crossref PubMed Scopus (723) Google Scholar), a variant of the naturally secreted luciferase of the deep-sea shrimp Oplophorus gracilirostris, which has made BRET suitable to detect ligand binding of NanoLuc-tagged receptors. However, the high cost of furimazine, the proper substrate of NanoLuc, seriously hinders the widespread application, especially in high-throughput experiments. Therefore, we aimed to establish a system using an alternative coelenterazine-utilizing luciferase that is also secreted naturally; thus, it may not induce incorrect folding of proteins tagged on the extracellular side. The luciferase of the marine copepod Gaussia princeps (Gaussia luciferase, GLuc) has the same molecular weight as NanoLuc (19 kDa), but its appropriate substrate is a natural substance, the native coelenterazine (Fig. 1A) (15Kaskova Z.M. Tsarkova A.S. Yampolsky I.V. 1001 lights: Luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine.Chem. Soc. Rev. 2016; 45: 6048-6077Crossref PubMed Google Scholar). Since GLuc is rapidly inactivated, we used a mutant of GLuc (GLucM23) (16Lindberg E. Mizukami S. Ibata K. Fukano T. Miyawaki A. Kikuchi K. Development of cell-impermeable coelenterazine derivatives.Chem. Sci. 2013; 4: 4395Crossref Scopus (15) Google Scholar), which emits light substantially longer and was shown to be even 10-fold brighter than the wildtype enzyme. First, we compared the properties of GLuc and NanoLuc with constructs that label the extracellular surface of the PM (GLuc‒PM and NanoLuc‒PM) by fusing them to a transmembrane domain. We also created versions of the constructs that were intracellularly tagged with Venus to perform expression-normalized comparisons (Fig. 1B). Using bioluminescent and fluorescent image acquisition, we verified that all constructs had proper PM localization (Fig. 1C). As shown in Figure 1, D and E, GLuc was found to have extreme brightness, was even brighter than NanoLuc, and both luciferases produced long-lasting (glow-type) luminescence (Fig. 1D). We tested the effect of different substrates in multiple concentrations. In agreement with previous studies, GLuc was the brightest when native coelenterazine was used (15Kaskova Z.M. Tsarkova A.S. Yampolsky I.V. 1001 lights: Luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine.Chem. Soc. Rev. 2016; 45: 6048-6077Crossref PubMed Google Scholar, 16Lindberg E. Mizukami S. Ibata K. Fukano T. Miyawaki A. Kikuchi K. Development of cell-impermeable coelenterazine derivatives.Chem. Sci. 2013; 4: 4395Crossref Scopus (15) Google Scholar). The GLuc-emitted luminescence was already sufficiently detectable in the presence of 5 μM coelenterazine; therefore, this concentration was used in the experiments. Similar to previous reports (17Inouye S. Sato J. Sahara-Miura Y. Yoshida S. Kurakata H. Hosoya T. C6-Deoxy coelenterazine analogues as an efficient substrate for glow luminescence reaction of nanoKAZ: The mutated catalytic 19kDa component of Oplophorus luciferase.Biochem. Biophys. Res. Commun. 2013; 437: 23-28Crossref PubMed Scopus (34) Google Scholar, 18Yano H. Cai N.S. Javitch J.A. Ferré S. Luciferase complementation based-detection of G-protein-coupled receptor activity.Biotechniques. 2018; 65: 9-14Crossref PubMed Scopus (8) Google Scholar), we found that NanoLuc efficiently utilizes some other coelenterazine derivatives as well, such as 2-deoxycoelenterazine (coelenterazine h). Although coelenterazine h was shown to be inferior as a substrate of NanoLuc compared with furimazine, it still induced sufficient brightness to perform BRET measurements in our setup. We compared the emission spectra of GLuc and NanoLuc, and the emission of the former was right-shifted by approximately 20 nm (Fig. 1F), which may be beneficial for the excitation of red-emitting acceptors. To test the performance of GLuc in BRET assays, we measured receptor‒ligand BRET in a similar manner as it was described in a previous study with NanoLuc (Fig. 2A) (12Stoddart 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: 661-663Crossref PubMed Scopus (147) Google Scholar). We expressed GLuc- or NanoLuc-tagged AT1 angiotensin receptor (AT1R) in HEK 293T cells as BRET donors and treated them with red fluorophore–conjugated angiotensin II (TAMRA‒AngII) as BRET acceptor for 2 h at room temperature to reach equilibrium binding (Fig. 2, B and C). In both cases, the binding of TAMRA‒AngII to the tagged receptors resulted in an increase of the BRET signal, as the molecular proximity between the donor and the acceptor caused resonance energy transfer. The increase of the BRET ratio was higher with GLuc‒AT1R, for which a possible explanation could be the greater overlap between the excitation spectrum of TAMRA and the emission spectrum of GLuc than that of NanoLuc. The specificity of the signal was verified by competitive ligand binding measurements. The competitive AT1R antagonist candesartan prevented a great portion