A NanoBRET-Based H3R Conformational Biosensor to Study Real-Time H3 Receptor Pharmacology in Cell Membranes and Living Cells

Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H3 receptor (H3R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H3R ligands. In the current study, we have further characterized this H3R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H3R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H3R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay.


Introduction
G protein-coupled receptors (GPCRs) are membrane-associated seven transmembrane (7TM) proteins that trigger intracellular signaling upon binding of extracellular messengers such as hormones and neurotransmitters. GPCR activation by agonists involves conformational changes in the 7TM domain with most significantly an outward movement of the intracellular side of TM6 to create a pocket at the intracellular interface of TM3, TM5, TM6 and intracellular loop (IL)2 to engage the coupling of heterotrimeric G proteins, GPCR kinases, or β-arrestins, as revealed by X-ray crystallography and more recent cryo-electron microscopy (cryo-EM) structures [1][2][3]. This outward movement of TM6 is smaller for partial agonists as compared to full agonists resulting in a sub-efficient coupling of intracellular signaling transducers and consequently submaximal cellular responses despite full receptor occupancy [4][5][6].
Real-time agonist-induced changes in GPCR conformations have been dynamically measured in living cells using intramolecular resonance-energy transfer (RET)-based biosensors by incorporating a RET acceptor molecule into (the truncated) intracellular loop (IL)3 of the GPCR and fusing a RET donor molecule to its C-terminal tail, or vice versa, allowing for the real-time monitoring of the distance between the two GPCR domains [7,8]. In addition, ligand-induced changes in the distance between TM4 and TM6 has also been measured by labeling-introduced cysteines at the intracellular end of these TMs with optimized Cy3B and Cy7 fluorophores followed by single molecule fluorescent RET imaging [6]. In line with structural studies, full agonists induce a larger change in basal RET in these intramolecular biosensors as compared to partial agonists. Moreover, an opposite change of RET can be observed upon the addition of inverse agonists confirming that GPCRs can adopt a conformation with some basal activity in the absence of ligands [9][10][11][12][13]. Initially, cyan and yellow fluorescent proteins (CFP and YFP) were used as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively, in intramolecular GPCR conformation sensors to measure the distance/re-orientation between IL3 and the C-terminal tail, with the substitution of YFP with the much smaller Fluorescein Arsenical Hairpin Binder (FlAsH) as an improved alternative due to its reduced effect on the GPCR structure [14][15][16]. More recently, bioluminescent luciferases such as Renilla luciferase or the engineered NanoLuc in combination with fluorescent proteins, FlAsH, or the self-labeling fluorescent HaloTag have been employed in BRET-based GPCR conformation sensors to allow for the measurements of conformational changes in GPCRs in a microplate reader assay format [11,12,[17][18][19][20][21].
We have recently reported on the development and initial characterization of a NanoBRET-based H 3 R conformational biosensor (∆icl3-H 3 R Nluc/Halo(618) ) that was based on an earlier reported FRET-based H 3 R biosensor with CFP and YFP [22]. In the NanoBRETbased H 3 R sensor the IL3 was substituted from residues Arg 230 to Arg 347 with a HaloTag that was subsequently self-labeled with 'NanoBRET 618' dye, and NanoLuc was fused in frame to the C-terminal tail ( Figure 1A) [12]. The histamine H 3 receptor (H 3 R) is associated with various neurological disorders such as Alzheimer's disease, Parkinson's disease, narcolepsy, and sleeping and learning disorders due to its important role in the central nervous system (CNS) by pre-synaptically controlling the release of histamine and other neurotransmitters including acetylcholine, dopamine, noradrenaline, serotonin, γ-aminobutyric acid, and glutamate [23]. The H 3 R is a constitutively active GPCR that display increased basal signaling in the absence of histamine [24,25]. Moreover, this spontaneous H 3 R activity can be inhibited in native mouse brains by inverse agonists resulting in reduced G protein activation and a consequently increased release of histamine from synaptosomes [26]. Several H 3 R-targeting antagonists/inverse agonists have entered pre-clinical trials for different CNS disorders in the last decade [27,28]. Moreover, pitolisant (Wakix ® ) has been approved as H 3 R inhibitor in 2017 and 2019 by the European Medicine Agency (EMA) and the Food and Drug Administration in the United States (FDA), respectively, to treat patients with narcolepsy [29,30].
The new NanoBRET-based H 3 R conformational biosensor accurately discriminates between H 3 R ligands with different efficacies, including full and partial agonists but also inverse agonists, suggesting that it adopts a constitutive active conformation in the absence of ligands [12]. In this study, we have used the ∆icl3-H 3 R Nluc/Halo(618) biosensor to pharmacologically characterize a small selection of pre-clinical H 3 R antagonists/inverse agonists and two recently reported photo-switchable H 3 R tool ligands on living cells [31,32]. In addition, we have explored for the first time the function of a GPCR conformational biosensor in membrane preparations instead of intact cells to potentially further increase the assay's flexibility and throughput. upon stimulation with 10 μM H3R ligands measured as ΔBRET ratio in time. (C) Concentrationresponse curves measured after 30 min stimulation of the H3R biosensor with H3R ligands. Data are displayed as mean ± SD from 4 independent experiments performed in duplicate. (D-E) the photoswitchable agonist VUF15000 (D) and inverse agonist VUF14738 (E) switch from trans (cyan) to cis (magenta) upon illumination with 360 nm and from cis to trans by illumination with 430 nm. (F) Concentration-response curves measured after 20 min stimulation of the H3R biosensor with dark (trans) or pre-illuminated (cis) photo-switchable VUF15000 and VUF14738. Data are displayed as the mean ± SD from 3 independent experiments performed in duplicate.
Next, the compatibility of the BRET-based H3R biosensor with azobenzene-containing photo-switchable ligands was evaluated. The previously reported photo-switchable H3R tool compounds, the agonist VUF15000 and antagonist VUF14738 ( Figure 1D-F), showed decreased (cis-off) and increased (cis-on) binding affinities for the wild type H3R upon photo-switching from trans into the PSS-cis isomer by illumination at 365 nm [31,32]. (D-E) the photo-switchable agonist VUF15000 (D) and inverse agonist VUF14738 (E) switch from trans (cyan) to cis (magenta) upon illumination with 360 nm and from cis to trans by illumination with 430 nm. (F) Concentration-response curves measured after 20 min stimulation of the H 3 R biosensor with dark (trans) or pre-illuminated (cis) photo-switchable VUF15000 and VUF14738. Data are displayed as the mean ± SD from 3 independent experiments performed in duplicate.

