Synthesis and Pharmacological Characterization of New Photocaged Agonists for Histamine H3 and H4 Receptors

The modulation of biological processes with light-sensitive chemical probes promises precise temporal and spatial control. Yet, the design and synthesis of suitable probes is a challenge for medicinal chemists. This article introduces a photocaging strategy designed to modulate the pharmacology of histamine H3 receptors (H3R) and H4 receptors (H4R). Employing the photoremovable group BODIPY as the caging entity for two agonist scaffolds—immepip and 4-methylhistamine—for H3R and H4R, respectively, we synthesized two BODIPY-caged compounds, 5 (VUF25657) and 6 (VUF25678), demonstrating 10–100-fold reduction in affinity for their respective receptors. Notably, the caged H3R agonist, VUF25657, exhibits approximately a 100-fold reduction in functional activity. The photo-uncaging of VUF25657 at 560 nm resulted in the release of immepip, thereby restoring binding affinity and potency in functional assays. This approach presents a promising method to achieve optical control of H3R receptor pharmacology.


Introduction
Photopharmacology represents a new and cutting-edge area in the field of chemical biology, offering precise spatiotemporal control over biological processes [1][2][3][4].Photopharmacology involves the use of light to modulate the activity of bioactive compounds, providing a non-invasive means to influence cellular functions with high precision [1][2][3][4].Photocaging is one of the important strategies employed in the field of photopharmacology and this approach involves the temporary inactivation of a bioactive molecule through a light-sensitive protecting group [5][6][7][8].Upon exposure to light, the caging group is removed, releasing the bioactive molecule to act on its respective target.This approach holds great potential for studying and modulating cellular signaling pathways, potentially offering unprecedented spatial and temporal control of receptor activation.
In this study, we report our efforts in the field of photopharmacology of G proteincoupled receptors (GPCR) by developing photocaged agonists specifically tailored for histamine H 3 -and H 4 receptors (H 3 R and H 4 R).The H 3 R and H 4 R are Gα i -coupled GPCRs and integral components of the histaminergic system [9,10].Both GPCRs play a pivotal role in regulating various physiological processes and are emerging as promising targets for therapeutic intervention [9,10].The H 3 R is primarily localized in the central nervous system, modulating the release of neurotransmitters such as histamine, dopamine, and acetylcholine.The intricate involvement of the H 3 R in cognitive functions, sleepwake cycles, and appetite regulation underscores its significance as a potential target for neurological and psychiatric disorders [11][12][13].Moreover, recently an H 3 R inverse agonist/antagonist (pitolisant) was approved for therapeutic use in narcolepsy (Wakix ® ) or in apnea patients suffering from excessive daytime sleepiness (Ozawade ® ) [14][15][16].On the other hand, the H 4 R, although more recently discovered, has sparked considerable interest due to its expression in the immune system [9,10].As an immune modulator, the H 4 R is implicated in the regulation of inflammatory responses, making it an attractive target for conditions involving immune dysregulation, such as allergies and autoimmune diseases [17,18].Recently, its role in neurological disorders has also gained attention [19].
To allow spatio-temporal studies of the function of H 3 R and H 4 R, we focused on the design, synthesis, and characterization of novel photocaged agonists to enable spatiotemporal control of histamine receptor activity.Previously, caging groups such as nitrobenzyl, coumarin, and BODIPY (boron-dipyrromethene) have been successfully employed in the field of photocaging [2,[6][7][8]20].Especially, BODIPY dyes have gained interest as caging groups in view of their photostability and strong absorbance in the visible region [21].This last property makes BODIPY compounds well-suited for light-triggered applications in biological context, as UV (ultraviolet) light is well-known to damage biomolecules.In this study, we report on the synthesis and characterization of BODIPY-caged immepip and 4-methylhistamine, two small molecule agonists known to act on H 3 R and H 4 R [22,23].Caged compounds were analyzed for their photochemical properties, aggregation, and molecular pharmacological properties in radioligand binding and GPCR signaling studies.