of the BRET signal, proving that this part of the signal originated from specific interaction between TAMRA‒AngII and AT1R. The remaining nonspecific signal reflects random collisions between the donor and acceptor molecules (bystander BRET), whose amplitude is linearly proportional to the acceptor concentration. Accordingly, increasing concentrations of TAMRA‒AngII elevated the nonspecific signal linearly, whereas the specific binding was saturable (Fig. 2D). To avoid possible receptor internalization–related changes in the signal, we always overexpressed an internalization inhibitor protein, the dominant negative form of dynamin2A (DN-Dyn). We confirmed that its application does not disturb the ligand binding properties of the receptor (Fig. S1, A and B). We also tested GLuc in receptor‒ligand BRET measurements for another GPCR, the α1A adrenergic receptor (α1AAR) (Fig. 2, E and F). The green-emitting fluorescent BODIPY FL‒prazosin was applied as the labeled ligand, the α1AAR antagonist prazosin and carvedilol or the α1AAR agonist A61603 were used as unlabeled ligands. Again, the ligand binding of the tagged receptor could be detected with GLuc. The tracer ligand was displaced by all α1AAR ligands, representing that binding of any orthosteric ligand can be measured. It must be emphasized that the phenomenon of bystander BRET not only denotes a background signal but can also be beneficially exploited. Bystander BRET measurements are widely used to detect enrichment of a protein in a particular compartment, where a compartment-targeted molecule and the protein of interest are labeled with BRET partners. The labeling can even be indirect by tagging an interaction partner of the protein, which has the advantage that no molecular modification of the protein under investigation is required. Using bystander BRET, cellular redistribution of proteins, such as receptor internalization and β-arrestin recruitment, or lipid levels in the PM were previously successfully monitored (19Lan T.-H. Liu Q. Li C. Wu G. Lambert N.A. Sensitive and high resolution localization and tracking of membrane proteins in live cells with BRET.Traffic. 2012; 13: 1450-1456Crossref PubMed Scopus (59) Google Scholar, 20Balla A. Tóth D.J. Soltész-Katona E. Szakadáti G. Erdélyi L.S. Várnai P. Hunyady L. Mapping of the localization of type 1 angiotensin receptor in membrane microdomains using bioluminescence resonance energy transfer-based sensors.J. Biol. Chem. 2012; 287: 9090-9099Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 21Gyombolai P. Tóth A.D. Tímár D. Turu G. Hunyady L. Mutations in the "DRY" motif of the CB1 cannabinoid receptor result in biased receptor variants.J. Mol. Endocrinol. 2015; 54: 75-89Crossref PubMed Scopus (21) Google Scholar, 22Namkung Y. Le Gouill C. Lukashova V. Kobayashi H. Hogue M. Khoury E. Song M. Bouvier M. Laporte S.A. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.Nat. Commun. 2016; 7: 12178Crossref PubMed Scopus (112) Google Scholar, 23Tóth J.T. Gulyás G. Tóth D.J. Balla A. Hammond G.R.V. Hunyady L. Balla T. Várnai P. BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.Biochim. Biophys. Acta. 2016; 1861: 177-187Crossref PubMed Scopus (30) Google Scholar). In these experiments, the acceptor accumulation in the donor's proximity results in the elevation of the BRET signal. We applied a strategy based on similar principles to assess ligand binding of unmodified receptors. Instead of measuring BRET between luciferase-tagged receptors and their fluorescent ligands ("receptor‒ligand BRET"), we detected bystander BRET between luciferase-labeled PM and fluorescent ligands bound to their receptors ("PM‒ligand BRET") (Fig. 2G). We coexpressed untagged AT1R with GLuc‒PM or NanoLuc‒PM constructs that label the extracellular side of the PM (Fig. 2, H and I). Treatment of cells with TAMRA‒AngII induced an increase of the BRET signal between the PM-targeted donor and TAMRA‒AngII. This signal originated from two types of bystander BRET. A significant portion of the signal could be prevented by the AT1R antagonist candesartan, showing that this signal reflects the specific interaction between TAMRA‒AngII and untagged AT1R, which caused the enrichment of TAMRA‒AngII at the PM. Accordingly, this signal was absent when no receptor was expressed (Fig. 2, J and K). The candesartan-insensitive part of the signal was caused by the other (nonspecific) type of bystander BRET (Fig. 2L), which is due to random collisions between PM–tagged luciferases and unbound acceptor-conjugated ligands. This candesartan-insensitive signal is similar and equally high as in receptor‒ligand BRET measurement (see Fig. 2, D and L). PM‒ligand BRET, in comparison with receptor‒ligand BRET, showed lower amplitude of the specific signal (ΔBRET values of the different setups are shown in Fig. 2, B, C, J, and K). This was in agreement with the fact that resonance energy transfer is greatly