Ligand
Intact Cells Membrane Preparations Next, the compatibility of the BRET-based H 3 R biosensor with azobenzene-containing photo-switchable ligands was evaluated. The previously reported photo-switchable H 3 R tool compounds, the agonist VUF15000 and antagonist VUF14738 ( Figure 1D-F), showed decreased (cis-off) and increased (cis-on) binding affinities for the wild type H 3 R upon photo-switching from trans into the PSS-cis isomer by illumination at 365 nm [31,32]. These affinity shifts were readily translated into a shifted potency (pEC 50 ) or antagonizing potency (pIC 50 ), respectively, in functional H 3 R assays such as [ 35 S]-GTPγS binding to activated G proteins and downstream G protein-coupled inwardly rectifying potassium (GIRK) channel activity [31,32]. First, binding affinities for the photo-switchable ligands were determined in a competition binding assay with [ 3 H]NAMH on cell membranes expressing the ∆icl3-H 3 R Nluc/Halo(618) . The Photo-switchable agonist VUF15000 and antagonist VUF14738 displayed an 8.0-fold decrease and a 31.6-fold increase in binding affinity for the ∆icl3-H 3 R Nluc/Halo(618) sensor, respectively, upon photoisomerization from transto PSS-cis-isomer (Table 1), which is comparable to their light-induced affinity shifts on wild type H 3 R (Supplementary Table S1) [31,32].
Next, intact cells expressing the H 3 R biosensor were first incubated for 20 min with increasing concentration of the transor cis-isomers of VUF15000 and VUF14738 in the dark, followed by addition of the NanoGlo substrate and immediate detection of ∆BRET signal. Both transand cis-isomers of VUF15000 act as full agonists with higher intrinsic activities than histamine, whereas similar maximum responses were previously observed in a [ 35 S]-GTPγS binding [31]. cis-VUF15000 displayed a 7.9-fold lower potency as compared to trans-VUF15000 ( Figure 1F; Table 1). Oppositely, both VUF14738 isomers behave as inverse agonists with cis-VUF14738 having a 7.9-fold higher potency than trans-VUF14738 ( Figure 1F; Table 1). The smaller light-induced shifts in pEC 50 as compared to pK i values for VUF14738 might be the consequence of unintended cis to trans switching at 430 nm by the lower wavelength shoulder of the Nluc peak bioluminescence at 460 nm [33]. Hence, the use of red-shifted Nanoluc substrates in combination with far-red acceptor fluophores could be explored in future optimizations of the H 3 R biosensor for photopharmacology research to avoid interference with photoligand switching [34].