Design, Synthesis, and Photochemical Characterization of Photocaged Compounds
Following the discovery of immepip as a potent H 3 R agonist, various modifications have been reported in structure-activity relationship (SAR) studies.Among those, three earlier described modifications were instrumental in the design of photocaged immepip.First, the introduction of bulky N-alkyl substituents, such as an isopropyl group on the piperidine ring, by Kitbunnadaj et al. results in a remarkable >100-fold decrease in affinity [24].Next, N-phenyl piperidine substituted immepip analogs prepared by Ishikawa et al. showed a pK i range of 1-60 nM, representing 3 to 200-fold lower activity compared to immepip [25].Lastly, the investigation by Vaccaro et al. of the basicity of the piperidine nitrogen revealed that analogs with a carbonyl group were 10 to >1500-fold less active towards H 3 R [26].In contrast, so far very few immepip analogs with substituents on the imidazole N-atom have been reported [26].Thus, in the design of our BODIPY-based photocaged H 3 R agonist, we considered photocaging of the piperidine amine functional group as the most viable approach.
In contrast to H 3 R agonists, there are significantly fewer reported H 4 R agonists, and many of them also exhibit activity towards H 3 R.In 2005, 4-methylhistamine was reported as a selective H 4 R agonist over H 3 R [22], making it an ideal candidate for the design of a photocaged H 4 R agonist.Within the structure of 4-methylhistamine, there are two potential positions for the introduction of the photocage group (the amino and imidazole groups).However, SARs of these two positions have not been well explored.We opted for attaching the required carbamate to the amino group rather than to the imidazole in view of its anticipated higher stability in the dark.Indeed, the recently published cryo-EM structures of histamine-bound H 4 R [27,28] indicate that the primary amino group of histamine direct or indirectly forms a salt bridge with Asp 943.32 in transmembrane domain 3 and thus holds promise as a site for photocaging.From the three main classes of photocages in the literature, BODIPY was chosen in this study as photocaging group for the H 3 R/H 4 R ligands due to its lower-energy and rapid uncaging compared with o-nitrobenzyl and coumarin cages [29][30][31].
(VUF25678) were prepared from 4 by a substitution reaction with immepip and 4methylhistamine, respectively.Next, the photochemical properties of the caged VUF25657 and VUF25678 were investigated.Due to the large conjugation system of the BODIPY cage, the aqueous solubility of two caged compounds is limited to 3.2 µM, as determined by the nephelometry measurements (Figure S1).Based on the photochemical stability results in Figure S2, photo characterizations of VUF25657 and VUF25678 were performed under red LED light.Due to the low absorbance of immepip and 4-methylhistamine at 230 and 254 nm, a quantitative analysis method based on mass (MS) detection was employed.First, calibration curves of monitored compounds with the corresponding reference compound of the same molecular weight were built (Figure S3).Then, after illumination, the reference compounds were added to LCMS samples as internal standards.Based on the MS signal areas, the concentrations of caged compounds and parent compounds could be monitored.In Tris-HCl buffer (50 mM, pH 7.4), the absorption maximum (λmax) was determined to be 551 nm for VUF25657 (Figure 1A) and 546 nm for VUF25678 (Figure S4A).Within 20 min of continuous illumination of VUF25657 at 3.2 µM in Tris-HCl buffer with 560 ± 5 nm, the absorption centered around 550 nm decreased significantly (Figure 1A), and the active ligand immepip could be obtained (Figure 1B).The same results were observed for caged-4-methylhistamine (VUF25678) (Figure S4).After 180 s of illumination, the concentration of immepip reached 0.92 µM, amounting to 29% uncaging efficiency.Further illumination led to a decreased immepip concentration (for example, 0.53 µM at 20 min).One potential explanation for this could be the reaction of immepip with side products or intermediates formed during the uncaging process.A similar uncaging profile was observed for VUF25678 (Figure S4B).Next, the photochemical properties of the caged VUF25657 and VUF25678 were investigated.Due to the large conjugation system of the BODIPY cage, the aqueous solubility of two caged compounds is limited to 3.2 µM, as determined by the nephelometry measurements (Figure S1).Based on the photochemical stability results in Figure S2, photo characterizations of VUF25657 and VUF25678 were performed under red LED light.Due to the low absorbance of immepip and 4-methylhistamine at 230 and 254 nm, a quantitative analysis method based on mass (MS) detection was employed.First, calibration curves of monitored compounds with the corresponding reference compound of the same molecular weight were built (Figure S3).Then, after illumination, the reference compounds were added to LCMS samples as internal standards.Based on the MS signal areas, the concentrations of caged compounds and parent compounds could be monitored.In Tris-HCl buffer (50 mM, pH 7.4), the absorption maximum (λ max ) was determined to be 551 nm for VUF25657 (Figure 1A) and 546 nm for VUF25678 (Figure S4A).Within 20 min of continuous illumination of VUF25657 at 3.2 µM in Tris-HCl buffer with 560 ± 5 nm, the absorption centered around 550 nm decreased significantly (Figure 1A), and the active ligand immepip could be obtained (Figure 1B).The same results were observed for caged-4-methylhistamine (VUF25678) (Figure S4).After 180 s of illumination, the concentration of immepip reached 0.92 µM, amounting to 29% uncaging efficiency.Further illumination led to a decreased immepip concentration (for example, 0.53 µM at 20 min).One potential explanation for this could be the reaction of immepip with side products or intermediates formed during the uncaging process.A similar uncaging profile was observed for VUF25678 (Figure S4B).
(VUF25678) were prepared from 4 by a substitution reaction with immepip and 4methylhistamine, respectively.Next, the photochemical properties of the caged VUF25657 and VUF25678 were investigated.Due to the large conjugation system of the BODIPY cage, the aqueous solubility of two caged compounds is limited to 3.2 μM, as determined by the nephelometry measurements (Figure S1).Based on the photochemical stability results in Figure S2, photo characterizations of VUF25657 and VUF25678 were performed under red LED light.Due to the low absorbance of immepip and 4-methylhistamine at 230 and 254 nm, a quantitative analysis method based on mass (MS) detection was employed.First, calibration curves of monitored compounds with the corresponding reference compound of the same molecular weight were built (Figure S3).Then, after illumination, the reference compounds were added to LCMS samples as internal standards.Based on the MS signal areas, the concentrations of caged compounds and parent compounds could be monitored.In Tris-HCl buffer (50 mM, pH 7.4), the absorption maximum (λmax) was determined to be 551 nm for VUF25657 (Figure 1A) and 546 nm for VUF25678 (Figure S4A).Within 20 min of continuous illumination of VUF25657 at 3.2 μM in Tris-HCl buffer with 560 ± 5 nm, the absorption centered around 550 nm decreased significantly (Figure 1A), and the active ligand immepip could be obtained (Figure 1B).The same results were observed for caged-4-methylhistamine (VUF25678) (Figure S4).After 180 s of illumination, the concentration of immepip reached 0.92 μM, amounting to 29% uncaging efficiency.Further illumination led to a decreased immepip concentration (for example, 0.53 μM at 20 min).One potential explanation for this could be the reaction of immepip with side products or intermediates formed during the uncaging process.A similar uncaging profile was observed for VUF25678 (Figure S4B).