sensitive to the distance between donor and acceptor molecules, which is larger when the PM is labeled and not the target receptor. Remarkably, the half-maximal inhibitory concentration of candesartan for the high-affinity binding site (IC50_Hi) was significantly lower in the case of the receptor‒ligand BRET than that of the PM‒ligand BRET (Fig. S1, C and D). Accordingly, the dissociation constant of TAMRA‒AngII for the high-affinity binding site (KD_Hi) of the untagged receptor was smaller than the KD_Hi for the GLuc-labeled receptor (Fig. 2, D and L), demonstrating that receptor tagging altered the ligand binding properties of the receptor. These results are in good agreement with previous studies showing altered ligand binding properties of AT1R upon N-terminal modification (24Zhang H. Unal H. Desnoyer R. Han G.W. Patel N. Katritch V. Karnik S.S. Cherezov V. Stevens R.C. Structural basis for ligand recognition and functional selectivity at angiotensin receptor.J. Biol. Chem. 2015; 290: 29127-29139Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 25Wingler L.M. McMahon C. Staus D.P. Lefkowitz R.J. Kruse A.C. Distinctive activation mechanism for angiotensin receptor revealed by a synthetic nanobody.Cell. 2019; 176: 479-490.e12Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and suggest that, although receptor‒ligand BRET is a sensitive approach to detect ligand binding, the results obtained with this method should be interpreted cautiously. Since we achieved larger BRET signal changes with GLuc than with NanoLuc in our system (compare Fig. 2, H with I and Fig. 2, J with K), only GLuc BRET measurements were performed in the further experiments. In principle, a ligand binding assay should be able to detect the binding of any orthosteric ligands regardless of their functional effects. In accordance, we were able to determine the binding of both the high-affinity full agonist angiotensin II (AngII) and the low-affinity β-arrestin‒biased agonist [Sar1,Ile4,Ile8]-angiotensin II (SII) (26Szakadáti G. Tóth A.D. Oláh I. Erdélyi L.S. Balla T. Várnai P. Hunyady L. Balla A. Investigation of the fate of type I angiotensin receptor after biased activation.Mol. Pharmacol. 2015; 87: 972-981Crossref PubMed Scopus (22) Google Scholar) with our assay (Fig. 2M). We also measured the ligand binding of α1AAR with the PM‒ligand BRET setup (Fig. 2, N and O). With this approach, we were able to measure the competition binding of all three unlabeled ligands, and no significant shift of competition binding curves was observed compared with GLuc‒α1AAR, suggesting that the effect of N-terminal receptor tagging on receptor conformation may vary between GPCRs. We tested if the amplitude of the specific signal correlates with the amount of the receptor DNA transfected (Fig. S2). We found a positive correlation for both AT1R and α1AAR, suggesting that a high level of receptor expression is advantageous for good signal-to-noise separation. Theoretically, our PM‒ligand BRET system could be utilized for ligand binding detection of any cell surface receptors, if an appropriate fluorescent ligand is available. The approach is quick and easy, as it requires only the coexpression of the GLuc‒PM extracellular surface biosensor and the unmodified receptor, and no further protein fusion procedure is needed. First, we adjusted our system to other GPCRs. We could detect the binding of BODIPY FL‒propranolol to the β2 adrenergic receptor (β2AR). Of interest, we found a consistent increase of the BRET ratio at low concentrations of the β2AR inverse agonist ICI118,552 instead of the expected drop in the signal (Fig. S3). We speculated that the 2-h incubation with the drug at room temperature may induce cell responses, for example, receptor externalization, which could influence the ligand binding results; therefore, we performed incubations on ice. Accordingly, we got regular competition binding curves (Fig. 3A). We could also successfully examine the ligand binding of the D1 dopamine receptor (D1R) or the AT2 angiotensin receptor (AT2R) (Fig. 3, B and C). A study of the latter with our assay could be especially useful in drug screening applications, because AT2R cannot be investigated with receptor signaling assays, as it does not signal in heterologous expression systems (27Zhang H. Han G.W. Batyuk A. Ishchenko A. White K.L. Patel N. Sadybekov A. Zamlynny B. Rudd M.T. Hollenstein K. Tolstikova A. White T.A. Hunter M.S. Weierstall U. Liu W. et al.Structural basis for selectivity and diversity in angiotensin II receptors.Nature. 