Dynamics of H 3 R Biosensor in Intact Cells
To further explore the dynamics of monitoring conformational changes in the H 3 R biosensor, we first stimulated ∆icl3-H 3 R Nluc/Halo(618) -expressing cells with 10 µM histamine followed by a second injection after 20 min with vehicle or the competitive inverse agonist pitolisant (0.1 to 10 µM). Pitolisant rapidly antagonized the histamine-induced conformational change of the ∆icl3-H 3 R Nluc/Halo(618) biosensor in a concentration-dependent manner and stabilized a more inactive receptor conformation at 1 and 10 µM as compared to vehicle (only)-stimulated cells, indicating that pitolisant fully displaced histamine from the biosensor within the measured timeframe at these concentrations ( Figure 2A). In addition, stimulation of ∆icl3-H 3 R Nluc/Halo(618) -expressing cells by consecutive injections of increasing concentrations of histamine in the same well with 15 min time intervals resulted in a concentration-dependent increase in BRET ( Figure 2B). The concentration-response curve (pEC 50 = 6.6 ± 0.07) generated from the ∆BRET ratios that were taken 15 min after each consecutive injection had a comparable amplitude to the concentration-response curve that was obtained from wells that were each stimulated with a different histamine concentration (pEC 50 = 6.4 ± 0.1) ( Figure 2C).

Dynamics of H3R Biosensor in Intact Cells
To further explore the dynamics of monitoring conformational changes in the H3R biosensor, we first stimulated Δicl3-H3RNluc/Halo(618)-expressing cells with 10 μM histamine followed by a second injection after 20 min with vehicle or the competitive inverse agonist pitolisant (0.1 to 10 μM). Pitolisant rapidly antagonized the histamine-induced conformational change of the Δicl3-H3RNluc/Halo(618) biosensor in a concentration-dependent manner and stabilized a more inactive receptor conformation at 1 and 10 μM as compared to vehicle (only)-stimulated cells, indicating that pitolisant fully displaced histamine from the biosensor within the measured timeframe at these concentrations ( Figure 2A). In addition, stimulation of Δicl3-H3RNluc/Halo(618)-expressing cells by consecutive injections of increasing concentrations of histamine in the same well with 15 min time intervals resulted in a concentration-dependent increase in BRET ( Figure 2B). The concentration-response curve (pEC50 = 6.6 ± 0.07) generated from the ΔBRET ratios that were taken 15 min after each consecutive injection had a comparable amplitude to the concentration-response curve that was obtained from wells that were each stimulated with a different histamine concentration (pEC50 = 6.4 ± 0.1) ( Figure 2C).

Behavior of the H3R Conformational Biosensor in Membrane Preparations
We have previously shown in radioligand binding experiments on cell membrane preparations that the Δicl3-H3RNluc/Halo(618) conformational biosensor binds ligands with comparable affinities to wild type H3R [12]. To evaluate whether the conformational biosensor can also detect ligand efficacy, as ΔBRET changes in membrane preparations, the H3R biosensor was first labeled with the HaloTag 618 dye, followed by the addition of NanoGlo ® substrate and stimulation with a small selection H3R ligands that have also been (previously) tested on intact cells. The agonists histamine and imetit (10 μM) induced an increase in the ΔBRET ratio that peaked 15-20 min after stimulation followed by a gradual decrease ( Figure 3A), which contrasts with the (previously) observed steady-state response for at least 45 min on intact cells expressing this H3R conformational sensor (see Figure 1B) [12]. The agonist peak response in membranes, however, is comparable with the observed steady-state amplitude in intact cells. All tested the inverse agonists (10 μM) steadily reduced the basal ΔBRET signal without reaching a clear steady-state plateau within the 1 h detection timeframe ( Figure 3A), whereas stable bottom plateaus were