Photopharmacological Characterization
The photocaged H 3 R and H 4 R agonists VUF25657 and VUF25678, either kept in the dark or pre-irradiated at 560 nm to achieve an uncaged state, were evaluated for their binding affinities by competition binding of the radioligands N-α-[methyl-3 H] histamine ([ 3 H]NAMH) and [ 3 H]histamine for the human H 3 R and H 4 R, respectively.Both GPCRs were transiently expressed in HEK293T cells.Previously, we have shown that [ 3 H]NAMH and [ 3 H]histamine saturably bind to the H 3 R and H 4 R, respectively, with K D values of 1.37 and 4.40 nM [36].The parent compounds immepip and 4-methylhistamine (4-MeHA) potently displaced radioligand binding to H 3 R (Figure 2A) or H 4 R (Figure 2B).Moreover, pre-irradiation of the parent agonist solution at 560 nm did not lead to any difference in affinity (Figure 2A,B).The BODIPY-caged immepip (VUF25657) displayed a 125-fold decreased affinity for H 3 R. Illumination of VUF25657 at 560 nm partially restored the observed H 3 R affinity, with a 12-fold increase in comparison to its caged state (Table 1).Caging 4-methylhistamine with BODIPY also led to a decreased affinity for H 4 R.However, the displacement of radioligand binding by VUF25678 could not be measured at concentrations > 1 µM due to solubility issues.Therefore, the accurate affinity shift between caged and uncaged VUF25678 could not be determined and pharmacological analysis of VUF25678 was halted.