2017; 544: 327-332Crossref PubMed Scopus (119) Google Scholar). It is interesting that AT2R had a TAMRA‒AngII affinity approximately two orders of magnitude higher than that of AT1R (Fig. S4), which explains the observed relatively higher IC50 of AngII to AT2R. We also performed experiments with non-GPCR cell surface receptors. We examined the epidermal growth factor receptor (EGFR), a prototypical tyrosine kinase receptor (Fig. 3D), and the transferrin receptor (TfR), as an example of cell surface nutrient receptors (Fig. 3E). In both cases, we could detect the specific binding of the Alexa488-conjugated ligands to their receptors, which was prevented by unlabeled ligands. These results show that PM‒ligand BRET using GLuc is a versatile tool for ligand binding measurement of cell surface receptors. A major advantage of resonance energy transfer–based ligand binding assays is the ability to detect ligand binding in real time and high temporal resolution, allowing simple determination of kinetic ligand parameters. We performed kinetic experiments on α1AAR in live cells with both GLuc BRET setups (receptor‒and PM‒ligand BRET). We measured the dissociation rate constant (koff) of the fluorescent ligand after its washout: the medium was replaced and supplemented with unlabeled ligand to prevent rebinding (Fig. 4A). We could follow the ligand dissociation in both setups. Next, we monitored the association of the fluorescent ligand to the receptor with real-time measurements (Fig. 4B) and determined the association rate constant (kon). Kinetic ligand parameters of unlabeled ligands can be assessed with the help of the Motulsky–Mahan equation (28Motulsky H.J. Mahan L.C. The kinetics of competitive radioligand binding predicted by the law of mass action.Mol. Pharmacol. 1984; 25: 1-9PubMed Google Scholar). The koff and kon values of the unlabeled ligand can be calculated by measuring the ligand binding after simultaneous treatment of the labeled ligand and the unlabeled ligand in different concentrations. With the GLuc BRET system we could successfully determine these parameters of prazosin for α1AAR (Fig. 4B). We also performed similar studies with AT1R. TAMRA‒AngII binds AT1R with two different affinities; however, the proportion of high- and low-affinity states shows a temporal change upon agonist binding because of the ternary complex formation with effectors. For the sake of simplicity, we fitted one site binding curves on the measured points. The koff values of TAMRA‒AngII differed between receptor‒ligand BRET and PM‒ligand BRET setups (Fig. 4C), in agreement with the observation that the N-terminal tagging of AT1R alters its binding properties. Thereafter, we assessed the kon values of TAMRA‒AngII (Fig. 4D) and calculated kon and koff for the unlabeled ligand candesartan in kinetic competitive ligand binding measurements (Fig. 4E). We tested whether our system could be applied for receptor ligand screening. As a model receptor, we chose the α1AAR, a major pharmacological target in the treatment of high or low blood pressure and benign prostate hyperplasia (29Alexander S.P.H. Christopoulos A. Davenport A.P. Kelly E. Mathie A. Peters J.A. Veale E.L. Armstrong J.F. Faccenda E. Harding S.D. Pawson A.J. Sharman J.L. Southan C. Davies J.A. CGTP CollaboratorsThe concise guide to pharmacology 2019/20: G protein-coupled receptors.Br. J. Pharmacol. 2019; 176 Suppl: S21-S141Google Scholar). In preliminary experiments, we compared the signal variance in equilibrium and kinetic binding measurements. From this viewpoint, the latter seemed to be more advantageous because of the possibility to apply baseline correction. To statistically characterize the suitability of our system for high-throughput screenings, we calculated the Z'-factor statistical parameter both for receptor‒ligand and PM‒ligand BRET (Fig. 5A) (30Zhang J.H. Chung T.D. Oldenburg K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J. Biomol. Screen. 1999; 4: 67-73Crossref PubMed Scopus (4981) Google Scholar). It was 0.919 and 0.774, respectively, proving that our system could also be applied in high-throughput screenings. Thereafter, we made a test screening with a compound library of 180 compounds (Fig. 5B and Table S1). As positive controls, four known α1AAR ligands were also tested. We defined those compounds as hits that induced at least 25% decrease of the signal (induced 25% displacement of the tracer ligand). Four compounds were found as hits (Fig. S5), which were previously not known to bind to α1AAR. The hits were further characterized. Their KD values were assessed using both ligand binding setups (Fig. 5C and Fig. S6A). Their functional effects on α1AAR, a receptor known to trigger phospholipase C–mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and concomitant calcium signaling, were also investigated. All four hits were able to prevent the cytosolic calcium release induced by the α1AAR agonist A61603 (Fig. 5D). Similarly, the hits abolished the A61603-induced serum response element (SRE)-luciferase activation, a reporter of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and serum response factor activity (Fig. 5E). Consistently with these results, they shifted the A61603 concentration‒response
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