Behavior of the H 3 R Conformational Biosensor in Membrane Preparations
We have previously shown in radioligand binding experiments on cell membrane preparations that the ∆icl3-H 3 R Nluc/Halo(618) conformational biosensor binds ligands with comparable affinities to wild type H 3 R [12]. To evaluate whether the conformational biosensor can also detect ligand efficacy, as ∆BRET changes in membrane preparations, the H 3 R biosensor was first labeled with the HaloTag 618 dye, followed by the addition of NanoGlo ® substrate and stimulation with a small selection H 3 R ligands that have also been (previously) tested on intact cells. The agonists histamine and imetit (10 µM) induced an increase in the ∆BRET ratio that peaked 15-20 min after stimulation followed by a gradual decrease ( Figure 3A), which contrasts with the (previously) observed steady-state response for at least 45 min on intact cells expressing this H 3 R conformational sensor (see Figure 1B) [12]. The agonist peak response in membranes, however, is comparable with the observed steady-state amplitude in intact cells. All tested the inverse agonists (10 µM) steadily reduced the basal ∆BRET signal without reaching a clear steady-state plateau within the 1 h detection timeframe ( Figure 3A), whereas stable bottom plateaus were previously observed on intact cells after approximately 30-45 min ligand stimulation (see Figure 1B) [12]. min stimulation (i.e., peak response) and remained above the required Z ≥ 0.5 up to 40 min indicating that the H3R biosensor in membranes is suitable for agonist screening within this timeframe [35]. However, the Z-factor decreased to 0.3 ± 0.15 after 60 min (Figure 3B,C). In contrast, the Z-factor gradually increased over time for the inverse agonist (10 μM pitolisant) ΔBRET window to Z = 0.4 ± 0.1 after 60 min and consequently did not qualify as a useful screening assay within the tested timeframe ( Figure 3B,C). Extrapolation of the observed Z-factor over time suggests that a longer incubation period (e.g., 90 min) is required for inverse agonist screening to obtain Z-factors ≥ 0.5 ( Figure 3C). Consequently, the simultaneous detection of agonist/inverse agonist-induced conformational changes will not be possible at one particular time-point in an end-point screening format using membranes. Full concentration-response curves on the H3R biosensor in membranes were measured 30 min after stimulation with H3R agonists and inverse agonists ( Figure 3D), resulting in intrinsic activity values that were largely comparable to those observed in intact The Z-factor for the agonist (10 µM histamine) ∆BRET window was 0.6 ± 0.1 after 10 min stimulation (i.e., peak response) and remained above the required Z ≥ 0.5 up to 40 min indicating that the H 3 R biosensor in membranes is suitable for agonist screening within this timeframe [35]. However, the Z-factor decreased to 0.3 ± 0.15 after 60 min ( Figure 3B,C). In contrast, the Z-factor gradually increased over time for the inverse agonist (10 µM pitolisant) ∆BRET window to Z = 0.4 ± 0.1 after 60 min and consequently did not qualify as a useful screening assay within the tested timeframe ( Figure 3B,C). Extrapolation of the observed Z-factor over time suggests that a longer incubation period (e.g., 90 min) is required for inverse agonist screening to obtain Z-factors ≥ 0.5 ( Figure 3C). Consequently, the simultaneous detection of agonist/inverse agonist-induced conformational changes will not be possible at one particular time-point in an end-point screening format using membranes.
Full concentration-response curves on the H 3 R biosensor in membranes were measured 30 min after stimulation with H 3 R agonists and inverse agonists ( Figure 3D), resulting in intrinsic activity values that were largely comparable to those observed in intact cells ( Figure 4A). Relative to the reference ligands histamine (IA = 1) and pitolisant (IA = −1), agonist imetit and all tested inverse agonists seemed to have a slightly increased amplitude (IA) on the H 3 R biosensor membranes as compared to intact cells ( Figure 4A; Table 1). sant and PF-3654746. The potency of thioperamide was not significantly different between intact cells and membrane preparations.