Photopharmacological Characterization
The photocaged H3R and H4R agonists VUF25657 and VUF25678, either kept in the dark or pre-irradiated at 560 nm to achieve an uncaged state, were evaluated for their binding affinities by competition binding of the radioligands N-α-[methyl-3 H] histamine ([ 3 H]NAMH) and [ 3 H]histamine for the human H3R and H4R, respectively.Both GPCRs were transiently expressed in HEK293T cells.Previously, we have shown that [ 3 H]NAMH and [ 3 H]histamine saturably bind to the H3R and H4R, respectively, with KD values of 1.37 and 4.40 nM [36].The parent compounds immepip and 4-methylhistamine (4-MeHA) potently displaced radioligand binding to H3R (Figure 2A) or H4R (Figure 2B).Moreover, pre-irradiation of the parent agonist solution at 560 nm did not lead to any difference in affinity (Figure 2A,B).The BODIPY-caged immepip (VUF25657) displayed a 125-fold decreased affinity for H3R.Illumination of VUF25657 at 560 nm partially restored the observed H3R affinity, with a 12-fold increase in comparison to its caged state (Table 1).Caging 4-methylhistamine with BODIPY also led to a decreased affinity for H4R.However, the displacement of radioligand binding by VUF25678 could not be measured at concentrations > 1 µM due to solubility issues.Therefore, the accurate affinity shift between caged and uncaged VUF25678 could not be determined and pharmacological analysis of VUF25678 was halted.Next, the photocaged H3R agonist VUF25657, which notably displays a significant alteration in binding affinity to H3R upon irradiation at 560 nm, was further evaluated for its functionality by a FRET-based EPAC cAMP (cyclic adenosine monophosphate) biosensor [37].H3R primarily couples to Gαi protein, which upon activation causes a decrease in intracellular cAMP levels via the inhibition of adenylate cyclase.Therefore, the HEK-EPAC cells were pre-treated with 10 nM of the beta2 adrenergic receptor (β2AR) agonist isoprenaline before H3R stimulation to enhance the basal intracellular cAMP levels via β2AR-Gαs activation.As anticipated, the H3R selective agonist immepip reduced the isoprenaline-induced cAMP accumulation, as detected by an increased FRET (fluorescence resonance energy transfer) signal in the EPAC (exchange protein directly activated by cAMP) biosensor (Figure 3A).In line with the binding affinity results, caging immepip with BODIPY (VUF25657) resulted in a decrease potency of cAMP inhibition (316-fold) as compared to immepip.Furthermore, as expected, irradiation of VUF25657 at 560 nm

Compound pK i pEC 50
In Next, the photocaged H 3 R agonist VUF25657, which notably displays a significant alteration in binding affinity to H 3 R upon irradiation at 560 nm, was further evaluated for its functionality by a FRET-based EPAC cAMP (cyclic adenosine monophosphate) biosensor [37].H 3 R primarily couples to Gα i protein, which upon activation causes a decrease in intracellular cAMP levels via the inhibition of adenylate cyclase.Therefore, the HEK-EPAC cells were pre-treated with 10 nM of the beta2 adrenergic receptor (β 2 AR) agonist isoprenaline before H 3 R stimulation to enhance the basal intracellular cAMP levels via β 2 AR-Gα s activation.As anticipated, the H 3 R selective agonist immepip reduced the isoprenaline-induced cAMP accumulation, as detected by an increased FRET (fluorescence resonance energy transfer) signal in the EPAC (exchange protein directly activated by cAMP) biosensor (Figure 3A).In line with the binding affinity results, caging immepip with BODIPY (VUF25657) resulted in a decrease potency of cAMP inhibition (316-fold) as compared to immepip.Furthermore, as expected, irradiation of VUF25657 at 560 nm restored the activity for cAMP inhibition, demonstrated a significantly higher potency than its caged state (20-fold) (Figure 3B, Table 1).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 13 restored the activity for cAMP inhibition, demonstrated a significantly higher potency than its caged state (20-fold) (Figure 3B, Table 1).