One explanation for these observed potency differences is that ligand-induced H3R biosensor conformational changes were measured in two different buffers between membranes and intact cells, i.e., 50 mM Tris-HCl (pH 7.4) versus HBSS, respectively, and binding affinities for at least some H3R ligands are known to be considerably different between buffers that contain different salt concentrations [36][37][38]. Indeed, pEC50 values measured on intact cells expressing the H3R biosensor in HBSS containing 138 mM NaCl were lower for all the tested ligands, except for thioperamide, as compared to their pKi values measured on H3R biosensor-expressing membranes in 50 mM Tris-HCl buffer (pH 7.4) ( Figure  4C; Table 1). Measuring ligand binding and conformational H3R changes on membranes in the same 50 mM Tris-HCl buffer (pH 7.4) yielded a better correlation between binding affinities and potency values for most ligands, except for bavisant and PF-3654746.  Table 1). Differences between pEC50 values obtained from H3R biosensor in intact cells versus membrane preparations are indicated with grey arrows. Data are displayed as mean ± SD from at least 3 independent experiments performed in duplicate. Deming linear regression was used to compare the fitted affinity and/or potency values between the different assay formats, the dotted line represents line of unity (B,C). HA = histamine;  Table 1). Differences between pEC 50 values obtained from H 3 R biosensor in intact cells versus membrane preparations are indicated with grey arrows. Data are displayed as mean ± SD from at least 3 independent experiments performed in duplicate. Deming linear regression was used to compare the fitted affinity and/or potency values between the different assay formats, the dotted line represents line of unity (B,C). HA = histamine; ime = imetit; pit = pitolisant; clob = clobenpropit; thio = thioperamide; bav = bavisant; ABT = ABT-239; PF = PF-3654746.
Although some correlation was observed for the pEC 50 values on the H 3 R biosensor in membranes versus intact cells, the rank order was different (membranes: thioperamide < bavisant < histamine <pitolisant < PF-3654746 < imetit < ABT-239 < clobenpropit versus intact cells: histamine < thioperamide <pitolisant <clobenpropit < ABT-239 < bavisant <imetit < PF-3654746) ( Figure 4B; Table 1). Remarkably, histamine and clobenpropit showed a 16-and 32-fold higher potency, respectively, to change the H 3 R biosensor conformation in membranes preparations as compared to intact cells, whereas ABT-239 was 10-fold more potent on membrane preparations. Smaller potency differences (<5-fold) were observed for the other tested ligands with slightly increased potencies for pitolisant and imetit on H 3 R biosensor-expressing membranes but with decreased potency values for bavisant and PF-3654746. The potency of thioperamide was not significantly different between intact cells and membrane preparations.
One explanation for these observed potency differences is that ligand-induced H 3 R biosensor conformational changes were measured in two different buffers between membranes and intact cells, i.e., 50 mM Tris-HCl (pH 7.4) versus HBSS, respectively, and binding affinities for at least some H 3 R ligands are known to be considerably different between buffers that contain different salt concentrations [36][37][38]. Indeed, pEC 50 values measured on intact cells expressing the H 3 R biosensor in HBSS containing 138 mM NaCl were lower for all the tested ligands, except for thioperamide, as compared to their pK i values measured on H 3 R biosensor-expressing membranes in 50 mM Tris-HCl buffer (pH 7.4) ( Figure 4C; Table 1). Measuring ligand binding and conformational H 3 R changes on membranes in the same 50 mM Tris-HCl buffer (pH 7.4) yielded a better correlation between binding affinities and potency values for most ligands, except for bavisant and PF-3654746.