Discussion
As an important component of photopharmacology, the photocaging strategy has a long history, compared to the photoswitch strategy, in controlling bioactivity by light.Dating back to the 1980s [38], the photocage strategy had already proven its important role in chemistry, biology, and other related fields [39].To serve the purpose of spatiotemporal modulation of biological processes, several classes of photocages have been discovered and the concept has been well-developed so far [40].In this study, the BODIPY cage was successfully coupled to selective H3R/H4R agonists and the resulting photocaged ligands could rapidly release the parent agonists.The novel photocaged H3R ligand VUF25657 was successfully applied in pharmacological assays.This new chemical biology tool exhibits significant activity differences in both H3R binding affinity and functional H3R assay before and after uncaging with visible light.
Despite the successful application, certain limitations of the BODIPY cage were also noted in this study.Compared to o-nitrobenzyl and coumarin cages, the BODIPY scaffold has a larger conjugation system, resulting in a lower-energy uncaging.However, this larger conjugation system often leads to compounds with low solubility in aqueous envi-

Real time modulation of isoprenaline-induced cAMP acumination in HEK-EPAC cells by 1 µM immepip (A). Modulation of isoprenaline-induced cAMP acumination in HEK-EPAC cells by increasing concentrations of VUF25657 (B)
. HEK-EPAC cells stably expressing H 3 R were pre-stimulated with 10 nM isoprenaline for 10 min before ligand addition.The FRET signal of the EPAC-sensor was measured after 20 min treatment of indicated ligands.VUF25657 was either kept in dark or pre-irradiated at 560 nm for 3 min to release the BODIPY cage.Data are mean ± S.D. from four independent experiments performed, each in triplicate.

Discussion
As an important component of photopharmacology, the photocaging strategy has a long history, compared to the photoswitch strategy, in controlling bioactivity by light.Dating back to the 1980s [38], the photocage strategy had already proven its important role in chemistry, biology, and other related fields [39].To serve the purpose of spatiotemporal modulation of biological processes, several classes of photocages have been discovered and the concept has been well-developed so far [40].In this study, the BODIPY cage was successfully coupled to selective H 3 R/H 4 R agonists and the resulting photocaged ligands could rapidly release the parent agonists.The novel photocaged H 3 R ligand VUF25657 was successfully applied in pharmacological assays.This new chemical biology tool exhibits significant activity differences in both H 3 R binding affinity and functional H 3 R assay before and after uncaging with visible light.
Despite the successful application, certain limitations of the BODIPY cage were also noted in this study.Compared to o-nitrobenzyl and coumarin cages, the BODIPY scaffold has a larger conjugation system, resulting in a lower-energy uncaging.However, this larger conjugation system often leads to compounds with low solubility in aqueous environments.The compound concentrations of VUF25657 and VUF25678 in both binding and functional assay had to be limited to maximally 3.2 µM, as determined by nephelometry.Due to this low concentration and the low absorbance of immepip and 4-methylhistamine at a common UV detection wavelength (such as 254 and 230 nm), a more complex MS-based analytical method than the routine LC-MS was employed to track the uncaging process.In our experiments with the caged H 4 R ligand, the release of the parent compound 4-methylhistamine could be monitored.However, due to the low concentration in the experiment and low activity of 4-methylhistamine, no significant difference was observed in the binding affinity assay before or after irradiation.To improve the aqueous solubility of BODIPY cages, Kand et al. have developed a water-soluble MESNA-BODIPY (2-mercaptoethanesulfonic acid-BODIPY) cage by introducing a sulfonic acid group [41].However, the applicability of compounds with such a cage may be reduced depending on the target location, as deprotonation might impede the accessibility of their target.Nevertheless, for photocaging of GPCR ligands the future use of water-soluble BODIPY analogs might be a good way forward.
Unexpectedly, the concentration of the desired parent compounds showed a decreasing trend after prolonged illumination.As proposed by Goswami et al. [32], uncaging of BODIPY analogs in aqueous solutions will yield a BODIPY alcohol next to the desired products.However, this desired alcohol was not easily observed in our uncaging process.Instead, a mass corresponding to a BODIPY-Tris byproduct was detected.We also hypothesized that the photo-bleaching products and/or reactive intermediates of the uncaging process could potentially further react with our desired uncaged compounds under these illumination conditions, thus leading to the decreased concentration of parent compounds.
In conclusion, in the present study we report on the successful BODIPY caging of the H 3 R/H 4 R agonists immepip and 4-methylhistamine and their evaluation with respect to photo-uncaging and subsequent target engagement.Our results indicate that VUF25657, the caged immepip, is the first successful, caged photopharmacological agent for spatiotemporal control of H 3 R function.