Discussion
Detection of conformational changes in GPCRs using RET between donor and acceptor molecules that are inserted in between TM5/TM6 and the C-terminal tail allows for the direct quantification of agonist and inverse agonist potency and efficacy upon ligand binding to the receptor. The FRET-based H 3 R sensor in intact cells and cultured on cover slips allowed for the rapid detection of ligand-induced conformational receptor changes using a fluorescent microscope equipped with a perfusion system with high temporal resolution but a relatively low throughput [22]. Substitution of YFP and CFP with respectively a red fluorescent dye covalently bound to HaloTag and NanoLuc allows a NanoBRET-based detection of conformational H 3 R changes in living cells using a 96-well plate reader-based format to readily generate full concentration-response curves for multiple ligands in a single assay run [12], as also previously optimized and reported for the α 2A -adrenergic receptor, β 2 -adrenergic receptor, and the parathyroid hormone 1 receptor [11,21]. As a follow-up on our initial report on this sensor, we evaluated a number of well-known H 3 R tools (photo-switchable ligands or preclinical candidates) for their conformational effects.
Our data indicate that all the preclinical candidates indeed act as inverse agonists, with bavisant showing a clear partial inverse agonistic effect. Moreover, the sensor also allowed the evaluation of the recently developed photo-switchable agonist and antagonist [31,32], although the light generated by the NanoLuc donor might to some extent also affect the cis-trans ratio due to spectral overlap of the cis-isomer and the NanoLuc emission.
Although this 96-well assay format significantly increases the ligand screening throughput, most microplate readers are only equipped with one or two injectors and consequently do not allow much flexibility with respect to adding multiple ligands and/or concentrations during BRET measurements. In this study we show that measurements can be paused to remove the plate from microplate reader to manually add (consecutive) ligands to the assay plate and continue the readout. Indeed, the rapid addition of pitolisant to cells that were pretreated with histamine resulted in a concentration-dependent decrease of the BRET signal showing that histamine can be quickly displaced from H 3 R by pitolisant thereby switching the receptor from an active into an inactive conformation. This is in line with the complete dissociation of histamine from the FRET-based H 3 R sensor within approximately 15 s upon washout [22]. Such a washout experiment is difficult to repeat using the NanoBRET-based H 3 R sensor as this will also remove the NanoGlo substrate resulting in reduced reproducibility, which could not be easily restored even by supplementing fresh NanoGlo.
In contrast to G protein-mediated downstream signaling assays, the agonist concentration-response curves on a conformational GPCR are not subjected to signal amplification and the observed potency should be comparable to binding affinity of the ligand for the receptor [39]. This makes the NanoBRET-based H 3 R biosensor very useful for initial drug discovery as a measure for ligand affinity and efficacy can be simultaneously obtained. However, performing pharmacological assays on living cells requires the constant availability of cells in their exponential growth phase, which can limit the numbers of assays. Considering that the NanoBRET-based H 3 R biosensor displayed comparable binding affinities for all tested ligands as the wild type H 3 R in membrane preparations that were generated from frozen cell pellets, we decided to evaluate the ligand-induced conformational H 3 R changes in these membranes. Ligand potency (pEC 50 ) values measured on membranes were more in line with affinity values (pK i ) obtained from radioligand competition binding assays, as compared to the potencies measured on intact cells. This is most likely related to presence of NaCl in the HBSS medium that is used for the intact cell assay, and known to affect binding affinities of H 3 R ligands [36][37][38]. Yet, a good correlation between the intrinsic activity of both agonists and inverse agonists was observed between the intact and membrane H 3 R conformational sensor assays.
In conclusion, the H 3 R biosensor in membranes could be a useful alternative for radioligand binding assays and allows for the simultaneous measurement of ligand affinity (via its potency) and efficacy on the H 3 R. Moreover, membranes expressing the H 3 R biosensor can be prepared in a large batch and stored in the freezer as (nearly) ready-touse cell pellets to avoid prolonged and time-consuming culturing of an H 3 R biosensorexpressing stable cell line that is required for living cell assays. Discoveries (Amsterdam, the Netherlands). VUF15000 and VUF14738 were synthesized in house as described previously [31,32]. NanoGlo ® (N1130) and HaloTag ® NanoBRET™ 618 Ligand (G9801) were bought from Promega (Madison, WI, USA). All other reagents were of analytical grade and obtained from conventional commercial sources.