Nephelometry
Compounds under investigation were placed into clear, flat-bottom 96-well plates in the absence of light, each containing a distinct concentration (from 10 −4 M to 10 −7.5 M, with a blank as a baseline reference), and were left to stabilize in a phosphate buffer solution with 1% DMSO for a minimum of 60 min before measurement.A kaolin suspension was placed in each plate at varying concentrations (10 −4 M to 10 −7.5 M), and was subjected to identical experimental conditions as the test compounds [42].Utilizing a NEPHELO star Plus instrument from BMG Labtech, Germany, turbidity measurements were taken under a set of parameters: fours cycles of readings, initiating the measurement at 0.1 s, with each subsequent reading at 0.1 s interval time.The laser intensity was set to 80%, the beam focus was adjusted to 2.0 mm, and the plates were agitated using an orbital shaking mode at 200 rpm, with an additional 10 s of shaking before the commencement of each cycle.Data analysis was carried out using GraphPad Prism 8 software with the collected data points, generating a line chart that displays both the mean and standard deviation values.These results were compared with the kaolin control to assess the tested compounds.

(Photo)chemical Stability Assay
Compound aqueous stability and room light stability tests (Figure S2) were carried out with 10 µM samples in 50 mM phosphate buffer plus 1% DMSO at room temperature in clear glass vials.Aqueous stability was checked hourly using LC detection at 254 nm, while room light stability was monitored every 5 min for an hour with identical LC settings.

Membrane Preparation
Membranes were collected from HEK293T cells transiently transfected with H 3 R or H 4 R by PEI method.In brief, 2 million HEK293T cells were seeded into a 10 cm dish one day before transfection.The next day, the cells were transfected with 2.5 µg cDNA encoding H 3 R (GenBank accession number AF140538) or H 4 R (GenBank accession number AY136745) supplemented with 2.5 µg empty pcDEF3 [45] plasmid using 20 µg PEI.Two days after transfection, the transfected HEK293T cells were detached using ice-cold PBS, and the membrane pellets were subsequently collected by centrifuging at 1932× g at 4 • C for 10 min.

Radioligand Binding Assay
For competition binding assays, the tested compounds were pre-irradiated to reach the uncaged state.In brief, 0.3 mM stock of photocaged ligand (dissolved in DMSO) was divided into two samples, one of which was subsequently diluted in Tris-HCl buffer (50 mM, pH 7.4) to 3 µM and then irradiated by 560 nm to reach the uncaged state, as confirmed by LC-MS, and the other sample was kept in dark to retain its caged state.The prepared membrane pellets expressing H 3 R or H 4 R were resuspended in Tris-HCl buffer (50 mM, pH 7.4) and disrupted by 5 s sonication to make membrane suspension.Next, all the following handling was performed under near-infrared light.For testing the binding to H 3 R, membrane suspension expressing H 3 R was incubated with 2 nM N-α-[methyl-3 H] histamine and increasing concentrations of unlabeled ligands prepared in Tris-HCl buffer.For testing the binding to H 4 R, binding assays were performed by displacing 4 nM [ 3 H] histamine with increasing concentrations of unlabeled ligands prepared in Tris-HCl buffer on membrane suspension expressing H 4 R. Following one hour of incubation with continuous shaking at 225 rpm at 25 • C, the reaction was terminated by transferring the mixture to GF/C filter plates that were pre-soaked with a solution containing 0.5% PEI and washed with cold Tris-HCl buffer.After drying for 30 min at 55 • C, the radioactivity remaining on the filters was quantified using a Microbeta Wallac Trilux scintillation counter, following the addition of scintillation liquid with a two-hour delay.

Figure 1 .
Figure 1.Photo-uncaging followed by UV-Vis (ultraviolet-visible) and LC-MS analysis.(A) VUF25657 (3.2 µM) was illuminated under 560 nm in Tris-HCl buffer (50 mM, pH 7.4)/1% DMSO at room temperature and a UV-Vis spectrum was measured at intervals.Only the 350-750 nm region is shown; (B) VUF25657 (3.2 µM) was illuminated under green LED light in Tris-HCl buffer (50 mM, pH 7.4)/1% DMSO at 37 °C and LC-MS analysis was performed at different time points using an internal standard.

Figure 1 .
Figure 1.Photo-uncaging followed by UV-Vis (ultraviolet-visible) and LC-MS analysis.(A) VUF25657 (3.2 μM) was illuminated under 560 nm in Tris-HCl buffer (50 mM, pH 7.4)/1% DMSO at room temperature and a UV-Vis spectrum was measured at intervals.Only the 350-750 nm region is shown; (B) VUF25657 (3.2 μM) was illuminated under green LED light in Tris-HCl buffer (50 mM, pH 7.4)/1% DMSO at 37 °C and LC-MS analysis was performed at different time points using an internal standard.

Figure 1 .
Figure 1.Photo-uncaging followed by UV-Vis (ultraviolet-visible) and LC-MS analysis.(A) VUF25657 (3.2 µM) was illuminated under 560 nm in Tris-HCl buffer (50 mM, pH 7.4)/1% DMSO at room temperature and a UV-Vis spectrum was measured at intervals.Only the 350-750 nm region is shown; (B) VUF25657 (3.2 µM) was illuminated under green LED light in Tris-HCl buffer (50 mM, pH 7.4)/1% DMSO at 37 • C and LC-MS analysis was performed at different time points using an internal standard.

Figure 2 .
Figure 2. Displacement binding curves of parent, caged, and uncaged ligands for H3R (A) and H4R (B).Binding affinities were determined by displacement of the binding of 2 nM [ 3 H]NAMH and 4 nM [ 3 H]histamine to H3R and H4R, respectively.Caged ligands were either kept in dark or preirradiated at 560 nm for 3 min for VUF25657 and 5 min for VUF25678 to achieve the uncaged state.Data shown are mean ± S.D. from three or four independent experiments, each performed in triplicate.

Figure 2 .Table 1 .
Figure 2. Displacement binding curves of parent, caged, and uncaged ligands for H 3 R (A) and H 4 R (B).Binding affinities were determined by displacement of the binding of 2 nM [ 3 H]NAMH and 4 nM [ 3 H]histamine to H 3 R and H 4 R, respectively.Caged ligands were either kept in dark or pre-irradiated at 560 nm for 3 min for VUF25657 and 5 min for VUF25678 to achieve the uncaged state.Data shown are mean ± S.D. from three or four independent experiments, each performed in triplicate.

Figure 3 .
Figure 3. Optical modulation of VUF25657-induced cAMP inhibition in EPAC-H3R cells.Real time modulation of isoprenaline-induced cAMP acumination in HEK-EPAC cells by 1 μM immepip (A).Modulation of isoprenaline-induced cAMP acumination in HEK-EPAC cells by increasing concentrations of VUF25657 (B).HEK-EPAC cells stably expressing H3R were pre-stimulated with 10 nM isoprenaline for 10 min before ligand addition.The FRET signal of the EPAC-sensor was measured after 20 min treatment of indicated ligands.VUF25657 was either kept in dark or pre-irradiated at 560 nm for 3 min to release the BODIPY cage.Data are mean ± S.D. from four independent experiments performed, each in triplicate.

Figure 3 .
Figure 3. Optical modulation of VUF25657-induced cAMP inhibition in EPAC-H 3 R cells.Real time modulation of isoprenaline-induced cAMP acumination in HEK-EPAC cells by 1 µM immepip (A).Modulation of isoprenaline-induced cAMP acumination in HEK-EPAC cells by increasing concentrations of VUF25657 (B).HEK-EPAC cells stably expressing H 3 R were pre-stimulated with 10 nM isoprenaline for 10 min before ligand addition.The FRET signal of the EPAC-sensor was measured after 20 min treatment of indicated ligands.VUF25657 was either kept in dark or pre-irradiated at 560 nm for 3 min to release the BODIPY cage.Data are mean ± S.D. from four independent experiments performed, each in triplicate.

Table 1 .
Pharmacological data of photocaged H3R and H4R agonist.Statistical difference (p < 0.05) of pKi and pEC50 values for the pre-irradiated compound in comparison to that in dark state was determined by unpaired t-test with Welch's and indicated with an asterisk (*).Data are mean ± S.D. from three or four independent experiments performed in triplicate.N.D. = not determined.