Photochemistry
The photo-switchable compounds (VUF15000, VUF14738) were synthesized in-house and their in-depth photochemical properties were previously reported [31,32]. Briefly, both compounds have an λ max value for the π-π* transition of the trans-isomer of 360 ± 20 nm and an n-π* transition of the cis-isomer of 430 ± 17 nm. Photo-switchable compounds (10 mM in DMSO) were illuminated with 360 ± 20 nm light for 300 s to reach a photostationary state (PSS) containing over 86% of the cis-isomer or kept in dark to ensure more than 99% of the trans-isomer. The illumination was carried out in cylindrical clear glass vials of 4.5 mL volume, with a typical distance of 2 cm from the light source. All subsequent experimental steps were conducted in the dark or under near-infrared light. Both cis-VUF15000 and cis-VUF14738 have thermal relaxation half-lives of >100 days at room temperature.

BRET-Based H 3 R Biosensor Detection on Intact Cells
Cells were collected in culture medium supplemented with 50 nM HaloTag NanoBRET 618 dye, transferred into white bottom 96-well plates (50,000 cells/well) and cultured for another 24 h. Next, the culture medium was replaced by a 1/1000 dilution of NanoGlo ® stock solution in HBSS. Subsequently, ligand solution or vehicle control was added and the stimulated BRET ratio was recorded at 37 • C using a BRETplus1 luminescence module (610 nm and 460 nm) of the PHERAstar FS (BMG labtech GmbH, Ortenberg, Germany). To avoid unintended backswitching of PSS-cis into the trans-isomer of the photo-switchable ligands VUF15000 and VUF14738 (λ max = 427) by the Nluc luminescent peak emission at 460 nm, the cells were first incubated for 20 min with the photo-switchable tool ligands followed by the addition of NanoGlo ® solution and direct luminescence detection at 460 and 610 nm [31][32][33].

Membrane Preparation
HEK293A cells that stably express the ∆icl3-H 3 R Nluc/Halo(618) biosensor were collected from 10 cm dishes (90% confluency) as previously described [12]. Briefly, cells were detached using cold phosphate-buffered saline (PBS) and centrifuged at 1900× g for 15 min at 4 • C. The supernatant was discarded and the cell pellet was stored at freezer (−20 • C) for further experiments. On the day of the experiment, cell pellets were resuspended (4-6 mL/10 cm dish) in 50 mM Tris-HCl (pH 7.4) and disrupted using a Branson 250 sonifier (Boom B.V., Meppel, The Netherlands).

[ 3 H]NAMH Competition Binding Assay on Membranes
Membrane suspensions (50 µL/well) were incubated with 2 nM [ 3 H]NAMH in combination with increasing concentrations of unlabeled ligands for 2 h at 25 • C with gentle agitation. Incubation was stopped by harvesting the homogenates onto 96-well GF/C plates pre-soaked with 0.5% (v/v) PEI using a 96-well Filtermate harvester (PerkinElmer, Groningen, The Netherlands). The GF/C filter plates were then washed three times with cold wash buffer (50 mM Tris-HCl, pH 7.4, 4 • C) and dried for 30 min. Filter-bound radioactivity was quantified by a Microbeta Wallac Trilux scintillation counter (Perkin-Elmer) after addition of 25 µL/well scintillation liquid.

BRET-Based H 3 R Biosensor Detection on Membranes
Membrane suspensions (50 µL/well) were incubated with 50 nM HaloTag NanoBRET 618 dye for 2 h at 25 • C. Next, NanoGlo ® stock solution (1/1000 dilution) was added per well and the basal BRET ratio was measured. Subsequently, ligand solution or vehicle control was added, and the stimulated BRET ratio was recorded at 25 • C.

Data Analysis
GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) was used for data analysis and statistics.
BRET ratios were calculated by dividing the BRET signal at 610 nm by the Nluc signal at 460-480 nm. ∆BRET was used for quantifying ligand-induced changes in BRET ratio using the following equation: Concentration-response curves were fitted using the "log (agonist) vs. response (three parameters)" model: Intrinsic activity (IA) value is calculated as: IA = fitted maximum response agonist or inverse agonist fitted maximum response histamine or pitolisant where agonist and inverse agonists were compared to histamine and pitolisant, respectively, and inverse agonism is indicated by (−).